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WO2011130895A1 - Promoteur siubi1, son procédé de préparation et ses utilisations - Google Patents

Promoteur siubi1, son procédé de préparation et ses utilisations Download PDF

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Publication number
WO2011130895A1
WO2011130895A1 PCT/CN2010/001774 CN2010001774W WO2011130895A1 WO 2011130895 A1 WO2011130895 A1 WO 2011130895A1 CN 2010001774 W CN2010001774 W CN 2010001774W WO 2011130895 A1 WO2011130895 A1 WO 2011130895A1
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Prior art keywords
rice
japonica
promoter
seq
nucleotide sequence
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Chinese (zh)
Inventor
张耕耘
倪雪梅
黄刚
张印新
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters

Definitions

  • the present invention belongs to the field of plant molecular biology and relates to a promoter, particularly a promoter of a plant such as millet, and a preparation method and use of the promoter. Background technique
  • a promoter is a component of a gene, usually located on the structural gene 5, which is a DNA sequence that RNA polymerase recognizes, binds to and initiates transcription.
  • the promoter can direct the holoenzyme to properly bind to the template, activate the RNA polymerase, and initiate gene transcription to control the start and expression of the gene expression (transcription).
  • promoters are one of the important factors affecting the efficiency of transgene expression. Selecting a highly efficient promoter is the key to efficient expression of foreign genes.
  • constitutive promoter means that under the control of the constitutive promoter, there is no significant difference in gene expression between different tissues and developmental stages, so it is called a constitutive promoter.
  • a constitutive promoter widely used at present is CaMV35S, which produces high efficiency expression in both monocotyledonous and dicotyledonous plants, but Ajith Anand et al. transferred the chitinase gene of rice to wheat and found that CaMV35S was used. As a promoter, the transferred plants showed gene silencing in the second generation, and the maize ubiquitin Ubiquitin promoter was used. In the fourth generation, the gene expression was still large (Ajith Anand et al., Plant). Biotechnology Journal, 2003 (1): 241-251) ⁇
  • the ubiquitin (Ub iqui t in, Ub i ) promoter is widely distributed in eukaryotes and has significant effects in enhancing the long-term and stability of gene expression, and has high activation efficiency, low methylation level, and genetic It is favored by factors such as stable traits (Xie Wei, Le Chaoyin. Journal of China Three Gorges University, 2007, 29 ( 2 ) : 176-179 ).
  • promoter sequences have been isolated from many ubiquitin genes, including: Ubi-1 promoter in the maize genome, rice ubiquitin RUBQ2 promoter, Arabidopsis ubiquitin promoter, sunflower ubiquitin UbBl promoter, tobacco Ubiquitin Ub i.
  • the maize ubiquitin Ubi-1 promoter has been widely used in monocotyledonous plants such as corn, wheat and rice.
  • the rice ubiquitin RUBQ2 promoter is also widely used in rice and sugar cane.
  • the Ub i promoter is effective in promoting long-term stable and efficient expression of foreign genes, and many Ub i promoters have been used in monocots and dicots. Moreover, the Ub i promoter has a higher level of transcriptional regulation than the promoters of nos, ocs, mas derived from T-DNA and CaMV35S and CsVMV derived from viruses. Guo Dianjing et al. (Acta Genetics, 1999, 26 (2): 168-173) found that the Ub i promoter was the most efficient in transgenic wheat callus, 4-5 times more than the CaMV35S promoter. Genschik et al.
  • the inventors In order to provide a new tool and choice for the regulation of target gene expression in plant research, the inventors have provided a new ubiquitin promoter through in-depth study of the millet genome, which can be used to regulate plants. Target gene expression.
  • a plant promoter having the nucleotide sequence set forth in SEQ ID NO: 1 is provided.
  • the specific base sequence length of the promoter is 2020 bases, as shown in SEQ ID NO: 1:
  • promoter sequence shown by SEQ ID NO: 1 is referred to as promoter S iUbi l , also referred to as promoter P603 for short.
  • the promoter is a constitutive promoter, rice callus with the promoter and GUS, and transgenic rice seedlings after GUS staining experiments, the rice callus and The roots, stems, leaves, etc. of the transgenic rice seedlings turn blue.
  • a further aspect of the invention relates to a promoter having a sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1.
