WO2015042737A1 - Leucine zipper protein bzip-4 from thellungiella halophila, and coding gene and use thereof - Google Patents

Leucine zipper protein bzip-4 from thellungiella halophila, and coding gene and use thereof Download PDF

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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
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seq
plant
gene
plants
expression vector
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PCT/CN2013/001156
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French (fr)
Chinese (zh)
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孙超
陈文华
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 PCT/CN2013/001156 priority Critical patent/WO2015042737A1/en
Priority to CN201380078599.4A priority patent/CN105452280A/en
Publication of WO2015042737A1 publication Critical patent/WO2015042737A1/en

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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

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  • 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).

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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

Provided are a leucine zipper protein bZIP-4 from thellungiella halophila, a coding gene thereof, and a use thereof in breeding a transgenic plant with improved drought tolerance.

Description

一种小盐芥亮氨酸拉链蛋白 6ZJP-¥及其编码基因与应用  A small salt mustard leucine zipper protein 6ZJP-¥ and its coding gene and application
技术领域 本发明涉及植物蛋白及其编码基因与应用，特别是涉及一个来源于小盐芥的亮氨酸 拉链蛋白/^ 及其编码基因， 以及其在培育耐旱性提高的转基因植物中的应用。 背景技术 温度、 盐渍和干旱等逆境胁迫会对高等植物的生长发育造成严重危害， 导致作物产量 降低， 品质下降， 严重威胁农业生产和自然环境。 其中干旱对作物产量的影响， 在诸多自 然逆境中占首位， 其危害相当于其它灾害之和， 是许多地区是农业发展的瓶颈。 据统计， 世界干旱、 半干旱地区占陆地面积的 34%; 我国干旱、 半干旱地区约占国土面积的 52%， 年受旱面积达 200 - 270万公顷 ， 全国灌溉区每年缺水约 30亿立方米， 因缺水而少收粮食 350 - 400亿公斤； 特别是我国主要产粮区如华北、东北和西北， 是我国缺水最严重的地区， 春旱频繁达到十年九遇。 FIELD OF THE INVENTION 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. 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. According to statistics, 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.
植物耐旱性大多属于多基因控制的数量性状， 利用常规育种方法改良作物的抗旱性受 到周期长、 优异种质资源缺乏的限制。 近年来的转录组学、 蛋白组学和基因表达调控的研 究初步揭示了植物干旱胁迫的作用分子机理。 目前， 利用干旱胁迫相关基因提高植物的抗 旱能力， 已经成为植物抗逆分子生物学的研究热点和植物抗逆基因工程重要的研究方向。  Most of the drought tolerance of plants belongs to the quantitative traits controlled by multiple genes. The improvement of drought resistance of crops by conventional breeding methods is limited by the long cycle and the lack of excellent germplasm resources. Recent studies on transcriptomics, proteomics and gene expression regulation have revealed the molecular mechanism of plant drought stress. At present, the use of drought stress-related genes to improve the drought resistance of plants has become a research hotspot of plant resistance to molecular biology and an important research direction of plant stress resistance genetic engineering.
植物受到逆境胁迫时会产生相应的应答反应， 以降低或消除逆境胁迫给植物带来的危 害。 植物的这种应答反应是一个涉及多基因、 多信号途径及多基因产物的复杂过程。 但就 目前的研究状况而言， 由于其机制十分复杂， 许多植物对逆境下的生物化学和生理学上的 响应机制仍有待深入研究。 在抗逆应答基因的功能及表达调控方面的研究将对植物抗逆相 关的信号传递途径之间的联系以及整个信号传递网络系统的研究提供重要的基础。 发明内容 本发明人利用 SSH (抑制差减杂交）与 RACE ( cDNA末端快速扩增）相结合的方法克 隆出了小盐芥的一种亮氨酸拉链蛋白 (本文命名为 bZIP-4) 的编码基因， 并测定了其 DNA 序列。 并且发现将其导入植物超量表达后， 可明显改善转基因植株的耐旱性， 而且这些性 状可稳定遗传。 本发明第一方面提供小盐芥的一种亮氨酸拉链蛋白 bZIP-4 的编码基因 （本文命名为 ThbZIP-4) ； 优选地， 其序列为 SEQ ID N0: 2。 When plants are stressed by stress, they will respond accordingly to reduce or eliminate the damage caused by stress. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. However, as far as the current research situation is concerned, due to the complexity of its mechanism, the biochemical and physiological response mechanisms of many plants to stress have yet to be further studied. Studies on the function and expression regulation of stress-responsive genes will provide an important basis for the link between plant stress-resistance-related signaling pathways and the study of the entire signaling network system. SUMMARY OF THE INVENTION 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.
本发明第二方面提供一种重组表达载体， 其含有本发明第一方面所述的基因， 其是通 过将所述基因插入到一种用于构建所述重组表达载体的基础载体而获得的， 并且所述基 因的核苷酸序列与所述基础载体的表达控制序列可操作地连接； 优选地， 所述基础载体为 pCAMBIA2300; 优选地， 所述重组表达载体为附图 2所示的 35S-r )Z/P-¥-2300载体。 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.
本发明第三方面提供一种重组细胞， 其含有本发明第一方面所述的基因或者本发明第 二方面所述的重组表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。  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.
本发明第四方面提供一种改善植物耐旱性的方法， 包括： 将本发明第一方面所述的基 因或者本发明第二方面所述的重组表达载体导入植物或植物组织并使所述基因表达； 优选 地， 所述植物是拟南芥。  A fourth aspect of the present invention provides a method for improving drought tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and causing the gene Expression; Preferably, the plant is Arabidopsis thaliana.
本发明第五方面提供一种制备转基因植物的方法， 包括： 在有效产生植物的条件下培 养含有本发明第一方面所述的基因或者本发明第二方面所述的重组表达载体的植物或植物 组织； 优选地， 所述植物是拟南芥。  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; Preferably, the plant is Arabidopsis thaliana.
本发明第六方面提供本发明第一方面所述的基因、 本发明第二方面所述的重组表达载 体或者本发明第三方面所述的重组细胞用于改善植物耐旱性以及用于植物育种的用途； 优 选地， 所述植物是拟南芥。  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; Preferably, the plant is Arabidopsis thaliana.
本发明第七方面提供本发明第一方面所述的基因编码的蛋白质， 其氨基酸序列如 SEQ ID NO: 1所示。 附图说明 图 1是 ThbZIP-4的植物表达载体 (358-Γ )Ζ/Ρ-¥-2300)的构建流程 （图 la-lb)。  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. Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector (358-Γ)Ζ/Ρ-¥-2300 of ThbZIP-4 (Fig. la-lb).
图 2是 ThbZIP-4的植物表达载体 <;358-Γ )Ζ/Ρ-¥-2300)的质粒图。  Figure 2 is a plasmid map of the plant expression vector <;358-Γ )Ζ/Ρ-¥-2300 of ThbZIP-4.
图 3是 ThbZIP-4 T1代转基因拟南芥植株 （图中， T1A2) 和作为对照的非转基因拟南 芥植株（图中， CK) 的耐旱模拟实验结果。 （图 3a为正常生长 20天的拟南芥植株； 图 3b 为正常生长 20天后干旱处理 14天的拟南芥植株）。  Figure 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).
图 4干旱胁迫和正常生长条件下的 T1代转基因拟南芥植株及对照植株 ABA含量变 化检测结果。 1-7依次为株系（从左至右）： T1A1、 T1A2、 T1A3、 T1A4、 T1A5、 T1A6、 CK, 其中 T1A1、 T1A2、 T1A3、 T1A4、 T1A5、 T1A6为转基因植株， CK为对照植株。  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.
图 5是转基因 T1代拟南芥植株和非转基因对照植株在转录水平上的蛋白表达验证结 果。 M为 DNA Ladder Marker (DL2000, TakaRa), 1-7耐旱转基因拟南芥 Tl代植株 （依 次为： T1A1、 T1A2、 T1A3、 T1A4、 T1A5、 T1A6、 T1A7), 8-11为非转基因拟南芥对照； 12-16 为不耐旱转基因拟南芥 T1代植株。 Figure 5 is a graphical representation of the protein expression at the transcriptional level of transgenic T1 Arabidopsis plants and non-transgenic control plants. fruit. 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.
