CN1425772A - Novel plant DNA virus carrier and its use - Google Patents

Abstract

The invention discloses a novel plant DNA virus vector and its application. The plant DNA virus vector provided by the invention includes the infective vectors of Chinese tomato yellow leaf curl virus DNA-A and DNAβ, and plant gene silencing vectors and virus expression vectors derived from them. The invention also provides a novel satellite DNA molecule for constructing plant DNA virus vector, which has at least 72% homology with Seq ID No.1-12. The invention also provides an application and a method of using the plant DNA virus vector to carry out viral functional genome research, plant functional genome research and exogenous protein expression. The present invention provides a crucial platform for the study of virus genome structure, function and expression regulation mechanism as well as the interaction between virus and plants, provides an excellent tool for the study of plant functional genomes, and provides a basis for the use of plants as bioreactors Expressing foreign proteins establishes a technical platform.

Description

A kind of novel plant DNA virus carrier and application thereof

Technical field

The present invention relates to a novel plant DNA virus vector and its application, specifically, the present invention relates to the infectious vector of Chinese tomato yellow leaf curl virus DNA-A and DNAβ and the plant gene silencing vector and virus expression vector derived from it . The invention also relates to a DNA beta satellite molecule for constructing a plant DNA virus vector and a whole genome sequence thereof. The invention also relates to an application and method of using the plant DNA virus vector to determine genome functions, plant genome functions and express foreign proteins.

Background technique

Whitefly-transmitted geminivirus (WTG) is a kind of plant single-stranded DNA virus that occurs widely in the world. loss. WTG belongs to the Begomovirus genus (Begomovirus) in the Geminiviridae family (Geminiviridae) in taxonomy, including more than 100 independent species. Under natural conditions, WTG is transmitted by whitefly (Bemisia tabaci). Its virions are in the shape of twin particles with a size of 18×30nm. The genome is single-stranded circular DNA with a size of 2.5-3.0kb. Most WTG viruses contain two DNA components of similar size, called DNA-A and DNA-B. Most of the sequences of DNA-A and DNA-B are different, but there are about 200 nucleotide sequences in the non-coding region that are almost identical (homology greater than 95%), called the common region (common region, CR), common region Contains sequences required for viral replication, transcription initiation and packaging, and can form a stem-loop structure. DNA-A encodes the replication-associated protein (Rep), replication-enhancing protein (Ren), coat protein (CP) and transcriptional activator protein (TrAP) that control late gene expression necessary for viral replication, which are associated with viral replication and mediators Related to transmission; DNA-B encodes a nuclear shuttle protein (NSP) and a motor protein (MP), which are related to the transport of the virus in plants and the host range of the virus. Currently, a few viruses in WTG, such as tomato yellow leaf curl virus (TYLCV) (except TYLCV in Thailand), tomato leaf curl virus (TLCV), cotton leaf curl virus (CLCuV) and red thistle yellow vein virus (AYVV), Only DNA-A was found, and pathogenicity assays for TYLCV and TLCV showed that DNA-A can infect tomato, Nicotiana benthamiana and N. tabacum, and show symptoms similar to those in the field , infected plants can be spread by whiteflies. And different from TYLCV and TLCV, although the infective clone of DNA-A of the red thistle yellow vein virus (AYVV) can infect the red thistle systematically, it rarely forms typical yellow vein symptoms. Further research found that AYVV DNA-A requires a new type of DNA molecule co-infection to cause typical symptoms. The present invention discovers this type of novel DNA satellite molecule on Chinese tomato yellow leaf curl virus.

Plant viral infectious vectors are an important platform for studying the function of viral genomes. The construction of viral infectious vectors can artificially manipulate viral genomes at the molecular level to obtain a uniform population of wild-type molecules or mutant molecules, and then Using DNA recombination technology to carry out mutation, deletion, insertion, substitution and complementation experiments to locate the functional region of the virus genome, so that the expression of gene function can be reflected and evaluated at the level of host plant phenotype. This technology is an effective means for in-depth study of the gene function of the virus and the interaction between the virus and the host plant. This technology was first successful in the bacteriophage virus Qβ and poliovirus. The infective clone of the plant DNA virus was established in Tomato Golden in 1982. Mosaic virus (TGMV) was successfully constructed by rubbing inoculation, and plant RNA virus was obtained for the first time in 1983 from brome mosaic virus (BTV) infective cDNA clone. Although the technology was produced relatively late, it developed very rapidly and has been widely used in the analysis of genetic expression and replication of viruses. On the basis of obtaining novel satellite molecules, the present invention constructs the infectious vector of the satellite molecules, so as to be applied to the functional genome research of viruses.

Virus-induced gene silencing vector is a very effective way to study the function of plant genome through forward genetics. It inserts plant endogenous genes into viral vectors, and forms dsRNA intermediates as the virus replicates. These dsRNAs are An enzyme called Dicer degrades 21-23nt RNA and combines with RNAase to form a complex. This complex specifically attacks its homologous sequence under the guidance of 21-23nt RNA, so that homologous genes in plants Dysfunction, so the function of the plant genome can be studied in terms of the plant's phenotype as well as biochemical characteristics. In the current situation of a large number of plant genomes being determined, more and more sequences do not know their functions. The next focus of molecular biology is functional genomics research, which needs to open up a variety of ways to study the functions of plant genomes, and virus-induced Gene silencing is a very efficient approach from genome to function.

Putting exogenous genes into plant cells can produce exogenous proteins, so plants are a bioreactor with broad application prospects. Plants have mechanisms for protein synthesis, folding, post-translational modification, and assembly. Foreign proteins can reproduce in large quantities in plants. Therefore, plants can be used to produce important medicinal proteins (especially vaccines) in large quantities and at low cost. There are two main ways to grow and use plants to produce drugs: (1) exogenous genes temporarily exist in plant cells (such as using plant virus vectors), and plants can express proteins encoded by exogenous genes; (2) exogenous genes exist for a long time In plant cells, the introduced exogenous gene will become a part of the plant genome, and then continuously produce the protein encoded by the exogenous gene. However, the expression level of exogenous genes in transgenic plants is relatively low, generally less than 1% of the soluble protein, and the process of gene transformation and plant regeneration is laborious and time-consuming, which largely affects the overall industry of using transgenic plants to produce medicinal proteins process. Plant viruses generally have a strong ability to reproduce. Based on this characteristic of plant viruses, since the 1980s, researchers around the world have begun to explore the feasibility of using plant virus vectors to express foreign genes efficiently, and successfully expressed Various proteins. On the basis of the constructed plant DNA virus infective clone, the present invention establishes an expression vector for expressing foreign protein by using the plant DNA virus.

Contents of Invention

Another object of the present invention is to provide a novel plant DNA virus vector, which includes a plant DNA virus infective vector and plant gene silencing vectors and viral expression vectors derived from it;

An object of the present invention is to provide a novel satellite DNA molecule for constructing plant DNA virus vector;

Another object of the present invention is to provide a use of the plant DNA virus vector to study virus functional genome, plant functional genome and express foreign protein.