  • a further aspect of the invention also relates to a variant of the promoter of SEQ ID NO: 1 having a promoter function selected from the group consisting of:
  • hybridization conditions are classified according to the degree of "stringency” of the conditions used in the measurement of hybridization.
  • the degree of stringency can be based, for example, on the melting temperature (Tm) of the nucleic acid binding complex or probe.
  • Tm melting temperature
  • “maximum stringency” typically occurs at about Tm-5 ⁇ (below probe Tm 5 °C); “higher stringency” occurs about 5-10*C below Tm; “medium tightness” occurs Approximately 10 - “low stringency” below the needle Tm occurs approximately 20 - 25 below Tm.
  • hybridization conditions can be based on hybridized salt or ionic strength conditions and/or one or more stringency washes.
  • nucleic acid sequence that is identical or nearly identical to the hybridization probe can be determined using the conditions of maximum stringency; and nucleic acids having about 80% or more sequence identity to the probe are determined using conditions of high stringency. sequence.
  • suitable moderately stringent conditions for detecting hybridization of a polynucleotide of the invention to other polynucleotides include: pre-washing with 5xSSC, 0.5% SDS, 1. OmM EDTA (pH 8.0) solution; Hybridization was carried out in 5x SSC at -65 °C overnight; then washed twice with 2x, 0.5x and 0.2x SSC containing 0.1% SDS at 65 °C for 20 minutes.
  • hybridization stringency can be readily manipulated, such as varying the salt content and/or hybridization temperature of the hybridization solution.
  • suitable high stringency hybridization conditions include the above conditions, except that the hybridization temperature is raised to, for example, 60-65'C or 65-70 °C.
  • the nucleotide sequence which hybridizes under high stringency conditions to the nucleotide sequence shown by SEQ ID NO: 1 has the same or similar nucleotide sequence as SEQ ID NO: Promoter activity.
  • the nucleotide sequence which has one or more base substitutions, deletions, and additions to the nucleotide sequence shown by SEQ ID NO: 1 means that the nucleotides are respectively or simultaneously
  • the 5, the end and/or the 3, the end, and/or the sequence of the sequence are, for example, no more than 2 - 45, or no more than 2 - 30, or no more than 3 - 20, or no more than 4 - 15, or no more than 5 - 10, or no more than 6-8 consecutive integers are represented respectively by one base substitutions, deletions, additions, modifications 0
  • nucleotide sequence shown in SEQ ID NO: 1 is substituted with a nucleotide sequence represented by SEQ ID NO: 1 by a substitution, deletion or addition modification of one or more of the above bases.
  • the nucleotide sequence has the same or similar promoter activity.
  • a polynucleotide having a nucleotide sequence such as at least 95% "same" to the reference nucleotide sequence of SEQ ID NO: 1 means: a reference core of SEQ ID NO: 1.
  • the polynuclear per 100 nucleotides of the nucleotide sequence The nucleotide sequence of the nucleotide has the same nucleotide sequence as the reference sequence except that it contains up to 5 nucleotides. In other words, in order to obtain a nucleotide sequence and a reference nucleotide sequence of at least 95 ° /.
  • nucleotides in the reference sequence can be deleted or replaced by another nucleotide; or some nucleotides can be inserted into the reference sequence, wherein the inserted nucleotides can be as many as 5% of the total nucleotides of the reference sequence; or in some nucleotides, there is a combination of deletions, insertions and substitutions, wherein the nucleotides are up to 5% of the total nucleotides of the reference sequence.
  • mutations in the reference sequence may occur at the 5, or 3, terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, either alone in the nucleotides of the reference sequence, or as an OR Multiple adjacent groups are present in the reference sequence.
  • BLAST and BLAST 2.0 algorithms for determining sequence identity and percent sequence similarity are, for example, BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1977) Nucl. Acid. Res. 25: 3389-3402 and Altschul et al. 1990) J. Mol. Biol. 215: 403-410.
  • BLAST and BLAST 2.0 can be used to determine the percent nucleotide sequence identity of the invention, as described, for example, in the literature or by default parameters.
  • Software for performing BLAST analyses is available to the public through the National Center for Biotechnology Information (NCBI).
