AU2007203352A1 - Nucleic acid sequences and methods for the modification of plant gene expression - Google Patents

Description

19Jil. 2007 15:36 Baldwins 64 4 4736712 No. 4146 P. 3 -1-

AUSTRALIA

Patents Act 1990

SPECIFICATION

Name of Applicant: Actual Inventors: Address for Service: Invention Title: Rubicon Forests Holdings Limited and Arbogen, LLC Ranjan Perera, Stephen Rice, Clare Eagleton, Annette Lasham Baldwins Intellectual Property 16 Chisholm Street North Ryde Sydney Nucleic acid sequences and methods for the modification of plant gene expression The following statement is a full description of this invention, including the best method of performing it known to us:- 100820172 lDOC:JC:kd COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:36 Baldwins 64 4 4736712 No, 4146 P. 4 WO 01/98485 PCT/NZ01/00115 0 SNucleic adcid sequences and methods for the modifleation of plant gne expression "3 Technical Field of the Invention This invention relates to the regulation of polynucleotide transcription and/or expression.

More specifically, this invention relates to polynucleotide regulatory sequences isolated from Splants that are capable of initiating and driving the transcription of polynucleotides, and the use of IC) such regulatory sequences in the modification of transcription of endogenous and/or heterologous en polynucleotides and production of polypeptides. Polypeptide sequences arc also disclosed.

0 Any discussion of the prior art throughout the specification should in no way be Sconsidered as an admission that such prior art is widely known or forms part of common general 0knowledge in the field.

Background of the Invention Gene expression is regulated, in part, by the cellular processes involved in transcription.

During transcription, a single-stranded RNA complementary to the DNA sequence to be transcribed is formed by the action of RNA polyrerases. Initiation of transcription in eukaryotic cells is regulated by complex interactions between cas-acting DNA motifs, located within the gene to be transcribed, and frw-acting protein factors. Among the cis-acting regulatory regions are sequences of DNA, termed promoters, to which RNA polymerase is first bound, either directly or indirectly. As used herein, the term "promoter" refers to the 5' untranslated region of a gene that is associated with transcription and which generally includes a transcription start site. Other cisacting DNA motifs, such as enhancers, may be situated further up- and/or down-stream from the initiation site.

Both promoters and enhancers are generally composed of several discrete, often redundant elements, each of which may be recognized by one or more trons-acting regulatory proteins, known as transcription factors. Promoters generally comprise both proximal and more distant elements. For example, the so-called TATA box, which is important for the binding of regulatory proteins, is generally found about 25 basepairs upstream from the initiation site. The so-called CAAT box is generally found about 75 basepairs upstream of the initiation site. Promoters generally contain between about 100 and 1000 nucleotides, although longer promoter sequences are possible.

For the development of transgenic plants, constitutive promoters that drive strong transgene expression are preferred. Currently, the only available constitutive plant promoter that is widely used is derived from Cauliflower Mosaic Virus. Furthermore, there exists a need for plantderived promoters for use in transgenic food plants due to public conceptions regarding the use of viral promoters. Few gymnosperm promoters have been cloned and those derived from la COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. J l. 2007 15:37 Baldwins 64 4 4736712 No. 4146 P. WO 01/98485 PCT/NZ01/00115 0 0 angiosperms have been found to function poorly in gymnospems. There thus remains a need in the art for polynucleotide promoter regions isolated from plants for use in modulating transcription Sand expression of polynucleotides in transgenic plants.

Summary of the Invention SUnless the context clearly requires otherwise, throughout the description and the claims, It the words 'comprise', 'comprising' and the like are to be construed in an inclusive sense as Ce eC opposed to an exclusive or exhaustive sense; that is to say in the sense of "including but not limited Sto".

SBriefly, isolated polynucleotide regulatory sequences from eucalyptus and pine that are o involved in the regulation of gene expression are disclosed, together with methods for the use of such polynucleotide regulatory regions in the modification of expression of endogenous and/or hetrologous polynucleotides in transgenic plants. In particular, the present invention provides polynucleotides promoter sequences from 5' untranslated, or non-coding, regions of plant genes that initiate and regulate transcription of polynucleotides placed under their control, together with isolated polynucleotides comprising such promoter sequences.

In a first aspect, the present invention provides isolated polynucleotide sequences comprising a polynucleotide selected from the group consisting of: sequences recited in SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88.127; complements of the sequences recited in SEQ ID NO: 1-14, 20,22-62, 81-86, and 88-127; reverse complements of the sequences recited in SEQ ID NO: 1.14, 20, 22-62, 81-86 and 88-127; reverse sequences of the sequences recited in SED ID NO: 1-14, 20, 22-62, 81-86 and 88-127; sequences having either 40% 60%, 75% or identical nucleotides, as defined herein, to a sequence of probes and primers corresponding to the sequences set out in SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, polynucleotides comprising at least a specified number of contiguous residues of any of the polynucleotides identified as SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127; and extended sequences comprising portions of the sequences set out in SEQ ID NO: 1-14,20,22-62,81-86 and 88-127.

All of these are referred to herein as "polynucleotides of the present invention." In another aspect, the present invention provides genetic constructs comprising a polynucleotide of the present invention, either alone, or in combination with one or more additional polynucleotides of the present invention, or in combination with one or more known polynucleotides, together with cells and target organisms comprising such constructs.

In a related aspect, the present invention provides genetic constructs comprising, in the 3' direction, a polynucleotide promoter sequence of the present invention, a polynucleotide to be transcribed, and a gene termination sequence. The polynucleotide to be transcribed may comprise 2 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:37 Baldwins 64 4 4736712 No. 4146 P. 6 WO 01/98485 PCTINZO1/00115 an open reading frame of a polynucleotide that encodes a polypeptide of interest or it may be a non-coding, or untranslated, region of a polynucleotide of interest. The open reading frame may be orientated in either a sense or antisense direction. Preferably, the gene termination sequence is functional in a host plant Most preferably, the gene termination sequence is that of the gene of interest, but others generally used in the art, such as the Agrobaterium tumefaciens nopalin synthase terminator may be usefully employed in the present invention. The genetic construct may further include a marker for the identification of transformed cells.

In a further aspect transgenic cells, plant cells, comprising the genetic constructs of the present invention are provided, together with organisms, such as plants, comprising such transgenic cells, and fruits, seeds and other products, derivatives, or progeny of such plants.

Propagules of the inventive transgenic plants are included in the present invention. As used herein, the word "propagule" means any part of a plant that may be used in reproduction or propagation, sexual or asexual, including cuttings.

Plant varieties, particularly registerable plant varieties according to Plant Breeders' Rights, may be excluded from the present invention. A plant need not be considered a "plant variety" simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.

In yet another aspect, methods for modifying gene expression in a target organism, such as a plant, are provided, such methods including stably incorporating into the genome of the organism a genetic construct of the present invention. In a preferred embodiment, the target organism is a plant, more preferably a woody plant, most preferably selected from the group consisting of Eucalyptus grandis and Pinus radiata.

In another aspect, methods for producing a target organism, such as a plant, having modified polypeptide expression are provided, such methods comprising transforming a plant cell with a genetic construct of the present invention to provide a transgenic cell, and cultivating the transgenic cell under conditions conducive to regeneration and mature plant growth.

In another aspect the present invention provides a method for modifying a phenotype of a target organism, for example, a plant, comprising stably incorporating into the genome of the target organism a genetic construct comprising: a. a promoter sequence comprising a 5' region of a sequence of claim 1; b. a DNA sequence of interest and c. a gene termination sequence.

In yet a further aspect, the present invention provides isolated polynucleotides that encode ubiquitin. In specific embodiments, the isolated polynucleotides comprise a polynucleotide selected from the group consisting of: sequences recited in SEQ ID NO: 1 and 34; (b) complements of the sequences recited in SEQ ID NO: I and 34; reverse complements of the 3 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:37 Baldwins 64 4 4736712 No. 4146 P. 7 WO 01/98485 PCT/NZ010115 0 sequences recited in SEQ ID NO: 1 and 34; reverse complements of the sequences recited in SEQ ID NO: 1 and 34; reverse sequences of the sequence recited in SEQ ID NO: 1 and 34; and c' sequences having either 40%, 60%, 75% or 90% identical nucleotides, as defined herein, to a sequence of Polypeptides encoded by such polynucleotides are also provided, together with genetic constructs comprising such polynucleotides, and host cells and transgenic organisms, C^ for example plants, transformed with such genetic constructs. In specific embodiments, such Spolypeptides comprise a sequence provided in SEQ ID NO: 80 or 67, nr In yet further aspects, the present invention provides isolated polynucleotides comprising Ci the DNA sequence of SEQ ID NO: 21, or a complement, reverse complement or variant of SEQ ID SNO: 21, together with genetic constructs comprising such polynucleotides and cells transformed o with such sequences. As discussed below, removal of the sequence of SEQ ID NO: 21 from a polynucleotide that comprises the sequence of SEQ ID NO: 21 may enhance expression of the polynucleotide. Conversely, the inclusion of the sequence of SEQ ID NO: 21 in a genetic construct comprising a polynucleotide of interest may decrease expression of the polynucleotide.

In other aspects, methods for identifying a gene responsible for a desired function or phenotype are provided, the methods comprising transforming a plant cell with a genetic construct comprising a polynucleotide promoter sequence of the present invention operably linked to a polynucleotide to be tested, cultivating the plant cell under conditions conducive to regeneration and mature plant growth to provide a transgenic plant; and comparing the phenotype of the transgenic plant with the phenotype of non-transformed, or wild-type, plants.

In another aspect, the present invention provides a polynucleotide comprising a promoter sequence comprising a 5' region of a sequence of claim 1 operably linked to a heterologous polynucleotide. In specific embodiments, the heterologous polynucleotide comprises an open reading frame.

The above-mentioned and additional features of the present invention and the manner of obtaining them will become apparent, and the invention will be best understood by reference to the following more detailed description. All references disclosed herein are hereby incorporated by reference in their entirety as if each was incorporated individually.

4 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:38 Baldwins 64 4 4736712 No. 4146 P. 8 WO 01/98485 PCTINZ01/00115 0 0 SBrief Descrition of the Drawins Fig. 1 shows the expression in A. thaliana of the GUS gene in promoter reporter constructs containing either the supeubiquitin promoter with introns, the supemrbiquitin promoter without introns, or the CaMV 35S promoter. The GUS expression was measured l' by fluorimetic detemination of 4-methyl-umblliferone (MU) in protein extracts from these r n plants.

O Pig. 2 shows the expression of the GUS gene in tobacco plant protoplasts by. deletion constructs containing the superubiquitin promoter with or without the intron. The constructs o contained 1,103; '753; 573; 446; 368; and 195 bp upstream of the TATA sequence (bp C numbers 1,104-1,110 of SEQ ID NO: The GUS expression was measured by fluorimetric determination of 4-methyl-umbelliferone (MU) in protein extracts from these protoplasts, Fig. 3 shows the expression of the GUS gene in tobacco plant protoplasts by constructs containing P. radiara either the constitutive promoters Elongation factor-1 alpha, synthetase or-the superubiquitin promoter without the intron. The GUS expression was, measured by fluorimetric determination of 4-methyl-umbelliferone (MU) in protein extracts from these protoplasts.

Fig. 4 shows the expression of the GUS gene in tobacco plant protoplasts by a deletion construct containing a fragment of the E. grandis constitutive promoter Elongation factor-1 alpha.

Fig. 5 shows the expression in A. thaliana of the GUS gene in promoter reporter constructs containing the 3' UTR of the superubiquitin promoter in sense or antisense orientation together with either the supembiquitin promoter with intron, the superubiquitin promoter without intron, or the CaMV 35S promoter. The GUS expression was measured by fluorimetric determination of 4-methyl-umbelliferone (MU) in protein extracts from these plants.

Detailed Descrition of the Invention The present invention provides isolated polynucleotide regulatory regions that may be employed in the manipulation of plant phenotypes, together with isolated polynucleotides comprising such regulatory regions. More specifically, polynucleotide promoter sequences isolated from pine and eucalyptus are disclosed. As discussed above, promoters are components of the cellular "transcription apparatus" and are involved in the regulation of COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19Jul. 2007 15:38 Baldwins 64 4 4736712 NUo. 4146 P. 9 WO 01/98485 PCT/NZ01/00115 0 0 gene expression. Both tissue- and temporal-specific gene expression patterns have been Sshown to be initiated and controlled by promoters during the natural development of a plant.

The isolated polyncleotide promoter sequences of the present invention may thus be employed in the modification of growth and development of plants, and of cellular responses to external stimuli, such as environmental factors and disease pathogens.

if Using the methods and materials of the present invention, the amount of a specific e polypeptide of interest may be increased or reduced by incorporating additional copies of 0 genes, or coding sequences, encoding the polypeptide, operably linked to an inventive promoter sequence, into the genome of a target organism, such as a plant. Similarly, an Sincrease or decrease in the amount of the polypeptide may be.obtained by transforming the c. target plant with antisense copies of such genes.

The polynucleotides of the present invention were isolated from forestry plant sources, namely from Eucalyptus grandis and Pinus radiata, but they may alternatively be synthesized using conventional synthesis techniques. Specifically, isolated polynucleotides of the present invention include polynucleotides comprising a sequence selected from the group consisting of sequences identified as SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127; complements of the sequences identified as SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127; reverse complements of the sequences identified as SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127; at least a specified number of contiguous residues (x-mers) of any of the abovementioned polynucleotides; extended sequences corresponding to any of the above polynucleotides; antisense sequences corresponding to any of the above polynucleotides; and variants of any of the above polynucleotides, as that term is described in this specification.

In another embodiment, the present invention provides isolated polypeptides encoded by the polynucleotides of SEQ ID NO: 63-80, 87 and 130, The polynucleotides and polypeptides of the present invention were putatively identified by DNA and polypeptide similarity searches. In the attached Sequence Listing, SEQ ID NOS. 1-14, 20, 22-62, 81-86 and 88-127 are polynucleotide sequences, and SEQ ID NOS. 63-80, 87 and 130 are polypeptide sequences. The polynucleotides and polypeptides of the present invention have demonstrated similarity to promoters that are known to be involved in regulation of transcription and/or expression in plants. The putative identity of each of the inventive polynucleotides is shown below in Table 1, together with the untranslated region UTR) or putative promoter region (identified by residue number).