One aspect of the present invention provides a novel satellite DNA molecule, specifically a whitefly-transmitted geminivirus Chinese tomato yellow leaf curl virus DNAβ molecule, the DNAβ full-length nucleotide sequence and Seq ID No1-12 have at least 72% homology. DNAβ is half the size of DNA-A and has no sequence homology with DNA-A. Compared with different DNAβ, there is a very conserved region of about 115bp, which contains a highly conserved replication initiation sequence TAATATT/AC. Except for a common region, DNAβ has very low homology with known sequences. There is also a very conserved open reading frame among the known DNAβ, and there is an A-rich region in the genome.

Another aspect of the present invention provides an infectious vector, more specifically constructs the infectious vector of Chinese tomato yellow leaf curl virus DNA-A and novel satellite molecule DNA β, and is characterized in that the system is a set of The complementary vector, DNA-A, was able to infect plants systemically, but did not cause typical symptoms. A single DNAβ cannot replicate independently, it must rely on DNA-A, and DNAβ will also act on DNA-A in turn, only when DNA-A and DNAβ exist together, the plant can cause the typical severe pathogenic type, and DNAβ Can greatly boost the genomic content of DNA-A. The infectious vector of the present invention can be used for virus functional genome and virus and plant interaction research, and its method is:

a. Design mutation primers for the target gene of the virus, and mutate the target gene of the virus by Overlap-PCR,

b. Construct two mutant repeats of the forward copy,

c. insert the mutant repeats of the two forward copies constructed into the Agrobacterium vector,

d. import the Agrobacterium vector constructed in c into the Agrobacterium vector,

e. Inoculate the virus into the plant by Agrobacterium injection,

f. Observe the symptoms and detect the virus molecular level.

The invention also provides a plant gene silencing vector derived from an infectious vector, which induces plant gene silencing so as to study the function of the plant gene. The plant gene silencing carrier provided by the present invention can be used to study the function of plant genome, and its method is:

a. RNA extraction is carried out on plants, and the primers of plant endogenous genes are used to synthesize the first strand of cDNA, followed by RT-PCR amplification to obtain the cDNA fragment of the target plant gene,

b. Delete the C1 gene of DNAβ, and design primers to add multiple cloning sites,

c. Insert the cDNA fragment of the plant target gene into the multiple cloning site of DNAβ,

d. Construct two copies of DNAβ with plant endogenous genes,

e. Insert the β of the two copies constructed in d into the Agrobacterium vector,

f. Introduce the Agrobacterium vector constructed in e into Agrobacterium,

e. Inoculate the host plant with the Agrobacterium in f together with the Agrobacterium containing the DNA-A invasive clone,

g. Observing plant phenotypes or detecting changes in plant physiology and biochemistry.

The present invention also provides an exogenous gene expression vector derived from an invasive vector, which can be used to express an exogenous gene, and its expression construction method includes:

a. Design specific primers to pull out the β genome that accurately deletes C1, and construct it into pGEM-T easyVector,

b. Use another pair of C1-deleted β pulled out with specific primers to insert into the vector constructed in a. After splicing, two direct repeats and C1-deleted DNA β are obtained, and multiple genes are introduced at the position of C1. cloning site,

c. Introduce the direct repeat DNAβ constructed in b into the plant expression vector by enzyme digestion,

d. The exogenous gene can be directly inserted into the plant expression vector for expression.

In the present invention, the term "plant DNA virus vector" includes the infectious vectors of Chinese tomato yellow leaf curl virus DNA-A and DNAβ and the plant gene silencing vectors and viral expression vectors derived therefrom.

Advantages of the present invention:

1. The infectious carrier constructed by the present invention is a very valuable molecular tool, which can easily obtain a uniform population of natural molecules or mutant molecules. Using this molecular tool, the molecular biology research of the virus can be carried out, and can be obtained through Mutation, deletion, insertion, and recombination of viral genes are used to study the structure, function, and expression regulation mechanism of viral genomes. It is a crucial platform for studying viral functional genomes and the interaction between viruses and plants. In the present invention, there is an open reading frame (open reading frame, ORF) with unknown function on the DNAβ, and the present invention mutates the gene and clarifies the function of the gene. For the functional genome research of the invasive vector, functional research of other coding regions and non-coding regions may also be included.

2. The plant gene silencing vector of the present invention is a very good tool for studying plant functional genomes with forward genetics. The traditional reverse genetics method for screening mutants includes the method of T-DNA and transposon insertion, which is a very effective method for discovering new genes, but these methods also have some shortcomings. Traditional forward genetics It is not possible to study genes that cause lethal mutations during early development, and it is also impossible to study genes in gene families. Virus-induced gene silencing is silenced after adult plants, so some lethal genes can be studied. In addition, for multi-gene families, gene silencing not only attacks the exact homologous genes, but also attacks homologous genes with 10-20% fluctuation, so the genes in the gene family can also be studied. Compared with the forward genetics method of transgene transposon insertion, the invention has the advantages of small workload, simple construction, short period, and large-scale silencing can be carried out to study the function of the plant genome. In some viral vectors, the virus itself causes severe pathogenicity on the host, thereby interfering with the symptoms after silencing phenotype. The plant gene silencing vector in the present invention deletes the gene that plays a key role in pathogenicity, However, it can still perform replication and transcription, and DNA-A infects plants without any symptoms. The phenotype induced by plant endogenous gene fragments inserted into DNAβ is completely the phenotype after plant genes are silenced. Virus infection has the greatest impact on plants. narrowed to a limited extent.

3. Constructed a plant DNA virus expression vector with independent intellectual property rights, which can insert foreign protein genes into the virus vector. After the virus infects plants, the foreign gene can be expressed along with the propagation and amplification of the virus. The amount of exogenous genes expressed by transgenic methods is relatively low, generally below 1% of soluble protein, and the process of gene transformation and plant regeneration is laborious and time-consuming, which largely affects the use of transgenic plants to produce medicinal proteins. Comprehensive industrialization process. The viral expression system has a large replication capacity, simple construction, small workload, and short periodicity. The plant virus vector of the present invention can be amplified and propagated in a large amount in plants generally at 15-30dpi.