  • the nucleotide sequence having at least 90% sequence identity to the nucleotide sequence shown by SEQ ID NO: 1 comprises a polynucleotide sequence substantially identical to the sequence disclosed in SEQ ID NO: 1,
  • it contains at least 90% sequence identity, preferably at least 91%, 92%, 93%, 94%, 95, as compared to the polynucleotide sequences of the present invention.
  • sequences of sequence identity preferably at least 96%, 97%, 98% or 99% or higher.
  • the nucleotide sequence having at least 90% sequence identity with the nucleotide sequence shown by SEQ ID NO: 1 has the same or similar nucleotide sequence as SEQ ID NO: 1.
  • Promoter activity is derived from a monocotyledonous plant, specifically, the monocotyledonous plant is a millet, for example, the millet is a modified ⁇ Zhanggu No. 1 (Improved ⁇ Zhanggu No. 1 seed in April 2010 On the 1st, it was deposited at the Wuhan University of Wushan City, Hubei province, Wushan City, Wuhan, China, the China Center for the Collection of Cultures (CCTCC), the preservation number is CCTCC P201 006).
  • a further aspect of the invention also relates to a recombinant vector comprising a promoter of the invention.
  • the recombinant vector can be obtained by inserting the above promoter into a cloning vector or an expression vector.
  • Cloning vectors suitable for construction of the recombinant vectors of the invention include, but are not limited to, for example: pUC18, pUC19, pUC118, pUC119, pMD19-T, pMD20-T, pMD18-T S imp le Vec ter, pMD19-T S imp le Vec ter and so on.
  • Expression vectors suitable for construction of the invention include, but are not limited to, for example: pBI 121, pl 3W4, pGEM, and the like.
  • the recombinant vector is a P8+P603 recombinant vector.
  • a further aspect of the invention also relates to a recombinant cell comprising said recombinant vector of a promoter of the invention.
  • the recombinant cell can be obtained by transforming the recombinant vector containing the promoter of the present invention into a host cell.
  • Host cells suitable for constructing the recombinant cells of the present invention include, but are not limited to, for example, Agrobacterium tumefaciens cells LBA4404, EHA105, GV3101 and the like.
  • the recombinant cell is Agrobacteriu tumefaciens EHA105-P603.
  • the invention relates to a monocot callus transformed with a promoter of the invention.
  • the monocot is rice.
  • the rice includes, but is not limited to, for example: Zhonghua 9, Zhonghua 10, Zhonghua 11, Taipei 309, Danjiang 8, Yundao 2, Yuyou 63, Yanyou 608, Fengyou 22, Yanyou 88, Yuyou 416, Yanyou 107, Yuyou 128, Yanyou 718, Zhunliangyou 527, Chuanong No.
  • a further aspect of the invention also relates to a method of preparing a promoter of the invention, comprising the steps of:
  • PCR amplification primer pair can be designed according to the nucleotide complementation principle according to the nucleotide sequence of interest to be amplified.
  • the PCR amplification primer pair is as set forth in SEQ ID NO: 2 and SEQ ID NO: 3.
  • a further aspect of the invention also relates to a method of regulating expression of a gene of interest in a plant, the method comprising the step of transforming a callus of a plant with the promoter of the invention.
  • the transformation of the monocot callus utilizes recombinant cells comprising a promoter of the invention.
  • the aforementioned recombinant Agrobacterium tumefaciens EHA105-P603 is utilized in the transformation of the monocot callus.
  • the callus of the monocotyledonous plant is a rice callus, and specifically, the rice is Nipponbare.
  • plant gene transformation technology can be used to insert the gene of interest into the plant.
  • the genome includes Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, gene gun transformation, and electroporation. It is well known in the art that Agrobacterium-mediated gene transformation is often used for gene transformation of monocots and dicots, but other transformation techniques can also be used for gene transformation of the monocots of the present invention.
  • Agrobacterium-mediated gene transformation is often used for gene transformation of monocots and dicots, but other transformation techniques can also be used for gene transformation of the monocots of the present invention.
  • particle bombardment microgold or tungsten particle coated transformed DNA
  • protoplast transformation a method for transforming monocots that can also be used is protoplast transformation. After gene transformation, a general method is used to select and regenerate plants integrated with expression units.
  • plants which can utilize the monocot promoter to regulate expression of a gene of interest include, but are not limited to, rice, wheat, corn, millet, sugar cane, sorghum, barley, and the like.