6 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:38 Baldwins 64 44736712 No. 4146 P. PCT/NZDI/OO115 WO 01/98485

TABLEI

so 1-2064 Rupee WquttlacodIngtecwoP adUTfRs 2 1-2w6 Suw Ubido poictr with insro 1-1226 SupertUiquidnprnmserWff4SoUfnuo 4 -1-431 Cal dMuon ontrol S -1-167 JX4cgsaeaia -pmffiC 6 -1-600 4-Cwz~msc-OcA Uigne (ACLa -1-363 5T and, Callufe inndias., 11 -1-248 Ladt specific 121-634 0-methyl nsftasc -396 Root spEcific 14 -P-73 nt Epecifin 22 61 1-406 Pollan CMaxprotin 1.350 Pollen Ilwpuge 24 -1-49 Miecn oargen 64 1-284 Fale allerpen 26 65 1-77 Awdeo-inducad protei: 27 1-74 Auzln-Wdc pnmat 28 (A 1-99 Auxio-iuducedwpotein 29 -1-92? Plower gpecffic i 1-411 Flower opecfic 31 -1-178 Plower specific COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19-Jul. 2007 15:39 Baidwins 64 4 4736712 No. 4146 P. 11 PICT/NZ1/0O115 WO O1193485 32 1-179 flower SPecMfc 33 -1-178 Plowor spedl 63 1-81 Glerleys3'hah± uy-rosas 36 S9 1.04ni C wuhydicss 37 -1-648 Iflthivon redua 31 1-2IG Isoflsonsraductaa 39 -1-382 Cy en dc-3-pbosphMt debydrogmea 70 1-943 Bad apacif 41 -1-313 Uxya-apscfi 42 -1-715 ZYle-apeclfi 44 1-35 Xylom-epeeiflo 71 1-180 Modutem-speclf 46 72 1-238 Snwcoc-flwproui 47 -1-91 Saoasonc-likpozf 48 -1-91 SnaOMAnccllkpot 49 ISP pollen-peaiflo so -148 pcllcn-apcit 51 73 1-55 PoUgnapeci&e 52 74 1.575 Pafln-Specifi 53. 75 1-35 ol-seia 54 *1-335 NadulM bnmalo paflen speifi 53 1-336 Nadialic bonolog polle opec~fic 56 76 1-157 Sac=*e synthas 57 77 1-44 Sucrne yntan 58 -320 1 S==wsyntua COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. El. 2007 15:39 Baidwins 64 44736712 No. 4146 P. 12 WO 011984M5 PCT/NZO1/0115 59 -1-311 Plower speaifice 78 1-64 0-metbyl trUdarns 61 79 1-112 Mongatc fen=iA 62 1-420 ElOepdon arorA 81 -I 1~hozaologa 32 MW? homologne 83 MU homoo 84 -O oro Gpe 36 87 1-87 MU' hoMningU IN8 1-1156 Cbsleonesyrha ID- 1-2590 UW mown fio~wspeifi -1-1172 Unkwnwn flowet specifi 91 -446 SUMMos ynthms 92 -1-2119 Uulumown xylem spento 93 -1-2371 Ulwaldcbydc-3-Pouphata5 dahydrogensz 94 -1-1406 tlnhowcu pollen speoific -2546 Pinu radIat fmnl.peic prnin (PrULE) 96 -1-4726 ptnidia4 wn1.-apcifie protein (PrbLALEI) 97 -1-655 UD? glucose glycwyhrnaifaru 93 1-462 Mrpiln Peor Al 99. 1.222 Eonpdon~owA1 200 -1-410 8-sdmoos~medon hernba 101 -1-482 S-MdansyIltwOtlmS aymhutue 1-230 8-adsnOsylhionlz synrubrss 103 -S6 'CDP glucos 6 daydrogm 104 I -M5 Hypotbcd rtin COMS ID No: ARCS-i 53531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jil. 2007 15:39 Baldwin 64 44736712 No. 4146 P. 13 PCT/NZ01100115 WO 0119843 PWIYNf 1. N _M 1-32 Iis 106 1-342 InSaw 106-1-4 Anbfopatau-Wcs 104 1-362 AxaEWzulnM-fc 2 109 -1-326 Anbinopinunhikse-2 110 -1-296 Amok Abcptr-Ukokiuuau 111 1-723 ROOt R"Wpm-Uke klei 112 -1-1301 Pinwr4aLipidTfff&Pfl2ChrLTfl) 113 1 1-1008 Caflo add O-mtylnAFisu 114 -1-850 MP glucose Slcsyltmftrn 115 1-916 lID? glncoa6 dchdrpnw 116 -1-947 Laa 1 117 -1-1766 Arabinogalaotnllh-1 Ila- 1-1614 Corntua lip .0 w~Pmmnfcg Pator I O?1) 120 -1-901 Apmous.

121) 1-1w24 flt IA Tmmmciptiouktttt~ 122 -1-959 flmughtlmdadimdin 19 123 -1L140 Sut ToleroretPfto M2 130 89 Low Temparae Induced LTI-1G 125 -1143 xY1*mapsCMCrccppr-M lnma 226 1-1,D47 Root upedic 127 -1-3,U32 B~UmplozFmotoz 1-alphs In erIO a~bodjment, the preset invention provides polynuo~de seqUences isolted from Pbmw radia and hca~ypzw rwzd that encode a ribiqulin polypeptide. The fallength aequenoc of the ubiquitin polynucoocdes isolated from Pmw radixa is provided in SEQ ID NO! 1, with the egnetios of the prmoW region including anilion being provided COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:39 Baldwins 64 4 4736712 No.4146 P. 14 WO 01/98485 PCT/NZ01/0D115 0 0 in SEQ ID NO: 2 and the sequence of the promoter region excluding the intron being Sprovided in SEQ ID NO: 3. The sequence of the ubiquitin polyoucleotide isolated from Eucalyptus grandis is provided in SEQ ID NO: 34. In a related embodiment, the present invention provides isolated polypeptides encoded by the isolated polynucleotides of SEQ ID NO: 1 and 34, including polypeptides comprising the sequences of SEQ ID NO: 80 and 67.

C The term "polynucleotide(s)," as used herein, means a single or double-stranded C polymer of deoxyribonucleotide or ribonucleotide bases and includes DNA and Scorresponding RNA molecules, including HnRNA and mRNA molecules, both sense and anti-sense strands, and comprehends cDNA, genomic DNA and recombinant DNA, as well as Swholly or partially synthesized polynucleotides. An EnRNA molecule contains introns and l corresponds to a DNA molecule in a generally one-to-one manner. An mRNA molecule corresponds to an HnRNA and DNA molecule from which the introns have been excised. A polynucleotide may consist of an entire gene, or any portion thereof. Operable anti-sense polynucleotides may comprise a fragment of the corresponding polynucleotide, and the definition of "polynucleotide" therefore includes all such operable anti-sense fragments.

Anti-sense polynucleotides and techniques involving ani-sense polynucleoides are well known in the art and are described, for example, in Robinson-Benion et al. "Antisense techniques," Methods in Enzymol. 254(23):363-375, 1995; and Kawasakl et al., in Arjfic.

Organs 20(8):836-848, 1996.

All of the polynucleotides and polypeptides described herein are isolated and purified, as those tems are commonly used in the art. Preferably, the polypeptides and polynucleotdes are at least about 80% pure, more preferably at least about 90% pure, and most preferably at least about 99% pure.

The definition of the terms "complement", "reverse complement" and "reverse sequence", as used herein, is best illustrated by the following example. For the sequence AOOACC the complement, reverse complement and reverse sequence are as follows: Complement 3' TCCTGG Reverse complement 3' GOTCCT Reverse sequence 5' CCAGGA 3' Some of the polynucleotides of the present invention are "partial" sequences, in that they do not represent a full-length gene encoding a full-length polypeptide. Such partial sequences may be extended by analyzing and sequencing various DNA libraries using primers and/or probes and well known hybridization and/or PCR techniques. Partial 11 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:40 Baldwins 64 4 4736712 No. 4146 P. WO 011/948 PCT/NZO1/0011S 0 0 c, sequences may be extended until an open reading frame encoding a polypeptide, a full-length Spolynucleotide and/or gene capable of expressing a polypeptide, or another useful portion of the genome is identified. Such extended sequences, including full-length polynucleotides and genes, are desdbed as "corresponding to" a sequence identified as one of the sequences of SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, or a variant thereof, or a portion of one of c the sequences of SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, or a variant thereof, when C the extended polynucleotide comprises an identified sequence or its variant, or an identified o contiguous portion (x-mer) of one of the sequences of SBQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, or a variant thereof. Such extended polynucleotides may have a length of from Sabout 50 to about 4,000 nucleic acids or base pairs, and preferably have a length of less than Sabout 4,000 nucleic acids or base pairs, more preferably yet a length of less than about 3,000 nucleic acids or base pairs, mor preferably yet a length of less than about 2,000 nucleic acids or base pair. Under some circumstances, extended polynucleotides of the present invention may have a length of less than about 1,800 nucleic acids or base pairs, preferably less than about 1,600 nucleic acids or base pairs, more preferably less than about 1,400 nucleic acids or base pairs, more preferably yet less than about 1,200 nucleic acids or base pairs, and most preferably less than about 1,000 nucleic acids or base pairs.

Similarly, RNA sequences, reverse sequences, complementary sequences, antisense sequences, and the like, corresponding to the polynucleotides of the present invention, may be routinely 'ascertained and obtained using the cDNA sequences identified as SEQ ID NO: 1-14, 20,22-62, 81-86 and 88-127.

The polynucleotides identified as SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, and their extensions, may contain open reading frames ("ORFs") or partial open reading frames encoding polypeptides. Additionally, open reading frames encoding polypeptides may be identified in extended or full length sequences coresponding to the sequences set out as SEQ ID NO; 1-14, 20, 22-62, 81-86 and 88-127. Open reading frames may be identified using techniques that are well known in the art. These techniques include, for example, analysis for the location of known start and stop codons, most likely reading frame identification based on codon frequencies, etc. Suitable tools and software for ORF analysis include, for example, "GeneWise", available from The Sanger Center, Welcome. Trust Genome Campus, Hinxton, Cambridge, CB10 ISA, United Kingdom; "Diogenes", available from Computational Biology Centers, University of Minnesota, Academic Health Center, .UMHG Box 43, lMinneapolis MN 55455 and "GRAIL", available from the Informatics 12 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:40 Baldwins 64 4 4736712 No.4146 P. 16 WO 01/98485 PCT/NZ01100115 0 0 Group, Oak Ridge National Laboratories, Oak Ridge, Tennessee TN. Open reading frames and portions of open reading frames may be identified in the polynacleotidea of the present Sinvention. Once a partial open reading frame is identified, the polynucleotide may be extended in the area of the partial open reading frame using techniques that are well known in the art until the polynucleotide for the full open reading frame is identified. Thus, open reading frames encoding polypeptides may be identified using the polynucleotides of the c n present invention.

O Once open reading frames are identified in the polynacleotides of the present invention, the open reading frames may be isolated and/or synthesized. Expressible genetic Sconstructs comprising the open reading frames and suitable promoters, initiators, terminators, Setc., which are well known in the art, may then be constructed. Such genetic constucts may be introduced into a host cell to express the polypeptide encoded by the open reading frame.

Suitable host cells may include various prokaiyotic and eukaryotic cells, including plant cells, mammalian cells, bacterial cells, algae and the like.

Polypeptides encoded by the polynucleotides of the present invention may be expressed and used in various assays to determine their biological activity. Such polypeptides may be used to raise antibodies, to isolate corresponding interacting proteins or other compounds, and to quantitatively detrmine levels of interacting proteins or other compounds.

The term "polypeptide", as used herein, encompasses amino acid chains of any length including full length proteins, wherein amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention may be isolated and purified natural products, or may be produced partially or wholly using recombinant techniques. The term "polypeptide encoded by a polynucleotide" as used herein, includes polypeptides encoded by a nacleotide sequence which includes the partial isolated DNA sequences of the present invention.

In a related aspect, polypeptides are provided that comprise at least a functional portion of a polypeptide having a sequence selected from the group consisting of sequences provided in SEQ ID NO: 63-80, 87 and 130, and variants thereof. As used herein, the "functional portion" of a polypeptide is that portion which contains the active site essential for affecting the function of the polypeptide, for example, the portion of the molecule that is capable of binding one or more reactants. The active site may be made up of separate portions present on one or more polypeptide chains and will generally exhibit high binding affinity.

13 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:40 Baldwins 64 4 4736712 No. 4146 P. 17 WO 01/98485 PCT/NZ01/00115 0 0 Functional portions of a polypeptide may be identified by first preparing fragments of Z the polypeptide by either chemical or enzymatic digestion of the polypeptide, or by mutation analysis of the polynucleotide that encodes the polypeptide and subsequent expression of the resulting mutant polypeptides. The polypeptide fragments or mutant polypeptides are then tested to determine which portions retain biological activity, using for example, the representative assays provided below. A functional portion comprising an active site may be c made up of separate portions present on one or more polypeptide chains and generally Sexhibits high substrate specificity.

Portions and other variants of the inventive polypeptides may be generated by o synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino NC acids, and generally fewer than about 50 amino acids, may be generated using techniques well known to those of ordinary skill in the art. For example, such polypeptides may be synthesized using any of the commercially available solid-phase techniques, such as the Merifield solid-phase synthesis method, wherein amino acids are sequentially added to'a growing amino acid chain. (Merrifeld, J. Am. Chem. Soc. 85: 2149-2154, 1963). Equipment for automated synthesis of polypeptides is commercially available from suppliers such as Perin Elmer Applied Biosystems, Inc. (Foster City, California), and may be operated according to the manufacturer's instructions. Variants of a native polypeptide may be prepared using standard mutagenesis techniques, such as oligonucleotide-directed sitespecific mutagenesis (Kunkel, Proc. Nal. Acad. Sci, USA 82:488492, 1985). Sections of DNA sequences may also be removed using standard techniques to permit preparation of truncated polypeptides.