Specific Implementation Example 1 Cloning and sequencing of novel DNA molecules

Collect Chinese tomato yellow leaf curl virus samples, extract the total plant DNA, use this as a template, and use beta01 (5'- GGTACC ACTACGCTACGCAGCAGCC-3', containing KpnI site) and heta02 (5'- GGTACC TACCCTCCCAGGGGTACAC-3', KpnI site) was used as a primer to amplify the full-length genome of DNAβ by PCR, and the PCR product was purified by QIAquick Gel Extraction Kit and cloned into pGEM-T Easy Vector to obtain a full-length copy of the clone pGEM-T-β(New) And sequence determination was carried out to obtain the full-length genome sequence of DNA-β 1.3kp, such as No.1-12. The homology between Chinese tomato yellow leaf curl virus DNAβ is 72%-99%, and the homology with other reported DNAβ (AYVVβ, CLCuMVβ, CLCuRVβ and BYVMVβ) is only 35-43%. DNAβ is half the size of DNA-A, and has no sequence homology with all geminivirus DNA-A except for the highly conserved replication initiation sequence TAATATT/AC. Although the homology between DNA β is very low, all DNA β has a highly conserved sequence of nearly 115 bases. This region may contain a replicase binding site, but there is no TATA box region, so it may be different from DNA - Another unique promoter region of the bidirectional transcriptional promoter of -A and DNA-B. All DNAβs have an A-rich region (>56%) at ±760-±1000 bases. DNAβ has multiple open reading frames, but the ORFs of different DNAβs vary greatly in size and position, and only one of the ORFs (C1) is conserved in length, size and position. Example 2 Construction of Infectious Vectors and Establishment of Viral Functional Genome Research Methods

1. Construction of Infectious Vectors

Construct 1.7 copies of the genome of the cloned Chinese tomato yellow leaf curl virus full-length DNA-A and insert it into the plant expression vector pBinPLUS, and then introduce it into Agrobacterium EHA105 by three-parent mating method, and construct 2 copies of the genome insert of DNAβ by the same method The plant expression vector pBinPLUS was introduced into EHA105. The recombined Agrobacterium was inoculated into N. benthamiana, and the inoculation was divided into single DNA-A inoculation, single DNAβ inoculation and DNA-A+DNAβ inoculation. Single DNA-A inoculation was able to infect systemically, but did not cause the typical leaf curl symptoms observed in the field; plants inoculated with single DNAβ showed no symptoms, and no viral DNAβ was detected in the upper leaves, indicating that DNAβ could not replicate independently. When the two components of DNA-A and DNAβ are inoculated together, after 10dpi, the upper leaves begin to roll down, and the symptoms develop to the peak at 25-35dpi, and the stems appear, the leaves shrink, the plants are dwarfed, and ears appear in the later stage DNA β was detected by Southern Blot, and the amount of DNA-A genome was significantly higher than the amount of DNA inoculated with single DNA-A, so DNA-A and DNA β were interdependent, DNA β must rely on DNA-A replication, and DNA β The genomic DNA content of DNA-A can be greatly enhanced, and DNAβ is necessary for the pathogenicity of the virus to the host.

2. Establishment of virus functional genome research methods

a. Design primers for the target gene of the virus, recombine, mutate, insert or delete the target gene of the virus by Overlap-PCR and enzyme digestion, insert the mutated genome into pGEM-T Vector,

b. Insert another full-length mutant viral genome into pGEM-T Vector,

c. Insert the constructed two direct repeat mutant genomes into the plant expression vector pBINplus,

d. Introduce the recombinant vector constructed in c into Agrobacterium EHA105,

e. Inoculating mutant invasive clones into plants by Agrobacterium injection,

f. Observe the symptoms and detect the virus molecular level.

DNA-A must be present in the presence of DNAβ to cause disease, and DNAβ has only one gene C1 that is conserved in length and position, so mutation of the C1 gene will clarify the function of C1. The method for introducing mutant bases in the primers of the present invention mutates the C1 gene of Y10 DNAβ, so that the gene cannot be transcribed. When this mutant was inoculated with Nicotiana benthamiana together with DNA-A, it did not show the typical symptoms of curved leaves. Further Southern Blot detection of the DNA of the leaves of the plants found that DNAβ was detected in plants without symptoms, indicating that after the mutation of the C1 gene No longer causing pathogenicity, but still able to replicate, indicating that the gene does play an important role in pathogenicity. Example 3 Construction of virus-induced gene silencing vector and establishment of method

DNAβ can still replicate after the C1 gene in DNAβ is mutated, so if the C1 gene is replaced with a foreign fragment, it can also replicate, and the foreign gene can be transcribed by using the promoter and terminator of the C1 gene, while virus-induced gene silencing only needs Plant endogenous gene silencing can be induced with a small amount of dsRNA. The present invention inserts the cDNA of the plant phytoene desaturase (PDS) gene into DNAβ. This gene is a key enzyme in the plant carotenoid synthesis pathway. If the pathway is blocked, the leaves of the plant will produce a white effect. Its methods include:

a. Carry out RNA extraction to the plant, carry out the synthesis of cDNA first strand with the primer of plant PDS gene, then carry out RT-PCR amplification, obtain the 409bp cDNA fragment of PDS gene;

b. Delete the C1 gene of DNAβ, and switch to the multi-cloning site to design primers to join the multi-cloning site GGATC CAAGCTTGATATCCTGCAGCTCGAG TCTAG A, the restriction sites are BamHI , HindIII, EcoRV PstI, XhoI, XbaI , and use New Bet01 Pull out its full length with Beta02 and insert it into pGEM-T Easy Vector;

c. Insert the cDNA fragment of the PDS gene into the multiple cloning site of DNAβ;

d. Construct two direct repeats of DNAβ with PDS cDNA in pGEM-T Easy Vector;

e. Use EcoRI on both sides of pGEM-T Easy Vector to insert two direct repeats of DNAβ with PDS into the plant expression vector pBinPLUS;

f. Introduce the constructed vector into Agrobacterium EHA105 by the method of three-parent mating;

e. The recombinant Agrobacterium is inoculated with the host plant together with the Agrobacterium containing DNA-A;

f. Observe the plant phenotype.

25-30 days after inoculation, the upper leaves of the inoculated plant begin to turn white along the veins, and gradually expand, so that all the upper leaves along the veins are bleached. After the plants become silent, they can last for a long time. The inoculated virus does not show any symptoms on the plant due to the deletion of the C1 gene, and the phenotype displayed on the host plant is the phenotype after the plant is induced to silence. Example 4 Construction of Plant Expression Vector and Establishment of System

The C1 gene of DNAβ is replaced by foreign protein, and the transcription and expression of the foreign gene are initiated by the promoter and terminator of the C1 gene during the replication of the virus. Geminivirus-based vectors require two forward-repeating genome sequences, so after the introduction of multiple cloning sites, foreign fragments still need to be inserted twice. Therefore, further transformations have been carried out on the basis of the above vector construction method, and more Facilitate the expression of foreign proteins.

a. Y10-beta Substition Sal/F (5'- GTCGAC ATACATATATACGTATTC-3', containing SalI site) and Y10-beta Substition Xba/R ( TCTAGA GTTTATTTGTTGTGGATGATACATG, containing XbaI site) accurately delete the DNAβ genome of C1, PCR product Insert pGEM-T Easy Vector,

b. Use another pair of specific primers Y10-beta Substition XbaSma Bam/F (5' TCTAGA CCCGGGGGATCCATACATATATACGTATTCAAATATATG, containing XbaI, SmaI, BamHI) and Y10-beta Substition Xba/R to pull out the DNAβ of the deletion of C1, and pass XbaI The site was spliced with the vector constructed in a. After splicing, two direct repeats β were obtained, a C1 gene was deleted, and the multi-cloning site XbaI, SmaI, BamHI.SmaI was introduced at the position of C1. Dots can be used for cloning of PCR products,

c. Insert the direct repeat sequence constructed in b into the plant expression vector pBINplus using SalI and EcoRI sites,

d. The exogenous gene can be directly inserted into the plant expression vector for expression.