  • a further aspect of the invention also relates to the use of a promoter according to the invention for regulating the expression of a gene in a plant.
  • the plant is a monocot.
  • the gene of interest regulated by the promoter of the invention is GUS.
  • the monocot is rice, and in particular the rice is Nipponbare.
  • the promoter of the present invention may be used in the form of a single copy and/or multiple copies, or may be used in combination with a promoter and/or enhancer known in the art.
  • a further aspect of the invention relates to the use of a promoter according to the invention in rice breeding.
  • the rice is Nipponbare, Zhonghua 9, Zhonghua 10, Zhonghua 11, Taipei 309, Danjiang 8, Yundao 2, Yanyou 63, Yanyou 608, Fengyou 22, Yanyou 88, Qiyou 416 , Youyou 107, Youyou 128, Yanyou 718, Zhunliangyou 527, Chuanong 1 , Miscellaneous 0152, Japonica 88, Japonica 90, Japonica 92, Japonica 94, Japonica 96, Japonica 185, Indica 187, japonica 189, japonica rice 191, japonica rice 193, japonica rice 195, japonica rice 197, japonica rice 199, japonica rice 199, japonica rice 203, japonica rice 205, japonica rice 207, and Tsuwara 101.
  • the rice is Nipponbare.
  • the promoter of the present invention can be a novel promoter, as a plant: for example, rice, a transgenic tool promoter. Since the promoter of the present invention is a constitutive promoter and can regulate the expression of a gene of interest across species, the promoter of the present invention can facilitate a variety of plant breeding, such as low-expression gene transformation seedling screening, plant flower organ failure. Research on molecular breeding such as breeding. It will greatly shorten the breeding time of excellent varieties.
  • the promoter of the present invention can be widely used for cultivating plants such as rice, wheat, corn, millet, sugar cane, sorghum, barley, and the like.
  • the inventors obtained a ubiquitin promoter derived from millet by bioinformatics research, and verified the function of the promoter P603 by biological experiments, which has the function of regulating the expression of the target gene across species.
  • the constitutive promoter Specifically, the promoter can regulate the expression of GUS gene in rice: the efficient expression of the gus gene can be regulated in the root, stem and leaf of rice callus and rice transgenic seedlings.
  • F ig. 1 is the result of PCR amplification detection of promoter P603, wherein lane M: 200 bp DNA Ladder Marker, the number on the left side indicates the size of the strip of the ladderer pointed to, in bp; lane 1 : PCR amplification products.
  • F ig. 2 is a schematic representation of the pCAMBIA-1301 plasmid used to construct the p8 plasmid.
  • F ig. 3 is a schematic representation of the multiple cloning site and the GUS sequence portion of the p8 plasmid map.
  • F ig. 4 is a schematic representation of the p8 plasmid.
  • F ig. 5 is the result of GUS staining of transformed rice callus.
  • rice callus transformed with recombinant Agrobacterium tumefaciens p8+P603 having the promoter sequence P603 of the present invention exhibits blue color after GUS staining; recombinant Agrobacterium tumefaciens p8 without the promoter sequence of the present invention
  • the rice callus of the plasmid did not change color after GUS staining.
  • F ig. 6 is the result of GUS staining of transformed transgenic rice seedlings.
  • the rice seedling transformed with the recombinant Agrobacterium tumefaciens P8+P603 having the sequence of the promoter P603 of the present invention (right) is stained with GUS, and the roots, stems and leaves thereof are blue;
  • the sub-sequence of recombinant Agrobacterium tumefaciens P8 transformed rice seedlings did not change in color of roots, stems and leaves after GUS staining.
  • Example 1 Promoter PCR amplification of P603 fragment and construction of pMD18-T+P603 recombinant vector
  • the genomic DNA (gDNA) of the improved sorghum Zhanggu No. 1 seed was extracted using the plant genomic DNA extraction kit (TIANGEN new plant genomic DNA extraction kit, catalog number: DP320-02), and the improved ⁇ ⁇ ⁇ 1 was modified according to the promoter.
  • the sequence in gDNA is designed with a pair of PCR-specific amplification primers at the beginning and the end (upstream primer Fl, restriction enzyme site EcoR I and protective thiol, downstream primer R1, restriction enzyme site Sbf I and Protect bases).
