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WO2015042740A1 - Protéine de type calcineurine b cbl-4 de thellungiella halophila, gène codant pour celle-ci et application associée - Google Patents

Protéine de type calcineurine b cbl-4 de thellungiella halophila, gène codant pour celle-ci et application associée Download PDF

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WO2015042740A1
WO2015042740A1 PCT/CN2013/001159 CN2013001159W WO2015042740A1 WO 2015042740 A1 WO2015042740 A1 WO 2015042740A1 CN 2013001159 W CN2013001159 W CN 2013001159W WO 2015042740 A1 WO2015042740 A1 WO 2015042740A1
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seq
plant
gene
expression vector
plants
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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 plant proteins and their coding genes and applications, and more particularly to a calcineurin B-like protein CBL-4 derived from small salt mustard and a gene encoding the same, and a transgenic plant thereof for improving drought tolerance Application in .
  • 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. Among them, 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 first aspect of the present invention provides a gene encoding a calcineurin B-like protein gene ThCBL-4 of a small salt mustard (herein named ThCBL-4); 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 7 3 ⁇ 4J- ⁇ -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 the plant expression vector C35S-7 3 ⁇ 4J- ⁇ -2300 of ThCBL-4 (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector C35S-7 3 ⁇ 4J- ⁇ -2300 of ThCBL-4.
  • FIG. 3 shows the drought tolerance simulation results of a ThCBL-4 T1 transgenic Arabidopsis plant (in the figure, T1K2) and a non-transgenic Arabidopsis plant as a control (in the figure, CK).
  • 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
  • Figure 4 is a graph showing the results of protein expression verification at the transcriptional level of transgenic T1 Arabidopsis plants and non-transgenic control plants.
  • M is DNALadderMarker (DL2000, TakaRa)
  • 1-7 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant (in order: T1K1, ⁇ 1 ⁇ 2, ⁇ 1 ⁇ 3, ⁇ 1 ⁇ 4, ⁇ 1 ⁇ 5, ⁇ 1 ⁇ 6, T1K7)
  • 8-11 is a drought-tolerant transgenic Arabidopsis thaliana T1 plants
  • 12-15 are non-transgenic Arabidopsis controls.
  • 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 small salt mustard seedlings was used as a sample 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 ⁇ , 100 ⁇ 3 ⁇ 3 , 100 MMnSO 4 , 30 ⁇ ZnS0 4 , 1 ⁇ 2 ⁇ 0 4 , 0.1 ⁇ CoCl 2 , 100 ⁇ Na 2 EDTA, 100 MFeSO 4 ). When the seedlings were cultured for about 1 month, they were used for experiments.
  • 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, light cycle 16 hours light / 8 hours dark condition, 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.
  • the leaves of the small salt mustard leaves of the control group and the drought treatment group were respectively 0.1 g, and the total RNA of the leaves of the small salt mustard was extracted with a plant RNA extraction kit (purchased from Invitrogen).
  • a plant RNA extraction kit purchased from Invitrogen.
  • HITACHI's UV spectrophotometer U-2001 The absorbance values of total RNA at 260 nm and 280 nm were measured, and the ratio of OD 260 / OD 280 was 1.8-2.0, indicating that the total RNA purity was high; the integrity of total RNA was detected by 1.0% agarose gel electrophoresis, 28S band The brightness is about 2 times 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 was purchased from Sinopharm. It was placed in a 37 ° C water bath, shaken at 225 rpm for 30 minutes, and 200 ⁇ of bacterial solution was applied to 50 g/mL ampicillin.
  • LB ibid.
  • X-gal/IPTG purchased from TAKARA, lg packaged into 20 mg/ml mother liquor, 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. The working concentration was: above mother liquor ⁇ ⁇ /lOOml LB medium) on solid culture plate, cultured at 37 ° C 18 hour. 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).
  • sequence was SEQ ID No: 3. Sequence analysis indicated that the encoded protein of the sequence belonged to calcineurin B-like protein.
  • the full-length coding gene corresponding to the clone YLS-66 was cloned herein. Named as ThCBL-4, the corresponding protein was named CBL-4.
  • SEQ ID No: 3 is the 3 terminal sequence of the coding gene CBL-4. 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.
  • YLS-66GSP1 SEQ ID NO: 4