As used herein, the term "variant" comprehends nucleotide or amino acid sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added. Variants may be naturally occuring allelic variants, or non-naturally occurring variants. Variant sequences (polynucleotide or polypeptide) preferably exhibit at least 50%, more preferably at least 75%, and most preferably at least 90% identity to a sequence of the present invention, The percentage identity is determined by aligning the two sequences to be compared as described below, determining the number of identical residues in the aligned portion, dividing that number by the total number of residues in the inventive (queried) sequence, and multiplying the result by 100.

14 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jil. 2007 15:41 Baldwins 64 4 4736712 No.4146 P. 18.

WO 01/98485 PCT/NZO1/00115 0 0 Polynucleotide and polypeptide sequences may be aligned, and percentage of identical residues in a specified region may be determined against other polynucleotide and 0polypeptide sequences, using computer algorithms that are publicly available. Two exemplary algorithms for aligning and identifying the similarity of polynuclotide sequences are the BLASTN and PASTA algorithms. Polynucleotides may also be analyzed using the t BLASTX algorithm, which.compares the six-frame conceptual translation products of a c n nucleotide query sequence (both strands) against a protein sequence database. The similarity O of polypeptide sequences may be examined using the BLASTP algorithm. The BLASTN algorithm Version 2.0.4 [Feb-24-1998], Version 2.0.6 [Sept-16-1998] and Version 2.0.11 S[Jan-20-2000], set to the default parameters described in the documentation and distributed with the algorithm, are preferred for use in the determination of polynucleotide variants according to the present invention. The BLASTP algorithm, is preferred for use in the determination of polypeptide variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul, et at,, "Gapped BLAST and PSI-BLAST' a new generation of protein database search programs," Nucleic Acids Res. 25: 3389-3402, 1997. The BLASTN software is available on the NCBI anonymous FTP server (ftpl:/ncbi.nlm.nih.gov) under /blast/executables/ and is available from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, MD 20894

USA,

The FASTA software package is available from the University of Virginia (University of Virginia, PO Box 9025, Charlottesville, VA 22906-9025). Version 2.0u4, February 1996, set to the default parameters described in the documentation and distributed with the algorithm, may be used in the determination of variants according to the present invention.

The use of the PASTA algorithm is described in Pearson and Lipman, Improved Tools for Biological Sequence Analysis," Proc. Natl. Acad. Sci. USA 85: 2444-2448, 1988; and Pearson, "Rapid and Sensitive Sequence Comparison with FASTP and PASTA," Method in Enzymol. 183: 63-98, 1990..

The following running parameters are preferred for determination of alignments and similarities using BLASTN that contribute to the E values and percentage identity for polynucleotide sequences: Unix running command: blastall -p blastn -d embldb -e 10 -00 ED -r 1 -v 30 -b 30 -i queryseq -o results; the parameters are: -p Program Name [String]; d Database [String]; -e Expectation value [Real]; -0 Cost to open a gap (zero invokes COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:41 Baldwins 64 4 4736712 No. 4146 P. 19 WO 01/98485 PCTINZ01/O0115 0 0 rdefault behavior) [nteger]; -E Cost to extend a gap (zero invokes default behavior) [Integer]; Z -r Reward for a nucleotide match (BLASTN only) [Integer]; -v Number of one-line descriptions [Integer]; -b Number of alignments to show [Integpr]; -i Query File File In]; and -o BLAST report Output File [File Out] Optional, The following running parameters are preferred for determination of alignments and iy similarities using BLAST.P that contribute to the B values and percentage identity of c n polypeptide sequences: blastall -p blastp -d swissprotdb -e 10 -G 0 -B 0 -v 30 -b 30 -i O queryseq -o results; the parameters are: -p Program Name [String]; -d Database [String]; -e Expectation value [Real]; -G Cost to open a gap (zero invokes default behavior) [Integer]; o -E Cost to extend a gap (zero invokes default behavior) [Integer]; -v Number of one-line

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descriptions [Integer]; -b Number of alignments to show [Integer]; -I Query File [Pile In]; -o BLAST report Output File [File Out] Optional.

The "hits" to one or more database sequences by a queried sequence produced by BLASTN, PASTA, BLASTP or a similar algorithm, align and identify similar portions bf sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence, The BLASTN, FASTA and BLASTP algorithms also produce "Expect" values for alignments. The Expect value.(E) indicates the number of hits one can "expect" to see over a certain number of contiguous sequences by chance when searching a database of a certain size. The Expect value is used as a significance threshold for determining whether the hit to a database, such as the preferred EMBL database, indicates true similarity. For examplei an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the EMBL database, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance, By this criterion, the aligned and matched portions of the polynucleotide sequences then have a probability of 90% of being the same. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in the EM BL database is 1% or less using the BLASTN or FASTA algorithm.

According to one embodiment, "variant" polynucleotides and polypeptides, with reference to each of the polynucleoddes and polypeptides of the present invention, preferably comprise sequences having the same number or fewer nucleic or amino acids than each of the polynucleotides or polypeptides of the present invention and producing an E value of 0.01 or 16 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:41 Baldwins 64 4 4736712 No. 4146 P. WO 01/98485 PCT/NZ01/00115 0 0

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_less when compared to the polynucleotide or polypeptide of the present invention. That is, a Svariant polyncleotide or polypeptide is any sequence that has at least a 99% probability of being the same as the polynucleotide or polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTN, FASTA, or BLASTP algorithms set at parameters described above. According to a prefered embodiment, a variant polynucleotide lt is a sequence having the same number or fewer nucleic acids than a polynucleotide of the e 'present invention that has at least a 99% probability of being the same as the polynucleotide o of the present invention, measured as having an E value of 0.01 or less using the BLASTN or PASTA algorithms set at parameters described above. Similarly, according to a preferred o embodiment, a variant polypeptide is a sequence having the same number or fewer amino acids than a polypeptide of the present invention that has at least a 99% probability of being the same as a polypeptide of the present invention, measured as having an E value of 0.01 or less using the BLASTP algorithm set at the parameters described above.

Alternatively, variant polynucleotides of the present invention hybridize to the polynucleotide sequences recited in SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, or complements, reverse sequences, or reverse complements of those sequences under stringent conditions. As used herein, "stringent conditions" refers to prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65 6 C, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in lX SSC, 0.1% SDS at 65" C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at The present invention also encompasses polynucleotides that differ from the disclosed sequences but that, as a consequence of the discrepancy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. Thus, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, or complements, reverse sequences, or reverse complements thereof, as a result of conservative substitutions are contemplated by and, encompassed within the present invention.

Additionally, polynucleotides comprising sequences that differ from the polynucleotide sequences recited in SBQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, or complements, reverse complements or reverse sequences thereof, as a result of delotions and/or insertions totaling less than 10% of the total sequence length are also contemplated by and encompassed within the present invention. Similarly, polypeptides comprising sequences that differ from the polypeptide sequences recited in SEQ ID NO: 63-80, 87 and 130, as a result of amino 17 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19-Jul. 2007 15:42 Baldwins 64 4 4736712 No. 4146 P. 21 WO 01/98485 PCT/NZ01/00115 0 0 acid substitutions, insertions, and/or deletions totaling less than 10% of the total sequence Slength are contemplated by an encompassed within the present invention. In certain embodiments, variants of the inventive polypeptides and polynucleotides possess biological activities that are the same or similar to those of the inventive polypeptides or polynucleotides. Such variant polynucleotides function as promoter sequences and are thus tf) capable of modifying gene expression in a plant.

en The polynacleotides of the present invention may be isolated from various libraries, O or may be synthesized using techniques that are well known in the art. The polynucleotides may be synthesized, for example, using automated oligonucleotide synthesizers SBeckman Oligo 1000M DNA Synthesizer) to obtain polynucleotide segments of up to 50 or more nucleic acids. A plurality of such polynucleotide segments may then be ligated using standard DNA manipulation techniques that are well known in the art of molecular biology.

One conventional and exemplary polynucleotide synthesis technique involves synthesis of a single stranded polynucleotide segment having, for example, 80 nucleic acids, and hybrdizing that segment to a synthesized complementary 85 nucleic acid segment to produce a 5-nucleotide overhang. The next segment may then be synthesized in a, similar fashion, with a 5-nucleotide overhang on the opposite strand. The "sticky" ends ensure proper ligation when the two portions are hybridized In this way, a complete polynucleotide of the present invention may be synthesized entirely in vitro.

Polynucleotides of the present invention also comprehend polynucleotides comprising at least a specified number of contiguous residues (x-mers) of any of the polynucleotides identified as SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, complements, reverse sequences, and reverse complements of such sequences, and their variants. Similarly, polypeptides of the present invention comprehend polypeptides comprising at least a specified number of contiguous residues (x-mers) of any of the polypeptides identified as SEQ ID NO: 63-80, 87 and 130, and their variants. As used herein, the ten "x-mer," with reference to a specific value of refers to a sequence comprising at least a specified number of contiguous residues of any of the polynucleotides identified as SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, or the polypeptides identified as SEQ ID NO: 63-80, 87 and 130. According to preferred embodiments, the value ofx is preferably at least 20, more preferably at least 40, more preferably yet at least 60, and most preferably at least 80. Thus, polynucleotides and polypeptides of the present invention comprise a 20-mer, a 40-mar, a mer, an 80-mar, a 100-mer, a 120-mer, a 150-mer, a 180-mer, a 220-mer, a 250-mer, a 300- 18 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:42 Baldwins 64 4 4736712 No.4146 P. 22 WO 01/98485 PCT/NZ01/0011 0 0

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mer, 400-mar, 500-mer or 600-mer of a polynucleotide or polypeptide identified as SEQ ID SNO: 1-14, 20, 22-62, 81-86 and 88-127, and variants thereof.

As noted above, the inventive polynucleotide promoter sequences may be employed in genetic constructs to drive transcription and/or expression of a polynucleotide of interest.

The polynuoleotide of interest may be either endogenous or heterologous to an organism, for In example a plant, to he transformed. The inventive genetic constructs may thus be employed en to modulate levels of transcription and/or expression of a polynucleotide, for example gene, O that is present in the wild-type plant, or may be employed to provide transcription and/or expression of a DNA sequence that is not found in the wild-type plant.

SIn certain embodiments, the polynucleotide of interest comprises an open reading frame that encodes a target polypeptide. The open reading frame is inserted in the genetic construct in either a sense or antisense orientation, such that transformation of a target plant with the genetic construct will lead to a change in the amount of polypeptide compared to the wild-type plant. Transformation with a genetic construct comprising an open reading franie in a sense orientation will gnerally result in over-expression of the selected polypeptide, while transformation with a genetic construct comprising an open reading frame in an antisense orientation will generally result in reduced expression of the selected polypeptide.

A population of plants transformed with a genetic construct comprising an open reading frame in either a sense or antisense orientation may be screened for increased or reduced expression of the polypeptide in question using techniques well known to those of skill in the art, and plants having the desired phenotypes may thus be isolated.

Alternatively, expression of a target polypeptide may be inhibited by inserting a portion of the open reading frame, in either sense or antisense orientation, in the genetic construct. Such portions need not be full-length but preferably comprise at least 25 and.more preferably at least 50 residues of the open reading frame. A much longer portion or even the full length DNA corresponding to the complete open reading frame may be employed. The portion of the open reading frame does not need to be precisely the same as the endogenous sequence, provided that there is sufficient sequence similarity to achieve inhibition of the target gene. Thus a sequence derived from one species may be used to inhibit expression of a gene in a different species.

In further embodiments, the inventive genetic constructs comprise a polynucleotide including an untranslated, or non-coding, region of a gene coding for a target polypeptide, or a polynucleotide complementary to such an untranslated region. Examples of untranslated 19 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:42 Baldwins 64 4 4736712 No.4146 P. 23 WO 01/98485 PCT/NZ01/00115 0 0 regions which may be usefully employed in such constructs include intron and Suntranslated leader sequences. Transformation of a target plant with such a genetic construct may lead to a redution in the amount of the polypeptide expressed in the plant by the process of cosuppression, in a manner similar to that discussed, for example, by Napoli et al., Plant Cell 2:279-290, 1990 and de Carvalho Niebel et al., Plant Cell 7:347-358, 1995.

SAlternatively, regulation of polypeptide expression can be achieved by inserting cn appropriate sequences or subsequences DNA or RNA) in ribozyme constructs (McIntyre o and Manners, Transgenic Res, 5(4):257-262, 1996). Ribozymes are synthetic RNA molecules that comprise a hybridizing region complementary to two regions, each of which o comprises at least 5 contiguous nucleotides in a mRNA molecule encoded by one of the C inventive polynucleotides. Ribozymes possess highly specific endonuclease activity, which autocatalytically cleaves the mRNA.

The polynucleotide of interest, such as a coding sequence, is operably linked to a polynucleotide promoter sequence of the present invention such that a host cell is able to transcribe an RNA from the promoter sequence linked to the polynucleotide of interest The polynucleotide promoter sequence is generally positioned at the 5' end of the polynucleotide to be transcribed. Use of a constitutive promoter, such as the Pinus radiata ubiquitin polynucleotide promoter sequence of SEQ ID NO: 2 and 3' or the Eucalyptus grandis ubiquitin polynucleotide promoter sequence contained within SEQ ID NO: 34, will affect transcription of the polynucleotide of interest in all parts of the transformed plant. Use of a tissue specific promoter, such as the leaf-specific promoters of SEQ ID NO: 9-11, the rootspecific promoters of SEQ ID NO: 13 and 14, the flower-specific promoters of SEQ ID NO: 29-33, 59 and 89-90, the pollen-specific promoters of SEQ ID NO: 49-55 and 94, the budspecific promoter of SEQ ID NO: 40 or the meristem-specific promoter of SEQ ID NO: will result in production of the desired sense or antisense RNA only in the tissue of interest.

Temporally regulated pmmoters, such as the xylogenesis-specific promotrs of SEQ ID NO: 41-44 and 92, can be employed to effect modulation of the rate of DNA transcription at a specific time during development of a transformed plant. With genetic constructs employing inducible gene promoter sequences, the rate of DNA transcription can be modulated by exteal stimuli, such as light, heat, anaerobic stress,.alteration in nutrient conditions and the like.