Description of SEQ ID NO.1-12: Information (i) sequence characteristics of SEQ ID NO.1

(A) Length: 1338 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(ii)分子型：基因组DNA(iii)序列描述：SEQ ID NO.1accggtggcg agcggggtct ttgccgttga ggtgggtccc acatgaaaaa agaaagagaa      60aatgggcctt ggtgatgcaa ttgggccttt aatttatggg ctttgagtat tggactttgt     120atattgggcc tacttaaaat aattcaaatg ggcaggaata acaggaattt tatttaattc     180atatatttga atacgtataa atacgtattc atacatctga attcataaat acatcatatt     240catctcctac atctatatct tcaactggtg cttcttccat catgattata tctataactt     300gtaccatatc ttcttgcctg aactctccaa ttgtagcttc tttgtacatt attttgaggc     360agtttttgat gccttcttct aggctgttga agtcgaatgg tgggattatc ccatggtgtc     420tgtatgggat catgaatttc ttcttggcta atgctggtga ctttgttgat gctagttcaa     480tctgtacaag gattgaagtg tcttcttgca acttcacatc tatgatgaac tccataccct     540tcatgttgtt gtatttgatt gtcatgttta cttgttgtgg atcattcatg agactggtct     600tcttaaataa tgctaggatt gatgagttat ggactggaat gatttaacgt ggttcattgt     660tgttcatatg cttagtagtg gagatatgta tgattgatag tgacaatact attaatcata     720agtactttta gtaatgacat aataatccaa attattatgg ttaattataa ttgaaatagg     780aatagaaaag gaaataaaaa gaaaaggaaa ataaaaagaa aaggaaataa gataataaac     840ataaacccaa acaaatattt attaagaaag aaaagggagc gcagcgaaaa aaaaaatcaa     900atttgaaaca accaaaatca aataaacaca aaaagagaaa acaaaagaat aaaataataa     960aattaaagca aaataaaata aaaacagtaa aaacgtcgtc acgattttac tgcgcggtaa    1020taaaaaaata ctttcactta ggaggttata aatcgggaca ccaatggtaa atgggacacc    1080gatacatcag tgccccctat tagtgtctaa tgtgcaattt ccaaaaatac ccttatccct    1140gtgtctgtta ggcgcgtggg agtgcgctga aaaagttaat tttctctctc ctaaactcgt    1200cggagaagac gattgaggca cttcccggca tcaatttgcg acacgcgcgg cggtgtgtac 1260ccctgggagg gtagaaacca ctacgctacg cagcagcctt agctacgccg gagcttagct 1320cgccaccgtt ctaatatt iv. NO sequence information ( )

(A) Length: 1340 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(v)分子型：基因组DNA序列描述：SEQ ID NO.2accggtggcg agcggggtct ttgccgttga ggtgggtccc acattaaaaa agaaagagaa      60aatgggcctt ggtgatgcaa ttgggccttt aatttatggg ctttgagtat tggactttgt     120atattgggcc tacttaaaat aattcaaatg ggcaggaata acaggaattt tatttaattc     180atatatttga atacgtataa atacgtattc atacatctga attcataaat acatcatatt     240catctcctac atctatatct tcaactggtg cttcttccat catgattata tctataactt     300gtaccatatc ttcttgcctg aattctccaa ttgtagcttc tttgtacatt attttgaggc     360agtttttgat gccttcttct aggctgttga agtcgaatgg tgggattatc ccatggtgtc     420tgtatgggat catgaatttc ttcttggcta atgctggtga ctttgttgat gctagttcaa     480tctggacaag gattgaagtg tcttcttgca acttcacatc tatgatgaac tccataccct     540tcatgttgtt gtatttgatt gtcatgttta cttgttgtgg atcattcatg agactggtct     600tcttaaataa tgctaggatt gatgagttat ggactggaat gatttacgtg gttcattgtt     660gttcatatgc ttagtagtgg agatatgtat gattgatagt gacaatacta ttaatcataa     720gtacttttag taatgacata ataatccaaa ttattatggt taattataat tgaaatagga     780atagaaaagg aaataaaaag aaaaggaaaa taaaaagaaa aggaaataag ataataaaca     840taaacccaaa caaatattta ttaagaaaga aaagggagcg cagcgaaaaa aaaaaactca     900aatttgaaac aagcaaaatc aaataaacac aaaaagagaa aacaaaaaaa taaaataata     960aaattaaagc aaaataaaat aaaaacagta aaaacgtcgt cacgatttta ctgcgcggta    1020ataaaaaaat actttcactt aggaggttat aaatcgggac accaatggta aatgggacac    1080cgatacatca gtgcccccta ttagtgtcta atgtgcaatt tccaaaaaat acccttatcc    1140ctgtgtctgt taggcgcgtg ggagtgcgct gaaaaagtta attttctctc tcctaaactc    1200gtcggagaag acgattgagg cacttcccgg catcaatttg cgacacgcgc ggcggtgtgt    1260acccctggga gggtaggtac cactacgcta Information (vi) sequence characteristics of cgcagcagcc ttagctacgc cggagcttag 1320ctcgccaccg ttctaatatt 1340SEQ ID NO.3