  • ddH 2 0 fills up to a total volume of 25 ⁇ 1
  • the PCR amplification procedure was: 94 pre-denaturation for 5 min, then denaturation at 94 for 45 s, 55 ⁇ annealing for 50 s, 72 extension for 90 s, 35 reaction cycles, and finally 72 X: extension for 7 min.
  • the upstream primer F1 GGGAATTCACTTAGCTTCTGGCCATCTCCAGA (SEQ ID NO: 2), wherein the underline represents the EcoR I restriction site.
  • the downstream primer R1 GCCCTGCAGGCTGTAGAAGAAAAAACAAGCAA (SEQ ID NO: 3), wherein the underline represents the Sbf I restriction site.
  • the PCR amplification product was separated by 1.0% agarose gel electrophoresis to obtain a band of 2038 bp (Fig. 1), which was purified by TIANGEN agarose gel DNA recovery kit (catalog number: DP209-03). .
  • the PCR amplification product obtained above was subjected to T/A cloning (PMD18-T plasmid, TaKaRa, D103A), transformed into Escherichia coli, and the positive clone was picked and sequenced (as shown in SEQ ID NO: 4), which proved to be correct.
  • connection conditions of the T/A clone are as follows: T/A connection system: 10 ⁇ ⁇
  • PCR amplification product 10 ng ⁇ 20 ng, according to its concentration, ddH 2 0 is added to 10 ⁇ 1
  • the promoter of the ⁇ 603 promoter of the present invention was extracted from the Escherichia coli DH 5 ⁇ - ⁇ 603 transformed with the promoter P603 constructed in Example 1 according to the manual of the TIANGEN Ordinary Plasmid Mini Kit (Cat. No. DP103-03).
  • the resulting ligated product P8+P603 recombinant vector was transformed into competent cells DH5 a , 37 X: inverted culture for 16-24 h according to the method of molecular cloning experiments (third edition, Science Press). After the transformant grows out of bacteria, the monoclonal is picked for PCR detection and enzyme digestion.
  • the p8 plasmid used in the present invention is provided by pCAMBIA-1301 plasmid (Dong Yang, Kunming Institute of Zoology, Chinese Academy of Sciences; or may be derived from, for example, Shanghai Guorui Gene Technology)
  • the company purchased, or can be obtained from The CAMBIA Bios (biological open source) Licensee, Austral ia), modified and constructed as follows, as follows:
  • the plasmid pCAMBIA-1301 was double-digested with Kpn I /Nco I (NEB) to recover large fragments.
  • the following sequence was synthesized according to the restriction enzyme site used: GGTACCAAGCTTACTAGTCCTGCAGGTCTAGAG GATCCGTCGACCATGG (SEQ ID NO: 5) (The cleavage site included is Kpn I /Hindm/Spe I /Sbf I /Pst I /Xba I /BamH I /Sal I /Nco I ), recovered by double digestion with Kpn I /Nco I, and ligated with the large fragment recovered above to transform topk cells (provided by Dong Yang of Kunming Institute of Zoology, Chinese Academy of Sciences; or from Beijing, for example: Beijing Solaibao Technology Co., Ltd.
  • the transformant was selected by PCR, and the transformant with the amplified fragment of 350 bp was the multiple cloning site containing the desired construction. And the transformant of the p8 plasmid of the GUS sequence (see Figure 4).
  • the multiple cloning site in the p8 plasmid and the length of the GUS sequence are 2353 bases, as shown in SEQ ID NO: 8 (see Figure 3):
  • the p8 plasmid constructed in the present invention as shown in the above sequence EcoR I /Sac I /Kpn I /H ind m /Spe I /Sbf I /Ps t I /Xba I /BamH I /Sa in the multiple cloning site l I / Nco I restriction sites are indicated by box and underline, respectively, and the primers used for screening transformants are GCTTCCGGCTCGTATGTTGT/GAGTCGTCGGTTCTGTA AC (ie SEQ ID NO: 6 and 7), which are indicated by double underline, and GUS sequences are indicated in italics.
  • the intron sequences are shown in italics and shading, respectively.
  • the cloning vector pMD18-T+P603 obtained in Example 1 and the p8 plasmid constructed as described above were treated according to the following conditions under restriction enzymes EcoR I (NEB) and Sbf I (NEB).