The inventive genetic constructs further comprise a gene termination sequence which is located 3' to the polynucleotide of interest, A variety of gene termination sequences which COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jl. 2007 15:43 Baldwins 64 4 4736712 No.4146 P. 24 WO 01/9845 PCTINZ01/00115 0 0 may be usefully employed in the genetic constructs of the present invention ar well known in the art. One example of such a gene termination sequence is the 3' end of the Agrobacterium tumefaciens nopaline synthase gene. The gene temination sequence may be endogenous to the target plant or may be exogenous, provided the promoter is functional in the target plant For example, the termination sequence may be from other plant species, plant viruses, bacterial plasmids and the like.

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n The genetic constucts of the present invention may also contain a selection marker o that is effective in cells of the target organism, such as a plant, to allow for the detection of transformed cells containing the inventive construct. Such markers, which are well known in Sthe art, typically confer resistance to one or more toxins. One example of such a marker is the NPTI gene whose expression results in resistance to kanamycin or hygromycin, antibiotics which are usually toxic to plant cells at a moderate concentration (Rogers et al., in Weissbach A and H, eds. Merhods for Plant Molecular Biology, Academic Press Inc.: San Diego, CA, 1988). Transformed cells can thus be identified by their ability to grow in media containing the antibiotic in question. Alternatively, the presence of the desired construct in transformed cells can be determined by means of other techniques well known in the art, such as Southern andWestern blots.

Techniques for operatively linting the components of the inventive genetic constructs are well known in the art and include the' use of synthetic linkers containing one or more restriction en'donuclease sites as described, for example, by Sambrook et (Molecular cloning: a laboratory manual, CSHL Press: Cold Spring Harbor, NY, 1989). The genetic Sconstruct of the present invention may be linked to a vector having at least one replication system, for example E. coil, whereby after each manipulation, the resulting construct can be cloned and sequenced and the correctness of the manipulation determined.

The genetic constructs of the present invention may be used to transform a variety of target organisms including, but not limited to, plants. Plants which may be transformed using the inventive constructs include both monocotylodonous angiosperms grasses, con, grains, oat, wheat and barley) and dicotyledonous angiosperms Arabidopsis, tobacco, legumes, alfalfa, oaks, eucalyptus, maple), and Gymnosperms Scots pine; see Aronen, Finnish Forest Res. Papers, Vol. 595, 1996), white spruce (Uis et Biotechnology 11:84- 89, 1993), and larch (Huang et al., In Vitro Cell 27:201-207, 1991). In a preferred embodiment, the inventive genetic constructs are employed to transform woody plants, herein defined as a tree or shrub whose stem lives for a number of years and increases in diameter 21 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19.Jul. 2007 15:43 19. Ju. 2%]15:43 Baidwins 64 44736712N.44 P.2 No. 4146 P WQD 01/98485 PICTINZO1OOI1S each year by the addition of woody fissue. Preferably the target plant is selected from the group consisting of eucalyptus and pine species, most preferably from the group consisting of Eucalyptus gmandis and Finus racda Other species which may be usefully transformed with the genetic construts of the present invention include, but ame not lim~ited to: pines such as FPinns bansang, Finn brusi, Fins caflbaea, Pius clausa, Pinus canto fla, Finus ccndtci*4 Finn echinat, Finna aldarica, Finus elliosi, Finus Jeff rei, Pinus lambeniana Frns en in~onticola, Finns n*ga, Pinus palustnu, Pinus pinaster, Finn ponderosa, Finus ,-euinosa, o FPinus igi4a Pbnus serotina, .Pinw strobx's, Pinus qylvestris, Finus taeda, Pimas virginiana; other gymnosperms, such as Able: ainabilis, Able: balramnea, Able: concolor, Able: grandis, o Abie, lasiocarp, Abies magnifica, Able: pro cera Chamaecypcri: lansoniona, Chamaecyparis noorkosersis, Charnaucypanis ttryoides, -Hunipents virginiano. Larix decidua, Larix laricina, Larix leptolepia, Laflx occidenalis, Larix siberica, Llbocednw dccun'ens, Plcera able, Pica engeirnanni, Ficca glasses, Fles marianA, Pica pungens, Fieea rubens, Piea uirchensis, Pseudotsuga menslesl, Sequoia gigansca, Sequoia .sempennrens, Taxodiwn distichrcn, Tsuga c~madensis, Tsugez heterophylla, Tsuga mertenriana, Mida accidentalUs, Thuja pliewa; and Eucalypti, such as Eucalyptus aIM, Eucalyptus bancrofhli Eucalyptus botyroides, Eucalypwu bridgesiana. Eucalyptus calophylla, Eucalyptus carnaldulenuis, Eucalyptus cin-iodorz. Eucalyptus ciadocalyx, E~ucalyptus coccifra, Eucalyptus cuisli, EPucalyptu cLib.wsplearuz, Eucalyptus deghupta, Eucalypni delagatensis, Eucalyptus diversicolar, 'Eucalyptus dunni4 EucalypWu flci/Mia, Eucalyptus globzdus, Eucalyptu gornphocephala, Eucalyptus guraii, Eucalyptus henry4 Eucalyptus laavopinea, Eucalyptus macardhurii, Eucalyptus macrorhyncha, Eucalyptus snacidata, Eucalyptus marginata, Eucalyptus rnegacwpa,' Euealypnw melliodora, Eucalyptus nicIzoWl, Eucalyptus nftens Eucalyptus nova-anglica, Eucalyptus obliqisa, Eucalyptus obusiflora, Eucalyptus oreades, Eucalyptu passoflora, Eucalyptus polybracee, Eucalyptus regnans, Eucalyptus resinifera Eucalyptus robusra. Eucalyptus nsdis, Eucalyptus saligna, Eucalyptus siderorjlos.

Eucalyptus stuartiana, Rualyptus teretiaornZr Eucalyptus torelliana, Eucalyptus urnigera, Eucalyptus isrophylla, Eucalyptus virninalis, Eucalyptu viridis, Eucalyptus wandoo and Eucayptus yournanni; and hybrids of any of these species.

Techniques for stably incorporating genet constructs into the geno=e of target plants ar well known in the art and include Agrobacterium tLOJ2efiZ~iefl mediated introduction, electroporatian, protoplast fusion, injection into reproductive organs, injection into immature embryos, high velocity projectile introduction and the like. The choice of 22 COMSIDNa: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul 2007 15:44 Baldwins 64 4 4736712 No.4146 P. 26 WO 01/98485 PCT/NZ01/00115 0 0 technique will depend upon the target plant to be transformed. For example, dicotyledonous Splants and certain monocots and gymnosperms may be transformed by Agrobacterimn Ti 0\ plasmid technology, as described, for example by Bevan, Nucleic Acids Res. 12:8711-8721, 1984. Targets for the introduction of the genetic constructs of the present invention include tissues, such as leaf tissue, dissociated cells, protoplasts, seeds, embryos, meristematic In regions; cotyledons, hypocotyls, and the like. The preferred method for transforming en eucalyptus and pine is a biolistic method using pollen (see, for example, Aronen, Finnish 0 Forest Res. Papers, Vol. 595, 5 3pp, 1996) or easily regenerable embryonic tissues.

Once the cells are transformed, cells having the inventive genetic construct 0 incorporated in their genome may be selected by means of a marker, such as the kanamycin resistance marker discussed above. Transgenic cells may then be cultured in an appropriate medium to regenerate whole plants, using techniques well known in the art. In the case of protoplasts, the cell wall is allowed to reform under appropriate osmotic conditions. In the case of seeds or embryos, an appropriate germination or callus initiation medium it employed For explants, an apprpriate regeneration medium is used. Regeneration of plants is well established for many species. For a review of regeneration of forest trees see Dunstan et al., "Somatic embryogenesis in woody plants," in Thorpe TA, ed., In Vitro Embryogenesis of Plants (Current Plant Science and Biotechnology in Agriculture Vol. 20), Chapter 12, pp. 471-540, 1995, Specific protocols for the regeneration of spruce are discussed by Roberts et al., "Somatic embryogenesis of spruce," in Redenbaugh K, ed., Synseed: applications of synthetic seed to crop improvement, CRC Press: Chapter 23, pp. 427-449, 1993).

STransformed plants having the desired phenotype may be selected using techniques well known in the art. The resulting transformed plants may be reproduced sexually or asexually, using methods well known in the art, to give successive generations of transgenic plants.

As discussed above, the production of RNA in target cells can be controlled by choice of the promoter sequence, or by selecting the number of functional copies or the site of integration of the polynucleotides incorporated into the genome of the target host. A target organism may be transformed with more than one genetic construct of the present invention, thereby modulating the activity of more than gene. Similarly, a genetic construct may be assembled containing more than one open reading frame coding for a polypeptide of interest or more than one untranslated region of a gene coding for such a polypeptide.

The isolated polynucleotides of the present invention also have utility in genome mapping, in physical mapping, and in positional cloning of genes. As detailed below, the 23 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:44 Baldwins 64 4 4736712 No. 4146 P. 27 WO01/98485 PCT/NZ01/O0115 0 0 polynuceotide sequences identified as SEQ ID NO; 1-14, 20, 22-62, 81-86 and 88-127, and their variants, may be used to design oligonucleotide probes and primers. Oligonucleotide Sprobes designed using the polynucleotides of the present invention may be used to detect the presence and examine the expression patterns of genes in any organism having sufficiently similar DNA and RNA sequences in their cells using techniques that are well known in the Ci Sart, such as slot blot DNA hybridization techniques, Oligonucleotide primers designed using Sthe polynucleotides of the present invention may be used for PCR amplifications.

SOligonuclootide probes and primers designed using the polynucleotides of the present invention may also be used in connection with various microarray technologies, including the o microarray technology of Affymtrix (Santa Clara, CA).

As used herein, the term 'oligonucleotide" refers to a relatively short segment of a polynucleotide sequence, generally comprising between 6 and 60 nucleotides, and comprehends both probes for use in hybridization assays and primers for use in the amplification of DNA by polymeraechain reaction.

An oligonucleotide probe or primer is described as "corresponding to" a polynucleotide of the present invention, including one of the sequences set out as SEQ ID NO: 1-14, 20, 22-62, 81-86 and 88-127, or a variant, if the oligonucleotide probe or primer, or its complement, is contained within one of the sequences set out as SEQ ID NO; 1-14, 22-62, 81-86 and 88-127, or a variant of one of the specified sequences. Oligonucleotide probes and primers of the present invention are substantially complementary to a polynucleotide disclosed herein.

Two single stranded sequences are said to be substantially complementary when the nucleotides of one strand, optimally aligned and compared, with the appropriate nucleotide insertions and/or deletions, pair with at least 80%, preferably at least 90% to 95% and more preferably at least 98% to 100% of the nucleotides of the other strand. Alternatively, substantial complementarity exists when a first DNA strand will selectively hybridize to a second DNA strand under stringent hybridization conditions. Stringent hybridization conditions for determining complementarity include salt conditions of less than about 1 M, more usually less than about 500 mM, and preferably less than about 200 mlM. Hybridization temperatures can be as low as 5°C, but are generally greater than about 22PC, more preferably greater than about 30C, and most preferably greater than about 37tC. Longer DNA fragments may require higher hybridization temperatures for specific hybridization. Since the stringency of hybridization may be affected by other factors such as probe composition, 24 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:44 Baldwins 64 4 4736712 No. 4146 P. 28 WO 01/98485 PCT/NZ01/00115 0 0 presence of organic solvents and extent of base mismatching, the combination of parameters Z is more important than the absolute measure of any one alone.

In specific embodiments, the oligonucleotide probes and/or primers comprise at least about 6 contiguous residues, more preferably at least about 10 contiguous residues, and most preferably at least about 20 contiguous residues complementary to a polynucleotide sequence of the present invention. Probes and primers of the present invention may be from about 8 to n 100 base pairs in length or, preferably from about 10 to 50 base pairs in length or, more O preferably from about 15 to 40 base pairs in length. The probes can be easily selected using cl procedues well lkown in the art, taking into account DNA-DNA hybridization stringencies, o annealing and melting temperatures, and potential for formation of loops and other factors, which are well known in the art., Preferred techniques for designing PCR primers are disclosed in Dieffenbach, CW and Dyksler, OS. PCR Primer: a laboratory manual, CSHL Press: Cold Spring Harbor, NY, 1995. A software program suitable for designing probes, and especially for designing PCR primers, is available from Premier Biosoft International, 3786 Corina Way, Palo Alto, CA 94303-4504, A plurality of oligonucleotide probes or primers corresponding to a polynucleotide of the present invention may be provided in a kit form. Such kits generally comprise multiple DNA or oligonucleotide probes, each probe being specific for a polynucleotide sequence.

Kits of the present invention may comprise one or more probes or primers corresponding to a polynucleotide of the present invention, including a polynucleotide sequence identified in SEQ ID NO: -14, 20, 22-62, 81-86 and 88-120.

In one embodiment useful for high-throughput assays, the oligonucleotide probe kits of the present invention comprise multiple probes in an array format, wherein each probe is immobilized at a predefined, spatially addressable location on the surface of a solid substrate.

Aray formats which may be usefully employed in the present invention are disclosed, for example, in U.S. Patents No. 5,412,087 and 5,545,451; and PCT Publication No. WO 95/00450, the disclosures of which are hereby incorporated by reference.

The polynucleotides of the present invention may also be used to tag or identify an organism or reproductive material therefrom. Such tagging may be accomplished, for example, by stably introducing a non-disruptive non-functional heterologous polynucleotide identifier into an organism, the polynucleotide comprising one of the polynucleotides of the present invention.

COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:45 Baldwins 64 4 4736712 No.4146 P. 29 WO 01/98485 PCT/NZ01/00115 0 0 The following examples are offered by way of illustration and not by way of Slimitation.

EXAMPLE 1 Isolation and Characterization of a Ubiqgitin Gene Promoter from Pinus rdiata Pinus radiata cDNA expression libraries were constructed and screened as follows.

