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WO2014172847A1 - Facteur de transcription myb1-2 de type myb de thellungiella salsuginea et gene codant, et son utilisation - Google Patents

Facteur de transcription myb1-2 de type myb de thellungiella salsuginea et gene codant, et son utilisation Download PDF

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WO2014172847A1
WO2014172847A1 PCT/CN2013/074581 CN2013074581W WO2014172847A1 WO 2014172847 A1 WO2014172847 A1 WO 2014172847A1 CN 2013074581 W CN2013074581 W CN 2013074581W WO 2014172847 A1 WO2014172847 A1 WO 2014172847A1
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plant
gene
seq
expression vector
tobacco
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陈文华
孙超
崔洪志
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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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 a MYB transcription factor and a coding gene thereof, and particularly to a MYB transcription factor MYB 1-2 derived from salt mustard. And its coding genes, as well as their use in the cultivation of transgenic plants with improved drought tolerance.
  • BACKGROUND OF THE INVENTION Abiotic stresses, such as drought, salt, extreme temperature, chemical pollution and oxygen damage, can cause serious damage to plant growth and development, causing great losses to crop yield, and the impact of drought on crop yield is It takes the first place in natural adversity, and its harm is equivalent to the sum of other disasters. It is the bottleneck of agricultural development in many areas.
  • the world's dry and semi-arid areas account for 34% of the land area; China's dry and semi-arid areas account for about 52% of the country's land area, and the annual drought area amounts to 200-2.7 million hectares. 30 billion cubic meters, due to lack of water, the amount of food is reduced by 35 to 40 billion kilograms; especially China's main grain-producing areas such as North China, Northeast China and Northwest China are the areas with the most water shortage in China, and the spring drought frequently reaches 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.
  • xerophytes and halophytes have yielded significant results for stress-related genes and signal transduction.
  • the system has a further understanding (Liu Q.1998.
  • Two transcription factors, DREB 1 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 present inventors cloned a MYB transcription factor of salt mustard using SSH (Suppression Subtractive Hybridization) in combination with RACE (rapid amplification of cDNA ends) (the DNA sequence of the coding gene designated herein) and found that When introduced into recipient plants, the drought tolerance of transgenic plants can be significantly improved, and these traits can be stably inherited.
  • the first aspect of the present invention provides a gene encoding a MYB-like transcription factor MYB1-2 of salt mustard (herein named ⁇ / ⁇ ⁇ -2) having the sequence of SEQ ID NO: 2.
  • a second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into an expression vector, preferably, the expression vector is pCAMBIA2300; And the nucleotide sequence of the gene is operably linked to the expression control sequence of the recombinant expression vector; preferably, the recombinant expression vector is the rd29A-ThMYB 1-2-2300 vector shown in Figure 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 tolerance 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 causing the gene Expression; Preferably, the plant is tobacco.
  • a fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or a plant comprising the gene of the first aspect of the invention, the recombinant expression vector of the second aspect of the invention, under conditions effective to produce a plant Tissue; Preferably, 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 tolerance of a plant and for use in plant breeding Use;
  • the plant is tobacco.
  • FIG. 1 is a construction flow of a plant expression vector (rd29A-ThMYB 1-2-2300) of ThMYB1-2.
  • Figure 2 is a plasmid map of the plant expression vector (rd29A-ThMYB 1-2-2300) of ThMYBl-2.
  • Figure 3 shows the results of drought tolerance simulation experiments of ⁇ 3 ⁇ 4 -2 ⁇ transgenic tobacco plants (T Q C 3 ; right, T Q C 9 ) and non-transgenic tobacco plants (left CK) as controls.
  • Figure 4 is a verification result of molecular level detection of the ⁇ ⁇ 3 ⁇ 4 -2 gene in T1 transgenic tobacco plants and non-transgenic control plants by reverse transcription PCR at the transcriptional level.
  • M is DNA Ladder Marker (DL2000, TakaRa)
  • 1-4 are non-transgenic control tobacco plants
  • 5-20 are transgenic tobacco T Q plants with significant drought tolerance
  • 21-24 are transgenic tobacco T with insignificant drought tolerance.