  • the cloning vectors pMD18-T+P603 and p8 plasmid were digested as follows: Enzyme digestion system: 50 ⁇ 1
  • the ⁇ 4 buffer was thawed on ice, and the p8 plasmid vector after digestion was added in an amount of about 20 ng, and the P603 fragment in the present invention was added in an amount of 10-20 ng.
  • the competent cell DH5ot prepared by the 100 ⁇ l calcium chloride method was taken out from the ultra-low temperature freezer, and after melting on ice, 10 ⁇ ⁇ of the above-mentioned ligation product was added, and the mixture was gently stirred, water bathed for 30 min, and heat shocked at 42 ° C for 60 s. Water bath for 5 min, add 600 ⁇ l 4 pre-cooled S0C, 37. C resuscitate at 220 rpm for 45 min, centrifugation at 8000 rpm for 30 s, remove the supernatant, and take 150 ⁇ , gently spread, glass beads coated with LB (kan), 37 X: inverted culture for 16-24 h. The recombinant vector p8+P603 was obtained.
  • the resulting recombinant vector P8+P603 was subjected to PCR detection using Fl (SEQ ID NO: 2) and Rl (SEQ ID NO: 3) as primer pairs, respectively, to confirm that the resulting recombinant vector P8+P603 contained the desired promoter P603.
  • the recombinant vector containing the recombinant vector P8+P6Q3 was screened by EcoR I /Sbf I digestion.
  • Example 3 Preparation of recombinant Agrobacterium tumefaciens EHA105-P603 cells
  • the p8+P603 recombinant vector constructed as described in Example 2 and the p8 plasmid as a control were separately transformed into root cancer prepared according to the calcium chloride method described in the Guide to Molecular Cloning (Third Edition, Science Press).
  • Agrobacterium EHA105 (2009 12 On the 24th of the month, it was deposited in the Wuhan University of Wuhan Wushan, Wuhan, Wushan, Hubei province, the China National Type Culture Collection (CCTCC), the accession number is CCTCC M 209315), the specific methods are as follows:
  • the Agrobacterium tumefaciens competent cell EHA105 was taken out in an ultra-low temperature water tank and thawed on ice. After thawing, 5 ⁇ of ⁇ 8+ ⁇ 603 recombinant vector and ⁇ 8 plasmid and ⁇ 8 empty vector as control were added, gently mixed, ice-bathed for 10 min, frozen in liquid nitrogen for 5 min, and thawed at 37 °C for 5 min.
  • the PCR was carried out using Fl (SEQ ID NO: 2) and Rl (SEQ ID NO: 3) as primers and the transformants were cleaved by Kpn l /Sbf I.
  • a recombinant Agrobacterium tumefaciens which is a recombinant vector p8+P603 was amplified by PCR and amplified with a band of about 2038 bp and a band of about 2027 bp.
  • the recombinant Agrobacterium tumefaciens having the recombinant vector P8+P603 obtained according to the above method is named Recombinant Agrobacterium tumefaciens EHA105-P603.
  • the obtained recombinant Agrobacterium tumefaciens harboring the p8 plasmid was designated as Recombinant Agrobacterium tumefaciens EHA105-p8.
  • Recombinant Agrobacterium tumefaciens EHA105-p8 was designated as Recombinant Agrobacterium tumefaciens EHA105-p8.
  • the rice callus was induced as follows, and the callus was transformed with recombinant Agrobacterium tumefaciens EHA105-P603 and recombinant Agrobacterium tumefaciens EHA105-p8, respectively.
  • Acetosyringone (Acetosyringone, acetosyringone) in 30 ml of A AM medium (see Table 5 for specific formulation), gently resuspend the recombinant Agrobacterium tumefaciens cells (recombinant Agrobacterium tumefaciens EHA105-P603 or recombinant Agrobacterium tumefaciens EHA105) -p8 ) ;
  • step 4 placing the subcultured callus in a sterilized culture dish; pouring the recombinant Agrobacterium tumefaciens suspension prepared in step 3 into the culture dish, and immersing the callus therein for 15 min;
  • GUS staining solution (1 ml): 610 ⁇ 1 0.2 ⁇ Na 2 HP0 4 solution (pH-7.0); 390 ⁇ 10.2MNaH 2 P0 4 solution and 10 ⁇ 10.1 MX-gluc will use recombinant Agrobacterium tumefaciens EHA105 -P603 or recombinant callus of Agrobacterium tumefaciens EHA105-P8 was immersed in GUS staining solution, 37 was incubated until blue appeared, and the results were recorded. The results are shown in Figure 5, and recombined with p8+P603 containing the promoter.
