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WO2014082285A1 - Protéine à doigt de zinc du coton (czf5) et son gène codant et son utilisation - Google Patents

Protéine à doigt de zinc du coton (czf5) et son gène codant et son utilisation Download PDF

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Publication number
WO2014082285A1
WO2014082285A1 PCT/CN2012/085628 CN2012085628W WO2014082285A1 WO 2014082285 A1 WO2014082285 A1 WO 2014082285A1 CN 2012085628 W CN2012085628 W CN 2012085628W WO 2014082285 A1 WO2014082285 A1 WO 2014082285A1
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Prior art keywords
plant
seq
gene
expression vector
ghczf5
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Chinese (zh)
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崔洪志
梁远金
田大翠
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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Priority to CN201280077197.8A priority patent/CN104812900A/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the present invention relates to plant proteins and their coding genes and applications, and more particularly to a cotton-derived zinc finger protein (Czf5) and a gene encoding the same, and its use in breeding transgenic plants having improved drought resistance.
  • Czf5 cotton-derived zinc finger protein
  • the impact of drought on crop yields ranks first in many natural adversities, and its harm is equivalent to the sum of other disasters. Many regions are the bottleneck of agricultural development.
  • the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid areas account for about 52% of the country's land area, and the annual drought-affected area amounts to 200-2.7 million hectares.
  • Cubic meters due to lack of water, less than 350-40 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the most severe areas in China, and spring droughts frequently reach 10 years.
  • genes and their expression products can be divided into three categories: (1) genes and products involved in signal cascade amplification systems and transcriptional control; (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) proteins associated with the uptake and transport of water and ions.
  • genes and products involved in signal cascade amplification systems and transcriptional control (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) proteins associated with the uptake and transport of water and ions.
  • the system has a further understanding (Liu Q. 1998.
  • Two transcription factors, DREB1 and DREB2 with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought- and low temperature-responsive gene expression, respectively, in Arabidopsis.
  • the first aspect of the invention provides a gene encoding a zinc finger protein Czf5 of cotton (this article is named
  • GhCzf5 the sequence of which is SEQ ID NO: 2.
  • a second aspect of the invention provides a recombinant expression vector comprising the gene of the first aspect of the invention, and the nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector; preferably, The vector is the rd29A-Gh Czf5-2300 vector shown in Fig. 2.
  • a third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.
  • a fourth aspect of the present invention provides a method for improving drought resistance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and expressing the gene;
  • the plant is tobacco.
  • a fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant
  • the plant is tobacco.
  • a sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving drought resistance of a plant and for use in plant breeding Use;
  • the plant is tobacco.
  • the seventh aspect of the present invention provides the gene-encoded protein of the first aspect of the present invention, which has an amino acid sequence as shown in SEQ ID NO: 1.
  • FIG. 1 is a construction flow of a plant expression vector (rd29A-GhCzf5-2300;) of GhCzf5.
  • Figure 2 is a plasmid map of the plant expression vector Crd29A-GhCzf5-2300 of GhCzf5.
  • FIG. 3 shows the drought resistance growth of control tobacco and transgenic tobacco.
  • CK left: control tobacco
  • T1D1 middle
  • T1D8 right: transgenic tobacco lines.
  • Figure 4 shows the results of verification of transcriptional levels of T1 transgenic tobacco plants resistant to drought and drought.
  • M is Marker
  • 1-8 is a drought-tolerant T1 transgenic tobacco plant
  • 9-12 is a drought-tolerant T1 transgenic tobacco plant.