  • SSH library A method according to PCR-select TM cDNA Clontech kit Subtraction Kit's instructions shown by suppression subtractive hybridization subtraction library was constructed (SSH library). Growing during the experiment The mRNA of the leaves of the salt-treated mustard seedlings in the process of drought treatment was used as a sample (Tester), and the mRNA of the leaves of the untreated salt mustard seedlings was used as a control. Specific steps are as follows:
  • Salt mustard seeds (purchased at the Yanlan Plant Breeding Center of Wulanbu and Desert Green Botanical Garden in Bayannao, Inner Mongolia, China) are planted on sterilized vermiculite at 16 °C, photoperiod 16 hours light/8 hours dark (light Incubate under conditions of 2000-3000 Lx), and pour 1/2MS medium per week (9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM H 4 N0 3 , 0.75 mM MgS0 4 , 1.5 mM CaCl 2 , 50 ⁇ ⁇ , 100 ⁇ ⁇ 3 ⁇ 3 , 100 M MnSO 4 , 30 M ZnSO 4 , 1 ⁇ ⁇ 2 ⁇ 0 4 , ⁇ . ⁇ CoCl 2 , ⁇ Na 2 EDTA, 10( ⁇ M FeSO 4 ) — times. When the seedling diameter is reached Used for experiments at 5-6 cm.
  • test seedlings were divided into two groups, each with 4 pots and 1 pot per pot.
  • the first group was a control group, cultured at 25 °C, 16 hours light/8 hours dark, and normally 1/2MS watered.
  • the second group was the drought treatment group, cultured at 25 °C, cycle 16 hours light/8 hours dark condition, stopped watering, treated for 10 days, then cut the leaves of the two groups in time, and quickly frozen with liquid nitrogen, at -70 °C is stored in the refrigerator.
  • the total mustard leaves of the salt mustard were extracted with the plant RNA extraction kit (Invitrogen) by taking 0.5 g of the salt mustard leaves of the control and drought treatment groups, respectively.
  • the absorbance of total RNA at 260 nm and 280 nm was determined by HITACHI's UV spectrophotometer U-2001.
  • the OD 260 / OD 280 ratio was 1.8-2.0, indicating a high total RNA purity with a 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 Oligotex mRNA Purification Kit (purified polyA+ RNA from total RNA).
  • the method according to Clontech's PCR-select TM cDNA Subtraction Kit kit instructions will be shown suppression subtractive hybridization.
  • Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and subtractive hybridization was performed using 2 Tester cDNA and 2 ⁇ g of Driver cDNA as starting materials.
  • the Tester cDNA and Driver cDNA were digested with Rsa I for 1.5 hours in a 37 ° C water bath, and then the digested Tester cDNA was divided into two equal portions, and the different linkers were ligated, and the Driver cDNA was not ligated.
  • Two tester cDNAs with different adaptors were mixed with excess Driver cDNA for the first forward subtractive hybridization. Mixing the products of the two first subtractive hybridizations with the new denatured
  • the Driver cDNA was subjected to a second positive subtractive hybridization, and then the differentially expressed fragments were amplified by two inhibitory
  • the second PCR amplification product of the combined forward subtractive hybridization cDNA fragment was purified according to the method described in the product specification of pGEM-T Easy kit (purchased from Promega kit) (purified by QIAquick PCR Purification Kit, purchased from QIAquick PCR Purification Kit) Qiagen) is ligated to the pGEM-T Easy vector.
  • the specific steps are as follows: The following components are sequentially added using a 200 ⁇ PCR tube: The second PCR product of the purified positive subtractive hybridization cDNA fragment 3 ⁇ l, 2 ⁇ 4 ligase buffer 5 l , pGEM-T Easy vector 1 ⁇ 1, ⁇ 4 ⁇ ligase ⁇ ⁇ , ligated overnight at 4 °C. Then, 10 reaction products were taken and added to 100 competent E.
  • coli JMI09 (purchased from TAKARA), ice bath for 30 minutes, heat shock for 60 seconds, ice bath for 2 minutes, and 250 L LB medium (1% Tryptone was purchased from OXOID, 0.5% yeast extract (Yeast Extract, purchased from OXOID), 1% NaCl (purchased from Sinopharm)), placed in a 37 ° C shaker, shaken at 225 rpm for 30 minutes, and then taken 200 ⁇ L from The bacterial solution was coated with 50 g/mL ampicillin, 40 g/mL X-gal (5-bromo-4-chloro-3-indolyl- ⁇ -D-galactoside), 24 g/mL IPTG (isopropyl LB ( ⁇ -D-thiogalactopyranoside) (X-gal/IPTG purchased from TAKARA) was incubated on a solid culture plate at 37 ° C for 18 hours.