具体实施方式 下面结合非限制性实施例对本发明进行进一步说明。 所述实施例仅出于示例性目的， 并非意在限制本发明的范围。 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.
下面实施例中提到的未注明来源的限制性内切酶均购自 New England Biolabs公司。 实施例 1 干旱胁迫下小盐芥 SSH文库构建  The unrecognized restriction enzymes mentioned in the examples below were purchased from New England Biolabs. Example 1 Construction of SSH library of small salt mustard under drought stress
具体方法为：  The specific method is:
利用 Clontech公司的 PCR-selectTM cDNA Subtraction Kit所示的方法通过抑制差减杂交 方法构建差减文库。在实验过程中以干旱处理的盐芥幼苗的叶片的 mRNA作为样本（Tester), 以未处理的小盐芥幼苗的叶片的 mRNA作为对照 （Driver 具体步骤简述如下： The method of PCR-select TM cDNA Subtraction Kit from Clontech shown by using the subtractive hybridization method suppression construct subtraction libraries. During the experiment, the mRNA of the leaves of the salt-treated mustard seedlings was used as a sample (Tester), and the mRNA of the leaves of the untreated small salt mustard seedlings was used as a control (the specific steps of the driver are briefly described as follows:
(1) 供试材料：  (1) Test materials:
小盐芥（ T ellungiella halophila, 购自中国内蒙古巴彦淖尔市乌兰布和沙漠绿色植物 园盐生植物繁育中心） ， 播种到灭过菌的蛭石上， 在 25°C、 光周期 16小时光照 /8小时 黑暗（光强 2000 - 3000 Lx)条件下培养， 每周浇 1/2MS培养基（9.39mMKN03， 0.625 mMKH2P04， 10.3 mMNH4N03, 0.75 mMMgSO4， 1.5 mMCaCl2， 50 μΜΚΙ, 100 μΜ H3B03， 100 MMnSO4，30 μΜ ZnS04, 1 μΜ Na2Mo04， 0.1 μΜ CoCl2, 100 μΜ Na2EDTA, 100 MFeSO4) —次。 当苗株高达 25 - 30 cm时用于实验。 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 μΜΚΙ per week). , 100 μΜ H 3 B0 3 , 100 MMnSO 4 , 30 μΜ ZnS0 4 , 1 μΜ Na 2 Mo0 4 , 0.1 μΜ CoCl 2 , 100 μΜ Na 2 EDTA, 100 MFeSO 4 ). It was used for experiments when the seedlings were as high as 25 - 30 cm.
(2) 材料处理：  (2) Material handling:
将供试幼苗分为 2组， 每组 4盆， 每盆 1株。 第一组为对照组， 在 25°C、 光周期 16小时光照 /8小时黑暗条件下培养， 正常浇灌。 第二组为干旱处理组， 25°C、 光周期 16小时光照 /8小时黑暗条件下培养， 停止浇灌， 处理 10天， 处理完毕后及时剪取两组 幼苗顶端 1/3的叶片， 用液氮迅速冷冻后， 于 -70°C冰箱中保存。  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.
(3) 总 RNA提取：  (3) Total RNA extraction:
分别取对照组和干旱处理组的小盐芥叶片 0.5 g， 用植物 RNA提取试剂盒 （购自 Invitrogen) 提取小盐芥叶片的总 RNA。 用 HITACHI公司的紫外分光光度计 U-2001测 定总 RNA在 260 nm和 280 nm的吸光度值， OD260/OD280比值为 1.8-2.0， 表明总 RNA 纯度较高； 用 1.0%的琼脂糖凝胶电泳检测总 RNA的完整性， 28S条带的亮度约为 18S 条带的 2倍， 表明 RNA的完整性良好。 使用 Qiagen公司的 Oligotex mRNA 纯化试剂 盒（purification of poly A+ RNA from total RNA)分离 mRNA。 0.5 g of small salt mustard leaves of the control group and the drought treatment group were taken, and the total RNA of the leaves of the small salt mustard was extracted with a plant RNA extraction kit (purchased from Invitrogen). Measured with HITACHI's UV spectrophotometer U-2001 Absorbance values of total RNA at 260 nm and 280 nm, OD 260 / OD 280 ratio 1.8-2.0, indicating higher total RNA purity; total RNA integrity detected by 1.0% agarose gel electrophoresis, 28S bands 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.
( 4 ) 抑制差减杂交：  (4) Suppression of subtractive hybridization:
按 Clontech公司的 PCR-selectTM cDNA Subtraction Kit试剂盒所示的方法进行抑制差减 杂交。 先将 Driver mRNA和 Tester mRNA分别反转录， 得到双链 cDNA, 再以 2 Tester cDNA和 2 g Driver cDNA作为起始材料进行差减杂交。 在 37°C水浴下分别将 Tester cDNA 和 Driver cDNA用 Rsa I 酶切 1.5小时， 然后将酶切后的 Tester cDNA分成两等份， 连接上 不同的接头， 而 Driver cDNA不连接头。 两种连有不同接头的 Tester cDNA分别与过量的 Driver cDNA混合， 进行第一次正向差减杂交。 将两种第一次差减杂交的产物混合， 再与新 变性的 Driver cDNA进行第二次正向差减杂交，然后通过两次抑制性 PCR扩增差异表达的 片段， 使其得到富集。 Suppression Subtractive Hybridization performed by PCR-select TM cDNA Clontech's method shown Subtraction Kit kit. Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and subtraction hybridization was performed using 2 Tester cDNA and 2 g Driver cDNA as starting materials. The Tester cDNA and Driver cDNA were digested with Rsa I for 1.5 hours in a 37 ° C water bath, and then the digested Tester cDNA was divided into two equal portions, and the different linkers were ligated, and the Driver cDNA was not ligated. Two tester cDNAs with different adaptors were mixed with excess Driver cDNA for the first forward subtractive hybridization. The products of the two first subtractive hybridizations were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and then the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.
为了增加获得表达序列标签 （Expressed sequence tag, EST) (Unigene)的有效性， 避免 基因无酶切位点及所获得序列在非翻译区，本实验同时用内切酶 Haelll按上述步骤对 Tester cDNA和 Driver cDNA进行酶切并先后进行两次正向差减杂交和两次抑制性 PCR扩增， 最 后合并两组正向差减杂交 cDNA片段的第二次抑制性 PCR产物。  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.
( 5 ) cDNA差减文库的构建与初步筛选、 克隆、 鉴定  (5) Construction and preliminary screening, cloning and identification of cDNA subtraction library
依照 pGEM-T Easy试剂盒的程序， 将上述合并的正向差减杂交 cDNA片段的第二次 PCR产物 （使用 QIAquick PCR Purification Kit纯化， 购自 Qiagen) 与 pGEM-T Easy (购自 Promega试剂盒)载体连接， 其具体步骤如下： 用 200 μΐ PCR管依次加入下列成分： 纯化的 正向差减杂交 cDNA片段的第二次 PCR产物 3 μ1， 2χΤ4连接酶缓冲液 5 μ1， pGEM-T Easy 载体 1 μ1， T4 DNA连接酶 1 μΐ ， 于 4°C连接过夜。取 10 连接反应产物， 加入到 100 感受态大肠杆菌 JM109 (购自 TAKARA) 中， 冰浴 30分钟、 热休克 60秒、 冰浴 2分钟， 另加 250 μL LB培养液（ 1% Tryptone购自 OXOID, 0.5% Yeast Extract购自 OXOID, 1% NaCl 购自国药)置 37°C水浴中，以 225转 /分钟振荡培养 30分钟，取 200 μL菌液涂布于含 50 g/mL 氨苄青霉素 （购自天根生化科技 (北京)有限公司） 的 LB (同上） /X-gal/IPTG (X-gal/IPTG 购自 TAKARA, lg包装配制成 20 mg/ml母液，其工作浓度为： 以上母液 200 μ 1/100 ml LB 培养基; IPTG购自 TaKaRa, 5g包装配制成 100 mM浓度的母液， 其工作浓度为： 以上母液 100 μΙ/lOOml LB培养基） 固体培养平板上， 37°C培育 18小时。 计数培养板中直径 > 1 mm 的清晰白色及蓝色菌落数， 随机挑取 198个白色菌落 (编号： Gh-B001至 Gh-B198)。将所有 白色克隆分别接种于含有 50 μ§/ηΛ 氨苄青霉素的 LB 液体培养基的 96 孔细胞培养板 (CORNING)中， 37°C培养过夜后加甘油至终浓度 20%， 于 -80°C保存备用。 以巢式 PCR 引 物 Primer 1和 Primer 2R ( Clontech公司的 PCR-select™ cDNA Subtraction Kit试剂盒自带） 进行菌液 PCR扩增验证，得到 166个阳性克隆，然后将所有阳性克隆在送英潍捷基（上海） 贸易有限公司测序。 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 μΐ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 μl, 2χΤ4 ligase buffer 5 μl, pGEM-T Easy vector 1 μl, T4 DNA ligase 1 μΐ, 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 μL 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 μL 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 μ 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: 100 μΙ/100 ml LB medium above the mother liquor), cultured on a solid plate for 18 hours at 37 ° C. . Count the plates in diameter > 1 mm 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 μ § /ηΛ 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.
