WO2015042738A1 - Protéine glissière à leucine homéotique hdbzip-3 issue de thellungiella halophila, gène codant pour cette protéine, et utilisation du gène - Google Patents

Protéine glissière à leucine homéotique hdbzip-3 issue de thellungiella halophila, gène codant pour cette protéine, et utilisation du gène Download PDF

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WO2015042738A1
WO2015042738A1 PCT/CN2013/001157 CN2013001157W WO2015042738A1 WO 2015042738 A1 WO2015042738 A1 WO 2015042738A1 CN 2013001157 W CN2013001157 W CN 2013001157W WO 2015042738 A1 WO2015042738 A1 WO 2015042738A1
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seq
plant
gene
plants
expression vector
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PCT/CN2013/001157
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陈文华
孙超
崔洪志
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创世纪转基因技术有限公司
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Priority to CN201380078597.5A priority Critical patent/CN105452278A/zh
Priority to PCT/CN2013/001157 priority patent/WO2015042738A1/fr
Publication of WO2015042738A1 publication Critical patent/WO2015042738A1/fr

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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 homeobox-leucine zipper protein HDbZIP-3 derived from Brassica napus and its coding gene, and its improvement in drought tolerance Application in transgenic plants.
  • BACKGROUND OF THE INVENTION Stresses such as temperature, salting and drought can cause serious damage to the growth and development of higher plants, resulting in reduced crop yields, degraded quality, and serious threats to 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 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.
  • the present inventors used SSH (Suppression Subtractive Hybridization) in combination with RACE (Rapid Amplification of cDNA Ends)
  • SSH Stress Subtractive Hybridization
  • RACE Rapid Amplification of cDNA Ends
  • HDbZIP-3 leucine zipper protein of small salt mustard
  • the first aspect of the present invention provides a gene encoding a homeotype-leucine zipper protein HDbZIP-3 of the small salt mustard (herein named ThHDbZIP-3; preferably, the sequence thereof 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 73 ⁇ 4HZ1 ⁇ 2Z/P-3-2300 carrier.
  • the 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 (358-7 ⁇ / ⁇ 3 ⁇ 4 ⁇ / ⁇ -3-2300;) of ThHDbZIP-3 (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector of ThHDbZIP-3 (358-7 ⁇ / ⁇ 1 ⁇ 2 ⁇ / ⁇ -3-2300;).
  • FIG. 3 shows the results of drought tolerance simulation experiments of 73 ⁇ 4HZ1 ⁇ 2Z/P-3 T1 transgenic Arabidopsis plants (T106 in the figure) and non-transgenic Arabidopsis plants (in the figure, CK) as controls.
  • Fig. 3a is an Arabidopsis plant that is normally grown for 20 days
  • Fig. 3b is an Arabidopsis plant that has been subjected to drought treatment for 14 days after normal growth for 20 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 a strain (from left to right): T101, ⁇ 102, ⁇ 103, ⁇ 104, ⁇ 105, ⁇ 106, CK, wherein ⁇ 101, ⁇ 102, ⁇ 103, ⁇ 104, ⁇ 105, T106 are transgenic plants, and CK is a control plant.
  • FIG. 5 shows the results of protein expression verification at the transcriptional level of transgenic T1 Arabidopsis plants and non-transgenic control plants.
  • M is DNA Ladder Marker (DL2000, TakaRa)
  • 1-6 is a drought-tolerant transgenic Arabidopsis T1 plant (in order: ⁇ 101, ⁇ 102, ⁇ 103, ⁇ 104, ⁇ 105, ⁇ 106)
  • 7-11 is a drought-tolerant transgenic Arabidopsis thaliana 1st generation plants
  • 12-16 are non-transgenic Arabidopsis controls.
  • BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.
  • a subtractive library was constructed by the method of inhibition subtractive hybridization using the method shown by Clontech's PCR-selectTM cDNA Subtraction Kit.
  • the mRNA of the leaves of the drought-treated salt mustard seedlings was used as a sample (Tester) during the experiment, and the mRNA of the leaves of the untreated small salt mustard seedlings was used as a control.
  • the specific steps are as follows:
  • 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 in 8 hours dark (light intensity 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 NH 4 N0 3 , 0.75 mM MgS0 4 , 1.5 mM CaCl 2 , 50 ⁇ KI, 100 ⁇ ⁇ 3 ⁇ 3 , 100 M MnSO 4 , 30 ⁇ ZnS0 4 , 1 ⁇ ⁇ 2 ⁇ 0 4 , 0.1 ⁇ CoCl 2 , 100 ⁇ Na 2 EDTA, 100 M FeSO 4 ). It was used for experiments when the seedlings were as high as 25-30 cm.