C

e mRNA was extracted from plant tissue using the protocol of Chang et al., Plant Molecular 0 Biology Reporter 11:113-116, 1993 with minor modifications. Specifically, samples were dissolved in CPC-RNAXB (100 mM Tris-Cl, pH 8,0; 25 mM EDTA; 2.0 M NaCl; 2%CTAB; 2% PVP and 0.05% Spermidine*3HCl) and extracted with chloroformisoamyl alcohol, 24:1.

C mRNA was precipitated with ethanol and the total RNA preparate was purified using a Poly(A) Quik mRNA Isolation Kit (Stratagene, La Jolla, CA). A cDNA expression library was constructed from the purified mRNA by reverse transcriptase synthesis followed by insertion of the resulting cDNA clones in Lambda ZAP using a ZAP Express cDNA Synthesis Kit (Stratagene), according to the manufacturer's protocol. The resulting cDNAs were packaged using a Gigapack I Packaging Extract (Stratagene) employing 1 pl of sample DNA from the 5 j±1 ligation mix. Mass excision of the library was done using XL1-Blue MRP' cells and XLOLR cells (Stratagene) with ExAssist helper phage (Stratagene). The excised phagemide were diluted with NZY broth (Gibco BRL, Gaithersburg, MD) and plated out onto LB-kanamycin agar plates containing X-gal and isopropylthio-beta-galactoside

(IPTQ).

Of the colonies plated and picked for DNA miniprsp, 99% contained an insert suitable for sequencing. Positive colonies were cultured in NZY broth with kanamycin and cDNA was purified by means of alkaline lysis and polyethylene glycol (PEG) precipitation.

Agarose gel at 1% was used to screen sequencing templates for chromosomal contamination.

Dye primer sequences were prepared using a Turbo Catalyst 800 machine (Periin Elmer/Applied Biosystems Division, Foster City, CA) according to the manufacturer's protocol.

DNA sequence for positive clones was obtained using a Perkin Elmer/Applied Biosystems Division Prism 377. sequencer. cDNA clones were sequenced first from the end and, in some cases, also from the 3' end. For some clones, internal sequence was obtained using subloned fragments. Subloning was performed using standard procedures of restriction mapping and subcloning to pBluescript II SK+ vector.

S26 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19Jul. 2007 15:45 Baldwins 64 4 4736712 iNo. 4146 P. WO 01/98485 PCT/NZOI/0115 0 0 C. As described below, one of the most abundant sequences identified was a ubiquitin Sgene, hereinafter refered to as the "Super-Ubiquitin or SU" gene.

O

Isolation of eDNA clones containing the ubiqultin gene Sequences of cDNA clones with homology to the ubiquitin gene were obtained from C high-throughput cDNA sequencing as described above. Sequences from several independent C clones were assembled in a contig and a consensus sequence was generated from overlapping Ce O clones. The determined nucleoride sequence of the isolated Super Ubiquitin clone, comprising the promoter region (including an intron), coding region and 3' untranslated Sregion (UTR) is provided in SEQ ID NO: 1, The 5' UTR is represented by residues 1 to NC 2,064, the intron by residues 1,196 to 2,033, and the coding region of the gene, which contains three direct repeats, by residues 2,065 to 2,751. The 3' UTR is 328 residues long (residues 2,755 to 3,083). The nucleotide sequence of the Super Ubiquitin promoter region only, including the intron, is given in SEQ ID NO: 2. The nucleotide sequence of the Super Ubiquitin promoter region only, excluding the intron, is given in SEQ ID NO: 3. The predicted amino acid sequence for the Pinus radiata Super Ubiquitin is provided in SEQ ID NO: Ubiquitin proteins function as part of a protein degradation pathway, in which they covalently attach to proteins, thereby targeting them for degradation (for a review, see Belknap and Garbarino, Trends in Plant Sciences 1:331-335, 1996). The protein is produced from a precursor polypeptide, encoded by a single mBiRA. The Super Ubiquitin mRNA contains three copies of the ubiquitin monomer.

Cloning of the Super Ubiquitin Promoter Fragments of the Super Ubiquitin promoter were cloned by two different PCR-based approaches.

Method 1: Long Distance One Walking PCR Using "Long Distance Gene Walking" PCR (Min and Powell, Biotechniques 24:398- 400, 1998), a 2 kb fragment was obtained that contained the entire coding region of the ubiquitin gene, a 900 bp intron in the 5' UTR and approximately 100 bp of the promoter.

To generate this fragment, 2 nested primers were designed from the 3' UTR of the Super Ubiquitin cDNA sequence isolated from pine. Generally, the 5' UTR is used for 27 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. J l. 2007 15:45 Baldwins 64 4 4736712 No. 4146 P. 31 WO 01/98485 PCT/NZ01/00115 0 0 primer design to amplify upstream sequence. However, the available 5' UTR of Super T Ubiquitin was very short, and two initial primers derived from this region failed to amplify any fragments. Therefore, the primers of SEQ ID NO: 15 and 16 were designed from the 3'

UTR.

The method involved an initial, linear PCR step with pine genomic DNA as template lt using the primer of SEQ ID 'NO: 15, and subsequent C-tailing of the single stranded DNA e product using terminal transferase. The second PCR-step used these fragments as template o for amplification with the primer of SEQ ID NO: 16 and primer AP of SEQ ID NO: 17. The AP primer was designed to bind to the polyC tail generated by the terminal transferass Both Sprimers (SEQ ID NO: 16 and 17) contained a 5'-NotI restriction site for the cloning of products into the Nod site of a suitable vector. The final PCR product contained fragments of different sizes. These fragments were separated by electrophoreais and the largest were purified from the gel, digested with restriction endonuclease Nod and cloned in the Nor site of expression vector pBK-CMV (Statagene, La Jolla, CA). The largest of these clones contained the complet6 coding region of the gene (no introns were found in the coding sequence) and a 5' UTR which contained a 900 bp intron.

Method 2: "Genome Walker" kit The Super Ubiquitin gene promoter was cloned using a "Genome Walker" kit (Clontech, Palo Alto, CA). This is also a PCR-based method which requires two PCR primers to be constrcted, one of which must be gene-specific. Although the ubiquitin coding region is highly conserved, the 5' UTR from different ubiquitin genes is not conserved and could therefore be used to design a gene-specific primer. A 2.2 kb fragment was amplified and subcloned in pGEM-T-easy (Promega, Madison, WI). Analysis by PCR and DNA sequencing showed that the clone contained 5' UTR sequence of the Super.Ubiquitin gene, including the 900 bp intron and approximately 1 kb of putative promoter region. An intron in the 5' UTR is a common feature of plant polyubiquitin genes and may be involved in determining gene expression levels.

The gene specific primers used for these PCR reactions are provided in SEQ ID NO: 18 and 19.

28 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:46 Baldwins 64 4 4736712 No.4146 P. 32 WO 01/98485 PCT/NZ01/00115 0 0 Expression of Super Ubiquitin Z Using primers derived from the genepecific 5' and 3' UTR sequences, expression levels of Super Ubiquitin in different plant tissues was examined by means of RT-PCR.

Super Ubiquitin was found to be expressed in all plant tissues examined, including branch phloem and xylem, feeder roots, fertilized cones, needles, one year old cones, pollen sacs, pollinated cones, root xylem, shoot buds, structural roots, trunk phloem and trunk.

cn Expression of Super Ubiquitin in plant tissues was also demonstrated in a Northern blot assay O using a PCR probe prepared from the SFunctional analysi of the Super Ubicuitin Promoter To test the function of the Super Ubiquitin promoter in plants, Arabidopsis thaliana was transformed with constructs containing the reporter gene for Green Fluorescent Protein (GOI) operably linked to either the Super Ubiqitin promoter of SEQ ID NO: 2 or SEQ ID NO: 3 either with or without the intron). Constructs lacking a promoter were used as a negative control, with a plant T-DNA vector carrying a CaMV 35S promoter cloned in front of GOFP being used as a positive control The constructs were introduced into Arabidopsi via Agrobacterium-mediated transformation.

All the plant culture media were according to the protocol of Valvokens and Van Montagu, Proc. Natl. Acad. Set. USA 85:5536-5540, 1988 with minor modifications. For root transforination, sterilized seeds were placed in a line on the surface of germination medium, the plates were placed on their sides to facilitate root harvesting, and the seeds were Sgrown for two weeks at 24 0 C with a 16 h photoperiod.

Expression of the constructs was measured by determining expression levels of the reporter gene for Green Fluorescent Protein (GFP), Preliminary OFP expression (transient) was detected in early transgenic roots during T-DNA transfer. Transgenic roots that developed green callus, growing on shoot-inducing medium containing 50 g/ml Kanamycin and 100 lg/ml Timentin, were further tested for GFP expression. After several weeks of stringent selection on Kanamycin medium, several independent transgenic Arabidopsis lines were engineered and tested for OFP expression.

Expression was seen both with the Super Ubiquitin promoter including intron and the Super Ubiquitin promoter without the intron. However, preliminary results indicated that the levels of expression obtained with the Super Ubiquitin intron-less promoter construct were 29 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:46 Baldwins 64 4 4736712 No. 4146 P. 33 WO 01/98485 PCT/NZO1/00115 0 0 significantly higher than those seen with the promoter including intron, suggesting that the intron may contain a repressor. The sequence of the intron is provided in SEQ ID NO: 21.

EXAMPLE 2 Isolation of a CDC Promoter from APi rmadtea ci r n Plant polynucleotide sequences homologous to the Cell Division Control (CDC) Sprotein gene were isolated from a Pinus radiata cDNA expression library as described in Example 1, Using the "Genome Walker" protocol described above and gene specific primers Sdesigned from these plant polynueleotide sequences, 5'UTR sequence containing the putative pC promoter of the P. radiata CDC gene was isolated from genomic DNA. The determined nucleotid sequence is given in SEQ ID NO: 4.

EXAMPLE 3 Isolation of a Xvloeneaia-Specific Promoter from Pins radita Plant polynucleotide sequences specific for plant xylogenesis were isolated from Pinus radiata cDNA expression libraries prepared from xylem, essentially as described in Example 1. Using the'"Genone Walker" protocol described above and gene specific primers designed from these plant polynucleotide sequences, sequences containing putative Pinus radiata xylogenesis-specific promoters were isolated from genomic DNA. The detomined nucleotide sequences are provided in SEQ ID NO: 5 and 41-44. An extended cDNA sequence for the clone of SEQ ID NO: 41-44 is provided in SEQ ID NO: 92, EXAMPLE 4 Isolation of a 4-Coumarate-CoA LiMase Promoter from Pinus radiat Plant polynucleotide sequences homologous to the 4-Coumarate-CoA Ligase (4CL) gene were isolated from a Pinus radiata cDNA expression library as described in Example 1.

Using the "Genome Walker" protocol described above and gene specific primers designed from these plant polynucleotde sequences, sequences containing the putative promoter of the P, radiaa 4CL gene was isolated from genomic DNA. The determined nucleotide sequence is given in SEQ ID NO: 6.

COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. J l. 2007 15:46 Baldwins 64 4 4736712 No. 4146 P. 34 WO 01/98485 PCT/NZ01/00115 0 0 Genetic constructs comprising the reporter gene for Green Fluorescnt Protein (GFP) Z or GUS reporter genes operably linked to the promoter of SEQ ID NO: 6 were prepared and used to transfon Arabidopsis thaliana plants.

EXAMPLE C Isolation of a Cellulose Svnthase Promoter from Eucavlytus mradis o Plant polynacleotide sequences homologous to the cellulose synthase gene were isolated from a Eucalyptus grandis cDNA expression library essentially as described in o Example 1. Using the "Genome Walker" protocol described above and gene specific primers C' designed from these plant polynucleotide sequences, 5'UTR sequences containing the .putative promoter of the E grandis cellulose synthase gene were isolated from genomic DNA. Independent PCR experiments using different DNA bands as templates yielded two sequences which contained a number of base differences. One band was 750 bp in length and the nucleotide sequence of this band is given in SEQ ID NO: 7. The other band was 3 kb in length. The sequence of the 3' end of this band corresponded to the sequence given in SEQ ID NO: 7, with a number of base pair differences. The sequence of this 3' end is given in SEQ ID NO: 8. The sequence of the 5' end of this band is given in SEQ ID NO: EXAMPLE 6 Isolation of a Leaf-Specific Promoter from Eucalvmts trandis Plant polynucleotide sequences specific for leaf were isolated from Eucalyptus grandis cDNA expression libraries prepared from leaf tissue, essentially as described in Example 1. Using the "Genome Walker protocol described above and gene specific priers designed from these plant polynucleotide sequences, 5'UTR sequence containing a leafspecific promoter of a novel E, grandis gene (of unknown function) was isolated from genomic DNA. Independent PCR experiments using different DNA bands as templates yielded three sequences which contained a number of base differences and deletions. The determined nucleotide sequences of the three PCR fragments are given in SEQ ID NO; 9-11.

XAMPLE 7 Isolation of an O-Methyl Transferase Promoter from EucalvDpurarandis Plant polynucleotide sequences homologous to an 0-methyl transferase (OMT) gene wer isolated from a Eucalyptus grandis cDNA expression library essentially as described in 31 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:47 Baldwins 64 4 4736712 No. 4146 P. WO 01/98485 PCTINZ10011S 0 0 Example 1. Using the "Genome Walker" protocol described above and gene specific primers Zdesigned from these plant polynucleotide sequences, 5'UTB. sequences containing the putative promote of the E. grarnsi OMT gene was isolated from genomic DNA. The determined nucleotide sequence is given in SEQ ID NO: 12. This promoter sequaence was extended by further sequencing. The extended cDNA sequences are given in SEQ ID NO: and 113.

en Genetic constructs comprising the zeporter gene for Green Fluorescent Protein (GP) o operably linked to the promoter of SEQ ID NO; 12 were prepared and used to transform Arabidopsis thaliana 0 N~ EXAMPI 8 Isolation of Root-Specific Promoters from Pinus radiata Plant polynucleotide sequences homologous to the root-specific receptor-like kinase gene were isolated fron a Pinus radiata cDNA expression library as described in Example i: Using the "Genome Walker" protocol described above and gene specific primers designed from these plant polynucleotide sequences, 5'TR sequence containing a putative P. radiata root-specific promoter was isolated from genomic DNA. Two independent PCR experiments yielded sequences that contained a number of base differences. The determined nucleotide sequences fromthe two experiments are given in SEQ ID NO: 13, 14, 110 and 111.

