WO2015024144A1 - 一种棉花锌指蛋白zpt5-1及其编码基因与应用 - Google Patents

一种棉花锌指蛋白zpt5-1及其编码基因与应用 Download PDF

Info

Publication number
WO2015024144A1
WO2015024144A1 PCT/CN2013/000989 CN2013000989W WO2015024144A1 WO 2015024144 A1 WO2015024144 A1 WO 2015024144A1 CN 2013000989 W CN2013000989 W CN 2013000989W WO 2015024144 A1 WO2015024144 A1 WO 2015024144A1
Authority
WO
WIPO (PCT)
Prior art keywords
plant
seq
gene
expression vector
ghzpt5
Prior art date
Application number
PCT/CN2013/000989
Other languages
English (en)
French (fr)
Inventor
崔洪志
梁远金
何云蔚
王婷婷
Original Assignee
创世纪转基因技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 创世纪转基因技术有限公司 filed Critical 创世纪转基因技术有限公司
Priority to CN201380074330.9A priority Critical patent/CN105102472B/zh
Priority to PCT/CN2013/000989 priority patent/WO2015024144A1/zh
Publication of WO2015024144A1 publication Critical patent/WO2015024144A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the present invention relates to plant proteins and their encoding genes and applications, and more particularly to a cotton-derived zinc finger protein ZPT5-1 and its encoding gene, and its use in the cultivation of transgenic plants having improved salt tolerance.
  • Technical Background Salt stress is one of the most important abiotic stress hazards in the world's agricultural production. Salinized soil is usually dominated by sodium, calcium or magnesium salts, which is the main factor affecting plant growth and causing food and economic crops to reduce production. The world's saline-alkali soil covers an area of about 400 million hectares, accounting for one-third of the irrigated farmland.
  • Saline-alkali land is widely distributed in China, and the existing saline-alkali land area is about 0.4 million hectares. With the increase of population in China and the reduction of cultivated land, the development and utilization of saline-alkali resources has extremely important practical significance.
  • the improvement of plant resistance to salt and alkali, drought-tolerant ability and the selection of plant species or strains suitable for growth on saline-alkali land with high economic and ecological value is an economical and effective measure to utilize saline-alkali land.
  • most plants are poorly tolerant to saline and alkali, and can only grow on soils with a sodium chloride content of less than 0.3%. Excess Na + in soil will be plant Normal growth metabolism produces toxic effects. Therefore, how to increase crop yield in a salted environment has become a very important issue in agricultural production worldwide.
  • the salt tolerance of plants is a very complex quantitative trait, and its salt tolerance mechanism involves various levels from plants to organs, tissues, physiology and biochemistry to molecules.
  • scientists from various countries have also done a lot of work for this purpose, and have made a lot of new progress, especially in the use of the higher model plant Arabidopsis to study the salt-tolerant molecular mechanism of plants, which has made breakthroughs in the research in this field ( Zhu JK. 2002. Salt and drought stress singal transduction in plants. Annu. Rev. Plant Biol. 53: 1247-1273; Zhang ZL. 2011.
  • Higher plant cells can sense changes in physicochemical parameters in the external environment through various pathways, thereby transforming extracellular signals into intracellular signals, and finally transmitting stress signals to the nucleus through a series of signal transductions.
  • the transcription factor is activated, and the activated transcription factor acts on the functional gene to initiate the expression of the stress response gene to increase the tolerance of the plant.
  • ZPT5-1 zinc finger protein
  • SSH suppression subtractive hybridization
  • RACE rapid amplification of cDNA ends
  • the first aspect of the invention provides a gene encoding a zinc finger protein ZPT5-1 of cotton (this article is named
  • GhZPT5-l the sequence of which 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 an expression vector, and the nucleotide sequence of the gene
  • the expression control sequence of the expression vector is operably linked; preferably, the vector is the 35S-GhZPT5-l-2300 vector shown in Figure 2.
  • a third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.
  • a fourth aspect of the present invention provides a method for improving salt 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 salt tolerance of a plant and for use in plant breeding Use;
  • the plant is Arabidopsis thaliana.
  • a seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, the amino acid sequence of which is set forth in SEQ ID NO: 1.
  • FIG. 1 is a construction flow of a plant expression vector (35S-GhZPT5-l-2300) of a gene (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector of the gene (35S-GhZPT5-l-2300).
  • Figure 3 shows the results of salt tolerance simulation experiments of transgenic Arabidopsis thaliana plants transgenic with GhZPT5-1 gene (right panel, T ⁇ F2-9) and non-transgenic Arabidopsis plants (left panel, CK) as controls.
  • Figure 4 shows the results of molecular detection of transcriptional levels of genes in transgenic Arabidopsis plants and non-transgenic control plants by reverse transcription PCR.
  • the upper and lower partial bands shown in the figure belong to G ⁇ / ⁇ 5-J and tac t - ⁇ iacii ?- ⁇ as internal parameters, respectively.
  • 1_4 is a non-tolerant non-transgenic control Arabidopsis plant
  • 5-12 is a salt-tolerant T1 transgenic Arabidopsis plant (subsequently belongs to the above T ⁇ F2-4, T ⁇ F2-9, T ⁇ F2_13 and T ⁇ ).
  • the results showed that there was no transcription of GhZPT5-1 in the salt-tolerant non-transgenic control Arabidopsis plants, and the transcription level of GhZPT5-1 in the salt-tolerant transgenic Arabidopsis thaliana 1 ⁇ generation plants was higher, and the salt-tolerant transgenic Arabidopsis plant GhZPT5 was not salt-tolerant.
  • the transcription level of -l is very low.
  • the subtraction subtraction 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 cotton seedlings treated with salt was used as a tester during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a control.
  • the specific steps are as follows:
  • Test materials African cotton (National Cotton Medium Term Bank, obtained by the China Cotton Research Institute, Uniform No.: ZM-06838) Seeded onto a sterilized vermiculite substrate, cultured at 25 ° C, light dark cycle 16 h / 8 h, each 1/2MS liquid medium (containing 9.39 mM KN0 3 , 0. 625 mM KH 2 P0 4 , 10. 3 mM N N0 3 , 0.75 mM MgS0 4 , 1. 5 mM CaCl 2 , 50 ⁇ M KI, 100 ⁇ ⁇ H 3 B0 3 , 100 ⁇ ⁇ MnS0 4 , 30 ⁇ M ZnS0 4 , 1 ⁇ M N3 ⁇ 4Mo0 4 , 0. 1 ⁇ ⁇ CoCl 2 , 100 ⁇ M N3 ⁇ 4 EDTA, 100 ⁇ M FeS0 4 ) Once. It was used for experiments when the seedlings were as long as 25-30 cm.
  • test seedlings were divided into 2 groups of 4 plants each.
  • the first group was a control group, cultured at 25 ° C under light, and placed in 1/2 MS liquid medium.
  • the second group is the processing group, 25. C. Incubate under light, place in 1/2 MS liquid medium supplemented with a final concentration of 200 mM NaCl, and treat for 6 hours. After the treatment, the leaves of the two groups of seedlings should be cut out in time and quickly frozen with liquid nitrogen. Store in a -70 ° C refrigerator.
