WO2015042737A1 - Protéine à glissière à leucine bzip-4 de thellungiella halophila, gène codant pour celle-ci et utilisation associée - Google Patents

Protéine à glissière à leucine bzip-4 de thellungiella halophila, gène codant pour celle-ci et utilisation associée Download PDF

Info

Publication number
WO2015042737A1
WO2015042737A1 PCT/CN2013/001156 CN2013001156W WO2015042737A1 WO 2015042737 A1 WO2015042737 A1 WO 2015042737A1 CN 2013001156 W CN2013001156 W CN 2013001156W WO 2015042737 A1 WO2015042737 A1 WO 2015042737A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
plant
gene
plants
expression vector
Prior art date
Application number
PCT/CN2013/001156
Other languages
English (en)
Chinese (zh)
Inventor
孙超
陈文华
崔洪志
Original Assignee
创世纪转基因技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 创世纪转基因技术有限公司 filed Critical 创世纪转基因技术有限公司
Priority to PCT/CN2013/001156 priority Critical patent/WO2015042737A1/fr
Priority to CN201380078599.4A priority patent/CN105452280A/zh
Publication of WO2015042737A1 publication Critical patent/WO2015042737A1/fr

Links

Classifications

    • 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 leucine zipper protein derived from small salt mustard and its coding gene, and its use in breeding transgenic plants with improved drought tolerance .
  • BACKGROUND OF THE INVENTION Adversity stress such as temperature, salting and drought can cause serious damage to the growth and development of higher plants, leading to a decrease in crop yield and a decline in quality, which seriously threaten agricultural production and the natural environment.
  • 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 regions account for 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 350-400 billion kilograms of grain; especially China's major 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.
  • the present inventors cloned a leucine zipper protein of small salt mustard (herein named bZIP-4) by combining SSH (suppression subtractive hybridization) with RACE (rapid amplification of cDNA ends).
  • the gene, and its DNA sequence was determined. Moreover, it was found that after being introduced into plants for over-expression, the drought tolerance of the 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 leucine zipper protein bZIP-4 of small salt mustard (herein named ThbZIP-4); preferably, the sequence is 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 a basic vector for constructing the recombinant expression vector, And the nucleotide sequence of the gene is operably linked to the expression control sequence of the base vector; preferably, the base vector is pCAMBIA2300 ; preferably, the recombinant expression vector is 35S- shown in Figure 2 r) Z/P- ⁇ -2300 vector.
  • 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;
  • the plant is Arabidopsis thaliana.
  • 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 or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant Tissue;
  • the plant is Arabidopsis thaliana.
  • 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 Arabidopsis thaliana.
  • the seventh aspect of the present invention provides the gene-encoded protein according to 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 (358- ⁇ ) ⁇ / ⁇ - ⁇ -2300 of ThbZIP-4 (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector ⁇ ;358- ⁇ ) ⁇ / ⁇ - ⁇ -2300 of ThbZIP-4.
  • FIG 3 shows the results of drought tolerance simulation experiments of ThbZIP-4 T1 transgenic Arabidopsis plants (in the figure, T1A2) and non-transgenic Arabidopsis plants (in the figure, CK) as controls.
  • Fig. 3a is an Arabidopsis plant that grows normally for 20 days
  • Fig. 3b shows an Arabidopsis plant that has been treated for 14 days after normal growth for 14 days).
  • Fig. 4 Results of changes in ABA content of T1 transgenic Arabidopsis plants and control plants under drought stress and normal growth conditions.
  • 1-7 is the strain (from left to right): T1A1, T1A2, T1A3, T1A4, T1A5, T1A6, CK, wherein T1A1, T1A2, T1A3, T1A4, T1A5, T1A6 are transgenic plants, and CK is a control plant.
  • FIG. 5 is a graphical representation of the protein expression at the transcriptional level of transgenic T1 Arabidopsis plants and non-transgenic control plants.
  • M is DNA Ladder Marker (DL2000, TakaRa), 1-7 drought-tolerant transgenic Arabidopsis T1 plants (in order: T1A1, T1A2, T1A3, T1A4, T1A5, T1A6, T1A7), 8-11 is non-transgenic South Mustard control; 12-16 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant.