EXAMPLE 9 Isolatio of an M 1-alpha Promoter from Eucalvytus Grandis Plant polynucleotide sequences homologous to the Eucalyptus Elongation Factoralpha (EFl-alpha) gene were isolated from a Eucalyptus grandis oDNA expression library and used to screen a Eucalyptus grandis genomnic DNA library as follows.

The Eucalyptus grandis genomic DNA library was constructed using genomic DNA extracted from Eucalyptus nitens x grandis plant tissue, according to the protocol of Doyle and Doyle, Focus 12:13-15, 1990, with minor modifications. Specifically, plant tissue was ground under liquid nitrogen and dissolved in 2X CTAB extraction buffer CTAB, hexadecyltrimethylammonium bromide; 1.4IM NaCI, 20mMEDTA pH 8.0, 100mM Tris.HCIQ. pH 8.0, 1% polyvinylpyrollidone). After extraction with chloroform;: isonmylalcohol 10% CTAB was added to the aqueous layer and the 32' COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. J l. 2007 15:47 Baldwins 64 4 4736712 No. 4146 P. 36 WO 01/98485 PCT/NZ01/015 0

O

chloroform:isoamylalcohol extraction repeated., Genomic DNA was precipitated with Sisopropanol.

The resulting DNA was digested, with restriction endonuclease Sa3Al following standard procedures, extracted once with phenol:chloroformisoamylalcohol (25:24:1) and ethanol precipitated. The digested fragments were separated on a sucrose density gradient using ultracentrifugation. Fractions containing fragments of 9-23 kb were pooled and ethanol tn precipitated. The resulting fragments were cloned into the lambda DASH WIIBamI vector

C

S(Stratagene, La Jolla, CA) following the manufacturer's protocol and packaged using a Gigapack II Packaging Extract (Stratagene). The library was amplified once.

o The library was screened with radiolabeled EST fragments isolated from a Eucalyptus Ci grandis library (as described in Example that showed homology to the Eucalyptus EF1alpha gene. Phage lysates were prepared from positive plaques and genomic DNA was extracted.

From this genomic DNA, the 5'UTR region containing the putative promoter of the Eucalyptus EFl-alpha gene was obtained using the ELONGASE Amplification System (Giboo BRL). A 10 kb fragment was amplified and restriction mapped. The putative promoter region of the Eucalyptus elongation factor A (EF1-alpha) gene was identified on a 4kb fragment, which was suboloned into a pUC19 vector (Gibco BRL) containing an engineered Notd-site. The determined genomic DNA sequences of the isolated fragment containing the promoter region are provided in SEQ ID NO: 61 and 62, with the amino acid encoded by SEQ ID NO: 61 being provided in SEQ ID NO: 79. An extended sequence of the clone of SEQ ID NO: 61 is provided in SBQ ID NO: 127.

EXAMPLE Isolation of Fower-Specific Promoters from Eucalyptus grandis Plant polynucleotide sequences specific for flower-derived tissue were isolated from Eucalyptus grandis cDNA expression libraries prepared from flower tissue, essentially as described in Example 1, Using the "Genome Walker" protocol described above and gene specific primers designed from these plant polynucleotide sequences, several sequences, each containing a putative Eucalyptus grandis hlower-specific promoter, were isolated from genomic DNA. The determined nucleotide sequences are given in SEQ ID NO: 29-33 and 59.

An extended sequence of the clone of SEQ ID NO: 30-33 is provided in SEQ ID NO: 89. An extended sequence of the clone of SBQ ID NO: 29 is provided in SEQ ID NO: 33 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jil. 2007 15:47 Baldwins 64 4 4736712 No. 4146 P. 37 WO 01198485 PCT/NZ01/00115 0 0 S EXALMPLE 11 Isolation of Pollen-Secific Promoters from Eucalypts garanis and Pinus radiata Plant polynucleotid sequences specific for pollen were isolated from Eucalyptus grandif and Pinus radiaa cDNA expression libraries prepared from pollen, essentially as described in Bxample 1. Using the "Genome Walker" protocol described above and gene e c specific primers designed from these plant polynucleotide sequences, several sequences, each

C

o containing a putative pollen-specific promoter, were isolated from genomic DNA. The determined nucleotide sequences isolated from Pinus radiata are given in SEQ ID NO: 49- O 53, with the predicted amino acid sequences encoded by. SEQ ID NO: 51-53 being provided C' in SEQ ID NO: 73-75, respectively. An extended sequence for the clone of SEQ ID NO: 49 is provided in SEQ ID NO: 94.

EXAMPLE 12 Isolation of BudSpecific and Meristem-Specific Pomoter from Pis radiata Plant polynucleotide sequences specific for bud and meristem were isolated from Pinus radiata cDNA expression libraries prepared from bud and meristem, essentially as described in Example 1. Using the "Genome Walker" protocol described above and gene specific primers designed from these plant polynucleotide sequences, two sequences, one containing a putative bud-specific promoter and the other containing a putative maristemspecific promoter, were isolated from genomic DNA. The detenined nucleotide sequences for these two promoters are given in SEQ ID NO: 40 and 45, respectively. The predicted amino acid'sequences encoded by the DNA sequences of SEQ ID NO: 40 and 45 are provided in SEQ ID NO: 70 and 71, respectively.

EXAMPLE 13 Isolation of Promoters from Eucalvpats Rrandis Plant polynucleotide sequences showing some homology to various known genes were isolated from Eucalyptus grandi cdDNA expression libraries essentially as described in Example 1. Using the "Genome Walkr" protocol described above and gene specific primers designed from these plant polynucleotide sequences, sequences containing the putative promoters for the following K grandis genes were isolated from genomic DNA: auxin induced protein (SEQ ID NO: 26-28); carbonic anhydrase (SBQ ID NO: 36); isoflavone 34 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:47 Baldwins 64 4 4736712 No. 4146 P. 38 WO 01/9845 PCT/NZ01/00115 0 0 reductase (SBQ 1D NO: 37 and 38); pollen allergen (SEQ ID NO: 23-25); pollen coat protein Z (SEQ ID NO: 22), sucrose'synthaase (SEQ ID NO: 56-58); ubiquiin (SEQ ID NO: 34); glyceraldehyde-3-phosphate dehydrogenase (SEQ ID NO: 35 and 39); O-methyl transferase (OMT SEQ ID NO: 60); macrophage migration inhibition factor from mamman (Wt; SEQ ID NO: 81-86); UDP glucose 6-dehydrogonase (SEQ ID NO: 103); laccase I (SBQ ID NO: 105, 106 and 116); arabinogalactan-lie 1 (SEQ ID NO: 107); arabinogalactan-like 2 (SEQ e )ID NO: 108, 109); a hypothetical protein (SEQ ID NO: 104); constns (SEQ ID NO: 118); o Flowering Promoting Factor 1 (FPF:l; SEQ ID NO: 119); transcription factor DREB-1 (SEQ ID NO: 121); salt tolerance protein (SE QID NO: 123); xylem-specific histidine kinase-like (SEQ ID NO: 125) and root specific (SEQ ID NO; 126). The amino acid sequences encoded by the lNA sequences of SEQ ID NO: 22, 25, 26, 28, 34, 35, 36, 56, 57, 60, 86 and 124 are ,provided in SEQ ID NO: 63, 64, 65, 66, 67, 68, 69, 76, 77, 78, 87 and 130, respectively.

Extended cDNA sequences for the clones of SEQ ID NO: 58, 35, 60, 103, 106 and 107 are provided in SEQ ID NO: 91, 93, 113 and 115-117, respectively.

EXAMPLE 14 Isolation of Promoters from Pinus radira Plant polynucleodtide sequences showing some homology to various known genes were isolated from Pinu radian oDNA expression libraries essentially as described in Example 1. Using the "CGenome Walker" protocol described above and gene specific primrers designed from these plant polynuceleotide sequences, sequences containing the putative promoters for the following Pinus radiata genes were isolated from genomic DNA: sanescence-like protein (SEQ ID NO: 46-48); nodulin homolog pollen specific (SEQ ID NO: 54 and 55); chalcone synthase (SEQ ID NO: 88); PrMA 21 (SEQ ID NO: 95, 96); UDP glucose glycosyltransferase (SEQ ID NO: 97); elogation factor 1 alpha (SBQ ID NO: 98, 99); S-adenosylmethionine synthase (SEQ ID NO: 100-102); Pias radiata lipid transfer protein 2 (PrLTP2; SEQ ID NO: 112); Pinws radiata agamous protein (SEQ ID NO: 120); Drought Induced DI-19.(SEQ ID NO: 122) and low temperature induced protein LfI (SEQ ID NO 124). The amino acid sequences encoded by the polynucleotide sequences of SEQ ID NOS; 46 and 124 are provided in SEQ D NOS: 72 and 130. An extended cDNA sequence for the clone of SEQ ID NO: 97 is provided in SEQ ID NO: 114.

COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:48 Baldwins 64 4 4736712 No. 4146 P. 39 WO 01/98485 PCT/NZ0/00115 0 0 c EXAMPLE SPolvnucleotide and Amino Acid Analysis The determined cDNA sequences described above were compared to and aligned with known sequences in the EMBL database (as updated to October 2000). Specifically, the polynucleotides identified in SEQ ID NOS: 22-62 and 88-120 were compared to I' polynucleotides in the EMBL, database using the BLASTN.algorithm Version 2.0.6 [Sept-16n 1998] and the polynucleotides identified in SBQ ID NOS: 121-127 were compared to O polynucleotides in the EMBL database using the BLASTN algorithm Version 2.0.11 2000] set to the following running parameters: Unix running command: blastall -p blastn -d o embldb -e 10 -GO -ED -rl -v30 -b30 -i queryseq -o results. Multiple alignments of c redundant sequences were used to build up reliable consensus sequences. Based on similarity to known sequences from other plant or non-plant species, the isolated polynucleotides of the present invention identified as SBQ ID NOS: 22-62 and 88-127 were putatively identified as having the functions shown in Table 1, above.

The cDNA sequences of SEQ-ID NO: 1-22, 23, 25-42, 45-49, 57-59, 62, 88-99, 101- 112 and 114-12 were determined to have less than 40% identity to sequences in the EMBL database using the computer algorithm BLASTN, as described above. The cDNA sequences of SEQ ID NO: 56 and 113 were determined to have less than 60% identity to sequences in the EMBL database using BLASTN, as described above. The cDNA sequences of SEQ ID NO: 43, 52, 60 and 61 were determined to have less than 75% identity to sequences in the BMBL database using BLASTN, as described above. The cDNA sequences of SEQ ID NO: 24, 51 and 100 were determined to have less than 90% identity to sequences in the EMBL database using BLASTN, as described above, EXAMPlE 16 Modification of a Rporter Gene under Control of the Superubiquitin Promotr Six independent Arabidopis thatiana tranagenic lines were transformed with Pinus rdiata superubiquitin prbmoter constructs to demonstrate the relative expression of a GUS reporter gene under control of different superubiquitin promoter constructs. The reporter constructs in the plasmid pBI-101 contained the GUS (p-D-glucuronidase) reporter gene in frame with the superubiquitin promoter with the intron (SEQ ID NO: the superabiquitin 36 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:48 Baldwins 64 4 4736712 No. 4146 P. WO 01198485 PCT/NZ01/0011S 0 0 promoter without the intron (SBQ ID NO: and the CaMV 35S promoter. A reporter gene Z construct without a promoter sequence was used as control.

Groups of six Arabidopsis thaliana plants were transformed with the reporter constructs described above, using Agrobacterirn tamefaciens transformation protocols. A.

tumefaciens was transformed with 100ng of the plasmid DNA according to standard Stechniques, as described, for example, by Bevan (Nucleic Acids Res. 12;8711-8721, 1984).

r r n Fresh plant material was collected from each plant, protein extracted from the whole plant, o and the protein concentration detemined (Bradford, Anal. Biochen 72:248-254, 1976). The protein samples were diluted with carier bovine serum albumin to 100 ng protein to maintain Sreadings on the fluorimeter in the linear part of the standard curve using 4-methyl- C umbolliferone GUS activity was quantified by fluorimetric analysis, using a Victo z 1420 multi-label counter (Wallac, Turku, Finland) as described by Jefferson (Plant Mol. Bio.

Rep. 5:387405, 1987). As shown in Fig. 1, the construct containing the uperubiquitin promoter without the intron showed seven times more GUS activity than the CaMV 355 promoter and the construct containing the superubiquitin promoter with the intron showed sixty two times more GUS activity than the CaMV 35S promoter. No activity was detected for the promoter-less control construct.

EXAMPLE 17 Determination of the Activity of Superubiuitin Promoter Constructs in Tobacco Plant Protoplasts Isolation of otoplasts Protoplasts were isolated from sterile tobacco (Nicotiana tabacun) leaf tissue and transformed with superubiquitin promoter constructs. MesophyD protoplasts were prepared according to the method of Bilang t al., Plant Molecular Biology Manual A1:1-16, 1994. A number of fully expanded leaves were removed from sterile wild type tobacco plants, sliced perpendicular to the midrib and submerged in a digestion enzyme solution containing 1.2% cellulase and 0.4% pectinase (Sigma, St. Louis MO). The leaves were left to incubate in the dark without agitation at 26C for approximately 18 hours. The leaf strips were then gently agitated for 30 min to release the protoplasts. Protoplasts were further purified by filtration through 100 rm nylon mesh. One ml of W5 solution (154 mMMgC12, 125 mM CaCl 2 mM KCL 5 mM glucose, pH5.8 6) was carefully layered on top of the filtrate and 37 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19 Jul. 2007 15:49 Baldwins 64 4 4736712 No. 4146 P. 41 WO 01/98485 PCT/NZ01/00115 0 0 2 centdfuged at 80 x g for 10 min. The live protoplast layer was removed 'with a wide bore Spipette, washed twice with 10 ml W5 solution using centrifugation at 70 x g for 5 min, with final resuspension in 5 ml W5,solution. Protoplasts were counted in a hemocytometer and viability was determined under the microscope after staining with 5 mg/ml fluoroacein diactate (FDA) in 100% acetone.