  • the cotton leaves of the control and the salt-treated group were each 0.5 g, and the total RNA of the cotton leaves was extracted with the plant RNA extraction kit (Invitrogen). Absorbance values of total RNA at 260 nm and 280 nm, 0D 26 , were determined using a HITACHI UV spectrophotometer U-2001. /0D 28 . The ratio of 1. 8-2 ⁇ 0 indicates that the total RNA purity is high. The integrity of total RNA is detected by 1.0% agarose gel electrophoresis. The brightness of the 28S band is about twice that of the 18S band, indicating The integrity of the RNA is good. The mRNA was isolated using Qiagen's Oligotex mRNA purification assay [JC (Purification of poly A+ RNA from total RNA).
  • JC Qiagen's Oligotex mRNA purification assay
  • Suppression subtractive hybridization was performed as indicated by Clontech's PCR-selectTM cDNA Subtraction Kit kit.
  • the Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain a double-stranded cDNA, and 2 ⁇ g of Tester cDNA and 2 ⁇ g of Driver cDNA were used as starting materials for subtractive hybridization.
  • the Tester cDNA and Driver cDNA were digested with Rsa I for 1.5 h 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.
  • E. coli JM109 competent cells purchased from TAKARA
  • ice bath for 30 min heat shock for 60 s
  • ice bath for 2 min then add 250 ⁇ L LB medium (including 1 % Tryptone was purchased from 0X0ID, 0. 5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm.
  • LB medium including 1 % Tryptone was purchased from 0X0ID, 0. 5% Yeast Extract was purchased from 0X0ID, 1% NaCl was purchased from Sinopharm.
  • cultured at 225 r/min for 30 min cultured at 225 r/min for 30 min, and 200 ⁇ L of culture solution after shaking culture was taken.
  • the sequence was SEQ ID NO: 3.
  • the full-length coding gene corresponding to SEQ ID NO: 3 was named as its corresponding protein named ⁇ 5-1.
  • GhZPT5- 1-1 GSP1 SEQ ID NO : 4:
  • GhZPT5-1-2 GSP2 SEQ ID NO: 5:
  • Reagents come with universal primers:
  • the experimental procedure was performed according to the kit instructions (3 'RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • SEQ ID NO: 4 and 3' primer SEQ ID NO: 7 (the AUAP primer provided by the kit), reverse transcription of SEQ ID NO: 6 primer (AP primer brought by the kit) and cotton mRNA.
  • the cDNA was used as a template for the first round of PCR amplification. Specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 1 2. 5 mM dNTP, 2. 0 ⁇ 1 mRNA reverse transcribed cDNA, 1. 0 ⁇ 1 Ex Taq (purchased from TAKARA), 10 ⁇ M of the primers SEQ ID NO: 4 and P SEQ ID NO: 7 each of 2.0 ⁇ l, and 35 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 53 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 50-fold with double distilled water, and then 2.0 ⁇ l was used as a template, and the second round of PCR amplification was carried out with SEQ ID NO: 5 and the 3'-end primer SEQ ID NO: 7, and the specific steps were as follows:
  • PCR reaction system 5 ⁇ 1 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 1 2. 5 mM dNTP, 2. 0 ⁇ 1 diluted first round PCR product, 1. 0 ⁇ 1 Ex Taq, 10 ⁇ ⁇ Primers SEQ ID NO: 5 and SEQ ID NO: 7 each of 2.0 ⁇ l, and 35 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • the second PCR product recovery fragment (Gel Extraction Kit was purchased from OMEGA) was ligated to pGEM-T Easy Vector, transformed into E. coli JM109 (the same method as above), and 8 white colonies were randomly picked up to contain 50 g/mL ampicillin. Incubate in LB liquid medium, incubate at 37 °C overnight, add glycerol to a final concentration of 20%, and store at -80 °C until use. SEQ ID NO: 5 and 3' primer SEQ ID NO: 7 were used for PCR amplification of bacterial cells, and 6 positive clones were obtained, which were sent to Yingjie Jieji (Shanghai) Co., Ltd. for sequencing and sequencing, and 3' cDNA of the gene was obtained. end.
  • TTGTCCCGAC AGTACCGGTG TTGAAGAGAT CAAATAGCAG TCGGAGGGTT GTTTGCTTGG
  • GhZPT5- 1-1 GSP1 SEQ ID NO: 9:
  • GhZPT5- 1-3 GSP SEQ ID NO: 11:
  • the experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • the first round of PCR amplification was carried out using SEQ ID NO: 10 and the universal primer AAP (provided with the kit), and the cDNA obtained by reverse transcription of cotton mRNA (reverse transcription primer SEQ ID NO: 9) was used as a template.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ 1 ⁇ Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ 1 mRNA reverse transcribed cDNA, 1.0 ⁇ 1 Ex Taq (purchased from TAKARA), 10 ⁇ M primer SEQ ID NO : 10 and AAP each with 2.0 ⁇ 1, and 35 ⁇ 1 of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • 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 with SEQ ID NO: 6 and the universal primer AUAP (provided by the kit), and the specific steps were as follows: 50 ⁇ 1 PCR reaction system: 5 ⁇ 1 ⁇ Buffer, 3 ⁇ 1 2.5 mM dNTP, 2.0 ⁇ l diluted first round PCR product, 1.0 ⁇ 1 Ex Taq, 10 ⁇ M primer SEQ ID NO: 11 and AUAP 2.0 ⁇ 1, and 35 ⁇ 1 of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72V for 2 min), extension at 72 °C for 10 min.
  • a band of approximately 650 bp in the second round of PCR product (purchased from OMEGA using the Gel Extraction Kit) was recovered and ligated into the pGEM-T Easy vector, which was then transformed into JM109 competent cells (see above).
  • the transformed bacterial solution was applied to LB solid medium containing 50 ⁇ g/mL ampicillin, 40 g/mL X-gal, and 24 ⁇ g/mL IPTG for screening.
  • AAAATGCAAA AGCACCCTCC TCACGTTTGA CTAAATCTAG ACGTATTTTA TTTAATTATT 61 TCAACACAAG ACGCGTGGGT TTCATTCCAA AACTCATGTT TTCGCTTTGT TGACTCCGCA
  • a pair of primers were designed according to the sequence of SEQ ID NO: 12 as follows:
  • GhZPT5-l F SEQ ID NO: 13:
  • GhZPT5-l R SEQ ID NO: 14:
  • the GhZPT5-1 full-length coding sequence was cloned by SEQ ID NO: 13 and SEQ ID NO: 14.
  • the PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template.
  • 50 ⁇ 1 PCR reaction system 10 ⁇ 1 5 X PS Buffer, 3 ⁇ 1 2 ⁇ 5 mM dNTP, 2. 0 ⁇ 1 cDNA, 1. 0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ ⁇ primer SEQ ID NO: 13 and SEQ ID NO: 14 each of 2.0 ⁇ l, and 30 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • PCR amplification product plus A tail PCR product plus 2.5 times the volume of absolute ethanol, placed at _20 °C for 10 minutes, centrifuged, supernatant, dried, dissolved in 21 ⁇ l of double distilled water. Add 2. 5 ⁇ 1 lO X Ex Buffer, 0.5 ⁇ l 5 mM dATP, 2. 5 ⁇ 1 lO X Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes.
  • a DNA fragment of about 900 bp was recovered (using the Omega recovery kit) and ligated into the pGEM T-easy vector (GhZPT5-l-D was obtained, and the resulting plasmid was transformed into JM109 competent cells (method as above) ), and the transformed bacterial solution was applied to LB solid medium containing 50 ⁇ g/mL ampicillin, 40 g/mL X-gal, 24 g/mL IPTG for screening. 10 white colonies were randomly picked. They were inoculated separately in LB liquid medium containing 50 ⁇ g/mL ampicillin, and cultured overnight at 37 °C, then glycerol was added to a final concentration of 20%, and stored at -80 °C until use.