  • the test seedlings were divided into 2 groups, 4 pots per group and 1 pot per pot.
  • the first group was a control group, cultured at 25 ° C, photoperiod of 16 hours light / 8 hours dark, and normal watering.
  • the second group is the drought treatment group, 25 °C, light
  • the cells were cultured under the conditions of 16 hours light/8 hours dark, stopped watering, and treated for 10 days. After the treatment, the leaves of the top 1/3 of the seedlings of the two groups were cut out in time, quickly frozen with liquid nitrogen, and stored in a refrigerator at -70 °C. .
  • the leaves of the small salt mustard leaves of the control group and the drought treatment group were respectively 0.5 g, and the total RNA of the leaves of the small salt mustard was extracted with the plant RNA extraction kit (purchased from invitrogen).
  • the absorbance of total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001.
  • the OD 260 / OD 280 ratio was 1.8-2.0, indicating a higher total RNA purity; 1.0% agarose gel
  • the total RNA was detected by electrophoresis, and the brightness of the 28S band was about twice that of the 18S band, indicating good RNA integrity.
  • mRNA was isolated using Qiagen's purification of poly A+ RNA from total RNA.
  • 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, and the specific steps are as follows: The following components are sequentially added by using a 200 ⁇ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 ⁇ 1, 2 ⁇ 4 ligase buffer 5 , pGEM-T Easy vector 1 11, 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 was purchased from OXOID, 1% NaCl purchased from Sinopharm) was 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 from Tiangen Biochemical Technology (Beijing).
  • LB ibid.
  • X-gal/IPTG purchased from TAKARA, lg packaged into 20 mg/ml mother liquor, working concentration: 200 ⁇ /100 ml LB medium above IPTG was purchased from TaKaRa, and 5 g was packaged into a mother liquor at a concentration of 100 mM. The working concentration was: 100 ⁇ /100 ml of LB medium above the mother liquor.
  • the cells were incubated at 37 ° C for 18 hours on a solid culture plate. Count the number of clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 198 white colonies (number: Gh-B001 to Gh-B198).
  • the sequence was SEQ ID No: 3. Sequence analysis indicated that the encoded protein of the sequence belonged to the leucine zipper protein.
  • the full-length coding gene corresponding to the clone YLS-120 was named as ThHDbZIP-3, its corresponding protein is named HDbZIP-3.
  • SEQ ID No: 3 is the 3-terminal sequence of the coding gene HZ1 ⁇ 2Z/P-3. 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.
  • the kit comes with universal primers:
  • AAP SEQ ID NO: 7 :
  • 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-120GSP1 (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 primer used was 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 ⁇ 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 (94 °C for 50 seconds, 58 °C for 50 seconds, 72 °C for 90 seconds), 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.
  • the specific steps are as follows:
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation for 50 seconds at 94 °C, annealing for 50 seconds at 58 °C, extension of 90 seconds at 72 °C), extension at 72 °C for 10 minutes. A strip of about l.
  • lKbp 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 randomly picked 6
  • Gel Extraction Kit was purchased from OMEGA
  • pGEM-T Easy Vector was purchased from OMEGA
  • JM109 the specific method is the same as above
  • One white colony was inoculated separately in LB liquid medium containing 50 g/mL ampicillin, and cultured overnight at 37 ° C, 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 6 positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing. Sequencing, a 5' end sequence of the cDNA of the gene was obtained.
  • the obtained 5' RACE product clone YL16-3 was sequenced to obtain the sequence of SEQ ID NO: 9:
  • SEQ ID NO: 10 is not the full length sequence of ⁇ / ⁇ 1 ⁇ 2 ⁇ / ⁇ -3. Further 5' RACE is required to obtain the full length of the gene. According to the sequence of SEQ ID NO: 10, the following three specific primers were designed as specific primers for reverse transcription primers and 5' RACE.
  • YLS-120GSP1 SEQ ID NO: 11 :