(A) Length: 1341 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(vii)分子型：基因组DNA序列描述：SEQ ID NO.3accggtggcg agcggggtct ttgccgttga ggtgggtccc acatgaaaaa agaaagagaa      60aatgggcctt ggtgatgcaa ttgggccttt aatttatggg ctttgagtat tggactttgt     120atattgggcc tacttaaaat aattcaaatg ggcaggaata acaggaattt tatttaattc     180atatatttga atacgtataa atacgtattc atacatctga attcataaat acatcatatt     240catctcctac atctatatct tcaactggtg cttcttcctt catgattata tctataactt     300gtaccatatc ttcttgcctg aattctccaa ttgtagcttc tttgtacatt attttgaggc     360agtttttgat gccttcttct aggctgttga agtcgaatgg tgggattatc ccatggtgtc     420tgtatgggat catgaatttc ttcttggcta atgctggtga ctttgttgat gctagttcaa     480tctgtacaag attggaagtg tcttcttgca acttcacatc tatgttgaac tccataccct     540tcatgttgtt gtatttgatt gtcatgttta cttgttgtgg atcattcatg agactggtct     600tcttaaataa tgctaggatt gatgagttat ggactggaat gatttacgtg gttcattgtt     660gttcatatgc ttagtagtgg agatatgtat gattgatagt gacaatacta ttaatcataa     720gtacttttag taatgacata ataatccaaa ttattatggt taattataat tgaaatagga     780atagaaaagg aaataaaaag aaaatgaaaa taaaaaagaa aaggaaataa gataataaac     840ataaacccaa acaaatattt attaagaaag aaaagggagc gcagcgaaaa aaaaaactca     900aatttgaaac aagcaaaatc aaataaacac aaaaagagaa aacaaaaaaa taaaataata     960aaattaaagc aaaataaaat aaaaacagta aaaacgtcgt cacgatttta ctgcgcggta    1020ataaaaaaat actttcactt aggaggttat aaatcgggac accaatggta aatgggacac    1080cgatacatca ggtgccccct attagtgtct aatgtgcaat ttccaaaaat acccttatcc    1140ctgtgtctgt taggcgcgtg ggagtgcgct gaaaaaggtt aattttctct ctcctaaact    1200cgtcggagaa gacgattgag gcacttcccg gcatcaattt gcgacacgcg cggcggtgtg    1260tacccctggg agggtagaaa ccactacgct acgcagcagc cttagctacg ccggagctta 1320gctcgccacc gttctaatat t 1341 Information (viii) sequence characteristics of SEQ ID NO.4

(A) Length: 1338 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(ix)分子型：基因组DNA序列描述：SEQ ID NO.4accggtggcg agcggggtct ttgccgttga ggtgggtccc acatgaaaaa agaaagagat      60aatgggcctt ggtgatgcaa ttgggccttt aatttatggg ctttaagtat tggactttgt     120atattgggcc tacttaaaat aattcaaatg ggcaggaata acaggaattt tatttaattc     180atatatttga atacgtataa atacgtattc atacatctga attcataaat acatcatatt     240catctcctac atctatatct tcaactggtg cttcttccat catgattata tctataactt     300gtaccatatc ttcttgcctg aattctccaa ttgtagcttc tttgtacatt attttgaggc     360agtttttgat gccttcttct aggctgttga agtcgaatgg tgggattatc ccatggtgtc     420tgtatgggat catgaatttc ttcttggcta atgctggtga ctttgttgat gctagttcaa     480tctgtacaag gattgaagtg tcttcttgca acttcacatc tatgatgaac tccataccct     540tcatgttgtt gtatttgatt gtcatgttta cttgttgtgg atcattcatg agactggtct     600tcttaaataa tgctaggatt gatgagttat ggactggaat gatttacgtg gttcattgtt     660gttcatatgc ttagtagtgg agatatgtat gattgatagt gacaatacta ttaatcataa     720gtacttttag taatgacata ataatccaaa ttattatggt taattataat tgaaatagta     780atagaaaagg aaataaaaag aaaaggaaaa taaaaagaaa aggaaataag ataataaaca     840taaacccaaa caaatattta ttaagaaaga aaagggagcg cagcgaaaaa aaaaactcaa     900atttgaaaca agcaaaatca aataaaagta aaaagagaaa acaaaaaaat aaaataataa     960aattaaagca aaataaaata aaaacagtaa aaacgtcgtt acgattttac tgcgcggtaa    1020taaaaaaata ctttcactta ggaggttata aatcgggaca ccaatggtaa ttgggacacc    1080gatacatcag tgccccctat tagtgtctaa tgtgcaattt ccaaaaatac ccttatccct    1140gtgtctgtta ggcgcgtggg agtgcgctga aaaagttaat tttctctctc ctaaactcgt    1200cggagaagac gattgaggca cttcccggca tcaatttgcg acacgcgcgg cggtgtgtac    1260ccctgggagg gtagaaacca ctacgctacg cagcagcctt agctacgccg gagcttagct 1320cgccaccgtt ctaatatt 1338SEQ ID NO.5 information (x) sequence feature

(A) Length: 1336 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(xi)分子型：基因组DNA序列描述：SEQ ID NO.5accggtggcg agcggggtct ttgccgttga ggtgggtccc acataaagaa aagtaaggaa      60aatgggcttt ggtaatgcaa ttgggccgtt aagttatggg cctatattta atggactttg     120tatattgggc ctatttaaaa taaataaatt aggtcagaat aataagaatt ttatttaatt     180catatatttg aatacgtata tatatgtatt catacatctg aatttgtaaa tacatcatac     240tcatccccta catctatatc ttctactggg gcttcttcca tcatgattat gtctataact     300tgtaccatat cttcttgcct aaattcccca attgtagact ccttatacat tattttgagg     360cagtttttga tgccttcttc taaaatgttg aagtcgaatg gtgggataat cccatggtgt     420ctgtatggga tcatgaattt cttcttggct aatgctggtg actttgttga tgctagttca     480atccttacaa ggattgaagt gtcttcctgt aacttgacat caatgatgaa ctccaaaccc     540ttcatgttgt tgtatttgat agtcatgttt atttgttgtg gatgatacat gagtctgttc     600tgcttaaata atgctaggaa ttgtgatata tggactgtaa tgatttacgt gtttcattgt     660tgttcatatg cctactagtg gagatatgta tgtttgatag tgacatgaat attaaccatg     720attactttta ctaatgacat aataatctgg attattatgt gtaaaaagaa atggaaagag     780gagtagaaag aagaagtaaa tatagaataa aataaaaaag aaaaggaaat aagataataa     840acataaaccc aaacaaatat atattaagaa agaaaaggga gcgcagcgga aaaaaaaaaa     900ggtaaatttg aaacaaataa aatcaacaag atagtaaaat aaaaaaataa aaagaataaa     960atcaaaacaa ataaactaaa atagtaaaat cgtcatgatg attttactgc gcggtaataa    1020aaaaactact ttcatctcag aggtaaataa ttgggacacc aatggtaatt gggacaccga    1080tacatcggtt cccctattag tgccccaatg tgcaatttcc aaaaataccc ttatccctgt    1140gtctgagggg cgcgtggtat tgcgctgaaa aagttaagtt tctctctcca aaaactcacc    1200ggaactccga ttgagggcct tccggtcatc aatttgcgac acgcgcggca gtgtgtaccc    1260ctgggagggt agaaaccact acgctacgca gcagccttag ctacgccgga gcttagctcg 1320ccaccgttct aatatt 1336SEQ ID NO.6 information (xii) sequence characteristics