  • African cotton (National Cotton Medium Term Bank, obtained by the China Cotton Research Institute, Uniform No.: ZM-06838) was sown on sterilized vermiculite at 25 ° C, photoperiod 16 h / 8 h (light intensity 2000 - 3000 Lx) conditions Under the culture, 1/2MS medium (containing 9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM NH 4 N0 3 , 0.75 mM MgSO 4 , 1.5 mM CaCl 2 , 50 ⁇ , 100 ⁇ 3 ⁇ 3 , 100 MMnSO 4 per week) , 30 ⁇ ZnS0 4 , 1 ⁇ 2 ⁇ 0 4 , 0.1 ⁇ CoCl 2 , 100 ⁇ Na 2 EDTA, 100 MFeSO 4 ). It was used for experiments when the seedlings were as high as 25-30 cm.
  • the test seedlings were divided into two groups, each with 4 pots and 1 pot per pot.
  • the first group was a control group, which was cultured at 25 ° C, photoperiod of 16 h/8 h (light intensity 2000-3000 Lx), and was normally watered.
  • the second group was the drought treatment group, cultured at 25 °C, photoperiod 16h/8h (light intensity 2000-3000 Lx), stopped watering, treated for 10 days, and cut 1/3 of the top of the two seedlings in time after treatment.
  • the leaves, which were rapidly frozen with liquid nitrogen, were stored in a -70 ° C refrigerator.
  • RNA extraction kit purchased from Invitrogen
  • the absorbance of total RNA at 260 nm and 280 nm was determined by HITACHI's UV spectrophotometer U-2001.
  • the OD 26Q / OD 28Q ratio was 1.8-2.0, indicating a high total RNA purity with 1.0% agarose gel.
  • the total RNA was detected by electrophoresis, and the 28S band was about twice as bright as the 18S band, indicating good RNA integrity.
  • mRNA was isolated using Qiagen's purification of polyA+ RNA from total RNA.
  • Two tester cDNAs with different adaptors were mixed with an excess of Driver for the first forward subtractive hybridization.
  • the products of the two first subtractive hybridizations were mixed, and a second forward subtractive hybridization was performed with the freshly denatured Driver cDNA, and the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.
  • the second PCR product of the forward subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) and pGEM-T Easy (purchased from Promega kit) according to the procedure of the pGEM-T Easy kit
  • the specific steps are as follows: The following components were sequentially added using a 200 ⁇ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 ⁇ 1, 2> ⁇ 4 ligase buffer 5 ⁇ l, pGEM-T Easy vector 1 ⁇ l, T4 DNA ligase 1 ⁇ l, ligated overnight at 4 °C. 10 ⁇ of the reaction product was added to 100 ⁇ of competent E.
  • coli JM109 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 seconds, ice bath for 2 min, and additional 250 ⁇ LB medium (containing 1%). Tryptone (purchased from OXOID), 0.5% Yeast Extract (purchased from OXOID), 1% NaCl (purchased from Sinopharm)), placed in a 37 ° C water bath, shaken at 225 rpm for 30 min, and inoculated with 200 ⁇ M bacteria in 50 g/ml Ampicillin was cultured on LB (ibid.)/X-gal/IPTG (X-gal/IPTG purchased from TAKARA) plates at 37 ° C for 18 h.
  • PCR primers Primer 1 and Primer 2R (Clontech's PCR-select TM cDNA Subtraction Kit kit) were used to carry out PCR amplification of the broth, and 452 positive clones were obtained, and then all the positive clones were sent to the British Shanghai) Trading Co., Ltd. sequencing.
  • GhCzfo GSP1 SEQ ID NO: I:
  • GhCzfo GSP2 SEQ ID NO: .5:
  • the experimental procedure was carried out according to the kit instructions (3, RACE System for Rapid Amplification of cDNA Ends kit was purchased from Invitrogen).
  • PCR reaction system 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA inverted cDNA, 1.0 ⁇ Ex Taq (purchased from TAKARA), 10 ⁇ primers SEQ ID NO: 4 and AUAP 2.0 1 ⁇ 1, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 33 cycles (94 °C for 30 s, 58 ° C for 30 s, 72 ° C for 1 min), 72 ° C for 10 min.