  • GGACACGAAATCTTTCCATTC YLS-66GSP2 SEQ ID NO: 5 :
  • the TCCATCCAGATGCATCTGTC 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-66GSP1CSEQ ID NO: 4 As a reverse transcription primer, reverse transcription using a small salt mustard mRNA as a template, obtain a cDNA template, and then add a Poly C tail according to the procedure in the above 5' RACE kit instructions. The tailed product was subjected to the first round of PCR amplification using the template.
  • the primer used was SEQ ID NO: 4 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: 4 and SEQ ID NO: 7 each of 2.0 ⁇ 1, and 35 ⁇ 1 of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation for 45 seconds at 94 ° C, annealing for 45 seconds at 58 ° C, 45 seconds at 72 ° C), 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.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 10 X Ex Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ l diluted first round PCR product, 1.0 ⁇ 1 Ex Taq, 10 ⁇ ⁇ primer SEQ ID NO: 5 and SEQ ID NO: 8 Each of 2.0 ⁇ l, and 35 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation for 45 seconds at 94 ° C, annealing for 45 seconds at 58 ° C, 45 seconds at 72 ° C), extension at 72 ° C for 10 minutes.
  • a strip of approximately 500 bp in the second PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into pGEM-T Easy Vector, then converted to JM109 (specifically the same as above), randomly picked 6 whites
  • 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 are the same as above), and 6 positive clones were obtained and sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing. Sequencing, a 5' end sequence of the cDNA of the gene was obtained.
  • the resulting 5' RACE product clone 04 was sequenced to obtain the sequence of SEQ ID NO: 9: 1 GGGGGGGGGG ATGGGCTGCT CTCCGTCGAA GAGGAAGAAG GCAACACGAC CGCCGGGATA
  • SEQ ID NO: 10 is the full length sequence of ThCBL-4. according to
  • ThCBL-4F SEQ ID NO: 1 1:
  • ThCBL-4R SEQ ID NO: 12:
  • TTAAAAATTTAGTTTTTGCAAC AP SEQ ID NO: 13:
  • ThCBL-4 full-length coding sequence was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.
  • 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 PCR reaction was carried out using the PfuUltra II Fusion HS DNA Polymerase of Stratagene as the template. 50 ⁇ ⁇ PCR reaction system: 5 ⁇ 1 10 X PfuUltra II reaction Buffer, 0.5 ⁇ 1 25 mM dNTP, 2.0 ⁇ 1 cDNA, 1.0 ⁇ 1 PfuUltra II Fusion HS DNA Polymerase 10 ⁇ M primer SEQ ID NO: 11 and SEQ ID NO: 12 each of 2.0 ⁇ l and 37.5 ⁇ M of double distilled water.
  • PCR inverse Conditions Pre-denaturation at 95 °C for 2 minutes, 35 cycles (denaturation at 95 °C for 25 seconds, annealing at 51 °C for 25 seconds, extension at 72 °C for 30 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 ⁇ l ⁇ ⁇ ⁇ ⁇ Buffer, 0.5 ⁇ 1 5 mM dATP, 1.0 ⁇ Ex Taq. Reaction conditions: reaction at 70 ° C for 30 minutes.
  • a DNA fragment of about 2.8 Kbp was recovered (Omega recovery kit), ligated into the pGEM T-easy vector (73 ⁇ 4d- ⁇ -pGEM plasmid was obtained), and then transformed into JM109, and 6 white colonies were randomly picked and inoculated to contain 50.
  • G/mL ampicillin was cultured in LB liquid medium, and cultured at 37 ° C overnight, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use.
  • the primers SEQ ID NO: 11 and SEQ ID NO: 12 were used for PCR amplification (reaction system and reaction conditions are the same 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.
  • Nucleotide sequence of the 7M3 ⁇ 4L- ⁇ coding gene SEQ ID NO: 2 1 ATGGGCTGCT CTCCGTCGAA GAGGAAGAAG GCAACACGAC CGCCGGGATA TGAGGATCCT
  • 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 ThCBL-4 gene, respectively.
  • Pnos was amplified using the primers SEQ ID NO: 14 and SEQ ID NO: 15 with the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction system 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ ⁇ 121, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID ⁇ : 14 and SEQ ID NO: 15 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), 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 Kpnl and EcoRI and ligated into the pCAMBIA2300-lCPromega T4 ligase cassette to obtain pCAMBIA2300-2.
  • SEQ ID NO: 18 and SEQ ID NO: 19 The Arabidopsis thaliana 35S promoter was amplified using the pCAMBIA2300 plasmid as a template.
  • PrimeSTAR HS DNA polymerase using TaKaRa 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: 18 and P SEQ ID NO: 19 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, extension at 72 ° C for 30 seconds;), extension at 72 ° C for 10 minutes.