  • Small salt mustard (T ellungiella halophila, purchased from the Yanlan Plant Breeding Center of Ulan Buh and Desert Green Botanical Garden, Bayannaoer City, Inner Mongolia, China), seeded on sterilized vermiculite, at 25 ° C, photoperiod 16 hours light / Incubate under 8 hours of darkness (light intensity 2000 - 3000 Lx), and pour 1/2 MS medium (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 ⁇ per week).
  • 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 hours light / 8 hours dark, and was normally watered.
  • the second group was the drought treatment group, cultured at 25 ° C, photoperiod of 16 hours light / 8 hours dark, stopped watering, and treated for 10 days. After the treatment, the leaves of the top two groups of the seedlings were cut in time. After the nitrogen was rapidly frozen, it was stored in a -70 ° C refrigerator.
  • this experiment In order to increase the validity of the Expressed sequence tag (EST) (Unigene), avoid the gene-free cleavage site and the obtained sequence in the untranslated region, this experiment simultaneously uses the endonuclease Haelll to tester cDNA according to the above steps.
  • the cDNA was digested with Driver cDNA and subjected to two forward subtractive hybridizations and two inhibitory PCR amplifications. Finally, the second inhibitory PCR products of the two groups of forward subtractive hybridization cDNA fragments were combined.
  • the second PCR product of the combined 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 vector is ligated as follows: The following components are sequentially added using a 200 ⁇ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 ⁇ l, 2 ⁇ 4 ligase buffer 5 ⁇ l, pGEM-T Easy vector 1 ⁇ l, T4 DNA ligase 1 ⁇ , ligated overnight at 4 °C. Take 10 ligation reaction products, add to 100 competent E.
  • coli JM109 (purchased from TAKARA), ice bath for 30 minutes, heat shock for 60 seconds, ice bath for 2 minutes, and add 250 ⁇ L of LB medium (1% Tryptone 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 minutes, and 200 ⁇ L of bacterial solution was applied to 50 g / mL ampicillin (purchased LB (ibid.) /X-gal/IPTG (X-gal/IPTG from TAKARA, lg packaged into 20 mg/ml mother liquor from Tiangen Biochemical Technology (Beijing) Co., Ltd.), the working concentration is: above mother liquor 200 ⁇ 1/100 ml LB medium; IPTG was purchased from TaKaRa, and 5 g was packaged into a mother liquor of 100 mM concentration.
  • LB medium 1% Tryptone from OXOID
  • the working concentration was: 100 ⁇ /100 ml LB medium above the mother liquor), cultured on a solid plate for 18 hours at 37 ° C. .
  • the number of clear white and blue colonies was randomly selected from 198 white colonies (number: Gh-B001 to Gh-B198). All white clones were inoculated separately in 96-well cell culture plates (CORNING) containing 50 ⁇ ⁇ / ⁇ ampicillin in LB liquid medium, and cultured overnight at 37 ° C with glycerol to a final concentration of 20% at -80 ° C Save spare.
  • the nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit kit) were used to verify the PCR amplification of the bacteria, and 166 positive clones were obtained, and all the positive clones were sent to the UK. Base (Shanghai) Trading Co., Ltd. sequencing.
  • SEQ ID No: 3 is the 3 terminal sequence of the coding gene 6Z/P- ⁇ . Based on the sequence of SEQ ID NO: 3 which has been obtained, the following three specific primers were designed as specific primers for reverse transcription primers and 5 'RACE.
  • GATCATCCGCCTCTGCCTTC YLS-16GSP2 SEQ ID NO: 5 :
  • GACCATCCACTACTCTTTTCC YLS-16 GSP3 ( SEQ ID NO: 6) :
  • the kit comes with universal primers:
  • AAP SEQ ID NO: 7 :
  • GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP SEQ ID NO: 8 :
  • the GGCCACGCGTCGACTAGTAC experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • YLS-16GSP1 (SEQ ID NO: 4) as a reverse transcription primer, reverse transcription using small salt mustard mRNA as a template to obtain a cDNA template, and then adding a Poly C tail according to the procedure in the above 5' RACE kit instructions.
  • the first round of PCR amplification was carried out using the product after tailing as a template.
  • the primers used were SEQ ID NO: 4 and the universal primer SEQ ID NO: 7 (the kit is self-contained, I is a hypoxanthine modified a, c, g or t), the specific steps are as follows:
  • PCR reaction system 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ l 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcribed cDNA, 1.0 ⁇ Ex Taq (purchased from TAKARA), 10 ⁇ primers SEQ ID NO: 4 and SEQ ID NO: 7 each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 50 seconds, annealing at 55 ° C for 50 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.
  • 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: 5 and the universal primer SEQ ID NO: 8, and the specific steps were 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 ⁇ primers SEQ ID NO: 5 and SEQ ID NO: 8 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 50 seconds, annealing at 55 ° C for 50 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.
  • the strip of about 500 bp in the second PCR product was recovered (Gel Extraction Kit was purchased from OMEGA), and it was ligated to pGEM-T Easy Vector, and then converted to JM109 (the specific method is the same as above), and 10 whites were randomly picked.
  • the colonies were inoculated separately in LB liquid medium containing 50 g/mL ampicillin, cultured at 37 ° C overnight, and glycerol was added to a final concentration of 20% (v/v), and stored at -80 ° C until use.
  • the primers SEQ ID NO: 5 and the 3' primer SEQ ID NO: 6 were used for PCR amplification (reaction system and reaction conditions as above), and four positive clones (YL16-1, YL16-3, YL16-5 and YL16-6), sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, obtaining a 5' end sequence of the cDNA of the gene.
  • the obtained 5 'RACE product clone YL16-3 was sequenced to obtain the sequence of SEQ ID NO: 9:
  • SEQ ID NO: 10 is 7) the full length sequence of Z/P- ⁇ .
  • a pair of primers were designed according to the sequence of SEQ ID NO: 10 as follows: ThbZIP-4F (SEQ ID NO: 1 1 ):
  • ThbZIP-4R SEQ ID NO: 12 :
  • AP (SEQ ID NO: 13): GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
  • the small salt mustard RNA was extracted, and the primer SEQ ID NO: 13 was used as the reverse transcription primer to obtain the cDNA of the small salt mustard.
  • the PfuUltra II Fusion HS DNA Polymerase of Stratagene was used to carry out the PCR using the cDNA of the small salt mustard. reaction. 50 ⁇ PCR reaction system: 5 ⁇ lO PfuUltra II reaction Buffer, 0.5 ⁇ 25 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PfuUltra II Fusion HS DNA Polymerase 10 ⁇ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 11, and 37.5 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 95 °C for 2 minutes, 35 cycles (denaturation at 95 °C for 25 seconds, annealing at 57 °C for 25 seconds, extension at 72 °C for 45 seconds), extension at 72 °C for 5 minutes.
  • PCR amplification product plus A tail PCR product hydration to 400 ⁇ 1, first remove the protein with chloroform once, add the supernatant to add 3 ⁇ sodium acetate solution 40 ⁇ l, add 2 times of absolute ethanol, -20 ° C for 10 minutes, Centrifuge, remove the supernatant, allow to dry, and dissolve in 21 ⁇ l of double distilled water. Add 2.5 ⁇ ⁇ ⁇ Buffer, 0.5 ⁇ 5 mM dATP, 1.0 l Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes.
  • a DNA fragment of about 700 bp was recovered (Omega recovery kit), ligated into the pGEM T-easy vector (to obtain 7) Z/P- ⁇ -pGEM plasmid), and then transformed into JM109, and 6 white colonies were randomly picked and inoculated separately. Incubate in LB liquid medium containing 50 g/mL ampicillin, incubate at 37 ° C overnight, add glycerol to a final concentration of 20%, and store at -80 ° C until use.
  • the primers SEQ ID NO: 1 1 and SEQ ID NO: 12 were used for PCR amplification (reaction system and reaction conditions as above), and 4 positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing, sequence.
  • SEQ ID NO: 2 the amino acid sequence of the encoded protein is the amino acid sequence of SEQ ID NO: 1 6Z/P- ⁇ protein (SEQ ID NO: 1):
  • nucleotide sequence of the 73 ⁇ 4 ⁇ / ⁇ - ⁇ coding gene (SEQ ID NO: 2):
  • 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 ⁇ . Expression of proteins in plants.
  • the 35S promoter containing the double enhancer and the terminator Tnos were selected as promoters and terminators of the ThbZIP-4 gene, respectively.
  • Pnos were amplified using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using primers SEQ ID NO: 14 and SEQ ID NO: 15, using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 minutes, 33 cycles (denaturation for 30 seconds at 94 °C, annealing for 30 seconds at 56 °C, extension of 30 seconds at 72 °C), extension at 72 °C for 10 minutes.