  • the resulting PCR product was diluted 50-fold with double distilled water and 2.0 ⁇ L was used as a template to replace the page with SEQ ID NO: 5 and 3' (Rule 26)
  • the first primer AUAP performs the first round of PCR amplification, and the specific steps are as follows:
  • PCR reverse 50 ⁇ PCR reverse;, V: system: 5 ⁇ ⁇ Buffer ⁇ 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ diluted first round PCR product, 1.0 l Ex Taq, 10 ⁇ primer SEQ ID NO: 5 and AUAP 2.0 1 ⁇ 1, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (94'C denaturation for 30 s, 58 °C for 30 s, 72 °C for 1 min), 72 °C for 10 min.
  • a strip of about 500 bp in the second PCR product was recovered (Gel Extraction Kit purchased ⁇ OMEGA), and it was ligated to pGEM-T Easy Vector, and then transformed into the large intestine rod JM109 (the specific method is the same as above), and randomly picked.
  • Ten white colonies were inoculated in LB liquid medium containing 50 g/mL ampicillin, cultured at 37 ° C overnight, and th oil was added to a final concentration of 20%, and stored at -80 ° C until use.
  • GhCzfS GSP3 SEQ ID NO: 6:
  • GhCzfS GSP I SEQ ID NO : 7:
  • GhCzfS GSP5 SEQ ID NO: 8:
  • the first round of PCR amplification was carried out using SEQ ID NO: 7 and 5' universal primer AAP (provided with the kit), and cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 6) was used as a template for the first round of PCR amplification.
  • SEQ ID NO: 7 and 5' universal primer AAP provided with the kit
  • cDNA reverse transcription cDNA reverse transcription primer SEQ ID NO: 6
  • PCR reaction system 5 ⁇ 10> ⁇ Ex Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcribed cDNA, 1.0 ⁇ ⁇ Taq (purchased from TAKARA), 10 ⁇ primer SEQ ID NO: 7 and AAP each with 2.0 ⁇ l and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (94'C variability 30 s, 55 'C annealing 30 s, 72 °C extension lmin), 72 "C extension for 10 min.
  • the obtained PCR product was diluted 50-fold with double distilled water and 2.0 ⁇ L was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 8 and the 3' primer AUAP, and the specific steps were as follows:
  • the resulting 5' RACE product was cloned and sequenced, and ligated with the 3' RACE product sequencing result and the SEQ ID No: 3 sequence.
  • the full-length cDNA sequence of GhCzf5 was obtained SEQ ID No: 9:
  • a TACAG TA T : QT AA GT TTT AAA AAAC GAT'S CGC ⁇ C ⁇ "_; -ACCCI GA'S ATGTGGT:3 ⁇ 4 G?TT ? TT ⁇
  • T A.TTTTTT AT -iC-TATTI : TT CAATTTA TTTAAAAA AAAAAAAAAA
  • GhCzfS SEQ ID XO: U:
  • the GhCzf5 full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11.
  • the PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template.
  • 50 l PCR reaction system 10 ⁇ 5xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 ⁇ 1, and 30 ⁇ double steamed water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • the PCR amplification product plus A tail 2.5 times the volume of absolute ethanol was added to the PCR product, placed at -20 ° C for 10 minutes, centrifuged, the supernatant was removed, air-dried, and then dissolved in 21 ⁇ l of double distilled water. Then, 2.5 ⁇ ⁇ Buffer, 0.5 ⁇ 5 mM dATP, 2.5 ⁇ ⁇ Taq were added thereto. Reaction conditions: 70 'C reaction for 30 minutes. The obtained DNA fragment of about 500 bp was recovered (Omega recovery kit), and ligated into pGEM T-easy vector, and then transformed into JM109 (method as above), and 10 white colonies were randomly picked and inoculated to contain 50 g/mL.
  • the plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ⁇ gene was replaced with the Pnos promoter to reduce the expression of NPTII protein in plants. .