  • X-gal isopropyl LB ( ⁇ -D-thiogalactopyranoside
  • the sequence is SEQ ID No: 3. Sequence analysis indicated that the encoded amino acid sequence of the sequence is higher than the MYB transcription factor.
  • ThMYBl-2 the full-length gene encoded by the clone Th-D118 was named as ThMYBl-2, and the corresponding protein was named ⁇ 1-2.
  • ThMYBl-2 gene fragment SEQ ID NO: 3
  • three specific primers were designed as reverse transcription primers and 3'-end specific primers for 5' RACE.
  • ThMYBl-2 GSP1 SEQ ID NO: 4:
  • ThMYBl-2 GSP2 SEQ ID NO: 5:
  • ThMYB 1 -2 GSP3 SEQ ID NO: 6:
  • CTTTCTTTTAATATGAGTGTTC The experimental procedure was performed according to the kit instructions (5, RACE System for Rapid Amplification of cDNA Ends kit was purchased from Invitrogen).
  • the reverse transcription cDNA (reverse transcription primer SEQ ID NO: 4) extracted from the leaves of the drought-treated group was used as a template for the first round of PCR amplification. Increase, the specific steps are as follows:
  • PCR reaction system 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcribed cDNA, 1.0 ⁇ Ex Taq (Ex Taq from TAKARA), 10 ⁇ primer SEQ ID NO: 5 and AAP is 2.0 ⁇ l each, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.
  • the obtained PCR product was diluted 50 times 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: 6 and the 3' primer AUAP.
  • the specific steps are as follows:
  • PCR reaction 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: 6 and AUAP 2.0 ⁇ l each, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, after 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.
  • a band of about 600 bp in size from the second PCR product (Gel Extraction Kit was purchased from OMEGA) was recovered and ligated into pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (specific method is the same as above) And screened on LB solid medium containing 50 g/mL ampicillin. Ten white colonies were randomly picked and cultured in LB liquid medium containing 50 g/mL ampicillin, and cultured at 37 ° C overnight, and then glycerin was added to a final concentration of 20% by volume, and stored at -80 ° C until use.
  • SEQ ID NO: 6 and the 3' primer AUAP were used for PCR amplification (reaction system and reaction conditions are the same as above), and three positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the gene was obtained. The 5' end of the cDNA.
  • a pair of primers were designed according to the sequence of SEQ ID NO: 17 as follows:
  • ThMYBl-2F SEQ ID NO: 7:
  • ThMYBl-2R SEQ ID NO: 8:
  • ThMYBl-2 The full length of ThMYBl-2 was cloned by SEQ ID NO: 7 and SEQ ID NO: 8.
  • PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase and cDNA reverse-transcribed cDNA extracted from the leaves of the salt-treated mustard.
  • 50 ⁇ PCR reaction system 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 ⁇ l, and 30 ⁇ double Steamed water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 minutes, 33 cycles (denaturation at 94 °C for 30 seconds, annealing at 54 °C for 30 seconds, extension at 72 °C for 2 minutes), extension at 72 °C for 10 minutes.
  • PCR amplification product plus A tail The PCR product was added with 2.5 volumes of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, de-cleared, dried, and dissolved in 21 ⁇ l of double distilled water. Add 2.5 ⁇ ⁇ Buffer, 0.5 ⁇ l 5 ⁇ dATP, 2.5 ⁇ ⁇ Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 850 bp was recovered (Omega recovery kit), ligated into pGEM T-easy vector, transformed into JM109 (method as above), and 10 white colonies were randomly picked up in LB liquid containing 50 g/mL ampicillin.
  • SEQ ID NO: 7 and SEQ ID NO: 8 were subjected to bacterial cell PCR amplification (reaction system and reaction conditions are the same as above), and 4 positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the sequence was SEQ ID. NO: 2.
  • the amino acid sequence of the ThMYBl-2 protein encoded by the gene is shown in SEQ ID NO: 1.
  • Amino acid sequence of MYB 1-2 transcription factor SEQ ID NO: l 1 MGTNLVKKSP PFILTQTLLT
  • 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 of the ⁇ gene containing the double enhancer was replaced with the Pnos promoter to reduce the expression of prion protein in plants. .
  • the inducible promoter rd29A and the terminator Tnos were selected as promoters and terminators of the gene.
  • the construction flow chart is shown in Figure 1.