  • Example 4 The callus obtained in Example 4 was transferred to MS-R differentiation medium containing 50 mg/1 hygromycin B (HmB) (see Table 7 for specific formulation).
  • the seedlings were sealed with a parafilm seal, 29.5°. C light culture for 3-4 weeks; when the seedling grows to 3-4 cm, transfer to 1/2 MS rooting medium containing 50 mg/1 hygromycin B (HmB) (see Table 8 for specific formulation) for rooting.
  • the GUS staining process of the transgenic rice seedlings was the same as that of the callus in Example 5. The results are shown in Figure 6.
  • the weight of the recombinant plasmid containing p8+P603 containing the promoter The roots, stems and leaves of rice seedlings mediated by Agrobacterium tumefaciens (Fig. 6 right) were stained blue, and the roots of rice transformed by Agrobacterium tumefaciens mediated by recombinant p8 plasmid without promoter , stems, leaves (as a control, Figure 6 left) The color did not change after GUS staining.
  • the results show that the P603 promoter of the present invention has a regulatory effect on GUS gene expression.
  • the formulation of the relevant medium used in the examples of the present invention is as follows:
  • the following "conventional sterilization" as used in the medium refers to sterilization under the following conditions:
  • Fe 2 EDTA stock solution 100X 10 ml 5 ml 20 ml
  • N 6 vitamin stock solution 1000X 1 ml 0.5 ml 2 ml Inositol ( 500X) 2 ml 1 ml. 4 ml
  • the pH was adjusted to 5.8 with IN potassium hydroxide, and then sterilized by a conventional method after sealing.
  • Iron salt (Fe 2 EDTA) stock solution (100X) 3.73 g EDTA Disodium (Na 2 EDTA ⁇ 2H 2 0 ) and 2.78 g of FeS0 4 .7H 2 0 were separately dissolved and mixed. Dilute to 100Q ml with distilled water, warm bath for 2 hours, and store for 4 months after cooling.
  • N 6 vitamin stock solution (1000X): Vitamin 0.10 g, vitamin B 6 0.05 g, niacin 0.05 g, glycine 0.20 g, add distilled water to 100 ml, filter sterilization, 4 X: save for less than 1 week.
  • Kanamycin (Kan) 50mg/ml 1 ml 0.5 ml
  • YM Broth dry powder 21 g 10. 5 g agar powder (Agar) 15 g 7.5 g conventionally sterilized, cooled to room temperature and added
  • AAM macro ( 1 OX) : 2.5 g magnesium sulfate heptahydrate ( MgS0 4 ⁇ 7H 2 0 ), 1.5 g calcium chloride dihydrate (CaCl 2 ⁇ 2H 2 0 ), 1.33 g sodium dihydrogen phosphate dihydrate (NaH 2 P0 4 .2 ⁇ 2 0) , dilute to 1 L of distilled water, and store at 4 °C for later use.
  • AAM micro ( 100X) 0.7 g manganese sulfate monohydrate ( MnS0 4 ⁇ H 2 0 ), 0.2 g zinc sulfate heptahydrate ( ZnS0 4 ⁇ 7H 2 0 ), 0.075 g potassium iodide (KI), 0.3 g boric acid (H 3 B0 3 ), 25 mg sodium molybdate dihydrate (Na 2 MoG 4 .2H 2 0 ), 2.5 mg copper sulfate pentahydrate (CuS0 4 .5H 2 0), 2.50 ⁇ cobalt chloride hexahydrate ((00:1 2 , 611 2 0), dilute to 1 L of distilled water, 4 ⁇ save for use.
  • Fe 2 EDTA stock solution 100X 10 ml 5 ml 20 ml
  • Niinicro mother liquor (1000X) 1 ml 0.5 ml 2 ml
  • N 6 vitamin stock solution 1000X 1 ml 0.5 ml 2 ml inositol ( 500X) 2 ml 1 ml 4 ml
  • Acetyl syringone (AS) 100 mM 1 ml 0.5 ml 2 ml Adjust the pH to 5.2.