  • the inducible M29A promoter and terminator Tnos were selected as promoters and terminators of the GhCzf5 gene, respectively.
  • Pnos were amplified using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using primers SEQ ID NO: 12 and SEQ ID NO: 13, using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s), and extension of 72'C for 10 min.
  • the resulting PCR product was cleaved by EcoRI and Bglll to pCAMBIA2300 (promega, T4 ligase cassette) to obtain pCAMBIA2300-1.
  • Tnos was amplified using primers SEQ ID NO: 14 and SEQ ID NO: 15 with pBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ PCR reaction system: 10 ⁇ 5 ⁇ PS Buffer. 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ pBI12 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 14 and SEQ ID 1 ⁇ 0: 15 each 2. ( ⁇ 1 And 31 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C 10 Min.
  • the resulting PCR product was ligated by Sacl and EcoRl (promega T4 ligase cassette) to pCAMBIA2300-1 to obtain pCAMBIA2300-2.
  • Arabidopsis thaliana rd29A Amplification of Arabidopsis thaliana rd29A with primers SEQ ID NO: 16 and H SEQ ID NO: 17 was initiated with Arabidopsis thaliana (Columbia type, available from www.arabidopsis.org) DNA (see Zeng J., et Al. 2002, Preparation of total DNA from "recalcitrant plant taxa", Acta Bot. Sin., 44(6): Method 694-697 for extracting Arabidopsis DNA). 3 ⁇ 4 uses TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reverse v system: 10 ⁇ 5 xPS Buffer 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ Arabidopsis DNA, 1.0 ⁇ M PrimeSTAR > 10 ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 2.0 ⁇ 1, and 31 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), and extension at 72 V for 10 min.
  • the resulting PCR product was ligated by HindIII and Pstl (connection method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3.
  • the full-length sequence of the GhCzf5-encoding gene was amplified using primers SEQ ID NO: 18 and SEQ ID NO: 19 (model: 3 ⁇ 4 GhCzf5 full-length coding gene obtained in Example 2), and PrimeSTAR HS DNA polymerization using TaKaRa
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), 72 "C extension” for 0 min. Passing Pstl, Sacl enzyme The resulting PCR product was ligated (ligation method) to pCAMBIA2300-3 to obtain plant expression ry29A-GhCzf5-2300.
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 was plated on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin 1-2 days in advance Line inoculation, culture at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin and 50 ⁇ ⁇ / ⁇ 1 streptomycin, and cultured overnight (about 12-16 h) to OD 6 at 28 °C with shaking. . A value of 0.4 forms a seed broth.
  • Transformation of Agrobacterium The competent cells were thawed on ice, and 1 ⁇ of the plasmid was added to 40 ⁇ of competent cells, and the mixture was mixed and ice bathed for about 10 minutes. Transfer the mixture of competent cells and DNA to a ice-cold electric shock cup (purchased from bio-md) with a pipette and tap to bring the suspension to the bottom of the electric shock cup, taking care not to have air bubbles. Place the electric shock cup on the slide of the electric shock chamber, and push the slide to place the electric shock cup to the base electrode of the electric shock chamber.
  • a ice-cold electric shock cup purchased from bio-md
  • the program of MicroPulser (purchased from bio-rad) is set to "Agr" and the electric shock is applied once. Immediately remove the electric shock cup and add the pre-warmed LB medium at 28 °C. Quickly and gently spread the cells with a pipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h.
  • the leaves of sterile seedlings were cut into 5 mmx5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing expression vector rd29A-GhCzf5-2300 in logarithmic growth phase for 10 min, and the bacterial culture was sucked up and co-cultured under dark conditions. 2 days (MS solid medium). Transfer the leaves to a differentiation solid medium (MS+1 mg/1 cytokinin (BA) + 0.1 mg/1 naphthaleneacetic acid (NAA) + 50 mg/1 kanamycin + 500 mg/1 cephalosporin) , using 2000 Lx of light for 16 hours per day for about 45 days. After the buds grow up, they are cut and transferred to rooting medium.