  • Pnos was amplified using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and PrimeSTAR HS DNA polymerase of TaKaRa was used.
  • 50 ⁇ PCR reaction system 10 ⁇ 5xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ PBI121, 1.0 ⁇ PrimeSTAR 10 ⁇ primers SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 ⁇ l, and 31 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes.
  • the resulting PCR product was ligated by EcoRI, Bglll and ligated into pCAMBIA2300 (Promega, T4 ligase kit) to obtain pCAMBIA2300-1.
  • TaKaRa's PrimeSTAR HS DNA Polymerase 50 ⁇ PCR reaction system: 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ pBI121, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 ⁇ l, and 31 ⁇ double Steamed water.
  • PCR reaction conditions pre-variability at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes.
  • the pCAMBIA2300-2 was obtained by restriction enzyme digestion with pCAMBIA2300-l (Promega T4 ligase kit) by Sacl and EcoRI.
  • Arabidopsis thaliana Amplification of Arabidopsis thaliana with primers SEQ ID NO: 13 and SEQ ID NO: 14 using Arabidopsis thaliana (Columbia type, purchased from the Arabidopsis Bioresources Center (www.arabidopsis.org) of the Ohio State University) as a template rd29A promoter (see Zeng J "et L. 2002, Preparation of total DNA from” recalcit rant plant taxa ", Acta Bot Sin, 44 (6):..
  • PCR reaction system 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ Arabidopsis DNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 13 and SEQ ID NO : 14 each of 2.0 ⁇ l, and 31 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds ), extending at 72 ° C for 10 minutes.
  • the resulting PCR was produced by digestion with HindIII and Pstl. Connected to the object (connection method is the same as above) pCAMBIA2300-2 obtained pCAMBIA2300-3
  • ThMYBl-2 was amplified with primers SEQ ID NO: 15 and SEQ ID NO: 16 (template was ThMYB1-2 obtained in Example 2, PrimeSTAR HS DNA polymerase using TaKaRa. 50 ⁇ PCR reaction system: 10 ⁇ 5xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ ThMYBl-2-pGEM, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 15 and SEQ ID NO: 16 each 2.0 ⁇ l, and 31 ⁇ of double distilled water.
  • PCR reaction conditions Pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes. After Pstl, Sad digestion, the result The PCR product was ligated (ligation method as above) pCAMBIA2300-3 to obtain the plant expression vector rd29A-ThMYB 1-2-2300.
  • Agrobacterium tumefaciens LBA4404 (available 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 Single spot 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 g/ml streptomycin, and cultured overnight (about 12-16 hours) at 28 °C until the OD600 value was 0.4. , forming a seed bacterial liquid.
  • Transformation of Agrobacterium Melt LBA4404 competent cells on ice, add 1 ⁇ of the positive rd29A-ThMYB 1-2-2300 plasmid obtained in Example 3 to 40 ⁇ of the competent cells, mix and mix, and then ice bath about 10 minute. Transfer the mixture of competent cells and rd29A-ThMYBl-2-2300 plasmid to a pre-cooled electric shock cup with a micropipette and tap to bring the suspension to the bottom, taking care not to have air bubbles. Place the electric shock cup (purchased from Bio-Rad) on the slide of the electric shock chamber and push the slide to place the electric shock cup on the base electrode of the electric shock chamber.
  • the program of the MicroPulser (purchased from Bio-Rad) is set to "Agr" and the shock is applied once.
  • the electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added. Quickly and gently mix the competent cells with a micropipette.
  • the suspension was transferred to a 1.5 ml centrifuge tube and incubated at 225 rpm for 1 hour at 28 °C.
  • the leaves of the sterile seedlings were cut into 5 mm ⁇ 5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing the expression vector rd29A-ThMYB 1-2-2300 in the logarithmic growth phase for 10 minutes to absorb the bacterial liquid in the dark condition.
  • the cells were cultured for 2 days (MS medium).
  • the leaves were transferred to differentiation medium (MS+1 mg/L cytokinin (BA) + 0.1 mg/L naphthaleneacetic acid (NAA) + 50 mg/L kanamycin + 500 mg/L cephalosporin).
  • the photoperiod was cultured for about 45 days under the conditions of 10 hours light/14 hours darkness (in which the light intensity was 2000-3000 Lx).
  • the buds were grown, they were cut and transferred to rooting medium (MS+50 mg/L kanamycin+).