(A) Length: 1350 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(xiii)分子型：基因组DNA序列描述：SEQ ID NO.6accggtggcg agcggggttt tttgcccaaa tgtgggtccc acattggaga tgaaagagaa      60agatgggttt ggttatgcaa ttgggccttt aatggtgggc cgaattttat ggacctcgta     120ttttgggcct aatgagtata aataaattgg gctgtaatag aaaggaattt tatttaattt     180atatatttga atacgtataa ctacgtattc atacacttga attcaaaaat acatcgtatt     240catctcctac atctatatct tcaactggtg cctcttccat catgagaatg tctacgactt     300gtatcatgtc ttcttgcctg aattcccata tggtagactc tttgtacatt attttgaggc     360agtgtctgat gccttcctct aagctattga agtcaagtgg tggtattatc ccattgtgcc     420tgtatgggat catgaatttc ttcttggcta gtgctggtga ccttgttgat gcgagttcaa     480tctgtactag gattgaagtg tcttcctgca acttcacatc tatgatgaat tctaaaccct     540tgttgttgtt gtatttgata gtcattttaa ctggtgtgga ttatacgtga gtctggtcct     600gttaaataat gtttgggatt gttatatatg gacttaaacg atttacgtgg ttcattgttg     660ttcatgtaac tactagtgga gatatttatg attgatagtg acataactat caatcatgat     720tactttaaca aattacatga taatctagat tatcataggc aaatagaatt ggaaagaaga     780aaagaagaat agaaaagaag aagaaagaaa taaaaaataa aaaatgaaaa taaagaagat     840aataaggata aacagaaaca aatatatatt aagaaagaaa gggagcgcag cgattaaaaa     900aaaattgaaa aaatgaaaac aacaaaagaa taaaataaca aaacaaaaaa aactaaaaag     960aaacaagaaa aaaatataaa tcaaataaaa taaaacagta aaaacgtaga aacggtttta    1020ctgcgcggta ataaaaaaag actttgacct aggaggttaa aaattgggac accaatggta    1080aatgagaccc cgatacatcg gtgcctctat tgggtccttg aaagttaaat tcccaaaata    1140cccttatccc tgtgtctgag aagcgcgtgg tattgcgctg aaaaagttaa gtttctctct    1200ccaaaaactt gccggaacgc cgattgaggg tcttccggtc atcaatttgc gacacgcgcg    1260gcggtgtgta cccctgggag ggtaggaaac Information (xiv) sequence characteristics of cactacgcta cgcagcagcc ttagctacgc 1320cggagcttag ctcgccaccg ttctaatatt 1350SEQ ID NO.7

(A) Length: 1333 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(xv)分子型：基因组DNA序列描述：SEQ ID NO.7accggtggcg agcggggtct ttgccgtaga cagtgggtcc cacatagaag aatacaagaa      60attgggcctt gggtatgcaa tttgggccta gaattatggg cttcatttaa cggcctttga     120atattgggcc taattaaaat aattaagttg gctttaataa aatgaagttt attcaataca     180tatagttgaa tacgtatagc tacgtattca tacatctgaa tttataaata catcatattc     240atcacctaca tctatatcct caactggtgc ttcttccatc atgagaatat ctattacttg     300taccatgtct tcttgcctga attcccaaat tgtagattct ttgtacatta ttttgagaca     360gtgtttgatg ccttcttcta agctgttaaa gtcgagtggt ggtattatcc cattgtgcct     420gtatgggatc atgaatttct tcttggctaa tgctggtgat cttgttgatg ctagttcgat     480ctgtacaagg attgaagtgt cttcctgcaa tttcacatct atgatgaatt ccaaaccctt     540gttgttgttg tatgtgatag tcattttatt tggtgtggat tatacatgac tctggtcctg     600ttaaataatg ctgggaattg ttatacatgg acctgaatga tttacgtgga tcattattgt     660tcatgtaact tctagtggag atatttatga ttgatagtga cataactagc aagcatgagt     720attttaatta attacataat aatctagatt atcataggta aatagaagtg gaaagaataa     780tagaagagga aattaaacat attaagaaaa gaaaaaatga aaagaaaaaa gacaataaac     840ataaacacaa acaaatatat attaagaaaa aaagggagcg cagcgaaaaa aaaaaaaatt     900gatacaatca aaagcaacta agaaagaaaa aacaaaatag aaaaagaata aaaaaaagaa     960taaaacaaat aaaataaaac agtaaaaacg ttgaaacggt tttactgcgc ggtaataaaa    1020aaatactttc atctggcaag ttaaaaattg ggacaccaat ggtaaatgag accccgatac    1080atcggtgcct ctattaggtc cttgaaagtc aatttccaaa aataccctta tccctgtgtc    1140taagaagcgc gtgttattgc gctgaaaaag ttaagtttct ctctccaaaa actcgccgga    1200acggcgattg agggtcttcc ggttatcaat ttgcgacacg cgcggcggtg tgtacccctg    1260ggagggtaga aaccactacg ctacgcagca GCCTTAGCTA CGCCGGAGCT TAGCTCCCCA 1320ccGtttaat Att 1333Seq ID No.8 information (XVI) sequence feature

(A) Length: 1340 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(xvii)分子型：基因组DNA序列描述：SEQ ID NO.8accggtggcg tgcggggttt tttgccattt tgtgggtccc acatagaaga agaaagagaa      60agtgggcctt ggctatgcaa ttgggccttt aatttatggg cctggagtta ttggactttg     120tatattgggt ttaatcagaa taaataaatt gggttaaaat aatatgaaat ttatttaatg     180tatatgattg aatacgtatg catacgtatt catacacttg agtttataaa tacatcgtat     240tcatccccta catctatatc ctcaactggt ggttcttcca tcatgaggat gtctataact     300tgtaccatat cttcttgcct gaattcccca attgtagact ctttgtacat gattttgagg     360cagtgtttga tgccttcttc taagctgttg aagtctattg gtgggatgat cccctggtgc     420ctgtatggta tcatgaactt cttcttggct aatgctggtg actttgttga tgttagttca     480atctgaacaa ggaatgaagt gtcgtcctgc aacttcacat ctatgatgaa ctccatgccc     540ttcatgttat tgtatgtgat tgtcatgttt tatgctgtgg attatacatg agtcgtgcct     600tcttaaataa tgtctggaat tgtgatatat ggatttgaat caattacgtg gttcattgtt     660gttcatatat atattagtgg agataggtat gtttgatagt gacattacta tgtaacctga     720ttacttttag taatgacata ataatctaga ttattatggc taatagaggt gtaaagagta     780ttatgaaaga aaaatcaaag aataataaaa taaaaaagaa aaaaaataca ataaacataa     840acccaaacaa atatatatta agaaaaaaag ggagcgcagc ggaaaaaaaa aaaaggaaag     900aataattaaa tgtgaaacaa gcaaatgcaa caaaaaagaa agagaaaaga aatagataaa     960aaaatataaa acaaataaaa caacacggta aaatcgttcc aacggtttta ccgcgcggta    1020ataaaaaaat actttaacct cataggtaat taattgggac accaaaggta aatgggacac    1080cgatacactg gttcccctat taggtgctca atattcaatt tccaaaaata cccttatccc    1140tgtgtctgag gggcgcgtgg aaatgcgctg aaaaagttaa gtttctctct ccaaaaactc    1200accggacctc cgattgaggg ccttccggtc atcaatttgc gacacgcgcg gcggtgtgta    1260cccctgggag ggtaggaaac cactacgcta cgcagcagcc ttagctacgc cggagcttag 1320ctcgccaccg ttctaatatt 1340SEQ ID NO.9 information (xviii) sequence characteristics