( 6) 差异克隆的 cDNA测序分析：  (6) cDNA sequencing analysis of differential clones:
将 DNA测序结果去除载体和不明确序列及冗余的 cDNA后， 共得到 123 个 EST (Unigene；)。 经分析有 22个重叠群， 有 101个单一的序列。 经 BlastN发现其中 53条 EST (Unigene) 在 GenBank 中有同源序列， 21条 EST功能未知或者为假定蛋白， 另有 27条 未获得同源匹配， 推测可能是处于 3'或 5'末端非翻译区的较短序列。 实施例 2 盐芥亮氨酸拉链蛋白编码基因 ThbZIP-4的克隆  After removing the vector and the ambiguous sequence and redundant cDNA from the DNA sequencing results, a total of 123 ESTs (Unigene;) were obtained. After analysis, there are 22 contigs with 101 single sequences. BlastN found that 53 ESTs (Unigene) have homologous sequences in GenBank, 21 ESTs are unknown or hypothetical proteins, and another 27 have not obtained homologous matches, presumably at the 3' or 5' end. A shorter sequence of regions. Example 2 Salt mustard leucine zipper protein coding gene Cloning of ThbZIP-4
克隆子 YLS-16 去掉冗余 DNA后， 序列为 SEQ ID No: 3， 序列分析表明该序列的编 码的蛋白质属于亮氨酸拉链蛋白， 本文将克隆子 YLS-16 对应的全长编码基因命名为 ThbZIP-4, 其对应的蛋白命名为 bZIP-4。  After the cloned YLS-16 was removed from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the encoded protein of the sequence belonged to the leucine zipper protein. In this paper, the full-length coding gene corresponding to the clone YLS-16 was named as ThbZIP-4, its corresponding protein is named bZIP-4.
SEQ ID No: 3 SEQ ID No: 3
GGGACCG TA AGCCCTGTTT CATCAGACGG GTTAGGACAT GGACAAGTGG ATAACATAGG AAGTCAGTAT GGAGTGGATA GGGAGGGTT AAGGGGAAGG AAAAGAGTAG GGATGGTCC AGTGGAGAAA GTAGTGGAAA GAAGGGAGAG GCGGATGATC AAGAACCGTG AGTCTGCTGC AAGGTCTAGA GCAAGAAAGC AGGCGTATAC AGTGGAATTA GAAGGGGAAT TGAACCAGTT GAAAGAAGAG AATGCGCAGC TAAAACA GC ATTGGGGGAG TTGGAGAGAA AGAGGAAGCA ACAGTAT TT GAGACTTTGA AGACAAGAGC ACAACCGAAG TTGCGGAAGG CGAGCGGGAG ATTGGGGACA TTGATGAGGA ACCCGAGT G TCCGCTCTGA bZIP-4全长编码基因的克隆  GGGACCG TA AGCCCTGTTT CATCAGACGG GTTAGGACAT GGACAAGTGG ATAACATAGG AAGTCAGTAT GGAGTGGATA GGGAGGGTT AAGGGGAAGG AAAAGAGTAG GGATGGTCC AGTGGAGAAA GTAGTGGAAA GAAGGGAGAG GCGGATGATC AAGAACCGTG AGTCTGCTGC AAGGTCTAGA GCAAGAAAGC AGGCGTATAC AGTGGAATTA GAAGGGGAAT TGAACCAGTT GAAAGAAGAG AATGCGCAGC TAAAACA GC ATTGGGGGAG TTGGAGAGAA AGAGGAAGCA ACAGTAT TT GAGACTTTGA AGACAAGAGC ACAACCGAAG TTGCGGAAGG CGAGCGGGAG ATTGGGGACA TTGATGAGGA ACCCGAGT G TCCGCTCTGA bZIP-4 encoding full-length Cloning of genes
根据已经获得的 SEQ ID No: 3序列分析， SEQ ID No: 3 为编码基因 6Z/P-¥的 3 端序列。 根据已经获得的 SEQ ID NO : 3序列， 设计如下三条特异性引物， 作为反转录引物 及 5 'RACE的特异性引物。  According to the sequence analysis of SEQ ID No: 3 which has been obtained, 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.
YLS-16GSP1 ( SEQ ID NO: 4 ) ： YLS-16GSP1 ( SEQ ID NO: 4 ) :
GATCATCCGCCTCTGCCTTC YLS-16GSP2 ( SEQ ID NO: 5 ) ： GATCATCCGCCTCTGCCTTC YLS-16GSP2 ( SEQ ID NO: 5 ) :
GACCATCCACTACTCTTTTCC YLS-16 GSP3 ( SEQ ID NO:6) ：  GACCATCCACTACTCTTTTCC YLS-16 GSP3 ( SEQ ID NO: 6) :
GGACCGTTAAGCCCTGTTTC  GGACCGTTAAGCCCTGTTTC
试剂盒自带通用引物：  The kit comes with universal primers:
AAP ( SEQ ID NO: 7 ) ：  AAP ( SEQ ID NO: 7 ) :
GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP ( SEQ ID NO: 8 ) ：  GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG AUAP ( SEQ ID NO: 8 ) :
GGCCACGCGTCGACTAGTAC 实验步骤按试剂盒说明书操作 （5' RACE System for Rapid Amplification of cDNA Ends 试剂盒购自 Invitrogen公司）。  The GGCCACGCGTCGACTAGTAC experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
以 YLS-16GSP1(SEQ ID N0: 4)为反转录引物， 以小盐芥 mRNA为模板进行反转录， 获 得 cDNA模板， 然后按照上述 5' RACE试剂盒说明书中的步骤加 Poly C尾， 以加尾后的产 物为模板进行第一轮 PCR扩增， 所用引物为 SEQ ID NO: 4与通用引物 SEQ ID NO: 7 (试 剂盒自带， I为次黄嘌吟修饰的 a、 c、 g或 t)， 具体步骤如下：  Using 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:
50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer, 3 μ1 2.5 mM的 dNTP， 2.0 μΐ mRNA反转录的 cDNA, 1.0 μΐ Ex Taq (购自 TAKARA)、 10 μΜ的引物 SEQ ID NO: 4和 SEQ ID NO: 7各 2.0 μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5分钟， 33个循环（94°C变性 50秒， 55°C退火 50秒， 72°C延伸 1分钟）， 72°C延伸 10分钟。  50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μl 2.5 mM dNTP, 2.0 μΐ mRNA reverse transcribed cDNA, 1.0 μΐ Ex Taq (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 4 and SEQ ID NO: 7 each of 2.0 μl, and 35 μΐ 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.