  • the cells were cultured for about 30 days in 500 mg/L cephalosporin. After the root system was developed, the seedlings were transferred to MS medium supplemented with 500 mg/L cephalosporin for number storage.
  • the obtained transgenic tobacco leaves were extracted, and the DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3) using SEQ ID NO: 7 and SEQ ID NO: 8 (50 ⁇ PCR reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq 10 ⁇ primer SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions 94 °C pre-denaturation 5 minutes, 33 cycles (denaturation at 94 °C for 30 seconds, annealing at 55 °C for 30 seconds, extension at 72 °C for 2 minutes), extension at 72 °C for 10 minutes), PCR identification, preservation of positive plants for numbering Tod-ToC ⁇
  • Example 6 Drought tolerance simulation experiment and functional identification of overexpressing ThMYBl-2 transgenic tobacco T Q The sterilized vermiculite was soaked in 1/2 MS medium.
  • Tod-ToC ⁇ and control tobacco tissue culture seedlings were transplanted to vermiculite, respectively, at 25 °C, 10 hours light culture/14 hours dark culture cycle, 1/2MS every 5 days, 15 days after strong seedling culture, drought Stress test, transgenic tobacco, control tobacco drought for 14 days (without watering), 25 °C, 10 hours light culture / 14 hours dark culture cycle.
  • T Q transgenic plants seed T Q transgenic plants grow into plants
  • drought resistance identification showed the control plants are wilting serious
  • T Q C 2, T 0 C 3 ToC 7, T 0 C 9 T 0 Cio T 0 Cii T Q C 14 29 total seven lines 20 can be tobacco have normal growth, an obvious significant drought tolerance (see FIG. 3, T.C 3, T.C 9 as an example, T.
  • Example 7 verified at the transcriptional level ⁇ ⁇ 3 ⁇ 4 -2 gene were taken control tobacco, no significant drought tolerant transgenic tobacco T Q generation plants significantly drought tolerant transgenic tobacco T Q-generation plants (grow well) arid 14 days leaves 0.05 g, Total RNA was extracted using a plant RNA extraction kit (Invitrogen). The absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the respective RNA concentrations.
  • Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay LIBOX Superscript III Reverse Transcriptase (.2 ⁇ ag total RNA as a template, reverse transcription primer SEQ ID NO: 8).
  • the relative expression of MYB 1 -2 protein was detected by amplifying ThMYB 1 -2 by SEQ ID NO: 7 and SEQ ID NO: 8.
  • the PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction system 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 ⁇ l, and 30 ⁇ double Steamed water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 29 cycles (denaturation at 94 ° C for 30 seconds, annealing at 55 ° C for 30 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.
  • M is DNALadder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-4 for control tobacco, 5-20 for significantly drought-tolerant transgenic tobacco T Q plants, 21-24 for not significantly drought-tolerant transgenic tobacco T Q plants .
  • the strip size shown in the figure is consistent with the size of ThMYBl-2 (about 900 bp).
  • the results show that the control tobacco no transcript was detected signal exogenous gene ThMYBl-2 is significantly drought transfected gene transcription tobacco T Q progenies exogenous gene ThMYBl-2 is strong, no significant drought tolerant transgenic tobacco T Q progenies exogenous gene ThMYBl-2 transcription is weak or not transcribed.

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Abstract

La présente invention concerne le facteur de transcription MYB1-2 de type MYB dérivé de Thellungiella salsuginea et un gène codant associé, et son utilisation dans la culture de plantes transgéniques présentant une tolérance améliorée à la sécheresse.
PCT/CN2013/074581 2013-04-24 2013-04-24 Facteur de transcription myb1-2 de type myb de thellungiella salsuginea et gene codant, et son utilisation Ceased WO2014172847A1 (fr)

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CN201380074513.0A CN105008385B (zh) 2013-04-24 2013-04-24 一个盐芥myb类转录因子myb1-2及其编码基因与应用
PCT/CN2013/074581 WO2014172847A1 (fr) 2013-04-24 2013-04-24 Facteur de transcription myb1-2 de type myb de thellungiella salsuginea et gene codant, et son utilisation

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CN109295069B (zh) * 2018-09-19 2021-08-20 昆明理工大学 珠子参转录因子基因PjMYB1的应用
CN110317816B (zh) * 2019-07-12 2022-03-08 云南省烟草农业科学研究院 一种能提高烟草抗旱的转录因子NtMYB44b及其定点突变方法与应用

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