  • SEQ ID NO: 13 and SEQ ID NO: 14 The PCR amplification of the bacterial liquid was carried out (the reaction system and the reaction conditions were the same as above), and four positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • the sequence is SEQ ID NO: 2, and its coded ZPT5-1
  • the amino acid sequence of the protein is shown in SEQ ID NO: 1.
  • the plant binary expression vector PCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ⁇ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. .
  • the constitutive promoter 35S containing the double enhancer and the terminator Tnos were selected as promoters and terminators of the gene, respectively.
  • the construction process is shown in Figure 1.
  • Primer SEQ ID NO: 15 and SEQ ID NO: 16 were used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ 1 PCR reaction system: 10 ⁇ ⁇ 5XPS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 ⁇ 121, 1.0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ M primers SEQ ID NO: 9 and SEQ ID NO: 10 each of 2.0 ⁇ 1, and 31 ⁇ 1 of double distilled water.
  • PCR reaction conditions pre-variability at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the obtained PCR product was ligated to pCAMBIA2300 (purchased from Promega, T4 ligase cassette) by coR I, chymase to obtain pCAMBIA2300_l.
  • Primers SEQ ID NO: 17 and SEQ ID NO: 18 were used to amplify Tnos using the pBI121 plasmid as a template, using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72V for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was ligated into pCAMBIA2300-1 by digestion with 53 ⁇ 4c I, ⁇ oR I to obtain pCAMBIA2300_2.
  • the CaMV 35S promoter was amplified using the primers SEQ ID NO: 19 and SEQ ID NO: 20 using the pCAMBIA2300 plasmid as a template.
  • PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 ⁇ 1 PCR reaction system: 10 ⁇ ⁇ 5XPS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 pCAMBIA 2300 plasmid DNA, 1.0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ primers SEQ ID NO: 19 and SEQ ID NO : 20 each of 2.0 ⁇ 1, and 31 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was ligated by ⁇ /2dIII, /3 ⁇ 4 ⁇ I (connection method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300_3.
  • GhZPT5-1 was amplified using primers SEQ ID NO: 21 and SEQ ID NO: 22 (template was the positive GhZPT5-l-G plasmid obtained in Example 2), using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ 1 PCR reaction system: 10 ⁇ ⁇ 5XPS Buffer, 3 ⁇ 1 2.5 mM dNTP, 1.0 ⁇ 1 GhZPT5-l-pGEM, 1.0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ M primer SEQ ID NO: 21 and SEQ ID NO: 22 each of 2.0 ⁇ l and 31 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • the resulting PCR product was ligated (ligation method is the same as above) to pCAMBIA2300-3, and the plant expression vector 35S-GhZPT5_l_2300 was obtained (Fig. 2).
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 in LB solid medium containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml streptomycin Single spotted inoculation, cultured at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 ⁇ g/ml rifampicin and 50 ⁇ g/ml streptomycin, and cultured overnight (about 12-16 h) to 0D 6 at 28 °C with shaking. . The value is 0.4, and a seed bacterial liquid is formed.
  • Transformation of Agrobacterium Thaw competent cells on ice, add 1 ⁇ l of the positive 35S-GhAZF2-l-2300 plasmid obtained in Example 3 to 40 ⁇ l of competent cells, mix and heat for about 10 min. . Transfer the mixture of the competent cells after the ice bath and the 35S-GhAZF2-l-2300 plasmid with a micropipette to an ice-cold 0. 1 cm size electric shock cup (purchased from bio-rad), tapping The suspension reaches the bottom, taking care not to have air bubbles. Place the electric shock cup on the slide of the electric shock chamber, and push the slide to place the electric shock cup on the base electrode of the shock chamber.
  • the bacterial solution of the Agrobacterium LBA4404-transformed positive clone containing the 35S-GhAZF2-l-2300 plasmid obtained in Example 4 was inoculated to an LB liquid medium containing 50 ⁇ g/ml kanamycin overnight, and the next morning.
  • the cells were inoculated at 1:50 into a new LB medium (1 L) containing 50 ⁇ g/ml kanamycin and cultured for about 8 hours to the Agrobacterium liquid 0D 6 . . Between 1. 0 and 1.2.
  • Seed disinfection Soak for 10 minutes with 70% ethanol, and occasionally suspend the seeds; then wash them four times with sterile water and occasionally suspend the seeds. Then, the treated seeds were uniformly coated on the surface of 1/2 MS solid screening medium containing 50 ⁇ g/ml kanamycin (a maximum of 1500 seeds were plated in a 150 mm diameter plate), and vernalized at 4 °C. day. The temperature was maintained at 22 ° C, the light intensity was 3500-4000 lx, and the light cycle was 8 h dark / 16 h light, and cultured for 7-10 days. Seeds that can germinate and grow normally are transgenic seeds.
  • Example 8 the transgenic Arabidopsis thaliana and the control Arabidopsis thaliana were each treated without treatment, and the 1/2 MS liquid medium was normally watered; in addition, each of the transgenic Arabidopsis thaliana and the control Arabidopsis thaliana was watered with 150 mM NaCl. 1/2 MS liquid medium, constant temperature 22 ° C, light intensity 3500-4000 lx, 12 hours light culture / 12 hours dark culture cycle, observe the experimental results after 10 days. Identification of salt tolerance of 1 ⁇ generation transgenic plants (T.
  • Example 9 Eight transgenic 1 ⁇ generation plants with good salt tolerance in Example 9 were randomly selected (in order of the above four salt-tolerant strains T ⁇ F2-4, T ⁇ F2-9, T ⁇ F2_13 and T ⁇ F2_15, For each of the two strains, the control plants of Example 6 were randomly selected from 4 plants, and each of the leaves treated with 150 mM NaCl for 14 days was 0.05 g, and total RNA was extracted using a plant RNA extraction kit (Invitrogen). The absorbance values of total RNA at 260 nm and 280 nm were determined by ultraviolet spectrophotometry, and the respective RNA concentrations were calculated. Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (1 total RNA as a template and reverse transcription primer as SEQ ID NO: 14).
  • a tactin-2 (http://www. uniprot.org/uniprot/Q96292) was amplified by primers SEQ ID NO: 23 and SEQ ID NO: 24, and the relative expression of the Actin-2 protein was detected as an internal reference. .
  • TaKaRa's PrimeSTAR HS DNA polymerase to reverse The recorded cDNA was used as a template for PCR reaction. 50 l PCR reaction system: 10 ⁇ 1 5 X PS Buf f er, 3 ⁇ 1 2. 5 mM dNTP, 2. 0 ⁇ 1 cDNA, 1.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 32 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • ZPT5-1 protein The relative expression of ZPT5-1 protein was detected by amplifying GhZPT5-1 by the primers of SEQ ID NO: 13 and P SEQ ID NO: 14.
  • PCR was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and reverse transcribed cDNA as a template.
  • 50 yl PCR reaction system 10 ⁇ 1 5 X PS Buffer, 3 ⁇ 1 2. 5 mM dNTP, 2. 0 ⁇ 1 cDNA, 1 ⁇ 0 ⁇ 1 PrimeSTAR HS DNA polymerase, 10 ⁇ M primer SEQ ID NO : 13 ⁇ SEQ ID NO: 14 each of 2.0 ⁇ l, and 30 ⁇ l of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 32 cycles (denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 10 min.
  • the electrophoresis results of the product are shown in Figure 4, and the size of the bands shown in the figure is consistent with the size of the positive control.