  • Fe 2 EDTA stock solution 100X 10 ml 5 ml 20 ml inositol ( 500X) 2 ml 1 ml 4 ml sucrose ( Sucrose ) 30 g 15 g 60 g Sorbitol 30 g 15 g 60 g plant gel ( Phytagel ) 4 g 2 g 8 g added after routine sterilization
  • MS macro ( 20X) Ammonium nitrate 33.
  • Q g potassium nitrate 38.0 g, potassium dihydrogen phosphate 3.4 g, magnesium sulfate 7.4 g, calcium chloride 8.8 g dissolved one by one, then dilute to 1 L with distilled water at room temperature, 4 X: Save.
  • MS micro ( 1000X) manganese sulfate 16.90 g, zinc sulfate 8 ⁇ 60 g, boric acid 6.20 g, potassium iodide 0.83 g, sodium molybdate 0.25 g, copper sulfate 0.025 g, cobalt chloride 0.025 g, the above reagents are dissolved at room temperature Dilute to 1 L of distilled water and store at 4 °C.
  • MS vitamin stock solution (1000X): vitamin 0.010 g, vitamin B 6 0.050 g, niacin 0.050 g, glycine 0.200 g, dilute to 100 ml with distilled water, filter sterilization, 4 'C for less than 1 week.
  • MS macro (20X) is shown in Table 7.
  • MS micro (1000X) MS Vitamin Storage Solution (10QQX) See Table 7.

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Abstract

L'invention concerne un promoteur du millet génétiquement modifié Jizhanggu No.1 et ses variants, un procédé de préparation du promoteur et ses utilisations, le promoteur possédant la séquence nucléotidique représentée en SEQ ID NO: 1. Les variants présentant des fonctions de promoteur sont choisis parmi : 1) des séquences nucléotidiques qui s'hybrident à la séquence représentée en SEQ ID NO: 1 dans des conditions de stringence élevée, 2) des séquences nucléotidiques qui comprennent une modification de substitution, délétion et/ou d'insertion d'une ou plusieurs bases de la séquence nucléotidique représentée en SEQ ID NO: 1, et 3) des séquences nucléotidiques qui ont au moins 90 % d'identité vis-à-vis de la séquence nucléotidique représentée en SEQ ID NO: 1. L'invention concerne également des procédés de préparation du promoteur et ses utilisations dans la régulation des expressions des gènes cibles dans des plantes et du riz génétiquement sélectionné.
PCT/CN2010/001774 2010-04-23 2010-11-04 Promoteur siubi1, son procédé de préparation et ses utilisations Ceased WO2011130895A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6528701B1 (en) * 1999-03-02 2003-03-04 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Rice ubiquitin-derived promoters
CN1757740A (zh) * 2005-08-22 2006-04-12 中国林业科学研究院林业研究所 一种植物二价抗逆基因双元表达载体
WO2008140766A2 (fr) * 2007-05-08 2008-11-20 The Ohio State University Promoteurs d'ubiquitine de soja très efficaces et utilisations correspondantes
CN101457233A (zh) * 2007-12-12 2009-06-17 中国科学院遗传与发育生物学研究所 构建小球藻表达载体、转化小球藻和破壁小球藻的方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1789422A (zh) * 2005-10-14 2006-06-21 山东农业大学 Pnzip启动子的序列及其克隆与应用
CN100510077C (zh) * 2006-11-03 2009-07-08 上海师范大学 一种水稻高效表达启动子及其应用
CN101063139B (zh) * 2007-05-15 2010-09-22 中国农业大学 一种种子特异性高效启动子及其应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6528701B1 (en) * 1999-03-02 2003-03-04 Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College Rice ubiquitin-derived promoters
CN1757740A (zh) * 2005-08-22 2006-04-12 中国林业科学研究院林业研究所 一种植物二价抗逆基因双元表达载体
WO2008140766A2 (fr) * 2007-05-08 2008-11-20 The Ohio State University Promoteurs d'ubiquitine de soja très efficaces et utilisations correspondantes
CN101457233A (zh) * 2007-12-12 2009-06-17 中国科学院遗传与发育生物学研究所 构建小球藻表达载体、转化小球藻和破壁小球藻的方法

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