所得的 PCR产物用双蒸水稀释 50倍后取 2.0 μΐ作为模板， 用 SEQ ID NO: 5与通用引 物 SEQ ID NO: 8进行第二轮 PCR扩增， 具体步骤如下：  The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μL 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:
50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ稀释的第一轮 PCR 产物， 1.0 l Ex Taq、 10 μΜ的引物 SEQ ID NO: 5和 SEQ ID NO: 8各 2.0 μ1， 以及 35 μΐ的 双蒸水。 PCR反应条件： 94°C预变性 5分钟， 33个循环 （ 94°C 变性 50秒， 55°C退火 50 秒， 72°C 延伸 1分钟）， 72 °C 延伸 10分钟。 回收第二次 PCR产物中约为 500bp大小的条 带 （Gel Extraction Kit购自 OMEGA), 并将其连接到 pGEM-T Easy Vector, 然后转化到 JM109 (具体方法同上）， 随机挑取 10个白色菌落分别接种于含有 50 g/mL氨苄青霉素的 LB 液体培养基中培养， 37°C培养过夜后加甘油至终浓度 20% (v/v), -80°C保存备用。 用 引物 SEQ ID NO: 5与 3'端引物 SEQ ID NO: 6进行菌液 PCR扩增（反应体系及反应条件同 上）， 得到 4个阳性克隆 （YL16-1、 YL16-3、 YL16-5和 YL16-6), 送英潍捷基 （上海） 贸 易有限公司测序测序， 获得该基因的 cDNA的一段 5'端序列。 所得的 5 'RACE产物克隆子 YL16-3测序获得序列为 SEQ ID NO : 9： 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 l Ex Taq, 10 μΜ primers SEQ ID NO: 5 and SEQ ID NO: 8 2.0 μl, and 35 μΐ 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:
1 GGGGGGGGGG CGGCGGTGAG ATCGCGGCTA GACAACCAAC TTTTGGGGAG ATGACGCTTG 1 GGGGGGGGGG CGGCGGTGAG ATCGCGGCTA GACAACCAAC TTTTGGGGAG ATGACGCTTG
61 AAGATTTCTT GGTCAAGGCA GGTGTGGTTA GAGAACATCC CACTAACCCT AACCCTAACC61 AAGATTTCTT GGTCAAGGCA GGTGTGGTTA GAGAACATCC CACTAACCCT AACCCTAACC
121 CTAACCCAAA CCCAAACTCA AACCAAAACC AATCTAGTGT TATACCCGCA GCTACGCAGC 181 AGCAGCTCTA GGTGTGTTT CAAGGAACTG GTGATCCTAC T TCCCCGGT CAGGCCATGA121 CTAACCCAAA CCCAAACTCA AACCAAAACC AATCTAGTGT TATACCCGCA GCTACGCAGC 181 AGCAGCTCTA GGTGTGTTT CAAGGAACTG GTGATCCTAC T TCCCCGGT CAGGCCATGA
241 GTGTAGGTGA CCCTTCAGGT TATGGTAAAA GGACAGGAGG AGGAGGAGGG TACCAGCAGG241 GTGTAGGTGA CCCTTCAGGT TATGGTAAAA GGACAGGAGG AGGAGGAGGG TACCAGCAGG
301 CACCACCAGG GTTTGCTAT GGAGGTGGTG GTGGTGGGTT TGGAGGTGGT GGGCAACAAA301 CACCACCAGG GTTTGCTAT GGAGGTGGTG GTGGTGGGTT TGGAGGTGGT GGGCAACAAA
361 TGGGGATGGT GGGACCGTTA AGCCCTGTTT CATCAGACGG GTTAGGACAT GGACAAGTGG361 TGGGGATGGT GGGACCGTTA AGCCCTGTTT CATCAGACGG GTTAGGACAT GGACAAGTGG
421 ATAACATAGG AAGTCAGTAT GGAGTGGATA GGGAGGGTT AAGGGGAAGG AAAAGAGTAG 481 TGGA GGTC 将 5 ' RACE获得的序列 SEQ ID NO : 9， 与获得的序列 SEQ ID NO : 3拼接， 获得 SEQ ID NO: 10 : 421 ATAACATAGG AAGTCAGTAT GGAGTGGATA GGGAGGGTT AAGGGGAAGG AAAAGAGTAG 481 TGGA GGTC The sequence obtained by 5 ' RACE SEQ ID NO: 9, was spliced with the obtained sequence SEQ ID NO: 3 to obtain SEQ ID NO: 10:
1 GGGGGGGGGG CGGCGGTGAG ATCGCGGCTA GACAACCAAC TTTTGGGGAG ATGACGCTTG 61 AAGATTTCTT GGTCAAGGCA GGTGTGGTTA GAGAACATCC CACTAACCCT AACCCTAACC 1 GGGGGGGGGG CGGCGGTGAG ATCGCGGCTA GACAACCAAC TTTTGGGGAG ATGACGCTTG 61 AAGATTTCTT GGTCAAGGCA GGTGTGGTTA GAGAACATCC CACTAACCCT AACCCTAACC
121 CTAACCCAAA CCCAAACTCA AACCAAAACC AATCTAGTGT TATACCCGCA GCTACGCAGC121 CTAACCCAAA CCCAAACTCA AACCAAAACC AATCTAGTGT TATACCCGCA GCTACGCAGC
181 AGCAGCTCTA TGGTGTGTTT CAAGGAACTG GTGATCCTAC TTTCCCCGGT CAGGCCATGA181 AGCAGCTCTA TGGTGTGTTT CAAGGAACTG GTGATCCTAC TTTCCCCGGT CAGGCCATGA
241 GTGTAGGTGA CCCTTCAGGT TATGGTAAAA GGACAGGAGG AGGAGGAGGG TACCAGCAGG241 GTGTAGGTGA CCCTTCAGGT TATGGTAAAA GGACAGGAGG AGGAGGAGGG TACCAGCAGG
301 CACCACCAGG TGTTTGCTAT GGAGGTGGTG GTGGTGGGTT TGGAGGTGGT GGGCAACAAA 361 TGGGGATGGT GGGACCGTTA AGCCCTGTTT CATCAGACGG GTTAGGACAT GGACAAGTGG301 CACCACCAGG TGTTTGCTAT GGAGGTGGTG GTGGTGGGTT TGGAGGTGGT GGGCAACAAA 361 TGGGGATGGT GGGACCGTTA AGCCCTGTTT CATCAGACGG GTTAGGACAT GGACAAGTGG
421 ATAACATAGG AAGTCAGTAT GGAGTGGATA GGGAGGGTT AAGGGGAAGG AAAAGAGTAG421 ATAACATAGG AAGTCAGTAT GGAGTGGATA GGGAGGGTT AAGGGGAAGG AAAAGAGTAG
481 TGGA GGTCC AGTGGAGAAA GTAGTGGAAA GAAGGGAGAG GCGGATGATC AAGAACTGTG481 TGGA GGTCC AGTGGAGAAA GTAGTGGAAA GAAGGGAGAG GCGGATGATC AAGAACTGTG
541 AGTCTGCTGC AAGGTCTAGA GCAAGAAAGC AGGCGTATAC AGTGGAATTA GAAGCGGAAT541 AGTCTGCTGC AAGGTCTAGA GCAAGAAAGC AGGCGTATAC AGTGGAATTA GAAGCGGAAT
601 TGAACCAGTT GAAAGAAGAG AATGCGCAGC TAAAACATGC ATTGGGGGAG TTGGAGAGAA 661 AGAGGAAGCA ACAGTATTTT GAGACTTTGA AGACAAGAGC ACAACCGAAG T GCCGAAGG601 TGAACCAGTT GAAAGAAGAG AATGCGCAGC TAAAACATGC ATTGGGGGAG TTGGAGAGAA 661 AGAGGAAGCA ACAGTATTTT GAGACTTTGA AGACAAGAGC ACAACCGAAG T GCCGAAGG
721 CGAGGGGGAG ATTGCGGACA TTGATGAGGA ACCCGAGT G TCCGCTCTGA 根据 SEQ ID NO: 10序列检索分析， SEQ ID NO: 10 为 7 )Z/P-¥的全长序列。 721 CGAGGGGGAG ATTGCGGACA TTGATGAGGA ACCCGAGT G TCCGCTCTGA According to the sequence search of SEQ ID NO: 10, SEQ ID NO: 10 is 7) the full length sequence of Z/P-¥.
根据 SEQ ID NO: 10 序列设计一对引物如下： ThbZIP-4F ( SEQ ID NO: 1 1 ) ： A pair of primers were designed according to the sequence of SEQ ID NO: 10 as follows: ThbZIP-4F (SEQ ID NO: 1 1 ):
ATGACGCTTGAAGATTTCTTG ATGACGCTTGAAGATTTCTTG
ThbZIP-4R ( SEQ ID NO: 12 ) ：  ThbZIP-4R ( SEQ ID NO: 12 ) :
TCAGAGCGGACAACTCGGGTCAGAGCGGACAACTCGGG
AP ( SEQ ID NO: 13 ) ： GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT 通过 SEQ ID NO: 11和 SEQ ID NO: 12来克隆 ThbZIP-4全长编码序列。 AP (SEQ ID NO: 13): GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT The ThbZIP-4 full-length coding sequence was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.