  • the resulting PCR product was digested with EcoRI and Bglll and ligated into pCAMBIA2300 (Promega, T4 ligase cassette) to obtain pCAMBIA2300-1.
  • SEQ ID NO: 16 and P SEQ ID NO: 17 Amplification of Tnos using pBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase.
  • 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), extension at 72 ° C for 10 minutes.
  • the resulting PCR product was digested with Sacl, EcoRI and ligated into pCAMBIA2300-1 (Promega T4 ligase cassette) to obtain pCAMBIA2300-2.
  • SEQ ID NO: 18 and SEQ ID NO: 19 amplify the Arabidopsis thaliana 35S promoter with the pCAMBIA2300 plasmid as a template.
  • PrimeSTAR HS DNA polymerase using TaKaRa 50 ⁇ PCR reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ diluted 50-fold pCAMBIA2300 plasmid, 1.0 ⁇ PrimeSTAR, 10 ⁇ primer 8 £0 10 ⁇ 0: 18 and 8 £0 10 ⁇ «): 19 each of 2.0 ⁇ , 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 50 ° 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 HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3 SEQ ID NO: 18:
  • ThbZIP-4 (template is positive for Example 2)
  • ThbZIP-4-pGEM plasmid using stratagene's PfuUltra II Fusion HS DNA Polymerase.
  • PCR reaction conditions pre-denaturation at 95 °C for 2 minutes, 35 cycles (denaturation at 95 °C for 25 seconds, annealing at 57 °C for 25 seconds, extension at 72 °C for 45 seconds, extension at 72 °C for 5 minutes. Digestion by Pstl, Sad The resulting PCR product was ligated (ligation method as above) to pCAMBIA2300-3 to obtain a plant expression vector 35S-7)Z/P- ⁇ -2300.
  • 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 ⁇ ⁇ / ⁇ 1 streptomycin, and cultured overnight (about 12-16 hours) to OD 6 at 28 °C with shaking.
  • the K) value is 0.4, and a seed bacterial liquid is formed.
  • Transformation of Agrobacterium The competent cells were thawed on ice, and 1 ⁇ M of the positive 35S-7M)Zff- ⁇ -2300 plasmid obtained in Example 3 was added to 40 ⁇ of the competent cells, and the mixture was mixed and ice-cooled for about 10 minutes.
  • the mixture of competent cells and plasmid DNA was transferred to an ice-cold electric shock cup with a pipette, and tapped 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.
  • Plants to be transformed Arabidopsis seeds (Columbia type, Arabidopsis thaliana Bioresource Center, Ohio State University) Seeded in peat soil, treated at 4 ° C for 3 days, placed at 23 ° C, 16 hours light Sprouting in an 8 hour dark incubator. After 7-10 days, transplanted into a plastic crucible with a diameter of 7.5 cm containing peat soil and vermiculite (3:1 by volume), 6 plants per pot, placed at 23 ° C, 16 hours light / 8 hours dark Growing in the incubator. 40 ml of nutrient solution per pot before transplanting, and the soil moisture should be replenished in time after transplanting. The nutrient solution is properly watered during the growth period.
  • Agrobacterium After removing the bacterial solution of the Agrobacterium-positive transformed clone preserved in Example 4, a single colony of Agrobacterium was picked and inoculated into 10 mL of sterile LB liquid medium (containing 75 mg/L rifampicin). 100 mg/L streptomycin and 100 mg/L kanamycin were shaken overnight at 250 rpm at 28 °C. Then, the obtained bacterial liquid was inoculated to 200 mL of LB liquid medium containing the above antibiotics at a ratio of 1% to 2% (v/v), and the concentration of Agrobacterium was reached at OD 6 (at a constant temperature of 28 ° C).
  • the infusion medium contains 5.0% (w/v) sucrose and 0.05% (500 ⁇ ) Silwet L-77) resuspended Agrobacterium and suspended to an OD 6QQ of about 0.80.
  • Inflorescence dip dyeing The above Agrobacterium-containing dyeing medium is added to a large-mouth container, and 200-300 mL of the Agrobacterium-containing dyeing medium is added to each container having a diameter of 9 cm for dip dyeing. Invert the plants so that the aboveground tissues are completely immersed in the Agrobacterium suspension for 3 - 5 seconds and gently agitated. There should be a liquid film on the plant after infiltration. Dip-infected plants are placed in plastic trays, covered with a clean plastic or plastic wrap to moisturize them, then placed in low light or dark places overnight, taking care to prevent direct sunlight from shining on the plants. Remove the cover approximately 12 - 24 hours after processing. The plants are cultured normally, and the plants are further grown for 3 - 5 weeks until the pods are browned and dried. The seeds were harvested and the seeds were dried and stored at 4 °C in a centrifuge tube.