STransformation with amotMer constrcts SThe isolated protoplasts were transformed with plasmid DNA using a polyethylene Sglycol protocol..After centrifugation of the purified protoplasts at 70 x g for 5 min, they were resuspended in MMM solution (15 mM MgCl 0.1% w/v 2[N-morpholino]ethanesulfonic o acid (MBS), 0.5 M mannitol pH 5.8) to a density of 2 x 106 protoplasts/ml. Aliquots containing 5 x i05 protoplasts/nl in 250 pC were distibuted to 15 ml tubes and mixed with pg plasmid DNA. 250 pl polyethylene glycol-4000 was gently added and incubated for 5 minutes at room temperature. Ten ml W5 solution was slowly added, the protoplasts centrifuged at 70 x g for 5 min and finally resuspended in 2 ml 13 mediuit (Bilang et al., Plant Molecular Biology Manual AI:1-16, 1994). The transformed protoplasts were incubated in the dark at 26"C for 24 hours before protein was extracted for reporter enzyme assays using 4-methyl-umbelliferyl-glucuronide (MUG).

Protein was extracted from the protoplasts using the following protocol. Transformed protoplast suspensions were centrifuged at 70 x g for 10 min, resuspended in 50 gl extraction buffer (Jefferson, Plant Mol. Biol Rep. 5:387-405, 1987) and vigorously mixed using a vortex. The homogenate was cleared by centrifugation at 4,300 rpm for 5 min, the supernatant removed and used for protein assays (Bradford, Anal, Biochem. 72:248-254, -1976).

The results shown in Fig. 2 demonstrate the promotr activity of deletion constructi of the superubiquitin promoter without the intron (SBQ ID NO: 3) and the superubiquitin promoter with the intron (SEQ ID NO: 2) in tobacco plant protoplasts transformed as described above. The deletion constructs were made in plasmid pBI-101 that contained the GUS reporter gene, using Endonuclease II (ibco BRL, Gaithersburg, MD) according to the manufacturer's protocols. The deletion constructs contained 1,103; 753; 573; 446; 368 and 195 bp of superubiquitin promoter sequence, respectively, upstream of the TATA sequence (bp numbers 1,104-1,110 of SEQ ID NO: A control construct containing no sequence upstream of the TATA sequence was also made. These results show that the construct 38 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:49 Baldwins 64 4 4736712 No. 4146 P. 42 WO 01/98485 PCT/NZI/00115 0 0 containing the entire superubiquitin promoter with the intron had the highest MU activity in Z the protoplasts.

In Tig 3, the tobacco protoplasts were transformed with four different promoter construcs in plasmid pBl-101 containing the GUS reporter gene. These included the superabiquitin promoter without the intron (SEQ ID NO: an elongation factor Ick S promoter (SEQ E) NO: 99) and a 5-adenosylmethionine synthetase promoter (SBQ ID NO: en 100). A prornoterless control was included in the expetiment, and is referred to in Pig. 3 as o pBI-E101.

EXAMPLE 18 Determination of the Activity of P. radiatd ollen-specific Prosrtr and E. prandi Pollen Specific Promoter Constructs in transformed Arabidosi:s thaliana cv Colngibia Arabidopsis sthaliana transgenic lines were transformed with A. tumfaciens containing constructs of the P. radiata pollen specific promoter (SEQ ID NO: 94) and E.

grandis pollen specific promoter (SEQ ID NO: 22) to demonstrate the relative expression of a GUS reporter gone under control of these promoter constructs. The promoter sequences were cloned into plasmid pBI-101 containing a OTS reporter gene.

Agrobacerium umefaclens trpansformation Agrobacterium tuwmfaeins strain 0V3101 was transformed with these constructs using electroporation. Electrocompetent A. sumefacins cells were prepared according to the method of Walkerpeach and Velten, Plat Mol. Biol. Man. 31:1-19, 1994. Construct DNA (4 ng) was added to 40 pl competent A. tumefaciens GV3101 cells and electroporation was done using a BTX Electro Cell Manipulator 600 at the following settings: Mode: T Resistance high voltage Set Capacitance: C (not used in HV mode), Set Resistance: BR (129 Ohm), Set charging voltage: S 1.44kV, Desired field strength; 14.4kV/cm and Desired pulse strength: t -5.O msec. 400 p1 YEP liquid media (20gI yeast, 20 g/l peptone and g/l sodium chloride) was added to the cuvotte and left to recover for one hour at room temperature. Thnsformed bacteria in YEP medium were spread out on solid YEP medium containing 50 mg/I kanamycin and 50 mg/i rifampicin and incubated at 29 0 C for two days to allow colony growth.

39 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:49 Baldwins 64 4 4736712 No, 4146 P. 43 WO 01/98485 PCT/NZ01/00115 0 0 Confirmation of transfomation of constructs into A. Unsmfa/Ciea Z To confirm that the constructs have been transformed into A. tmefadcens, DNA from the A. tumefacns colonies from the YBP plates were isolated using standard protocols and amplified using the polymerase chain reaction (PCR) with primer designed from the pBI- 101 vector sequence. The primer sequences are given in SEQ ID NOS: 128 and 129, PCR reactions were set up following standard protocols and 30 PCR cycles ware done with C extension temperature of 72C.

C

Transformadon of A. thaliana with transfo~rmed. .tumefacitns The optical density of the A. trwnfacdens bacterial culture was adjusted to 0.7 with Nq infiltration medium sucrose, 0.05% Silwett L-77 surfactant). A. thaliana cv. Columbia plants (6 punnets per construct and 10-12 plants per punnet) were pruned by removing secondary bolts. Pruned A. thaliana plants in punnets were dipped into infiltration solution and moved back and forth for 5 seconds. Punnets were put on their side to allow excess infiltration medium to drain covered with a top tray and wrapped in plastic wrap to maintain humidity. Plants were placed in a growth room at ambient conditions for 24 hours. After this period, the top tray and plastic wrap were removed and plants were set upright until siliques formed, Seeds were harvested and sterilized with a 5% sodium bypochlorite solution to destroy any residual A. tnuefaciens bacteria and fungal contamination.

Under sterile conditions, 100 p1 seeds from the transformed A. thaliana plants Swere placed into an Eppendorf tube. One ml sterile water was added and the seeds left to imbibe the water for no longer than an hour. The water was remove by centrifugation, 1 ml ethanol added to the seeds and gently mixed, This step was not allowed to last longer than one minute. The ethanol was removed by centrifugation, 1 ml 5% sodium hypochlorite solution was added to the seeds and gently mixed for up to 5 min. The sodium hypochlorite solution was removed by centrifugation and the seeds washed with sterile water for 1 min.

The washing step was repeated three more times with centrifugation. Seeds wer finally resuspended in sterile water. 500 p1 of seeds in solution were pipetted onto half-strength Murashige and Skoog medium (MS; Gibco BRL) agar plates containing 50 mg/i kanamycin and 250 mg/l timentin and spread evenly with a flamed wire-loop. The Pctri dishes were placed in a refrigerator for 3 days to allow the seeds to stratify. Thereafter the plates were placed in growth room and grown under lights at 22 0 C with a 14 hour photoperiod until COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:50 Baldwins 64 4 4736712 No. 4146 P. 44 WO 01/98485 PCT/NZ01/00115 0

O

c germination. Putative transformant seedlings were selected as those growing on the S antibiotic-containing medium, with large, healthy-looking dark green leaves and a strong root system. These transgenic plants were removed and placed into soil culture at 22°C with a 12 hour photoperiod.

C

Stainin of plant tisues Cn Tissue were taken from the flower, le, stem and root of A. thaliana transformed Swith constructs of P. radiatI unnown pollen specific promoter and E. grmadis pollen specific promoter and stained histochemically to determine the expression of the GUS gene under control of the pollen specific promoters. The GUS staining protocol is described by SCampisi et al., Plant J. 17:699-707, 1999.

A. thaliana flower, leaf, stem and root tissue were immersed in staining solution mM NaPO 4 pH 7.2; 0.5% Triton X-100; 1 mM X Glucuronide sodium salt (Cibco BRL)) for immunochemical staining. Vacuum was applied twice for 5 min to infiltrate the tissut with the staining solution. The tissue was left in the staining solution for 2 days (with agitation) at 37° for color development and then destained in 70% ethanol for 24 hours at 37°C (with agitation). The tissues were examined for blue GUS staining using a light microscope. GUS expression was observed only in the flower buds of plants transformed with the P. radiata pollen specific promoter construct, and not in the leaf, stem or root tissue.

With the E. grandis pollen specific promoter construct, Gus expression was observed in the floral buds as well as in the hydathodes of the leaves. No expression was observed in the stem or root tissues, To determine in which cell layers the GUS gene was expressed, flower buds were fixed for thin sectioning. The flower buds were fixed with formaldehyde acetic acid (FAA) in an Eppendorf tube and vacuum was applied twice for 15 min. After incubation for 2 hours at room temperature, vacuum was again applied for 15 min and the tissue left overnight at 4"C. The tissues were then dehydrated using a series of ei anol and then passed into a xylene series. Paraffin wax (Sigma) was added slowly and the tissues left for 72 hours with wax changes every 12 hours. Sections of 8 to 10 m thickness were prepared using a microtome.

The thin sections illustrated that GUS expression was restricted to the tapetum cell layer in the anther of the floral bud of A. thaliana transformed with the P. radiata construct (SEQ ID NO: 49). No staining was observed in other tissues fom the floral bud.

GUS expression was confined to the pollen grains within the flower'bud of A. thaliana 41 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:50 Baldwins 64 44736712 No. 4146 P. WO 01/848

PCTNZOVOOIZS

transformed with the E. prandis pollen specific promoter construct, with low levels of OTiS Z expression in the fibrous and connective tissue off tho anther No GUIS expression was observed in other organs of the floral bud.

EXAMPLIE19 Detearmiation of the Activity of oE. nandis.Efl alnpha Promoterfleletion Mn Cotiatruct in trnsfonned Arabidasi thaliana cv Colmi Protoplasts from Nicoziana tabacum Bright Yellow 2 (BY-2) cell suspension were o transformed with a deletion construct of the E. grandk W l-alpha prom'oter to determine ci GUS expresion. Base pairs 2,174 to 3,720 of SEQ MD NO: 127 were cloned into expression vector pART9, containing the reporter gene GUS and an OCS termination sequence.

PEparation of protopasmi Sterile Nicotiana tabrxurn Bright Ydllow-2 suspension cultures were prepared as desctbed in Example 17. After incubation ftr 3 to 5 days, 3 g of the N. abanm B3Y.2 cell suspension were suspended in an enzyme solution containing 1% cellulose, 0.3% Pecrinase and 0.5% driselase in 0.4 M mannitol. These were left to digest in the dark, with agitation at 2601 C, for 3-4 hours. Protoplasta were purified by filtration through a 63 pm nylon- mesh. Protoplasts were centrifuged at 80x g for 5 mein, washed twice,with 10 ml FMSB medium (Fulcuda. Murashige and Skcoog medium;, Easezwa, Syonio, Plant Cell Physiol.

24:127-132,12983) and finally resuspended in 5 ml FMS medium Protoplasts were counted in it hemocytometer and viability determined by staining with 5 mg/mi FDA (fluorescein deacetar; Sigma St Louis MI) in 100% acetone by viewing under the fluorescent microscope.

immsfoation Of Prmtonls Protoplasts were transformed according to the protocol described by Morgan and Ow (In: Methodsi in Plant Molecular Biology: a laboratory course manual, pp. 1-16. P. Maliga, D~lessig, A.R. 'Cashmoro, W. Onaissem, and LE.Vaxner, eds. Cold Spring Harbor Laboratory, CSHPF, NY). Briefly, the protocol is as follows. Following the counting step, protoplas were centifuged at B0x g fo: 5 mini and resuspended in Ix MaMg solution (0.4 M aqn-niotol, 15 mM MgCla.63 5 0. 0.1% 2-(N-Morpholino)ethane aulfonic acid (MvES)) to a density of 5x10 6 protoplasts/mi. Aliquots, of 100 gl1(0.5 x l0tprotoplaats) wer distributed to 42 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:50 Baidwins 64 4 4736?12 No.4146 P. 46 WO 01/J98485 PCTNZOIOO11S D tas an wmbedWit 5 1113 1IX MaM5 (200g, 5 Miu). Neutted protoplhiti =luserdd in SOul 1% MaMg sotitof, and heat ibockod bypwiga*5foS dts Aaeor Iibfion at room t=einptaf 5-10 Minutes, fme trmdcing WNa as added 2OMzgDNA ioug carierfDNA). To ti, 500 AO496PG-3 500 wagtYfMad incbaed r 5 xlmtMat room. teipere 51ml -W5 (154mM NOM. 125 mM (N ~~~CaC3a.

2 5m 5 mM Gluos) 00ohin was slowly added isB o? wdb ceutdfugatioll at 2001 g for 5 miB- PO!O&rtO PIt W, nSSPBnded iii Intl KSAM en znebzm at~proriately0.5 le protoplastsm Samjlca were t=anaeed to 6-well plates, and incbated jufte dark at 26 0 C for 48 hours oto axrac proteirk. pratOplaitfl weC centrfuged at 20D: g for 5 =min airfgo rsusened n.100 P, GMS anerationbiiffr (50nm NJPO4 pH 7.2s,10 =M KDTA PH B, 0.01% Sarcsyl, 0.1% Triton X-100) conaining PmeroaPtoethI~ (jefeontaPan Ma. EBiol. Rep. 53 87-405, 19 87) and vortexed for 1 mn4% Ttl homogMtC was cleared by ciftgalafl at 5,000 rpm for 5 minute's. The su'poraa otax-teprti a traufred to a fresh tAb. and stored at -80*C- Me protein==rdZ cnehWler eied by BioRad protein massy it (Bioptail Herules, CA) followving the Mauafactur&'5 Protocol$.