  • the upper and lower partial strips shown in the figure belong to GhZPT5-l and A tactin-2, respectively, with A tac tin-2 ⁇ as the internal reference.
  • 1-4 are non-tolerant non-transgenic control Arabidopsis plants
  • 5-12 are salt-tolerant T1 transgenic Arabidopsis plants (subsequently belong to the above T ⁇ F2-4, T ⁇ F2-9, T ⁇ F2_13 and T ⁇ F2_15 four salt-tolerant strains, 2 strains per strain)
  • 13 is 35S-GhZPT5-l-2300 plasmid PCR positive control
  • 14-17 is transgenic salt-tolerant Arabidopsis plants (subordinate to 2 strains) Department, 2 tests each)
  • 18 is a blank control without template.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

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棉花的锌指蛋白ZPT5-1及其编码基因，以及其在培育耐盐性提高的转基因植物中的应用。

Description

一种棉花锌指蛋白 ZPT5-1及其编码基因与应用
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棉花的锌 指蛋白 ZPT5-1及其编码基因， 以及其在培育耐盐性提高的转基因植物中的应用。 技术背景 盐胁迫是世界农业生产最重要的非生物逆境危害之一， 盐渍土壤通常以钠 盐、钙盐或镁盐为主，成为影响植物生长、导致粮食和经济作物减产的主要因素。 世界上盐碱土的面积约有 4亿公顷， 占灌溉农田的 1/3。盐碱地在中国分布广泛， 现有盐碱地面积约 0. 4亿公顷。 随着我国人口增加， 耕地减少， 盐碱地资源的 开发利用有着极其重要的现实意义。而植物抗盐碱、耐干旱能力的提高和适宜在 盐碱地上生长并具有较高经济和生态价值的植物种或品系的选育，则是利用盐碱 地经济、 有效的措施。 对绝大多数农作物来说， 大多数植物对盐碱、 干旱的耐受 性差， 只能生长在氯化钠含量为 0. 3%以下的土壤上， 土壤中过量的 Na+会对植物 体的正常的生长代谢产生毒害作用。因此如何在盐渍环境下提高作物产量就成为 全世界农业生产中十分重要的问题。
植物的耐盐性是一个十分复杂的数量性状， 其耐盐机制涉及从植株到器官、 组织、 生理生化直至分子的各个水平。各国的科学家也为此做了大量的工作， 并 取得了很多新进展，特别在利用高等模式植物拟南芥来研究植物的耐盐分子机理 方面， 使该领域的研究有了突破性的进展 （Zhu JK. 2002. Salt and drought stress singal transduction in plants. Annu. Rev. Plant Biol. 53： 1247-1273； Zhang ZL. 2011. Arabidopsis Floral Initiator SKB1 Confers High Salt Tolerance by Regulating Transcription and Pre— mRNA Spl icing through Altering Histone H4R3 and Smal l Nuclear Ribonucleoprotein LSM4 Methylation. Plant Cel l, 23 : 396 - 411 ) 。
高等植物细胞可通过多种途径感受外界环境中物化参数的变化，从而将胞外 的信号变为胞内信号， 通过系列的信号传导最后将胁迫信号传递至细胞核内， 激 活转录因子， 而激活转录因子再作用于功能基因， 启动逆境应答基因的表达从而 提高植物的耐逆性。尽管研究者已从不同侧面开展了大量研究， 但由于其机制十 分复杂， 植物抗盐中的许多重要问题仍有待探索。例如， 植物抗盐的关键因子仍 未找到； 植物耐盐的分子机制并不十分清楚。 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 的方法克隆了非洲棉 （ Gossypium herbaceum L. ) 的一个锌指蛋白 （本文命名为 ZPT5-1 )的编码基因， 并测定了其 DNA序列。 并且发现将其导入受体植株并使其 表达后， 可显著改善受体植株的耐盐性， 而且这些性状可稳定遗传。
本发明第一方面提供棉花的一个锌指蛋白 ZPT5-1的编码基因 （本文命名为
GhZPT5-l) , 其序列为 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表达载体而获得的， 并且所述基因的核苷 酸序列与所述表达载体的表达控制序列可操作地连接； 优选地， 所述载体为图 2 所示的 35S-GhZPT5-l-2300载体。
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。
本发明第四方面提供一种改善植物耐盐性的方法， 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。 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。
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盐性以及用于 植物育种的用途； 优选地， 所述植物是拟南芥。
本发明第七方面提供由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 基因的植物表达载体（35S-GhZPT5-l-2300)的构建流程（图 la- lb )。
图 2是 基因的植物表达载体（35S-GhZPT5-l-2300)的质粒图。 图 3是转 GhZPT5-l基因的 1\代转基因拟南芥植株 （右图， T\F2-9 ) 和作为 对照的非转基因拟南芥植株 （左图， CK) 的耐盐模拟实验结果。
图 4 是利用反转录 PCR对 1\代转基因拟南芥植株和非转基因对照植株中 基因在转录水平上的分子检测的验证结果。 图中所示上下两个部分条 带分别属于 G ^/^5-J与 tac t - ^ iacii ?-^作为内参。 1_4为不耐盐的非 转基因对照拟南芥植株， 5-12为耐盐的 T1代转基因拟南芥植株（依次属于上述 T\F2-4、 T\F2-9、 T\F2_13 与 T\F2_15 四个耐盐株系， 每个株系 2 株）， 13 为 35S-GhZPT5-l-2300质粒 PCR阳性对照， 14-17为转基因不耐盐拟南芥植株 （分 属 2个株系， 各检测 2株)， 18为无模板的空白对照。 结果表明， 不耐盐非转基 因对照拟南芥植株中无 GhZPT5-l 的转录，耐盐转基因拟南芥 1\代植株中 GhZPT5-l的转录水平较高，不耐盐转基因拟南芥植株 GhZPT5-l的转录水平很 低。 具体实施方式 以下结合非限制性实施例对本发明进行进一步说明。
以下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、 盐胁迫下棉花 SSH文库构建：
具体方法为：
利用 Clontech公司的 PCR-select™ cDNA Subtraction Kit所示的方法通过 抑制差减杂交方法构建抑制差减文库。在实验过程中以盐处理的棉花幼苗的叶片 的 mRNA 作为样本 （tester ) , 以未处理的棉花幼苗的叶片的 mRNA 作为对照 ( driver )。 具体步骤简述如下：
( 1 ) 供试材料： 非洲棉（国家棉花中期库，获取单位中国棉花研究所，统一编号： ZM-06838 ) 播种到经过灭菌处理的蛭石基质上， 在 25°C、 光暗周期 16h/8h条件下培养， 每 周浇 1/2MS液体培养基 （含 9. 39 mM KN03， 0. 625 mM KH2P04， 10. 3 mM N N03， 0. 75 mM MgS04， 1. 5 mM CaCl2， 50 μ M KI， 100 μ Μ H3B03， 100 μ Μ MnS04， 30 μ M ZnS04, 1 μ M N¾Mo04, 0. 1 μ Μ CoCl2, 100 μ M N¾EDTA， 100 μ M FeS04) 一次。 当苗株长高达 25-30 cm时用于实验。