  • the resulting PCR product was ligated by HindIII and Xbal (connection method as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3 SEQ ID NO: 18: ACTAAGCTTATGGTGGAGCACGACACTCT
  • SEQ ID NO: 20 and SEQ ID NO: 21 Amplified ThCBL-4 (template was the positive ThCBL-4-pGEM plasmid obtained in Example 2) using Strafugene's PfuUltra II Fusion HS DNA Polymerase. 50 ⁇ PCR reaction system: 5 ⁇ l O PfuUltra II reaction Buffer, 0.5 ⁇ l 25 mM dNTP, 2.0 ⁇ ThCBL-4-pGEM plasmid, 1.0 ⁇ PfuUltra II Fusion HS DNA Polymerase 10 ⁇ primers SEQ ID NO: 20 and SEQ ID NO: 21 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 52 °C for 25 seconds, extension at 72 °C for 30 seconds, extension at 72 °C for 5 minutes. Digestion by Xbal, Kpnl After the resulting PCR product is ligated (the connection method is the same as above)
  • pCAMBIA2300-3 obtained the plant expression vector 35 S-73 ⁇ 4CAL- ⁇ -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 ⁇ ⁇ / ⁇ 1 streptomycin, and cultured overnight (about 12-16 hours) to OD 6 at 28 °C. 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 ⁇ 4d- ⁇ -2300 plasmid obtained in Example 3 was added to 40 ⁇ of competent cells, and the mixture was mixed and ice bathed 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 room, and push the slide to place the electric shock cup on the electric shock room. At the base electrode.
  • 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 to 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).
  • Infiltration of inflorescence 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 the above ground 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, then placed in low light or dark places overnight, taking care to prevent direct sunlight from the plants. Remove the cover approximately 12 - 24 hours after processing.
  • the plants are cultured normally, and the plants are further grown for 3 to 5 weeks until the pods are browned and dried.
  • the seeds were harvested and the seeds were dried and stored 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 final concentration of the required amount to 50 ⁇ ! / 1 of kanamycin, shake well, pour 25 mL into each dish, set the bench to cool and solidify and then seed.
  • SEQ ID NO: 11 P SEQ ID NO: 12 (50 ⁇ PCR reaction system: 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primer SEQ ID NO: 11 and P SEQ ID NO: 12 each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions 94 °C pre-denaturation 5 minutes, 33 cycles (denaturation for 45 seconds at 94 °C, 45 seconds at 51 °C, extension for 30 seconds at 72 °C), extension at 72 °C for 7 minutes), numbers of plants identified as positive by PCR (T1K1-T1K12) ), and save.
  • T1K1-T1K12 Drought tolerance simulation experiment and functional identification of transgenic Arabidopsis thaliana T1 plants overexpressing ThCBL-4. The sterilized vermiculite was soaked with 1/2 MS medium.
  • T1K1-T1K6 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 seedlings of uniform size were kept in each pot for drought experiments.
  • T1 transgenic plants plants grown from seeds of T0 transgenic plants
  • T1K1, T1K2, T1K3, T1K4, T1K5 and T1K6 each strain 25 out of 4
  • Arabidopsis thaliana survived and continued to grow with obvious drought tolerance (see Figures 3a and 3b, using T1K2 as an example, T1K1, T1K3, T1K4, T1K5, T1K6 results with T1K2 Similarly, not shown here).
  • Example 7 validates 73 ⁇ 4CB- ⁇ protein expression 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: 13).
  • the relative expression of the protein was examined by amplifying T CBL-4 by SEQ ID NO: 11 and SEQ ID NO: 12.
  • the 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 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR 10 ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 ⁇ l, and
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 29 cycles (denaturation at 94 ° C for 45 seconds, annealing at 51 ° C for 45 seconds, extension at 72 ° C for 45 seconds), extension at 72 ° C for 10 minutes.
  • M is DNA Ladder Marker (DL2000, TakaRa), and 1-7 is a drought-tolerant transgenic Arabidopsis thaliana T1 plant (in order: ⁇ 1 ⁇ 1, ⁇ 1 ⁇ 2, ⁇ 1 ⁇ 3, ⁇ 1 ⁇ 4, ⁇ 1 ⁇ 5, ⁇ 1 ⁇ 6, T1K7) 8-11 is a drought-tolerant transgenic Arabidopsis thaliana first-generation plant, and 12-15 is a non-transgenic Arabidopsis control.
  • the size of the electrophoresis band of the PCR product shown in the figure is the same as the size of the 73 ⁇ 4CAL- ⁇ (about 660 bp).