  • Transgenic seed screening prepare an aqueous solution containing 1/4 MS of large elements, add 0.8% agar powder, and heat in a microwave oven Completely dissolve into agar, cool to about 50 °C, add the required amount of kanamycin at a final concentration of 50 ⁇ !/ 1 , shake well, pour 25 mL into each dish, set the bench for cooling After solidification, it can be sown.
  • ThbZIP-4 was amplified with SEQ ID NO: 11 and SEQ ID NO: 12 (50 ⁇ PCR reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 11 and SEQ ID NO: 12 each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • T1A1-T1A6 and control Arabidopsis seeds were planted on vermiculite, 10 seeds per pot, 25 °C, 10 hours light culture / 14 hours dark culture cycle, 1/2MS every 7 days, after 20 days of culture 4-5 seedlings of uniform size were kept in each pot for drought experiments.
  • the drought resistance of T1 transgenic plants (plants grown from seeds of T0 transgenic plants) showed that the control plants were wilting, while T1A1, T1 A2, T1A3, T1A4, T1A5, and T1A6 had 24 strains each.
  • Example 7 Determination of ABA changes after drought stress
  • ABA is recognized as a plant hormone associated with stress, which can act as a signaling molecule to regulate the expression of multiple stress-inducing genes, thereby improving the plant's resistance to stress.
  • T1A1, T1A2, T1A3, T1A4, T1A5, T1A6 transgenic plants
  • CK control plants
  • ELISA phytohormone abscisic acid
  • Example 8 Verification of ⁇ ⁇ / ⁇ - ⁇ protein expression at the transcriptional level Control Arabidopsis thaliana plants, drought-tolerant transgenic Arabidopsis T1 plants (one strain belonging to T1A1, T1A2, T1A3, Tl A4, Tl A5, T1A6, respectively) and the drought-tolerant transgenic Arabidopsis thaliana T1 plants Total RNA extracted by plant RNA extraction kit (Invitrogen) with 0.05 g of leaves per day for 10 days.
  • RNA concentration 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 (2 ⁇ g of total RNA as a template, reverse transcription primer SEQ ID NO: 13) was carried out according to the method shown by Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase. Amplification by SEQ ID NO: 11 and SEQ ID NO: 12.
  • ThbZIP-4 detects the relative expression of mp-4 protein.
  • PCR was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and reverse transcribed cDNA as a template.
  • 50 ⁇ l ⁇ Reaction system 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 l PrimeSTAR, 10 ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 ⁇ l, and 30 ⁇ Double steamed water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 29 cycles (denaturation for 45 seconds at 94 ° C, annealing for 45 seconds at 57 ° C, extension of 1 hour at 72 ° C), extension at 72 ° C for 10 minutes.
  • M is DNA Ladder Marker (DL2000, TakaRa), 1-7 drought-tolerant transgenic Arabidopsis thaliana T1 plants (in order: T1A1, T 1A2, T1A3, T1A4, T1A5, T1A6, T1A7)
  • 8-1 1 is a non-transgenic Arabidopsis control
  • 12-16 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant.
  • the size of the electrophoresis band of the PCR product shown in the figure is the same as the size of ⁇ ⁇ / ⁇ - ⁇ (about 720 bp).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne une protéine à glissière à leucine bZIP-4 de Thellungiella halophila, un gène codant pour celle-ci et une utilisation associée dans la sélection d'une plante transgénique présentant une meilleure tolérance à la sécheresse.
PCT/CN2013/001156 2013-09-26 2013-09-26 Protéine à glissière à leucine bzip-4 de thellungiella halophila, gène codant pour celle-ci et utilisation associée WO2015042737A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2013/001156 WO2015042737A1 (fr) 2013-09-26 2013-09-26 Protéine à glissière à leucine bzip-4 de thellungiella halophila, gène codant pour celle-ci et utilisation associée
CN201380078599.4A CN105452280A (zh) 2013-09-26 2013-09-26 一种小盐芥亮氨酸拉链蛋白bZIP-4及其编码基因与应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2013/001156 WO2015042737A1 (fr) 2013-09-26 2013-09-26 Protéine à glissière à leucine bzip-4 de thellungiella halophila, gène codant pour celle-ci et utilisation associée