(A) Length: 1334 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(xix)分子型：基因组DNA序列描述：SEQ ID NO.9accggtggcg agcggggatt ttccgaactt tgtgggcccc acatactgga agaaagagaa      60agtggactct ggttatgcaa ttgggccttt aatgaatggg ctttatctta atgtaaatgt     120ctattgggct tgagtaaaaa agaatagttg ggctcaaata aaatacaatt ttatttaatt     180atatatctga atacatactt tatacgtatt catacatctg aattcataaa tacatcgtat     240tcatcaccta catctatatc ttcaactggt gcctcttcca tcattagtat gtctataact     300tgtaccatat cttcttgcct gaattcccag atatttgagt gcttgtacat tattttgagg     360cagtgtttta tgccttcttc taggctgttg aagtcgattg gtgggatgag gccttggtgc     420ctgtaaggga tcatgaattt cttcttggct aatgctggtg accgtgttga gcttagttct     480acctgaacga ggaaggaagt gtctgcttcc aatttgacat ctattttgaa ttccagaccc     540ctcttgttgt tgtatttgat agtcattctt atttggtgtg gattgtacat gagtttgatc     600tgcttaaata atgcaagtaa ttttgataca tggacttgat tgatttacgt gtttcattgt     660tgttcatata ggttatagtg gaaatatttg tcattgatag tgacattact atcaatgtta     720agtactttta ctaatgacat tataatctag attataatga ataagtataa gtggaaagaa     780aaataggaaa taaaatttga agaaaataaa ataaaaaaag aaagaagaca attaacatga     840acacaaacaa acatatacta agaaaagaaa agggagcgca gcgaaaaaaa aaaacataga     900ttcaaaacca acagaatcaa ccaaacagaa aaaaagaaaa ataaaataaa caattaattc     960aaaaaaaaaa aaataatcag taaaaacata agcaggtttt tacggcgcgg taaaaaaata    1020aattacattc acctcgtagg taaataatta ggaccccaaa ggtaaatgag accccgatac    1080atcggggtct ctattaggtc ctcaataatt acattccaaa aataccctta tccctgtgtc    1140tgaaaggcgc gtggtattgc tctgaaaaag ttaagtttct ctctccaaaa accccccgga    1200gctccgattg agggccttcc ggtcgtcaat ttgcgacacg cgcggcggtg tgtacccctg    1260ggagggtagg aaaccactac gctacgcagc agccttagct acgccggagc ttagctcgcc 1320accgttataa tatt 1334SEQ ID NO.10 information (xx) sequence feature

(A) Length: 1344 bases

(B) type: nucleic acid

(C) chain type: single chain

(D)拓扑：环形(xxi)分子型：基因组DNA序列描述：SEQ ID NO.10accggtggcg agcggggatt ttccgaactt tgtgggcccc acatactgga agaaagagaa      60agtggactct ggttatgcta ttgggccttt aatgaatggg ctttatctta atgtaaattt     120atattgggct tgaataaaaa agaatagttg ggctcagata aaatacaatt ttatttaatt     180atatatctga atacgtataa gtacgtattc atacatctga attcataaat acatcgtatt     240catcacctac atctatatct tcaactggtg cctcttccat cattagtatg tctataactt     300gtaccatatc ttcttgcctg aattcccaaa tatttgagtg cttgtacatt attttgaggc     360agtgttttat gccttcttct aggctgttaa agtcgattgg tgggatgagc ccttggtgcc     420tgtaagggat catgaatttc ttcttggcta atgctggtga tcgtgttgag cttagttcga     480cctgaacaag gaaggaagtg tctgcttcca atttgacatc tattttaaat tccagacccc     540tcttgttgtt gtatttgata gtcattctta tttggtggtg gattgtacat gagtttgatc     600tgcttaaaaa atgcgagtaa tgttgataca tggactggat tgatttacgt gtttcattgt     660tgttcatata gggtatagtg gagatatttg tcattgatag tgacatgact atcaatgtta     720attactttta ctaatgacat tataatctag attataatga ataagtaaaa tttaaaagaa     780aaatagaaaa taaaataaaa aatgtgaaga aaataaaata aaaaaagaaa gaagacaata     840gacataaaca caaacaaaca tatactaaga aaagaaaagg gagcgcagcg gaaaaaaaaa     900acatagattc aaaagcaata gttgcaagaa aacagaaaaa aaagaaaaag acaaataaac     960aattaaaaca aaatgaataa aataaaacag taaaaacctg gccaggtttt taccgcgcgg    1020ttaaaaaaat aaattacatt cacctcaaag gtaaataatt aggaccccaa aggtaaatga    1080ggcaccgata tatcggtgcc tctatcaggt cctcaataat tacattccaa aaataccctt    1140atccctgtgt ctgaaaggcg cgtggtattg cgctgaaaaa gttaagtttc tctctccaaa    1200aaccccccgg agctccgatt gagggccttc cggtcatcaa tttgcgacac gcgcggcggt    1260gtgtacccct gggagggtag aaaccactac gctacgcagc agccttagct acgccggagc 1320ttagctcgcc accgttataa tatt 1344 Information (xxii) sequence characteristics of SEQ ID NO.11