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  • 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 de type calcineurine B de Thellungiella halophila ThCBL-4, un gène codant pour celle-ci et une application du gène dans la culture d'une plante transgénique présentant une tolérance accrue à la sécheresse.
PCT/CN2013/001159 2013-09-26 2013-09-26 Protéine de type calcineurine b cbl-4 de thellungiella halophila, gène codant pour celle-ci et application associée Ceased WO2015042740A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2013/001159 WO2015042740A1 (fr) 2013-09-26 2013-09-26 Protéine de type calcineurine b cbl-4 de thellungiella halophila, gène codant pour celle-ci et application associée
CN201380078600.3A CN105452453A (zh) 2013-09-26 2013-09-26 一种小盐芥钙调磷酸酶b类似蛋白cbl-4及其编码基因与应用

Applications Claiming Priority (1)

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PCT/CN2013/001159 WO2015042740A1 (fr) 2013-09-26 2013-09-26 Protéine de type calcineurine b cbl-4 de thellungiella halophila, gène codant pour celle-ci et application associée

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WO2015042740A1 true WO2015042740A1 (fr) 2015-04-02

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CN114634940B (zh) * 2022-04-07 2024-02-20 广西大学 一种玉米类钙调磷酸酶b蛋白基因及其在提高植物耐旱性和耐盐性的应用
CN115490762B (zh) * 2022-05-11 2023-06-27 西南大学 蜡梅CpCBL8基因及其编码的蛋白与应用

Citations (4)

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CN101054411A (zh) * 2007-04-09 2007-10-17 中国农业科学院作物科学研究所 玉米钙调磷酸酶b类似蛋白及其编码基因与应用
CN101979579A (zh) * 2010-10-20 2011-02-23 江苏省农业科学院 一种类钙调磷酸酶b亚基蛋白基因及其编码蛋白质和该基因的克隆
WO2012028646A1 (fr) * 2010-08-31 2012-03-08 Westfälische Wilhelms-Universität Münster Moyens destinés à améliorer des caractères agrobiologiques dans des plantes
KR20120052156A (ko) * 2010-11-15 2012-05-23 충북대학교 산학협력단 배추 유래의 ＢｒＣＩＰＫ3 유전자 및 이의 용도

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WO2012028646A1 (fr) * 2010-08-31 2012-03-08 Westfälische Wilhelms-Universität Münster Moyens destinés à améliorer des caractères agrobiologiques dans des plantes
CN101979579A (zh) * 2010-10-20 2011-02-23 江苏省农业科学院 一种类钙调磷酸酶b亚基蛋白基因及其编码蛋白质和该基因的克隆
KR20120052156A (ko) * 2010-11-15 2012-05-23 충북대학교 산학협력단 배추 유래의 ＢｒＣＩＰＫ3 유전자 및 이의 용도

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DATABASE GENBANK 5 June 2013 (2013-06-05), SWARBRECK, D. ET AL., accession no. M_122333.5 *
DATABASE GENBANK 5 June 2013 (2013-06-05), SWARBRECK, D. ET AL., accession no. P_197815.1 *
SUN, ZHIBIN ET AL.: "Overexpression of aThellungiella halophila CB19 homolog, ThCBL9, confers salt and osmotic tolerances in transgenicarabidopsis thaliana", JOURNAL OF PLANT BIOLOGY, vol. 51, no. 1, 1 January 2008 (2008-01-01), pages 25 - 34 *

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