Publications (1)

Publication Number Publication Date
WO2015042737A1 true WO2015042737A1 (fr) 2015-04-02

Family

ID=52741702

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/001156 WO2015042737A1 (fr) 2013-09-26 2013-09-26 Protéine à glissière à leucine bzip-4 de thellungiella halophila, gène codant pour celle-ci et utilisation associée

Country Status (2)

Country Link
CN (1) CN105452280A (fr)
WO (1) WO2015042737A1 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1472223A (zh) * 2002-07-30 2004-02-04 中国农业科学院生物技术研究所 一个玉米bZIP类转录因子及其编码基因与应用
US20050193443A1 (en) * 2002-07-30 2005-09-01 Ttu D-0426 Transcription factors, DNA and methods for introduction of value-added seed traits and stress tolerance
CN101220363A (zh) * 2008-01-25 2008-07-16 北京未名凯拓农业生物技术有限公司 水稻bZIP及其基因在提高植物耐逆性能上的应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102060919B (zh) * 2009-07-06 2013-09-11 中国农业科学院生物技术研究所 三个棉花abf/areb/abi5/dpbf类转录因子及其编码基因与应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1472223A (zh) * 2002-07-30 2004-02-04 中国农业科学院生物技术研究所 一个玉米bZIP类转录因子及其编码基因与应用
US20050193443A1 (en) * 2002-07-30 2005-09-01 Ttu D-0426 Transcription factors, DNA and methods for introduction of value-added seed traits and stress tolerance
CN101220363A (zh) * 2008-01-25 2008-07-16 北京未名凯拓农业生物技术有限公司 水稻bZIP及其基因在提高植物耐逆性能上的应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 15 March 2001 (2001-03-15), accession no. AK19599 *