  • YLS-120GSP2 SEQ ID NO: 12:
  • YLS-120 GSP3 SEQ ID NO: 13 :
  • the AACTTGGTTTTATCAGACATAG experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • YLS-120GSP1 (SEQ ID NO: 11) 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 tailed product as a template.
  • the primers used were SEQ ID NO: 11 and the universal primer SEQ ID NO: 7 (the kit is self-contained, and I is hypoxanthine modified a, c, g or t), 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 (purchased from TAKARA), 10 ⁇ primers SEQ ID NO: 11 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 for 50 seconds at 94 ° C, annealing for 50 seconds at 56 ° C, extension of 90 ° C for 90 seconds), extension at 72 ° C for 10 minutes.
  • the resulting PCR product was diluted 50-fold with double distilled water and 2.0 ⁇ L was used as a template, and a second round of PCR amplification was carried out using SEQ ID NO: 12 and the universal primer SEQ ID NO: 8, 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: 12 and SEQ ID NO: 8 each 2.0 ⁇ l, and
  • PCR reaction conditions pre-denaturation at 94 °C for 5 minutes, 33 cycles (denaturation for 50 seconds at 94 °C, 50 seconds for 50 seconds, extension for 90 seconds at 72 °C), and extension of 72 V for 10 minutes.
  • the primers SEQ ID NO: 12 and the 3 '-end primer SEQ ID NO: 13 were used for PCR amplification (reaction system and reaction conditions as above), and 6 positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing. Sequencing, a 5' end sequence of the cDNA of the gene was obtained.
  • SEQ ID NO: 14 The sequence of SEQ ID NO: 14, obtained by 5 'RACE, was spliced with the obtained splicing sequence SEQ ID NO: 10 to obtain SEQ ID NO: 15:
  • SEQ ID NO: 15 is: the full length sequence of ⁇ / ⁇ - ⁇ .
  • a pair of primers were designed according to the sequence of SEQ ID NO: 15 as follows: ThHDbZIP-3F (SEQ ID NO: 16): ATGGAGTTTCTCGGCGACAG
  • ThHDbZIP-3R (SEQ ID NO: 17): TCAAGCAGTTGAAGGACAGTTC AP (SEQ ID NO: 18):
  • ThHDbZIP-3 full-length coding sequence was cloned by SEQ ID NO: 16 and SEQ ID NO: 17.
  • the small salt mustard RNA was extracted, and the primer SEQ ID NO: 18 was used as the reverse transcription primer to obtain the cDNA of the small salt mustard.
  • the PCR reaction was carried out using the cDNA of the small salt mustard using the PfuUltra II Fusion HS DNA Polymerase of Stratagene. 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: 16 and SEQ ID NO: 17 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 58 °C for 25 seconds, extension at 72 °C for 1 minute), 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.
  • the primers SEQ ID NO: 16 and SEQ ID NO: 17 were used for PCR amplification (reaction system and reaction conditions as above), and 6 positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • SEQ ID NO: 2 the amino acid sequence of the encoded protein is SEQ ID NO: 1
  • 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 prion protein in plants. .
  • the 35S promoter containing the double enhancer and the terminator Tnos were selected as promoters and terminators of the ThHDbZIP-3 gene, respectively.
  • Primer SEQ ID NO: 19 and SEQ ID NO: 20 were used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and TaKaRa's PrimeSTAR HS DNA polymerase was used. 50 ⁇ l ⁇ Reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ ⁇ 121, 1.0 ⁇ PrimeSTAR, 10 ⁇ primer SEQ ID ⁇ : 19 and SEQ ID NO: 20 each 2.0 ⁇ l, and 31 ⁇ 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), and 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: 21 and P SEQ ID NO: 22 amplifies Tnos with pBI121 as a template, using TaKaRa
  • PrimeSTAR HS DNA polymerase 50 ⁇ PCR reaction system: 10 ⁇ 5 > ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ pBI121, 1.0 ⁇ Prime STAR, 10 ⁇ of primers SEQ ID NO: 21 and P SEQ ID NO:
  • 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 and EcoRI and ligated into the pCAMBIA2300-lCPromega T4 ligase cassette to obtain pCAMBIA2300-2.
  • TCAGAATTCCCAGTGAATTCCCGATCTAGTA SEQ ID NO: 23 and SEQ ID NO: 24 Amplify the Arabidopsis thaliana 35S promoter using the pCAMBIA2300 plasmid as a template.
  • PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 ⁇ ⁇ Reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ diluted 50-fold pCAMBIA2300 plasmid, 1.0 ⁇ PrimeSTAR, 10 ⁇ primer SEQ ID NO: 23 and P SEQ ID NO: 24 2.0 11, 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, elongation 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 obtained pCAMBIA2300-3