Protein exuacas ware diluted 1/10 witetato u GUS expresionc in t protoplast Catit was detemuned using a XUG (4-methyl uznboifaryl p~~lcXDd)assay, protein saples. containing 1 Ag protei nude up to a total volume of 46 Al with eCiol buft w=r aliqaoted Onto a mIdotitre plate and incuatedat 7'C.To ach ~pk Splof 0 mMIftG ws added o tat t final =ConflflratOf of UG was I the plate was incubated at 371C for 30 mein anld tetminatid by adding 150 Aj stop sulutjon (0.2 M NarCOs, pHul1.20), still keepfile, Pla at 37C Plates wore read in a Victor? 1420 MoltikbW cuJntt with excitation st at 365 Lm and emison at 455 em. The- COncS~tJradon, of 4-nmet$I1flbOMron0 W2J Was calCUltate against a standard =,rm and 6h 3o expression c1aatgeil *noeedwpnifof theGUS eport&ZO=0 lflN- tabactsBY-2 protopifasts tramformad a~ an K- grundz E1 alpba. deletion construct was seen conzpaed to the coutrol pWi mdWtbmt all inset.

COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:53 Baidwins 64 44736712 No.4146 P. 47 WO 01/98485 PCTINZO1100115 Cl EBXAIX. ROnnIadQP of the EIto o3VRSuru iuilin RHO (ST eu sOn GQM In thc polyateleotde, sequenoas t'y in SEQ ID NO: 1 encdiug P. radita ci mlupsiquidin (51. promoter ad g=n seqences, =n 3' =zatamaatd region W"s idaOted (nEt~eotdM 1,7S4 to 3,053). To detemine fteded Of this regiwi on the en expression of 250 bp oftto 3'M 11T (ualodes 2,755 td 3,073 from SEQ ir) No: 1).

o was cloned in te sens and aisouse orimitation into pasreld EBI-121 cantling the GUS gon uinder control of the 355 CaMV promoter and plasmid pRI-101 cntinng the GUS 0 geSe under control of the P. n4tata SUJ promoter Onobuidhg the intro) given in SEQ ED NO: Cl For controls, coiasffUts were made thaxt contained tho 8SU promoter witot an in (SEQ ID NO: 3) and without The SU FM31 sequmnc, thc 3SU promoter with an iotron (SEQ ID NO- 2) and without the SU3 31 1 sequence swell as A constrmct contaiin the CaM'V promoter but not &ae S133'U11 sequence.

Ak thaliana cv Columlbla were transformed wdit these constrcts usin t.0 floral dip protocol described in Example IS.

Six A. haliana plants wm erervested by trimming off to 6Mad tissue and then harvstig (he ret of the plant, includin the roots, The roots wore, &ised in tap water end thez samples immersed it liquid nitrogen before, stoiing at -SOT. Six plats fr=om eah coustract wore ound uinder liquid idfrogen and approimtoly 100 mg transferred to a microfage tubs. Piseo samples from each control were included in the assay. Extation mM NPO4 pH7.2, 10 zoM DTA pHS ,01% Sarcosyl, 0.1% TritonXC-J.0)was prepared. To 32 ml of eitraodoii bufe, S ml metbmrl and 28 #1 P-mercaptoetbanol was added. Of this buffer, 200 ILI was aded to each sample vortexd and stored on ice. Samples wer sp= at 4bC at 151 000 rpm for 15 n. The supeinatat was trafenoed to a fresh rub.

and dilated wit 800 Al of etaction buffer. Protein conceontration. was deemined, using the floRad Protein Assay Kit Dou expressio of GUS by the four constructs was determined using a MUG awsay, u follows. To 28 ml exraction buffer (as dcacribed i.Exawple 18), 8 MI meth&=l. 56 Alj3 mecaptootbano and 4 rl of 10 mg/ml bovin, serum albmi6n (BA) wore added. To COMS ID No: ARCS-153531 Received by IP Australia: Time (I-tm) 14:10 Date 2007-07-19 19. Jul. 2007 15:51 Baidwins 64 44736712 No. 4146 P. 48 WO 01I985 PCTINZO10O115 miorottrs plat wells, 100 and 10 ng of proten from each oowt was added as we! as ILI extreedon bufe contairdg BSA and 5 Al 10 mM MUG. The plat was coverd in foil ad incubated at 370C for eactly 20 =mtes. The reaction wa trminated by adding 150 Ad 0.2!iM WA2CO3 pH 11.2. FIsts wem read wit a Vaior' 1420 Muldhabel coazte ifii oxafl adct at 365 x= ad omidssion at 455 GUiS cpreasin levels w= detmined ci against a MU standard cum, tn ig. 5, conflt $134. bontafii ffie SU. YUfl intof smas ozieatfoji eabscd e tko expesion of the $13 withiout intron promoter almiost to The level oft 511ST promoter with 0 ~~~the iltro Ili ootnnfc 033 and SR35 contaning tho S t tlR ki t e aises oriantation, 0r~a Wdit Waf WMeurCd to basal lCVcls.

COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19

Claims (3)

19.Juli. 2007 15:51 Baidwins 64 4 4736712 No. 4146 P. 49 PCT/NZO1/0115 wo 01198485 .aim" 1. An isolated polyxm~ctdc copising a soqucc seeted from t group consisting of sequecms recited i SEQ ID NO: 2-14,20,22-33,35-43,45-49,51,52,56-62 and 88-127; complcmnaets 6f lbm sequacs rbCited i SEQ ID NO: 2-14,20.22-33,35-43,

45-49.51, 52,56-62 and 88-127; reverse complmes of the sequence tucked in SEQ ID NO 2-14, 20, 22-33, 3543,45-49, 51, 52,56-62 and 88-127; revese sequenwe of lt sequc recited in SBQ ID NO: 2-14, 20, 22-33, 35-43, 45-49, 51, 52, 56-62 and 88-127; sequences having at least 40% identical nuclootides to a sequence provided in SEQ ID NO: 2-14, 20,22-23, 25-33,35,42,45-49,57-59, 62, 85-99, 101-112 and 114-127 as deti:ned using the computaer algorifthm BLASTN; sequensca baitg at I=st 60% idealral nucicotides to a sequence provided in SEQ ID NO: 2-14, 20, 22-23, 25-33, 3542, 45-49, 56-59, 62, 18-99, 101-112 and 114-127 as dateilned using the computer algorithm BLASTN; sequences having at least 75% idemtical nleolidee to a sequene provided in SBQ H) NO: 2-14,20,22-23,25-33,35-49,52,56-61,62, 88-99, 101-112 and

114-127 as detmnted using the computer algorithm BLASIN; and sequces having at least 90% identical cletides to a sequence provided in SEQID NO: 2-14,20,22-33,35-49,51,52,56-61, 62,88-112 and 114-127 as determined using the computer algouitbmBLASlN. 2. An isolated polnucleotide comprising a sequemc e seleoted from the group consisnfg sequences redted in SEQ ID NO: 1 and 34; complements of semces recited InSEQID NO? I and 34; reverse complements of sequences recited in SEQ ID NO: 1 and 34: reverse sequece of sequenCes recited in SEQ ID NO: 1 and 34; sequencs haig at least 40% idntial nucleotides to sequences recited in SEQ ID NO: 1 and 34 as dtemined usg tw computer algoritbntBLAST; squenes having at' least 60% idenical rnicotides to sequces recited' in SEQ ID NO: I and 34 as detemaied using the oomtttw algoitl= BLAIN; 46 COMB ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19.Jul. 2007 15:51 Baidwins 64 4 4736712 No. 4146 P. WO OIL"485 PCTNZOI/00115 saqaences havbqg at least 75* ideaicul nucletides to seqauences recited in SEQ ID NO: 1 and 34 as determined fing to computn agorit=m ELASTN; and h) sequnce bring at leat 90* idandcal wuleotides to sequeces recited in SEQ ID NO: 1 and 34 as detrmined uing the computer algoitm ]BLAST& C-I 3. An isolatd pobrepdd encdedr by a pbmucleode selected from to group Cfl conisig of: Mn sequences reaited in flQ H) NO. 1,22,25,26,28, 34, 35,36,40,45,46,51- Cl 53, 56, 57 60, 61, 86 and 124; complements of the sequences of SEQ ID NO: 1, 22, 25, 25, 28, 34, 35, 36, 45, 46, 51-53, 56, 57, 60, 61, 86 and 124; cl revse complerents of a sequec of SEQ ID NO: 1.22, 25,26, 28334,35, 36,40, 45, 46,51-53, 56,57, 60, 61, 86 and 124; revese scquaesm= of a sequence of SBQM I NO: 1, 22, 25, 26,28, 34,35,36, 45, 46,51-53,56, 57, 60, 61, 86 and 124: sequences haing at last 40% idenical maekotides to a sequewe provided in SEQ ID NO: 1,22,25,26, 28, 34,35,36,40,45, 46,51-53,56,57, 60,61, 86 and 124; qunem having at least 60% idet ic =aleotides to a sequence provided SEQ ED NO: 1,22,25,26,28,34,35,36,40,45,46,51-53,'56,57,60,61, 86 and 124; Cs) seqenes having at least 75% identcal mleotidm to a sequence provided in SEQID NO: 1, 22,25,26,28,34,35,36,40, 45,46,51-53,56,57,60,61, 86 and 124; and sequences havig at leat 90% idemical imoleotides to a seqenc provided in SEQIDNO: 1,22,25,26,28,34,35,36,40,45,46, 51-53,56,57, 60, 61, 86 and 124. 4. Mhe isolated polypeptide of claim 3, wherin to polyptide oompisa a sequence selected from th group conssting of SEQ ED NO: 63-80, 87 and 130. S. A genetc conhtutu comprising a polyxmcleotide according to any one of cliditn I and 2. 6. A gentic conuuct compdiuing in the direction a promoter sequence, 47 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:52 Baidwins 64 44736712 No.4146 P. 51 WO 0M845 PCT/NZOI/GO115 a DXA sequence of inteest, and SZ aigenemtemtation. seqawzc, qflr the promoter sequence comprise ga isolated polyzucleotide according to 7. The gwe~do conatuct of clam 6, wbeen t DNA seqacuc of Intextst oprises an ri ~open reain fram encodin a polypeptide, of ismt en 8. Tb. =gsietic conaflct of claim 6, iwurain the, DNA ueano of Inteest co=dZD =C at non-codIng region of a gem enoding a polypcprida of lntees. Ci~9 A trinsgenic cea compdusing a geneti construct of mUy one of claim 5-8. o 10. An organism comprisin a rawsgeic cell accoring to claim 9. o 11. A plant comprisin a tranagecic cell according to claim 9. or a part or propagule or progeny thereof. 12. A method for modifyig gene expresson in a tage organism compisiok stably incozoratiug into tho gonzu of thie organis A genatic consruct; acordin to mny one of claims 5-8. 13. Mhe madhod of claim 12 wherein the organic~ is a plant 14. A method fur producing a plant aving modified g=n expression, comprising: trMIsfo±rding a plant coll wit a genefictoonstmict to provide a trmusgenic cell, wherein the genstc constuct comprises: CI) a promoter sequnce comprising a seque=c of SEQ ID NO: 1-14, 20, 2-62, 81-86 and 8&-127; (ii) a DNA sequa=c of intrest; and a gene teminton sequene; &A culivating te tnageci cel under condidioni conducive to regeneration gad ma~re plan growth. A method for modifying a phenotyei of a target organism, co.mpr.is..in--- g ably incorporating into the pioma of t targe organism a genetic constut comprising: a prmoter sequence compsing asaquence of SEQ ID) NO: 1-14,20,22-62, 81- 86 and 88-127, itfDNA seqence of Interest; and agn em winton sequence 16. The method of claim 15, witoein the targe organic is a pint 17. A method for identifying a, Sen responsible for a desired function or phenotype, 4, COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:52 Baidwins 64 44736712 No. 4146 P. 52 raTIZ0ovo0ns WO 0119848 fransfoxbg a plint cell wift a genetic oontcr comorising a promoter seqanc operably linked to a gene to beb teste, the promoter sequee comprisin a saewe of SEQ ID NO: 1-14,20,22-62, 81-86 aM 88-127;, ca~tizg the pla cell under condition conducive to mg==rton and mature plalnt growth to provid, a trasgnic plut and comparing the phezioty of tboe izangenic plan wit ft phenotype of nn franbmed ph=u. 18. An isolated polymicleotide comprising a sequnce selected from th. group cnist Of: a sequence recited in SEQ 3D NO: 21; complcntso of a saenc rocked iu SESQD NO: 2ti; revuse complement of a sequae, recited in SEQ ID NO: 21; revmre sequence of.i seqwenc recited int SEBQ ID NO: 21; sequecnces having, at least 40% identical nuclcotides to at sequec recd im SEQ ID NO; 21 as deterned using the computer algoriflm EIASTN; seqene having at least 60% identical meleotides to at sequenc recited in. SE3Q ID NO: 21 as determined using the cotputer algorithim BLAST?!; sequences having at last 75% idesnioW1 nacotidu. to a sequenreited in *SEQ ED NO, 21 as determined using the computer ulgctm BU MT?; arid sequences having at least 90% identical nucleotides to a sequence recited in -SEQ ID NO- 21 as detemined, usin the computer algoritm BLASN. 19. A genetic constrict comprisin a pobmncleoid =0codi4 to claim 18S. A tranagenic cel omprising a genetic construct accordinag to claim 19. 21. A =vtod for modffyg gene, expression in. a targt organism comprising stably inorporating into die gonome of the organism a genetic conanat according to clam 19. 22. A method for ,nodify~ng expression of a pojymzcleotide that compr4es tho sequence of SEQ ID NO: 21, the zeetd comprisin ragmoving the sequence of SEQ ED NQ; 21 from the poLnuItcloid. 23. A polyzncleotide, comprisin a sequenc selected from the gromp consisting of SEQ ID NO- 2-14, 20. 22-33, 35-43, 45-49, 51, 52, 56-62 and 88-127 operably finbd to a hetetologous polyrsacleotida. 4.9 COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19 19. Jul. 2007 15:52 Baidwins 64 44736712 No. 4146 P. 53 PCTINZO1/OO115 WO swe4es 24. Tho po1ymckcotde of o~am 23, wheren &o hetarologon pvlomloiide ooir an open readhg 6w.. COMS ID No: ARCS-153531 Received by IP Australia: Time 14:10 Date 2007-07-19