提取小盐芥 RNA， 以引物 SEQ ID NO: 13为反转录引物， 获取小盐芥 cDNA .采用 Stratagene的 PfuUltra II Fusion HS DNA Polymerase, 以小盐芥的 cDNA为模板进行 PCR 反应。 50 μΐ PCR反应体系： 5 μΐ lO PfuUltra II reaction Buffer, 0.5 μΐ 25 mM的 dNTP， 2.0 μΐ cDNA, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ的引物 SEQ ID NO: 11 和 SEQ ID NO: 12各 2.0 μ1， 以及 37.5 μΐ的双蒸水。 PCR反应条件： 95 °C预变性 2分钟， 35个循环 （95 °C 变性 25秒， 57 °C退火 25秒， 72 °C 延伸 45秒）， 72 °C 延伸 5分钟。 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 μΐ PCR reaction system: 5 μΐ lO PfuUltra II reaction Buffer, 0.5 μΐ 25 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 each 2.0 11, and 37.5 μΐ 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扩增产物加 A尾： PCR产物补水至 400 μ1， 先用氯仿抽一遍去除蛋白， 吸取上 清加入 3Μ 醋酸钠溶液 40 μ1， 加 2倍的无水乙醇， -20 °C放置 10分钟， 离心， 去上清， 晾干，用 21 μΐ双蒸水溶解。加入 2.5 μΐ Ι ΟχΕχ Buffer, 0.5 μΐ 5 mM的 dATP ， 1.0 l Ex Taq。 反应条件： 70°C反应 30分钟。将得到约 700bp的 DNA片段回收（Omega回收试剂盒）， 连接至 pGEM T-easy载体上（得到 7 )Z/P-¥-pGEM质粒） ， 然后转化 JM109, 随机挑取 6个白色菌落分别接种于含有 50 g/mL氨苄青霉素的 LB 液体培养基中培养， 37°C培养 过夜后加甘油至终浓度 20%， -80°C保存备用。 用引物 SEQ ID NO: 1 1与 SEQ ID NO: 12 进行菌液 PCR扩增（反应体系及反应条件同上），得到 4个阳性克隆，送至英潍捷基（上 海）贸易有限公司测序，序列为 SEQ ID NO: 2，其编码的蛋白的氨基酸序列为 SEQ ID NO: 1 6Z/P-¥蛋白的氨基酸序列 （SEQ ID NO: 1 ) ：  PCR amplification product plus A tail: PCR product hydration to 400 μ1, first remove the protein with chloroform once, add the supernatant to add 3 Μ sodium acetate solution 40 μl, add 2 times of absolute ethanol, -20 ° C for 10 minutes, Centrifuge, remove the supernatant, allow to dry, and dissolve in 21 μl of double distilled water. Add 2.5 μΐ ΟχΕχ ΟχΕχ Buffer, 0.5 μΐ 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. Is 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):
1 MTLEDFLVKA GWREHP NP NPNPNENPNS NQNQSSVIPA ATQQQLYGVF QGTGDPTFPG 1 MTLEDFLVKA GWREHP NP NPNPNENPNS NQNQSSVIPA ATQQQLYGVF QGTGDPTFPG
61 QAMSVGDPSG YGKRTGGGGG YQQAPPGVCY GGGGGGFGGG GQQMOVIVGPL SPVSSDGLGH 61 QAMSVGDPSG YGKRTGGGGG YQQAPPGVCY GGGGGGFGGG GQQMOVIVGPL SPVSSDGLGH
121 GQVDNIGSQY GVDMGGLRGR KRWDGPVEK WER QRR I KNRESAARSR AR QAYTVEL 181 EAELNQLKEE NAQLKHALGE LERKRKQQYF ESLK RAQPK LPKASGRLRT L RNPSCPL 121 GQVDNIGSQY GVDMGGLRGR KRWDGPVEK WER QRR I KNRESAARSR AR QAYTVEL 181 EAELNQLKEE NAQLKHALGE LERKRKQQYF ESLK RAQPK LPKASGRLRT L RNPSCPL
7¾^/^-¥编码基因的核苷酸序列 （SEQ ID NO: 2 ) ： The nucleotide sequence of the 73⁄4^/^-¥ coding gene (SEQ ID NO: 2):
1 ATGACGCTTG AAGATTTCTT GGTCAAGGCA GGTGTGGTTA GAGAACATCC CACTAACCCT 1 ATGACGCTTG AAGATTTCTT GGTCAAGGCA GGTGTGGTTA GAGAACATCC CACTAACCCT
61 AACCCTAACC CTAACCCAAA CCCAAACTCA AACCAAAACC AATCTAGTGT TATACCGGCA  61 AACCCTAACC CTAACCCAAA CCCAAACTCA AACCAAAACC AATCTAGTGT TATACCGGCA
121 GCTAGGCAGC AGCAGCTCTA TGGTGTGTTT CAAGGAACCG GTGATCCTAC T TCCCCGGT 121 GCTAGGCAGC AGCAGCTCTA TGGTGTGTTT CAAGGAACCG GTGATCCTAC T TCCCCGGT
181 CAGGCCATGA GTGTAGGTGA CCCTTCAGGT TATGGTAAAA GGACAGGAGG AGGAGGAGGG 181 CAGGCCATGA GTGTAGGTGA CCCTTCAGGT TATGGTAAAA GGACAGGAGG AGGAGGAGGG
241 TACCAGCAGG CACCACCAGG TGTTTGCTAT GGAGGTGGTG GTGG GGGTT TGGAGGTGGT241 TACCAGCAGG CACCACCAGG TGTTTGCTAT GGAGGTGGTG GTGG GGGTT TGGAGGTGGT
301 GGGCAACAAA GGGGA GGT GGGACCGTTA AGCCCTGTTT CATCAGACGG GTTAGGACAT301 GGGCAACAAA GGGGA GGT GGGACCGTTA AGCCCTGTTT CATCAGACGG GTTAGGACAT
361 GGACAAGTGG ATAACATAGG AAGTCAGTAT GGAGTGGATA TGGGAGGGTT AAGGGGAAGG 361 GGACAAGTGG ATAACATAGG AAGTCAGTAT GGAGTGGATA TGGGAGGGTT AAGGGGAAGG
421 AAAAGAGTAG GGATGGTCC AGTGGAGAAA GTAGTGGAAA GAAGGCAGAG GCGGA GATC 421 AAAAGAGTAG GGATGGTCC AGTGGAGAAA GTAGTGGAAA GAAGGCAGAG GCGGA GATC
481 AAGAACCGTG AGTCTGCTGC AAGGTCTAGA GCAAGAAAGC AGGCGTATAC AGTGGAATTA 481 AAGAACCGTG AGTCTGCTGC AAGGTCTAGA GCAAGAAAGC AGGCGTATAC AGTGGAATTA
541 GAAGGGGAAT GAACCAGTT GAAAGAAGAG AATGCGCAGC TAAAACATGC ATTGGGGGAG541 GAAGGGGAAT GAACCAGTT GAAAGAAGAG AATGCGCAGC TAAAACATGC ATTGGGGGAG
601 TTGGAGAGAA AGAGGAAGCA ACAGTAT TT GAGAGTTTGA AGACAAGAGC ACAACGGAAG601 TTGGAGAGAA AGAGGAAGCA ACAGTAT TT GAGAGTTTGA AGACAAGAGC ACAACGGAAG
661 TTGCGGAAGG GGAGCGGGAG ATTGCGGACA TTGATGAGGA ACCCGAGT G TCCGCTCTGA 实施例 3 因植物表达载体构建 661 TTGCGGAAGG GGAGCGGGAG ATTGCGGACA TTGATGAGGA ACCCGAGT G TCCGCTCTGA Example 3 Construction of plant expression vector
选择植物双元表达载体 pCAMBIA2300 (购自北京鼎国昌盛生物技术有限责任公司） 作为植物表达载体， 用 Pnos启动子替换 ΝΡΤΠ基因含双增强子的 35S启动子， 以降低 ΝΡΤΠ 蛋白在植物中的表达。选择含双增强子的 35S启动子及终止子 Tnos分别作为 ThbZIP-4基因 的启动子和终止子。 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 ΝΡΤΠ gene was replaced with the Pnos promoter to reduce ΝΡΤΠ. 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.