  • SEQ ID NO: 25 and SEQ ID NO: 26 Amplified ThHDbZIP-3 (template was the positive ThHDbZIP-3-pGEM plasmid obtained in Example 2) using Strafugene's PfuUltra II Fusion HS DNA Polymerase. 50 ⁇ PCR reaction system: 5 ⁇ lOxPfuUltra II reaction Buffer, 0.5 ⁇ 1 25 mM dNTP, 2.0 ⁇ ThHDbZIP-3-pGEM plasmid, 1.0 ⁇ PfuUltra II Fusion HS DNA Polymerase, 10 ⁇ primer SEQ ID NO: 25 and P SEQ ID NO: 26 each of 2.0 ⁇ l, 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 58 °C for 25 seconds, extension at 72 °C for 1 minute, extension at 72 °C for 5 minutes. Digestion by Sall, Smal The resulting PCR product was ligated (ligation method as above) to pCAMBIA2300-3 to obtain a plant expression vector 35S-73 ⁇ 4HZ1 ⁇ 2Z P-3-2300.
  • 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 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-73 ⁇ 4HZ1 ⁇ 2Z/P-3-2300 plasmid obtained in Example 3 was added to 40 ⁇ M of 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.
  • Bio-Rad purchased from Bio-Rad
  • 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 pot with a diameter of 7.5 cm containing peat 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 into 200 mL of LB liquid medium containing the above antibiotics at a ratio of 1% - 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) Resuspend Agrobacterium and suspend it to 0D 6 (K) about 0.80.
  • Inflorescence dip dyeing The above Agrobacterium-containing dyeing medium was added to a large-mouth container, and 200-300 mL of the Agrobacterium-containing dyeing medium was added to each container having a diameter of 9 cm for dip dyeing. Invert the plants so that all the aboveground tissues are immersed in the Agrobacterium suspension for 3 - 5 seconds and gently agitate. There should be a liquid film on the plant after infiltration. The immersed plants are placed in a plastic tray, covered with a clean plastic or plastic wrap to moisturize, and then placed in low light or dark overnight, taking care to prevent direct sunlight from the plant. 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. Seeds were harvested and the seeds were dried in a centrifuge tube at 4 °C.
  • Screening of transgenic seeds Prepare an aqueous solution containing 1/4 MS of large elements, add 0.8% agar powder, heat in a microwave oven until the agar is completely dissolved, and cool to about 50 °C. Add the required amount to a final concentration of 50 rn U 1 Kanamycin, shake well, pour 25 mL into each dish, and set the bench to cool and solidify.
  • ThHDbZIP-3 was amplified with SEQ ID NO: 16: and SEQ ID NO: 17 (50 ⁇ PCR reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • the sterilized vermiculite was soaked in 1/2 MS medium.
  • T101-T106 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.
  • T1 transgenic plants plants grown from seeds of TO transgenic plants
  • T101, ⁇ 102, ⁇ 103, ⁇ 104, ⁇ 105 and T106 were each. 22 out of 4
  • Arabidopsis thaliana survived and continued to grow with obvious drought tolerance (see Figures 3a and 3b, using T106 as an example, T101, T102, T103, TIC 4, T105 results and T106 is similar, not shown here).
  • 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.
  • T101, ⁇ 102, ⁇ 103, ⁇ 104, ⁇ 105, T106 transgenic plants
  • CK control plants
  • ELISA phytohormone abscisic acid
  • Example 8 verified the ThHDbZIP-3 protein at the transcriptional level.
  • RNA Extraction Kit Plant RNA Extraction Kit
  • the absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the individual RNA concentrations.
  • Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (2 ⁇ ⁇ total RNA as a template, reverse transcription primer SEQ ID NO: 18).
  • the relative expression of HDbZIP-3 protein was detected by amplification of ThHDbZIP-3 by SEQ ID NO: 16 and SEQ ID NO: 17.
  • PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase.
  • 50 ⁇ l ⁇ Reaction system 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR 10 ⁇ primers SEQ ID NO: 16 and SEQ ID ⁇ : 17 each 2.0 ⁇ l, and 30 ⁇ Double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 29 cycles (94 ° C denaturation for 45 seconds, 58 ° C Anneal for 45 seconds, 72 ° C for 2 minutes), and extend at 72 ° C for 10 minutes.
  • M is DNA Ladder Marker (DL2000, TakaRa)
  • 1-6 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant (in order: T101, T102, T103, T104, T105, T106).