( 2 ) 材料处理：
将供试幼苗分为 2组， 每组 4株。 第一组为对照组， 在 25°C、 光照下培养， 放置到 1/2 MS液体培养基中。 第二组为处理组， 25。C、 光照下培养， 放置到添 加有终浓度为 200 mM NaCl的 1/2 MS液体培养基中， 处理 6小时， 处理完毕后 及时剪取两组幼苗的叶片， 用液氮迅速冷冻后， 于 -70°C冰箱中保存。
( 3 ) 总 RNA提取：
分别取对照组和盐处理组的棉花叶片各 0. 5g， 用植物 RNA 提取试剂盒 ( Invitrogen)提取棉花叶片的总 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， 0D26。/0D28。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 Ol igotex mRNA 纯化试齐 [J盒（Purification of poly A+ RNA from total RNA)分离 mRNA。
( 4) 抑制差减杂交：
按 Clontech公司的 PCR-select™ cDNA Subtraction Kit试剂盒说明书所示 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 μ g Tester cDNA和 2 μ 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 h， 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。
( 5 ) cDNA差减文库的构建与初步筛选、 克隆、 鉴定 依照 pGEM-T Easy试剂盒 （购自 Promega) 的产品说明书所示方法， 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载体连接， 具体步骤如 下： 用 200 μ ΐ 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 μ 1， 2 Χ Τ4连接酶缓冲液 5 μ 1， pGEM-T Easy载体 1 μ 1， T4 DNA连接酶 1 μ ΐ ， 于 4°C连接过夜。 取 10 连接反应产物， 加入到 100 μ L大肠杆菌 JM109感受态细胞（购自 TAKARA)中，冰浴 30 min、 热休克 60 s、 冰 浴 2 min, 然后加入 250 μ L LB培养液（含 1% Tryptone购自 0X0ID, 0. 5% Yeast Extract购自 0X0ID, 1% NaCl购自国药） 置 37 °C水浴中， 以 225 r/min振荡培 养 30 min, 取 200 μ L振荡培养后的菌液种植于含 50 μ g/mL氨苄青霉素 （购 自北京拜尔迪）、40 μ g/mL X-gal ( 5-溴 -4氯 _3_吲哚 - β _D_半乳糖苷）、 24 μ g/mL IPTG (异丙基 - β -D-硫代吡喃半乳糖苷） （X-gal和 IPTG均购自 TAKARA) 的 LB (同上） 固体培养板上， 37 °C培育 18 h。 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 300个白色菌落 (编号： Gh-S2-001至 Gh-S2-300)。 将 所有白色菌落分别接种于含有 50 μ g/mL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 231个阳性克隆， 对所有阳性克隆在送英潍捷基 （上海） 贸易有限公司测序
( 6 ) 差异克隆的 cDNA测序分析：
将上述 231个差异克隆的 DNA测序结果去除载体和不明确序列及冗余的 cDNA 后， 共得到 203个有效 EST (Unigene)。 实施例 2 非洲棉锌指蛋白基因 GhZPT5-l的克隆
将克隆子 Gh-S2-010的测序结果去掉冗余 DNA后， 序列为 SEQ ID NO : 3， 本文将 SEQ ID NO : 3对应的全长编码基因命名为 其对应的蛋白命名 为 ΖΡΤ5-1。
SEQ ID NO: 3
1 ACAAAACCCA GATTTCATAG TAATCAGATC TGTGAATTAT ACCTGTCAGC TGAGCAATGA
AAAGAGAAAG AGAAAGTGTA GAGATTCAGG GGTTTGACAT TGCTAAATGT CTAATGCTGC 121 TATCTCAGGG CCTCCAAACC AAGTCTAAGG AGCATTCGGT GAACGAGGTT TTCGAGTGCA
181 AAACTTGTCA CCGTTGCTTC CCATCCTTCC AAACATTGGG AGGTCACCGG GCTAGCCACA
241 AGAGACCCAA ACTAATGGGG GACAAACCAA ATGAAACAAT ACAATTCCCT AGTTTATCAA
301 CCAAGCCTAA GACTCATGAG TGCTCCTTTT GCGGCCAAGA GTTTTCAATG GGGCAAGCTT
361 TGGGTGGCCA TATGAGGAGG CATCGAGTTG CCATGAACGA AAGCTTCTCA CCGTTTCCGC
421 TTGTCCCGAC AGT
GhZPT5-l全长编码基因的克隆
根据已经获得的 SEQ ID NO: 3序列， 设计两条特异性引物， 作为 3 ' RACE 的 5 ' 端特异性引物：
GhZPT5- 1—1 GSP1 : SEQ ID NO : 4：
CCAAGCCTAAGACTCATGAG
GhZPT5- 1—2 GSP2: SEQ ID NO : 5：
GGCATCGAGTTGCCATGAAC
试剂自带通用引物：
AP : SEQ ID NO : 6：
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT
AUAP : SEQ ID NO : 7：
GGCCACGCGTCGACTAGTAC
实验步骤按试剂盒说明书操作 （3 ' RACE System for Rapid Ampl ification of cDNA Ends试剂盒购自 Invitrogen公司）。
用 SEQ ID NO : 4与 3 ' 端引物 SEQ ID NO : 7 (试剂盒自带的 AUAP引物）， 以 SEQ ID NO : 6引物 （试剂盒自带的 AP引物） 和棉花 mRNA反转录得到的 cDNA 为模板进行第一轮 PCR扩增。 具体步骤如下：
50 μ 1 PCR反应体系： 5 μ 1 ΙΟ Χ Εχ Buffer, 3 μ 1 2. 5 mM的 dNTP， 2. 0 μ 1 mRNA反转录的 cDNA, 1. 0 μ 1 Ex Taq (购自 TAKARA)、 10 μ M的引物 SEQ ID NO : 4禾 P SEQ ID NO : 7各 2. 0 μ 1， 以及 35 μ 1的双蒸水。 PCR反应条件： 94°C 预变性 5 min， 33个循环（ 94°C 变性 30 s， 53°C退火 30 s， 72 °C 延伸 2 min)， 72 °C 延伸 10 min。 所得的 PCR产物用双蒸水稀释 50倍后取 2. 0 μ 1作为模板， 用 SEQ ID NO: 5与 3' 端引物 SEQ ID NO: 7进行第二轮 PCR扩增， 具体步骤如下：
50 μ 1 PCR反应体系： 5 μ 1 ΙΟ Χ Εχ Buffer, 3 μ 1 2. 5 mM的 dNTP， 2. 0 μ 1稀释的第一轮 PCR产物， 1. 0 μ 1 Ex Taq、 10 μ Μ的引物 SEQ ID NO: 5和 SEQ ID NO: 7各 2. 0 μ 1， 以及 35 μ 1的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 （94°C 变性 30 s， 56°C退火 30 s， 72 °C 延伸 2min)， 72 °C 延 伸 10 min。 第二次 PCR产物回收片段 (Gel Extraction Kit购自 OMEGA) 连接 于 pGEM-T Easy Vector, 转化到大肠杆菌 JM109 (具体方法同上）， 随机挑取 8个 白色菌落于含有 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。 SEQ ID NO: 5与 3' 端引物 SEQ ID NO: 7进行菌液 PCR扩增， 得到 6个阳性克隆， 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 3' 端。