用引物 SEQ ID NO: 14和 SEQ ID NO: 15以植物表达载体 pBI121 (购自北京华夏远 洋科技有限公司） 为模板扩增 Pnos， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 1.0 μΐ pBI121 , 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 14禾 P SEQ ID NO: 15各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反 应条件： 94°C预变性 5分钟， 33个循环（94°C 变性 30秒， 56°C退火 30秒， 72°C 延伸 30 秒）， 72 °C 延伸 10 分钟。 通过 EcoRI、 Bglll 酶切后将所得 PCR 产物连接到 pCAMBIA2300 (Promega, T4连接酶盒)获得 pCAMBIA2300-l。 SEQ ID NO: 14 ：  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. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 14 and P SEQ ID NO: 15 each 2.0 μl, and 31 μΐ Double steamed water. 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: 14:
GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 15：  GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 15:
ATCCAGATCTAGATCCGGTGCAGATTATTTG ATCCAGATCTAGATCCGGTGCAGATTATTTG
SEQ ID NO: 16禾 P SEQ ID NO: 17以 pBI121为模板扩增 Tnos, 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 1.0 μΐ pBI121 , 1.0 μΐ PrimeSTAR 10 μΜ的引物 SEQ ID NO: 16禾口 SEQ ID NO: 17各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5分钟， 33个循环 (94°C 变性 30 秒， 58 °C退火 30秒， 72°C 延伸 30秒）， 72 °C 延伸 10分钟。 通过 Sacl、 EcoRI酶切后 将所得 PCR产物连接到 pCAMBIA2300-1 (Promega T4 连接酶盒)获得 pCAMBIA2300-2。 SEQ ID NO: 16： 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. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ pBI121, 1.0 μΐ PrimeSTAR 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 μ1, and 31 μΐ Double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), 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: 16:
AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 17：  AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 17:
TCAGAATTCCCAGTGAATTCCCGATCTAGTA TCAGAATTCCCAGTGAATTCCCGATCTAGTA
SEQ ID NO: 18和 SEQ ID NO: 19以 pCAMBIA2300质粒为模板扩增拟南芥 35S启动 子。采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 1.0 μΐ稀释 50倍的 pCAMBIA2300质粒， 1.0 μΐ PrimeSTAR、 10 μΜ 的引物 8£0 10 ^^0: 18和 8£0 10 ^«): 19各2.0 ^， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5分钟， 33个循环 (94 °C 变性 30秒， 50°C退火 30秒， 72°C 延伸 30秒；)， 72 °C 延伸 10分钟。 通过 HindIII、 Pstl酶切后将所得 PCR产物连接到 （连接方法同上） pCAMBIA2300-2获得 pCAMBIA2300-3 SEQ ID NO: 18： 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 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ diluted 50-fold pCAMBIA2300 plasmid, 1.0 μΐ PrimeSTAR, 10 μΜ primer 8 £0 10 ^^0: 18 and 8 £0 10 ^«): 19 each of 2.0 ^, and 31 μΐ 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:
ACTAAGCTTATGGTGGAGCACGACACTCT SEQ ID NO: 19：  ACTAAGCTTATGGTGGAGCACGACACTCT SEQ ID NO: 19:
TGACTGCAGAGAGATAGATTTGTAGAGAGAGAC SEQ ID NO: 20和 SEQ ID NO: 21扩增 ThbZIP-4 (模板是实施例 2所获得的阳性 TGACTGCAGAGAGATAGATTTGTAGAGAGAGAC SEQ ID NO: 20 and SEQ ID NO: 21 Amplified ThbZIP-4 (template is positive for Example 2)
ThbZIP-4-pGEM质粒） ， 采用 stratagene的 PfuUltra II Fusion HS DNA Polymerase。 50 μΐ PCR反应体系： 5 μΐ lO PfuUltra II reaction Buffer, 0.5μ1 25 mM的 dNTP， 2.0 μΐ ThbZIP-4-pGEM质粒， 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase、 10 μΜ的引物 SEQ ID NO:20禾 P SEQ ID NO: 21各 2.0 μ1， 以及 37.5 μΐ的双蒸水。 PCR反应条件： 95 °C预变性 2分钟， 35个循环 (95 °C 变性 25秒， 57°C退火 25秒， 72°C 延伸 45秒， 72°C 延伸 5 分钟。通过 Pstl、 Sad酶切后将所得 PCR产物连接（连接方法同上）到 pCAMBIA2300-3， 获得植物表达载体 35S-7 )Z/P-¥-2300。 ThbZIP-4-pGEM plasmid), using stratagene's PfuUltra II Fusion HS DNA Polymerase. 50 μΐ PCR reaction system: 5 μΐ lO PfuUltra II reaction Buffer, 0.5 μl 25 mM dNTP, 2.0 μΐ ThbZIP-4-pGEM plasmid, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase, 10 μΜ primer SEQ ID NO: 20 and P SEQ ID NO: 21 each of 2.0 μl, and 37.5 μΐ 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. 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.
SEQ ID NO: 20：  SEQ ID NO: 20:
AACTGCAGATGACGCTTGAAGATTTCTTG SEQ ID NO: 21：  AACTGCAGATGACGCTTGAAGATTTCTTG SEQ ID NO: 21:
AAGGAGCTC TCAGAGCGGACAACTCGGG 实施例 4 35S-ThbZIP-4-23 表达载体转化农杆菌  AAGGAGCTC TCAGAGCGGACAACTCGGG Example 4 35S-ThbZIP-4-23 expression vector for transformation of Agrobacterium
农杆菌 LBA4404 (购自 Biovector Science Lab, Inc)感受态细胞的制备： 提前 1-2天将 农杆菌 LBA4404在含 50 g/ml利福平和 50 g/ml链霉素的 LB固体培养基上划单斑接种， 28°C培养 1至 2天。 挑取单菌落接种于 5 ml含 50 μ§/ιη1利福平和 50 μ§/ιη1链霉素的 LB液 体培养基中， 28°C下摇动培养过夜 (约 12-16小时)至 OD6(K)值为 0.4， 形成种子菌液。取 5 ml 活化后的菌液 （1 :20的比例） 接种于 100 ml同样浓度抗生素的 LB液体培养基中， 28°C摇 动培养 2-2.5小时至 OD6(K)=0.8。 冰浴菌液 10分钟， 每隔 3分钟摇匀一次， 使细菌均匀进入 休眠状态。 于 4°C下 4000 g离心 10分钟， 弃上清液； 加入 10 ml冰预冷的 10% (v/v)甘油重 悬浮菌体， 4°C下 4000 g离心 10分钟，收集沉淀；用冰预冷的 10% (v/v)甘油重复洗 3-4次； 加入 4 ml冰浴预冷的 10% (v/v)甘油重新悬浮细菌沉淀， 以 40 μΐ/管将其分装， 于 -70°C保存 备用。 Preparation of competent cells of Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc): 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 μ § /ιη1 rifampicin and 50 μ § /ιη1 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. 5 ml of activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium of the same concentration of antibiotics, and cultured at 28 ° C for 2 to 2.5 hours to OD 6 (K) = 0.8. The ice bath liquid was shaken for 10 minutes every 3 minutes to allow the bacteria to enter the dormant state evenly. Centrifuge at 4000 g for 10 minutes at 4 ° C, discard the supernatant; add 10 ml ice-cold 10% (v / v) glycerol resuspended cells, centrifuge at 4000 g for 10 minutes at 4 ° C, collect the precipitate; Ice pre-cooled 10% (v/v) glycerol was washed 3-4 times repeatedly; 4 ml (v/v) glycerol pre-cooled in 4 ml ice bath was added to resuspend the bacterial pellet and dispensed at 40 μΐ/tube. Store at -70 ° C for later use.