  • a differentiation solid medium MS+1 mg/1 cytokinin (BA) + 0.1 mg/1 naphthaleneacetic acid (NAA) + 50 mg/1 kanamycin + 500 mg/1 cephalosporin
  • MS+50 mg/l kanamycin+500 mg/l cephalosporin was cultured for about 30 days. After the root system was developed, the seedlings were transferred to MS medium supplemented with 500 mg/1 cephalosporin. The number is saved.
  • the leaves of the obtained transgenic tobacco plants were cut out, DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and PCR amplification was carried out using SEQ ID NO: 10 and SEQ ID NO: 11 (50 ⁇ ⁇ reaction system: 5 ⁇ ⁇ ⁇ Buffer 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • SEQ ID NO: 10 and SEQ ID NO: 11 50 ⁇ ⁇ reaction system: 5 ⁇ ⁇ ⁇ Buffer 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • the sterilized vermiculite was soaked in 1/2 MS medium.
  • T0D1-T0D20 transgenic tobacco and control tobacco seeds were separately sown on vermiculite, 15 seeds per pot, 25 ° C, 10 hours light culture / 14 hours dark culture cycle.
  • 1/2MS was poured every 5 days. After 25 days of culture, 4-5 seedlings of the same size were kept in each pot for drought test.
  • Transgenic tobacco and control tobacco were dried for 14 days (no watering), 25 °C, 10 Hour light culture / 14 hour dark culture cycle.
  • T1 transgenic plants plants grown from seeds of T0 transgenic plants
  • T1D1, T1D8, T1D10, T1D13 and T1D17 had 18 of 23 tobaccos. It can grow normally and exhibits obvious drought tolerance (see Figure 3, taking T1D1, T1D8 as an example.
  • the results of T1D10, T1D13, T1D17 are similar to T1D1, T1D8, not shown here).
  • Example 7 Verification of Czf5 protein expression at the transcriptional level In Example 6, 18 of the 1 ⁇ generation transgenic plants capable of growing normally under drought conditions were randomly selected, and 5 of the examples 6 were not able to grow normally under drought conditions.
  • PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase.
  • 50 ⁇ l ⁇ Reaction system 10 ⁇ 5xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 ⁇ l, and 30 ⁇ double Steamed water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 29 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • M is the DNA Ladder Marker (DL2000, purchased from Shenzhen Rui Zhen Biotechnology Co., Ltd.), 1-8 is a normal growing plant, and 9-12 is a plant that cannot grow normally.
  • the strip size shown in the figure is the same as the size of GhCzf5.
  • the results showed that the transcription of GhCzf5 in normal growing plants was stronger, and there was no GhCzf5 transcription or transcription in normal growth plants.

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Abstract

L'invention concerne une protéine à doigt de zinc (CzF5) provenant du coton et son gène codant, et son utilisation dans l'incubation de plantes transgéniques ayant une résistance accrue à la sécheresse.
PCT/CN2012/085628 2012-11-30 2012-11-30 Protéine à doigt de zinc du coton (czf5) et son gène codant et son utilisation Ceased WO2014082285A1 (fr)

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PCT/CN2012/085628 WO2014082285A1 (fr) 2012-11-30 2012-11-30 Protéine à doigt de zinc du coton (czf5) et son gène codant et son utilisation
CN201280077197.8A CN104812900A (zh) 2012-11-30 2012-11-30 一种棉花锌指蛋白(Czf5)及其编码基因与应用

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110791523A (zh) * 2019-12-13 2020-02-14 南京农业大学 一种棉花抗旱相关基因GhRCHY1及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101182520A (zh) * 2007-11-14 2008-05-21 南京农业大学 一个水稻锌指蛋白基因及其耐逆性基因工程应用
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