所得的 3' RACE产物克隆子测序获得序列与 SEQ ID NO: 3进行拼接， 获得 ID NO: 8：
ACAAAACCCA GATTTCATAG TAATCAGATC TGTGAATTAT ACCTGTCAGC TGAGCAATGA
AAAGAGAAAG AGAAAGTGTA GAGATTCAGG GGTTTGACAT TGCTAAATGT CTAATGCTGC
TATCTCAGGG CCTCCAAACC AAGTCTAAGG AGCATTCGGT GAACGAGGTT TTCGAGTGCA
AAACTTGTCA CCGTTGCTTC CCATCCTTCC AAACATTGGG AGGTCACCGG GCTAGCCACA
AGAGACCCAA ACTAATGGGG GACAAACCAA ATGAAACAAT ACAATTCCCT AGTTTATCAA
CCAAGCCTAA GACTCATGAG TGCTCCTTTT GCGGCCAAGA GTTTTCAATG GGGCAAGCTT
TGGGTGGCCA TATGAGGAGG CATCGAGTTG CCATGAACGA AAGCTTCTCA CCGTTTCCGC
TTGTCCCGAC AGTACCGGTG TTGAAGAGAT CAAATAGCAG TCGGAGGGTT GTTTGCTTGG
ACTTGAATTT GACTCCTTTG GAGAACGATT TGCAAGTTCT ATTTGGGAAC AAGGCTCCCA
AGGTTGATCT TTGCATTTGA TACAATACCA TCCATTCAAA TGCTTTTTCT TCTTCTTCTT
TTGTGTTCTA TTCTTTAATT TGTTTT
根据已经获得的 SEQ ID NO: 3序列， 设计三条特异性引物， 作为反转录引 物及 5' RACE的 3' 端特异性引物。
GhZPT5- 1—1 GSP1 : SEQ ID NO: 9：
GTTCACCGAATGCTCCTTAGAC GhZPT5-l_2 GSP2: SEQ ID NO: 10：
CAATGTCAAACCCCTGAATCTCTAC
GhZPT5- 1—3 GSP: SEQ ID NO: 11：
GGTATAATTCACAGATCTGATTAC
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）。
用 SEQ ID NO: 10与通用引物 AAP (试剂盒自带）， 以棉花 mRNA反转录所得 的 cDNA (反转录引物 SEQ IDN0:9)为模板进行第一轮 PCR扩增， 具体步骤如下：
50 μ 1 PCR反应体系： 5 μ 1 ΙΟΧΕχ Buffer, 3 μ 1 2.5 mM的 dNTP， 2.0 μ 1 mRNA反转录的 cDNA, 1.0 μ 1 Ex Taq (购自 TAKARA)、 10 μ M的引物 SEQ ID NO: 10和 AAP各 2.0 μ 1， 以及 35 μ 1的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 （94°C 变性 30 s， 55°C退火 30 s， 72 °C 延伸 2min)， 72 °C 延 伸 10 min。
将所得的 PCR产物用双蒸水稀释 50倍后取 2.0 μ 1作为模板，用 SEQ ID NO: 6与通用引物 AUAP (试剂盒自带） 进行第二轮 PCR扩增， 具体步骤如下： 50 μ 1 PCR反应体系： 5 μ 1 ΙΟΧΕχ Buffer, 3 μ 1 2.5 mM的 dNTP， 2.0 μ 1稀释的 第一轮 PCR产物， 1.0 μ 1 Ex Taq、 10 μ M的引物 SEQ ID NO: 11和 AUAP各 2.0 μ 1， 以及 35 μ 1的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环（94°C 变性 30 s， 58°C退火 30 s， 72V 延伸 2 min), 72 °C 延伸 10 min。 回收第二 轮 PCR产物中约为 650 bp大小的条带 （使用 Gel Extraction Kit购自 OMEGA), 并将其连接到 pGEM-T Easy载体， 然后转化到 JM109感受态细胞中（具体方法同 上）， 并将转化后的菌液涂布于含 50 μ g/mL氨苄青霉素、 40 g/mL X-gal, 24 μ g/mL IPTG的 LB固体培养基上进行筛选。 随机挑取 10个白色菌落分别接种于 含有 50 μ g/mL氨苄青霉素的 LB液体培养基中， 37°C培养过夜后加甘油至终浓 度 20%， -80°C保存备用。 使用 SEQ ID NO: 11与通用引物 AUAP进行菌液 PCR扩 增验证 （反应体系及反应条件同上） ， 得到 3个阳性克隆，送英潍捷基 （上海） 贸易有限公司测序，获得该基因的 cDNA的 5' 端。
将所得的 5' RACE产物克隆测序后所得序列与 SEQ ID NO: 3序列拼接， 获 得 SEQ ID NO: 12：
1 AAAATGCAAA AGCACCCTCC TCACGTTTGA CTAAATCTAG ACGTATTTTA TTTAATTATT 61 TCAACACAAG ACGCGTGGGT TTCATTCCAA AACTCATGTT TTCGCTTTGT TGACTCCGCA
121 GACAAATGGG AGTAGTTGTC TTTATGAATA GTCTATTTAT TAGTCAAACT TCTCCGCTGT
181 GAGCATTGAG AGGTGAGAGT TGGTTCCTCC TCTATATAAA ACTCAAACAT GAGCGATGCT
241 TGTCACCATT CACATCAAAA CAAAACCCAG ATTTCATAGT AATCAGATCT GTGAATTATA 301 CCTGTCAGCT GAGCAATGAA AAGAGAAAGA GAAAGTGTAG AGATTCAGGG GTTTGACATT
361 GCTAAATGTC TAATGCTGCT ATCTCAGGGC CTCCAAACCA AGTCTAAGGA GCATTCGGTG
421 AACGAGGTTT TCGAGTGCAA AACTTGTCAC CGTTGCTTCC CATCCTTCCA AACATTGGGA
481 GGTCACCGGG CTAGCCACAA GAGACCCAAA CTAATGGGGG ACAAACCAAA TGAAACAATA
541 CAATTCCCTA GTTTATCAAC CAAGCCTAAG ACTCATGAGT GCTCCTTTTG CGGCCAAGAG 601 TTTTCAATGG GGCAAGCTTT GGGTGGCCAT ATGAGGAGGC ATCGAGTTGC CATGAACGAA
661 AGCTTCTCAC CGTTTCCGCT TGTCCCGACA GTACCGGTGT TGAAGAGATC AAATAGCAGT
721 CGGAGGGTTG TTTGCTTGGA CTTGAATTTG ACTCCTTTGG AGAACGATTT GCAAGTTCTA
781 TTTGGGAACA AGGCTCCCAA GGTTGATCTT TGCATTTGAT ACAATACCAT CCATTCAAAT
841 GCTTTTTCTT CTTCTTCTTT TGTGTTCTAT TCTTTAATTT GTTTT
根据 SEQ ID NO : 12序列设计一对引物如下：
GhZPT5-l F: SEQ ID NO : 13：
ATGAAAAGAG AAAGAGAAAG TGTAGAGATT C
GhZPT5-l R: SEQ ID NO : 14：
TCAAATGCAA AGATCAACCT TG