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Abstract

L'invention concerne une protéine glissière à leucine, HDbZIP-3, issue de Thellungiella halophila. Elle concerne un gène codant pour ladite protéine, et une utilisation du gène dans la culture d'une plante transgénique présentant une tolérance accrue à la sécheresse.
PCT/CN2013/001157 2013-09-26 2013-09-26 Protéine glissière à leucine homéotique hdbzip-3 issue de thellungiella halophila, gène codant pour cette protéine, et utilisation du gène WO2015042738A1 (fr)

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CN201380078597.5A CN105452278A (zh) 2013-09-26 2013-09-26 一种小盐芥同源异型-亮氨酸拉链蛋白HDbZIP-3及其编码基因与应用
PCT/CN2013/001157 WO2015042738A1 (fr) 2013-09-26 2013-09-26 Protéine glissière à leucine homéotique hdbzip-3 issue de thellungiella halophila, gène codant pour cette protéine, et utilisation du gène

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106565833A (zh) * 2015-10-09 2017-04-19 中国科学院植物研究所 抗旱相关蛋白与其编码基因以及二者在调控植物抗旱性中的应用

Citations (1)

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Publication number Priority date Publication date Assignee Title
CN1641028A (zh) * 2004-01-15 2005-07-20 中国科学技术大学 一种拟南芥转录因子及其编码基因与应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1641028A (zh) * 2004-01-15 2005-07-20 中国科学技术大学 一种拟南芥转录因子及其编码基因与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
4 March 2003 (2003-03-04), accession no. A050448 *
DATABASE GENBANK 9 July 2013 (2013-07-09), accession no. EE29652 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106565833A (zh) * 2015-10-09 2017-04-19 中国科学院植物研究所 抗旱相关蛋白与其编码基因以及二者在调控植物抗旱性中的应用
CN106565833B (zh) * 2015-10-09 2019-09-06 中国科学院植物研究所 抗旱相关蛋白与其编码基因以及二者在调控植物抗旱性中的应用

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