转化农杆菌： 在冰上融化感受态细胞， 往 40 μΐ的感受态细胞中加入 1 μΐ实施例 3中所 得的阳性 35S-7M)Zff-¥-2300质粒， 混匀后冰浴约 10分钟。 将所述感受态细胞和质粒 DNA 的混合物用移液枪转移到冰预冷的电击杯中， 轻敲使悬浮液到达底部， 注意不要有气泡。 将电击杯（购自 Bio-Rad)放到电击室的滑道上，推动滑道将电击杯放至电击室基座电极处。 使用 0.1 cm规格的电击杯， MicroPuMUer (购自 Bio-Rad)的程序设置为 "Agr"， 电击一次 。 立即取出电击杯， 加入 28°C预热的 1 ml LB培养基。 快速而轻柔的用移液枪将细胞打匀。 将悬浮液转入 1.5 ml的离心管， 在 28°C下， 225 rpm培养 1小时。 取 100 - 200 μΐ的菌液涂 布于相应的抗性筛选培养基平板上（LB固体培养基，含 50 μ§/ιη1利福平、 50 μ§/ιη1链霉素、 50 μ§/ιη1卡那霉素）， 28°C培养。 筛选阳性转化克隆， 并将其菌液于 -70°C保存备用。 实施例 5利用农杆菌介导的转化法获得转基因拟南芥 Transformation of Agrobacterium: The competent cells were thawed on ice, and 1 μM of the positive 35S-7M)Zff-¥-2300 plasmid obtained in Example 3 was added to 40 μΐ 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. Using a 0.1 cm size electric shock cup, the program of MicroPuMUer (purchased from Bio-Rad) was set to "Agr" and the shock was applied once. Immediately remove the electric shock cup and add 1 ml of LB medium pre-warmed at 28 °C. Quickly and gently spread the cells with a pipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 225 rpm for 1 hour at 28 °C. 100-200 μL of bacterial solution was applied to the corresponding resistant selection medium plate (LB solid medium containing 50 μ § /ιη1 rifampicin, 50 μ § /ιη1 streptomycin, 50 μ § /ιη1 Kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Using Agrobacterium-mediated Transformation to Obtain Transgenic Arabidopsis
待转化植株培养： 拟南芥种子 （哥伦比亚型， 来自美国俄亥俄州立大学的拟南芥生物 资源中心） 播种在泥炭土中， 经 4°C低温处理 3天后， 置于 23 °C、 16小时光照 /8小时黑暗 的培养箱中发芽。 7-10天后移栽到装有泥炭土和蛭石（体积比 3: 1 )的口径为 7.5 cm的塑料 钵中， 每钵栽种 6株， 置于 23 °C， 16小时光照 /8小时黑暗的培养箱中生长。 移栽前每钵 浇营养液 40 ml， 移栽后视土壤湿度及时补充水分。 在生长期间适当浇灌营养液。 按需要每 3-4 周一次 （或者时间更长）。 为了在每个植株上得到较多的花芽， 当大多数植株第一个花 序形成后剪去第一个花序， 解除顶端优势， 促使多个次生花序的同步出现。 当大多数花序 约 1-10 cm高 （剪去第一个花序后 4 - 8天） 时准备浸染。  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. Every 3-4 weeks (or longer) as needed. In order to obtain more flower buds on each plant, when the first inflorescence of most plants is formed, the first inflorescence is cut off, and the apical dominance is removed, prompting the simultaneous appearance of multiple secondary inflorescences. Prepare for dip when most of the inflorescences are about 1-10 cm high (4-8 days after the first inflorescence is cut).
农杆菌的培养： 取出实施例 4中保种的农杆菌阳性转化克隆的菌液活化后， 挑取农杆 菌单菌落接种到 10 mL无菌 LB液体培养基中（含 75 mg/ L利福平、 100 mg/ L链霉素和 100 mg/L卡那霉素）， 28°C恒温下 250转 /分钟振摇过夜培养。 再将所得到的菌液按 1%-2% (v/v) 的比例接种到 200 mL同样含上述抗生素的 LB液体培养基中， 28°C恒温振摇使农杆菌的浓 度达到 OD6(K)=1.8，然后在 4°C下 3000转 /分钟离心 15分钟，弃去上清液后用浸染培养基（该 浸染培养基含有 5.0% (w/v)的蔗糖和 0.05% ( 500 μΙΤί)的 Silwet L-77)重新悬浮农杆菌， 悬浮至 OD6QQ约 0.80。 Culture of 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). K) = 1.8, then centrifuge at 3000 rpm for 15 minutes at 4 ° C, discard the supernatant and use the dip medium (the infusion medium contains 5.0% (w/v) sucrose and 0.05% (500 μΙΤί) Silwet L-77) resuspended Agrobacterium and suspended to an OD 6QQ of about 0.80.
花序的浸染： 将上述含农杆菌的浸染培养基加入大口容器中， 每个口径 9 cm的容器中 加入 200 - 300 mL所述含农杆菌的浸染培养基用于浸染。 将植株倒置， 使地上组织全部浸 没在农杆菌悬浮液中 3 - 5秒， 并要轻轻搅动。 浸润后植株上应该有一层液体膜。 浸染过的 植株放在塑料盘中， 用干净的塑料或保鲜膜覆盖以保湿， 然后放置在弱光或暗处过夜， 注 意小心防止阳光直射植株。 处理后约 12 - 24小时去掉覆盖。 正常培养植株， 植株进一步生 长 3 - 5周， 直至角果变褐变干。 收获种子， 并将种子用离心管在 4 °C下干燥贮存。  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.
转基因种子筛选:配制含 1/4 MS大量元素的水溶液， 加入 0.8 % 琼脂粉， 用微波炉加热 至琼脂完全溶化， 待冷却到 50°C左右， 加入所需量的终浓度为 50 η^·!/1的卡那霉素， 摇匀 后每培养皿中倒入 25 mL ， 置实验台冷却凝固后即可播种。 把称量好的种子倒在一张普通 复印纸上， 用手指轻敲复印纸， 将种子均匀地播种在琼脂胶上， 盖上培养皿盖， 置 4 °C冰 箱冷处理 72小时后， 移至 23 °C、 16小时光照 /8小时黑暗的培养箱中发芽， 定期统计种子 发芽和幼苗生长情况，将抗性幼苗及时移栽到营养土中。移栽后视土壤湿度及时补充水分。 在生长期间适当浇灌营养液。 取生长 20天的拟南芥叶片 0.1 g， 提取 DNA， 用 SEQ ID NO: 11和 SEQ ID NO: 12扩增 ThbZIP-4: ( 50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM 的 dNTP， 2.0 μΐ DNA, 1.0 μΐ Ex Taq、 10 μΜ的引物 SEQ ID NO: 11和 SEQ ID NO: 12 各 2.0 μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5分钟， 33个循环 （94°C变性 45 秒， 57°C退火 45秒， 72°C延伸 1分钟）， 72 °C 延伸 7分钟）， 将 PCR鉴定为阳性的植株进 行编号 （T1A1-T1A12), 并保存。 实施例 6 过表达 ThbZIP-4的转基因拟南芥 T1代植株的耐旱模拟实验及功能鉴定 灭过菌的蛭石用 1/2MS培养基浸透。 T1A1-T1A6及对照拟南芥种子分别播种在蛭石上， 每盆播种 10颗种子， 25 °C、 10小时光培养 /14小时暗培养循环， 每 7天浇一次 1/2MS, 培 养 20天之后， 每盆保留大小较一致的 4-5棵苗， 用于干旱实验。 转基因拟南芥、 对照拟南 芥干旱 14天 （不浇水)， 25 °C、 10小时光培养 /14小时暗培养循环。 T1代转基因植株 （T0 代转基因植株的种子长成的植株）的抗旱性鉴定表明，对照植株都萎蔫严重，而 T1A1、T1 A2、 T1A3、 T1A4、 T1A5、 T1A6 六个株系共 24 (每株系各 4-5棵） 棵拟南芥中 21棵能够存活 并继续生长显现出明显的耐旱性 （参见图 3a和 3b， 以 T1A2为例， T1A1、 T1A3、 T1A4、 TIC 5、 T1A6 的结果与 T1A3类似， 在此未示出）。 实施例 7干旱胁迫后 ABA变化的测定 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 η^·!/ 1 , shake well, pour 25 mL into each dish, set the bench for cooling After solidification, it can be sown. Pour the weighed seeds onto a piece of plain copy paper, tap the copy paper with your fingers, evenly sow the seeds on the agar gel, cover the Petri dish, and cool the chamber at 4 °C for 72 hours, then move to Germinating in a 23 ° C, 16 hour light / 8 hour dark incubator, regular seed germination and seedling growth, and timely transplanting the resistant seedlings into the nutrient soil. After transplanting, the soil moisture is added to the water in time. The nutrient solution is properly watered during the growth period. 0.1 g of Arabidopsis leaves grown for 20 days, DNA was extracted, and ThbZIP-4 was amplified with SEQ ID NO: 11 and SEQ ID NO: 12 (50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ DNA, 1.0 μΐ Ex Taq, 10 μΜ primers SEQ ID NO: 11 and SEQ ID NO: 12 each of 2.0 μl, and 35 μΐ 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 57 °C, extension for 1 minute at 72 °C), extension at 72 °C for 7 minutes), numbering the plants identified as positive by PCR (T1A1-T1A12), And save. Example 6 Drought tolerance simulation experiment and functional identification of transgenic Arabidopsis thaliana T1 plants overexpressing ThbZIP-4. The sterilized vermiculite was soaked in 1/2 MS medium. 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. Transgenic Arabidopsis thaliana, control Arabidopsis thaliana drought for 14 days (without watering), 25 °C, 10 hours light culture / 14 hours dark culture cycle. 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. 21 of the 4-5 Arabidopsis thaliana survived and continued to grow with obvious drought tolerance (see Figures 3a and 3b, with T1A2 as an example, T1A1, T1A3, T1A4, TIC 5, T1A6) Similar to T1A3, not shown here). Example 7 Determination of ABA changes after drought stress