通过 SEQ ID NO : 13和 SEQ ID NO : 14来克隆 GhZPT5-l全长编码序列。 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶， 以棉花的 cDNA为模板进行 PCR 反应。 50 μ 1 PCR反应体系： 10 μ 1 5 X PS Buffer, 3 μ 1 2· 5 mM的 dNTP， 2. 0 μ 1 cDNA, 1. 0 μ 1 PrimeSTAR HS DNA聚合酶、 10 μ Μ的引物 SEQ ID NO : 13和 SEQ ID NO : 14各 2. 0 μ 1， 以及 30 μ 1的双蒸水。 PCR反应条件： 94°C 预变性 5 min， 33个循环 （94°C 变性 30 s， 58 °C退火 30 s， 72 °C 延伸 2min)， 72 °C 延伸 10 min。
PCR扩增产物加 A尾： PCR产物加 2. 5倍体积的无水乙醇， _20 °C放置 10分 钟， 离心， 去上清， 晾干， 用 21 μ 1双蒸水溶解。 加入 2. 5 μ 1 lO X Ex Buffer, 0. 5 μ 1 5 mM的 dATP ， 2. 5 μ 1 lO X Ex Taq。 反应条件： 70 °C反应 30分钟。 将得到约 900 bp的 DNA片段回收（使用 Omega回收试剂盒）， 并将其连接至 pGEM T-easy载体上 （得到 GhZPT5-l- D ，然后将所得质粒转化到 JM109感受态细 胞中（方法同上）， 并将转化后的菌液涂布于含 50 μ g/mL氨苄青霉素、 40 g/mL X-gal、 24 g/mL IPTG的 LB固体培养基上进行筛选。 随机挑取 10个白色菌落 分别接种于含有 50 μ g/mL氨苄青霉素的 LB 液体培养基中， 37 °C培养过夜后加 甘油至终浓度 20%， -80 °C保存备用。 SEQ ID NO : 13与 SEQ ID NO : 14进行菌液 PCR扩增验证 （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 ZPT5-1蛋白氨基 酸序列如 SEQ ID N0 : 1所示。
ZPT5-1蛋白的氨基酸序列： SEQ ID NO : 1
1 KRERESVE I QGFDIAKCL
2 1 LLSQGLQTKS KEHSVNEVFE
4 1 CKTCHRCFPS FQTLGGHRAS
61 HKRPKL GDK PNETIQFPSL
81 STKPKTHECS FCGQEFS GQ
101 ALGGH RRHR VA NESFS PF
12 1 PLVPTVPVLK RSNSSRRVVC
14 1 LDLNLTPLEN DLQVLFGNKA
161 PKVDLCI *
GhZPT5-l编码基因的核苷酸序列： SEQ ID NO: 2
1 ATGAAAAGAG AAAGAGAAAG TGTAGAGATT CAGGGGTTTG ACATTGCTAA ATGTCTAATG
61 CTGCTATCTC AGGGCCTCCA AACCAAGTCT AAGGAGCATT CGGTGAACGA GGTTTTCGAG
121 TGCAAAACTT GTCACCGTTG CTTCCCATCC TTCCAAACAT TGGGAGGTCA CCGGGCTAGC 181 CACAAGAGAC CCAAACTAAT GGGGGACAAA CCAAATGAAA CAATACAATT CCCTAGTTTA
241 TCAACCAAGC CTAAGACTCA TGAGTGCTCC TTTTGCGGCC AAGAGTTTTC AATGGGGCAA
301 GCTTTGGGTG GCCATATGAG GAGGCATCGA GTTGCCATGA ACGAAAGCTT CTCACCGTTT
361 CCGCTTGTCC CGACAGTACC GGTGTTGAAG AGATCAAATA GCAGTCGGAG GGTTGTTTGC
421 TTGGACTTGA ATTTGACTCC TTTGGAGAAC GATTTGCAAG TTCTATTTGG GAACAAGGCT 481 CCCAAGGTTG ATCTTTGCAT TTGA 实施例 3 基因植物表达载体构建
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。 选择含双增强子的组成型启动子 35S 及终止子 Tnos分别作为 基因的启动子和终止子， 构建流程如图 1所 示。
使用引物 SEQ ID NO: 15和 SEQ ID NO: 16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 μ 1 PCR反应体系： 10 μ ΐ 5XPS Buffer, 3 μ 1 2.5 mM的 dNTP， 1.0 μ 1 ρΒΙ121, 1.0 μ 1 PrimeSTAR HS DNA聚合酶、 10 μ M的引物 SEQ ID NO: 9 和 SEQ ID NO: 10各 2.0 μ 1， 以及 31 μ 1的双蒸水。 PCR反应条件： 94°C预变 性 5 min， 33个循环（94°C 变性 30 s， 56°C退火 30 s， 72 °C 延伸 30 s)， 72 °C 延伸 10 min。通过 coR I、 Π酶切后将所得 PCR产物连接到 pCAMBIA2300 (购 自 Promega, T4 连接酶盒）获得 pCAMBIA2300_l。
SEQ ID NO: 15：
GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 16：
ATCCAGATCTAGATCCGGTGCAGATTATTTG
使用引物 SEQ ID NO: 17和 SEQ ID NO: 18以 pBI121质粒为模板扩增 Tnos， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μ 1 PCR反应体系： 10 μ 1 5XPS Buffer, 3 μ 1 2· 5 mM的 dNTP， 1.0 μ 1 ρΒΙ121, 1.0 μ 1 PrimeSTAR HS DNA 聚合酶、 10 μΜ的引物 SEQ ID NO: 17禾 P SEQ ID NO: 18各 2.0 μ 1， 以及 31 μ 1 的双蒸水。 PCR反应条件： 94°C预变性 5min， 33个循环（94°C 变性 30 s， 58 °C 退火 30 s， 72V 延伸 30 s)， 72 °C 延伸 10 min。 通过 5¾c I、 ^ oR I酶切后 将所得 PCR产物连接到 pCAMBIA2300-l获得 pCAMBIA2300_2。
SEQ ID NO: 17：
AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID NO: 18： TCAGAATTCCGCAGACGCTGCACTTGT
使用引物 SEQ ID NO: 19和 SEQ ID NO: 20以 pCAMBIA2300质粒为模板扩增 CaMV 35S启动子。 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μ 1 PCR反应 体系： 10 μ ΐ 5XPS Buffer, 3 μ 1 2.5 mM的 dNTP， 1.0 μ 1 pCAMBIA2300质 粒 DNA， 1.0 μ 1 PrimeSTAR HS DNA聚合酶、 10 μΜ的引物 SEQ ID NO: 19和 SEQ ID NO: 20各 2.0 μ 1， 以及 31 μ 1的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环 （94°C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 30 s)， 72 °C 延伸 10 min。 通过^ /2dIII、 /¾ί I酶切将将所得 PCR产物连接 （连接方法同上） 到 pCAMBIA2300-2获得 pCAMBIA2300_3。
SEQ ID NO: 19：
ACTAAGCTTATGGTGGAGCACGACACTCTC
SEQ ID NO: 20：
TGACTGCAGAGAGATAGATTTGTAGAGAGAGACTGGTG