(A) Length: 1341 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(xxiii)分子型：基因组DNA序列描述：SEQ ID NO.11accggtggcg agcggggttt ttccgaactt tgtgggaccc acatactgga agaaagagaa      60agtggactct ggttatgcaa ttgggccttt aataaatggg ctttatctta atggaaatgt     120gtattgggct tgaaaaaaaa aaaatagttg gactcaaata aaatacaatt ttatttaatt     180atatatctga atacgtataa gtacgtattc atacatctga attcataaat acatcatatt     240catcacctac atctatatct tcaactggtg cctcttccat cattagtatg tctataacct     300gtaccatatc ttcttgcctg aattcccaaa tatttgagtg tttgtacatt atcttgaggc     360attgttttat gccttcttct aggctgttga agtcgattgg tgggatgagc ccttggtgcc     420cgtaagggat catgaatttc ttcttggcta gtgctggtga ccgtgttgag cttagttcaa     480cctgaacaag gaaggaagta tctgcttcca atttgacatc tattttgaat tccagacccc     540tcttgttgtt gtatttgata gtcattctta tttggtgtgg attgtacatg agtttgatct     600gcttaaataa tgcaagtagt tttgatacat ggacttgatt gatttacgtg tttcattgtt     660gttcatatag gttatagtgg agatatttgt cattgatagt gacattacta tcaatgttaa     720gtacttttac taatgacata ataatctaga ttattatgaa taagtataag tgaaaggaaa     780aatagaaaaa aatataaaat ttgaagaaaa taaaataaaa aaagaaagaa gacaataaac     840ataaacacaa acaaacatat actaagaaaa gaaaagggag cgcagcgaaa aaaaaaaaca     900tatatccaaa accaatagaa gcaagaaaac agaataaaaa gaaaaagaaa aataaacaat     960taaaacaaat tgaataaaat aaaacagtaa aaacatgaca cggtttttac tgcgcggtaa    1020aaaaaataaa ttacattcac ctcgtaggta ataatttgga caccaaaggt aaatgagacc    1080ccgatacaat tggtgtacct attagtgtct caatattgaa tttccaaaaa tacccttatc    1140cctgtgtctg aaaggcgcgt gggagtgcgc tgaaaaagtt aagtttctct ctccaaaaac    1200tcaccggagc tccgattgag ggccttccgg tcatcaattt gcgacacgcg cggcggtgtg    1260tacccctggg agggtagaaa ccactacgct acgcagcagc cttagctacg ccggagctta 1320gctcgccacc gttataatat t 1341SEQ ID NO.12 information (xxiv) sequence feature

(A) Length: 1339 bases

(B) type: nucleic acid

(C) Chain type: single chain

(D)拓扑：环形(xxv)分子型：基因组DNA序列描述：SEQ ID NO.12accggtggcg agcggggttt ttccgaactt tgtgggcccc acatattgga agaaagagaa      60agtggactct ggttatgcaa ttgggccttt aatgaatggg ctttatctta atgtaaatgt     120ctattgggct tgagtaaaaa agaatagtag ggctcaaata aaatacaatt ttatttaatt     180atatatctga atacgtataa ttacgtattc atacatctga attcataaat acatcgtatt     240catcacctac atctatatct tcaactggtg cctcttccat cattagtata tctataactt     300gtaccatatc ttcttgcctg aattcccaaa tatttgagtg cttgtacatt attttgaggc     360agtgttttat gccttcttct aggctgttga agtcgattgg tgggatgagc ccttggtgcc     420tgtaagggat catgaatttc ttcttggcta atgctggtga ccgtgttgag cttatttcga     480cctgaacaag gaaggaagtg tctgcttcca acttgacatc tattttgaat tccagacccc     540ttttgttgtt gtatttgata gtcattctta tttggtgtgg attgtacatg agtttgatct     600gcttaaataa tgcaagtaat tttgatacat ggacttgatt gatttacgtg tttcattgtt     660gttcatatag gttatagtgg agatatttgt cattgatagt gacattacta tcaatgttaa     720gtacttttac taatgacata ataatctaga ttattatgaa taagtataat gggaaaaaga     780atagaaaaaa aataaaaaac ttaagataag aaaataaaaa aggaaagaag ataatacatg     840taagcacaaa caaaaatata tttagaaaag aaaaagggag cgcagcgaaa aaaaaaaaat     900aaattcaaaa ccaacagagt caacaaaaca taaaaaataa aaaaataaaa ataaagaatt     960aaagcaaaac aaattaaata aaatagtaaa aacatgagca ggtttttacg gcgttgttaa    1020aaaatagtta cattaaccgc ataggtaaat aatcgggaca ccaaaggtaa atgggacacc    1080gatacaattg gtgtacctat tagtgtctca aaattgaatt tccaaaaata cccttatccc    1140tgtgtctgaa aggcgggtgg tattgcgctg aaaaagttaa gtttttctct ccaaaaactc    1200cccggagctc cgattaaggg ccttccggtc atcaatttgc gacacgcgcg gcggtgtgta    1260cccctgggag ggtagaaacc actacgctac gcagcagcct tagctacgcc ggagcttagc 1320tcgccaccgt tataatatt 1339

Claims (10)

1. a novel plant dna viral vector is characterized in that, it comprises that the infectivity carrier of tomato yellow leaf curl china virus DNA-A and DNA β reaches by its deutero-plant gene silencing carrier and virus expression carrier.

2. the satellite DNA molecule of a novel structure plant DNA virus carrier, it is characterized in that, it comprises: a kind of whitefly-transmitted geminivirus tomato yellow leaf curl china virus DNA beta molecule, described nucleotide sequence and Seq ID No.1-12 have 72% homology at least.

3. the purposes of a novel plant dna viral vector is characterized in that, it is used for determining viral genome function, Plant Genome function and expresses foreign protein;

4. as the purposes of a kind of novel plant dna viral vector as described in the claim 3, it is characterized in that its method of determining viral genome function, Plant Genome function and expressing foreign protein comprises:

A. the DNA β with two forward multiple sudden changes or insertion plant cDNA or insertion foreign protein builds up to plant expression vector;

B. the carrier that makes up among a is imported the Agrobacterium host;

C. by injection the Agrobacterium among the b is inoculated host plant;

D. observe the detection of phenotype and molecular level.

5. a kind of novel plant dna viral vector as claimed in claim 1, it is characterized in that, it is the infectivity carrier of tomato yellow leaf curl china virus DNA-A and DNA β, this infectivity carrier inoculation host plant, DNA-A can systemic infection, causes typical symptoms when inoculating with DNA β but have only.

6. as the purposes of claim 3 or 4 described a kind of novel plant dna viral vectors, it is characterized in that utilizing the clear and definite viral genome function of DNA of plants infectivity carrier, on primer, introduce mutating alkali yl, virus genomic goal gene is realized sudden change by Overlap-PCR.

7. a kind of novel plant dna viral vector as claimed in claim 1 is characterized in that, it is an infectivity carrier deutero-plant gene silencing carrier, thereby the function of reticent research plant gene takes place this carrier inducing plant gene.

8. the purposes of a kind of novel plant dna viral vector as claimed in claim 3, it is characterized in that, utilize the clear and definite Plant Genome function of plant gene silencing carrier, its method is that an open reading frame to DNA β lacks and be changed by multiple clone site, inserts the cDNA fragment of plant endogenous gene and induce silence on its multiple clone site.

9. a kind of novel plant dna viral vector as claimed in claim 1 is characterized in that, it is an infectivity carrier deutero-dna virus expression vector, it is characterized in that this expression vector can expression alien gene.

10. as the purposes of described a kind of novel plant dna viral vector as described in the claim 3, it is characterized in that, utilize virus vector to express foreign protein, its method is to make up the DNA β of an open reading frame of two disappearances and have only one group of multiple clone site, and foreign protein directly inserts multiple clone site and expresses.