ABA是公认的与逆境胁迫相关的一种植物激素， 可以作为信号分子调控多个逆境诱导 基因的表达， 从而提高植物的抗逆能力。 我们取干旱胁迫 10天和正常生长条件下的转基因 植株 （T1A1、 T1A2、 T1A3、 T1A4、 T1A5、 T1A6) 及对照植株 （CK) 叶片各 0.2 g左右， 用植物激素脱落酸 (ABA)酶联免疫 （ELISA) 试剂盒 (购自上海锐谷生物科技有限公司)测定 ABA含量 （见图 4)。 实验结果表明， 无论干旱处理还是对照条件下， 转基因植株的 ABA含 量均高于对照 （CK1、 CK2), 证明 Γ )Ζ/Ρ-¥基因可以正调控植物内源的 ABA含量。 实施例 8在转录水平上验证 Γ Ζ/Ρ-¥蛋白表达 分别取对照拟南芥植株、耐旱转基因拟南芥 Tl代植株（分别属于 T1A1、T1A2、T1A3、 Tl A4、 Tl A5、 T1A6六个株系）和不耐旱转基因拟南芥 T1代植株的干旱 10天的叶片各 0.05 g， 用植物 RNA提取试剂盒 （Invitrogen) 提取的总 RNA。 用 HITACHI公司的紫外分光光 度计 U-2001测定总 RNA在 260 nm和 280 nm的吸光度值， 计算各个 RNA浓度。 依照 Invitrogen反转录试齐 Ll盒 Superscript III Reverse Transcriptase所示方法进行反转录 (2 μg总 RNA作为模板， 反转录引物 SEQ ID NO: 13 )。 通过 SEQ ID NO: 11和 SEQ ID NO: 12扩增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. We took about 0.2 g of transgenic plants (T1A1, T1A2, T1A3, T1A4, T1A5, T1A6) and control plants (CK) under drought stress for 10 days and normal growth conditions, and ELISA with phytohormone abscisic acid (ABA). The (ELISA) kit (purchased from Shanghai Ruigu Biotechnology Co., Ltd.) was used to determine the ABA content (see Figure 4). The results showed that the ABA content of the transgenic plants was higher than that of the control (CK1, CK2) under drought treatment and control conditions, which proved that the Γ)Ζ/Ρ-¥ gene could positively regulate the endogenous ABA content of plants. Example 8 Verification of Γ Ρ /Ρ-¥ 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. The absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the respective RNA concentrations. Reverse transcription (2 μg 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, 检测 mp-4蛋白相对表达情况。 ThbZIP-4, detects the relative expression of mp-4 protein.
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶， 以反转录的 cDNA为模板进行 PCR 反应。 50 μ1 ΡΟ 反应体系： 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ cDNA, 1.0 l PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 16禾 Ρ SEQ ID NO: 17各 2.0 μ1， 以及 30 μΐ 的双蒸水。 PCR反应条件： 94°C预变性 5分钟， 29个循环 （ 94°C 变性 45秒， 57°C 退火 45秒， 72°C 延伸 1分钟） ， 72°C 延伸 10分钟。  PCR was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and reverse transcribed cDNA as a template. 50 μl ΡΟ Reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 l PrimeSTAR, 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 μl, and 30 μΐ 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.
产物电泳结果如图 5所示： M为 DNA Ladder Marker ( DL2000, TakaRa) ， 1-7耐 旱转基因拟南芥 T1代植株（依次为： T1A1、 T 1A2、 T1A3、 T1A4、 T1A5、 T1A6、 T1A7 )， 8-1 1为非转基因拟南芥对照； 12-16为不耐旱转基因拟南芥 Tl代植株。 图中所示 PCR 产物电泳条带大小与 Γ Ζ/Ρ-¥的大小一致 （约 720 bp ) 。 结果表明， 对照拟南芥没有 ThbZIP-4转录， 耐旱转基因拟南芥 T1代植株中 7 )Z/P-¥的转录较强， 不耐旱转基因 拟南芥 T1代植株转录很弱或者没有转录。  The product electrophoresis results are shown in Figure 5: 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 Γ Ζ / Ρ - ¥ (about 720 bp). The results showed that there was no ThbZIP-4 transcription in Arabidopsis thaliana, and 7) Z/P-¥ transcription was stronger in drought-tolerant transgenic Arabidopsis thaliana T1 plants, and the drought-tolerant transgenic Arabidopsis T1 plants were weakly transcribed or not. Transcription.

Claims

权 利 要 求 书 claims
1. 小盐芥的一种亮氨酸拉链蛋白， 其序列为 SEQ ID N0: 1。 1. A leucine zipper protein from Salina chinensis, its sequence is SEQ ID N0: 1.
2. 编码 SEQ ID NO: 1的亮氨酸拉链蛋白的基因， 优选其序列为 SEQ ID NO : 2。 2. The gene encoding the leucine zipper protein of SEQ ID NO: 1, preferably its sequence is SEQ ID NO: 2.
3. 一种重组表达载体， 其含有权利要求 2所述的基因， 并且所述基因的核苷酸序 列与用于构建所述重组表达载体的基础载体的表达控制序列可操作地连接； 优选地， 所 述基础载体为 pCAMBIA2300。 3. A recombinant expression vector, which contains the gene of claim 2, and the nucleotide sequence of the gene is operably connected to the expression control sequence of the basic vector used to construct the recombinant expression vector; preferably , the basic vector is pCAMBIA2300.
4. 权利要求 3所述的重组表达载体， 其为附图 2所示的 35S-7 )Z/P-¥-2300载体。 4. The recombinant expression vector according to claim 3, which is the 35S-7)Z/P-¥-2300 vector shown in Figure 2.
5. 一种重组细胞， 其含有权利要求 2所述的基因或者权利要求 3或 4所述的重组 表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。 5. A recombinant cell containing the gene of claim 2 or the recombinant expression vector of claim 3 or 4; Preferably, the recombinant cell is a recombinant Agrobacterium cell.
6. 一种改善植物耐旱性的方法， 包括： 将权利要求 2所述的基因或者权利要求 3 或 4所述的重组表达载体导入植物或植物组织并使所述基因表达； 优选地， 所述植物是 拟南芥。 6. A method for improving plant drought tolerance, comprising: introducing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 into a plant or plant tissue and expressing the gene; preferably, The plant in question is Arabidopsis thaliana.
7. 一种制备转基因植物的方法， 包括： 在有效产生植物的条件下培养含有权利要 求 2所述的基因或者权利要求 3或 4所述的重组表达载体的植物或植物组织。 7. A method for preparing transgenic plants, comprising: cultivating plants or plant tissues containing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 under conditions that effectively produce plants.
8. 权利要求 7所述的方法， 其中所述植物是拟南芥。 8. The method of claim 7, wherein the plant is Arabidopsis thaliana.
9. 权利要求 2所述的基因、 权利要求 3或 4所述的重组表达载体或者权利要求 4 所述的重组细胞用于改善植物耐旱性以及用于植物育种的用途。 9. The use of the gene of claim 2, the recombinant expression vector of claim 3 or 4, or the recombinant cell of claim 4 for improving plant drought tolerance and for plant breeding.
10. 权利要求 9所述的用途， 其中所述植物是拟南芥。 10. The use of claim 9, wherein the plant is Arabidopsis thaliana.
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