Also Published As

Publication number Publication date
CN105452280A (zh) 2016-03-30

Similar Documents

Publication Publication Date Title
WO2013185258A1 (fr) Protéine hkt du coton et gène codant pour celle-ci, et leur utilisation
WO2014172852A1 (fr) Facteur de transcription myb myb1-1 du coton et son gène codant, et son utilisation
WO2015042742A1 (fr) Protéine de type calcineurine b cbl-3 du coton, gène codant pour celle-ci et utilisation associée
WO2015042740A1 (fr) Protéine de type calcineurine b cbl-4 de thellungiella halophila, gène codant pour celle-ci et application associée
WO2014169472A1 (fr) Protéine bzip-3 de coton de type glissière à leucine, gènes codant pour celle-ci, et application
WO2014172847A1 (fr) Facteur de transcription myb1-2 de type myb de thellungiella salsuginea et gene codant, et son utilisation
WO2014172826A1 (fr) Pyrophosphatase de tonoplaste vp1 issue de thellungiella halophila, ainsi que gène codant et application associés
WO2014205597A1 (fr) Hkt2 transporteuse présentant une affinité élevée pour les ions potassium et qui est dérivée de coton, gène codant et utilisation correspondante
WO2015042738A1 (fr) Protéine glissière à leucine homéotique hdbzip-3 issue de thellungiella halophila, gène codant pour cette protéine, et utilisation du gène
WO2014107863A1 (fr) Protéine à fermeture éclair de leucine homéotique du coton hdbzip-1 et gène codant et utilisation de celle-ci
WO2015042737A1 (fr) Protéine à glissière à leucine bzip-4 de thellungiella halophila, gène codant pour celle-ci et utilisation associée
WO2015042749A1 (fr) Protéine déhydrine dh5, provenant de thellungiella halophila, gène codant celle-ci et application correspondante
WO2015042741A1 (fr) Protéine glissière à leucine bzip-6de thellungiella halophila, gène codant pour cette protéine, et utilisation du gène
WO2015042739A1 (fr) Protéine glissière à leucine thellungiella halophila bzip-5, gène codant pour celle-ci, et application associée
WO2014101153A1 (fr) Isopentényl transférase ipt2 de coton, gène codant pour celle-ci et application de celle-ci
WO2015058323A1 (fr) Bétaïne aldéhyde déshydrogénase (badh) de bruguiear gymnorrhiza, gène codant pour celle-ci, et application du gène codeur
WO2014172842A1 (fr) Cystéine protéase cysp1-2 du coton et son gène codant, et son utilisation
WO2015042745A1 (fr) Protéine de type déhydrine dh2 de thellungiella halophila, gène codant pour celle-ci et application associée
WO2014082280A1 (fr) Protéine en doigt de zinc (czf4) de coton, gène la codant et son application
WO2015058322A1 (fr) Cofacteur à molybdène sulfurylase mcsu de bruguiera gymnorrhiza, gène codant pour celle-ci et utilisation
WO2014205598A1 (fr) Protéine de transport d'ions potassium haute affinité hkt1 obtenue à partir de thellungiella halophila, et gène codant et utilisation correspondante
WO2015042743A1 (fr) Protéine de type calcineurine b cbl-8 de thellungiella halophila, gène codant pour celle-ci et application associée
WO2014172825A1 (fr) Anti-protéine nhx2 sodium-hydrogène tonoplastique de thellungiella halophila, son gène codant et son utilisation
WO2015039272A1 (fr) Tréhalose synthase tps-2 de coton, gène codant et utilisation correspondants
WO2013185257A1 (fr) Facteur de transcription analogue à dreb1, issu du coton et gène codant pour celui-ci, et leur utilisation

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 201380078599.4

Country of ref document: CN

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13894258

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13894258

Country of ref document: EP

Kind code of ref document: A1