使用引物 SEQ ID NO: 21和 SEQ ID NO: 22扩增 GhZPT5-l (模板是实施例 2 所获得阳性 GhZPT5-l- G 质粒）， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μ 1 PCR反应体系： 10 μ ΐ 5XPS Buffer, 3 μ 1 2.5 mM的 dNTP， 1.0 μ 1 GhZPT5-l-pGEM, 1.0 μ 1 PrimeSTAR HS DNA聚合酶、 10 μ M的引物 SEQ ID NO: 21和 SEQ ID NO: 22各 2.0 μ 1， 以及 31 μ 1的双蒸水。 PCR反应条件： 94°C 预变性 5 min， 33个循环 （94°C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 2min)， 72 °C 延伸 10 min。 以 Pst I和 Sac I酶切后将所得 PCR产物连接 （连接方法同 上）到 pCAMBIA2300-3，并经验证获得植物表达载体 35S- GhZPT5_l_2300 (图 2)。
SEQ ID NO: 21：
TGACTGCAG ATGAAAAGAGAAAGAGAAAGTGTAGAGATTC SEQ ID NO: 22：
AAGGAGCTC TCAAATGCAAAGATCAACCTTG 实施例 4 35S-GhZPT5-l-2300表达载体转化农杆菌
农杆菌 LBA4404 (购自 Biovector Science Lab, Inc) 感受态细胞的制备： 将农杆菌 LBA4404在含 50 μ g/ml利福平和 50 μ 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 g/ml利福 平和 50 μ g/ml链霉素的 LB液体培养基中， 28 °C下摇动培养过夜（约 12-16 h) 至 0D6。。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含 50 μ g/ml利福平和 50 μ g/ml链霉素的 LB液体培养基中， 28 °C摇动 培养 2-2. 5 h至 0D6。。=0. 8。 冰浴菌液 10 min, 每隔 3 min摇匀一次， 令细菌均 匀进入休眠状态。 于 4°C下 4000 g离心 10 min, 弃上清液； 加入一定量预冷的 10%甘油重悬浮菌体， 4°C下 4000 g离心 10 min, 收集沉淀； 用冰预冷的 10%甘 油重复洗 3-4次；加入适量冰预冷的 10%甘油重新悬浮细菌沉淀，即制得 LBA4404 感受态细胞。 然后以 40 μ ΐ/管将其分装， 于 -70 °C保存备用。
转化农杆菌： 在冰上融化感受态细胞， 往 40 μ 1的感受态细胞中加入 1 μ 1 的实施例 3中所得的阳性 35S-GhAZF2-l-2300质粒， 混匀后冰浴约 10 min。 将 冰浴后的感受态细胞和 35S-GhAZF2-l-2300 质粒的混合物用微量移液器转移到 冰预冷的 0. 1 cm规格的电击杯 （购自 bio-rad) 中， 轻敲使悬浮液到达底部， 注意不要有气泡。将所述电击杯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。 将 MicroPul ser (购自 bio-rad) 的程序设置为 "Agr " ， 电击 一次 。 立即取出电击杯， 加入 1 ml 28 °C预热的 LB液体培养基。 快速而轻柔的 用微量移液器将细胞打匀。 将悬浮液转入 1. 5 ml的离心管， 28 °C、 225 rpm培 养 1 h。 取 100〜200 μ 1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 g/ml利福平、 50 μ g/ml链霉素、 50 μ g/ml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 用于转化的拟南芥种植
选择吸水性好， 土质松软的蛭石配合营养土 （1 : 1 ) 作为拟南芥种植土壤。 直径 9cm的花盆， 每盆播种 20-30颗拟南芥种子（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）。 播种以后在花盆上罩上薄膜， 给植株的生长 提供一个湿润的环境。 恒温 22 °C、 光照强度 3500-4000 lx、 光照周期为 12 h黑 暗 /12 h光照培养， 每 7天浇灌一次 1/2 MS液体培养基。 培养 30天后， 每盆保 留 4-5棵植株， 光照周期调整为 8 h黑暗 /16 h光照培养。 待大部分植株都抽苔 之后， 在花序基部剪掉整个主苔， 去其顶端优势， 约 1周后在腋芽部位长出 4-6 个新生侧苔， 待侧苔花序形成花蕾并部分开花或形成 1-2个角果时， 便可用于转 化。 实施例 6 拟南芥花浸转化
将实施例 4所得的含 35S-GhAZF2-l-2300质粒的农杆菌 LBA4404转化阳性克 隆的菌液接种至含有 50 μ g/ml卡那霉素的 LB液体培养基中培养过夜，第二天早 上以 1 : 50接种至含有 50 μ g/ml卡那霉素的新的 LB培养基 （1L) 中， 培养约 8 个小时，至农杆菌液 0D6。。在 1. 0到 1. 2之间。 5000rpm室温离心 5分钟，弃上清， 将农杆菌沉淀悬浮于相应体积的浸染培养基里 （1/2 MS; 含 5%蔗糖； 用 K0H调 至 pH5. 7 ; 并含有 200 μ Ι/L的 Si lwet L-77 )， 使 0D6。。在 0. 8左右。 将拟南芥 的上部缓缓、 螺旋式浸入所述含农杆菌的浸染培养基内， 轻轻顺时针晃动， 约 2 分钟， 用透明塑料罩盖严以保持湿度， 放入温室过夜。 24 小时后移去塑料透明 罩， 将水浇透。 浸泡后 2-3周， 保证植株水分充足。 当植株停止开花， 第一个果 荚成熟变黄时， 用纸袋套住， 当纸袋内的所有果荚变黄后， 停止浇水， 1-2周干 燥后收取种子， 进行转化子筛选。 实施例 7 拟南芥转基因阳性转化子的筛选
种子消毒： 先用 70%乙醇浸泡 10分钟， 并不时地使种子悬浮； 然后用无菌 水洗四次，并不时地使种子悬浮。然后，将处理后的种子均匀涂布在含 50 μ g/ml 卡那霉素的 1/2 MS固体筛选培养基表面上（一块 150mm直径的平皿最多播种 1500 棵）， 4°C春化 2天。 恒温 22 °C、 光照强度 3500-4000 lx、 光照周期为 8 h黑暗 /16 h光照条件下， 培养 7-10天。 能够正常萌发并生长的种子即为转基因种子。 所述转基因种子在含 50 μ g/ml卡那霉素的 1/2 MS固体平板上萌发 2周以后， 将能够生长的阳性植株转入土壤继续培养， 并每株剪取 1-2个叶片， 提取 DNA作 为模板， 用引物 SEQ ID N0 : 13和 SEQ ID N0 : 14进行 PCR检测。 去除 PCR阴性 植株， 收集阳性植株的种子， 分别标号为 LF2-1至 T。F2-20并保存。 实施例 8 过表达 GhZPT5-l转基因拟南芥 1\代植株的种植
选择吸水性好， 土质松软的蛭石配合营养土 （1 : 1 ) 作为拟南芥种植土壤。 将 T。F2-1至 T。F2-20每个转化株系播种 2盆， 非转基因对照拟南芥播种 2盆， 每 盆播种 20-30颗种子。播种以后在花盆上罩上薄膜，给植株的生长提供一个湿润 的环境。 恒温 22°C、 光照强度 3500-4000 lx、 光照周期为 12 h黑暗 /12 h光照 培养， 每 7天浇灌一次 1/2 MS液体培养基。 培养 25天后， 剪取转基因拟南芥株 系的叶片并提取其基因组 DNA作为模板， 用引物 SEQ ID NO : 13和 SEQ ID NO : 14 进行 PCR检测。 去除 PCR阴性植株， 保留 7-8阳性棵苗， 继续培养 10天后， 每 盆保留大小较一致的 4-5棵转基因拟南芥或非转基因对照拟南芥苗进行耐盐实 验。 实施例 9 过表达 GhZPT5-l转基因拟南芥 1\代植株的耐盐实验
取实施例 8中转基因拟南芥、对照拟南芥各一盆不作处理， 正常浇灌 1/2 MS 液体培养基； 另外将转基因拟南芥、 对照拟南芥各一盆浇灌含有 150 mM NaCl的 1/2 MS液体培养基， 恒温 22°C、 光照强度 3500-4000 lx、 12小时光培养 /12 小时暗培养循环， 10天后观察实验结果。 1\代转基因植株（T。代转基因植株的种 子长成的植株） 的耐盐性鉴定表明， T\F2-4、 T\F2-9、 T\F2_13与 T\F2_15四个株 系表现出显著的耐盐性（见图 3， 以 T\F2-9为例， 余者结果与之类似， 在此未图 示）。 实施例 10 在转录水平上验证 ZPT5-1蛋白表达
将实施例 9中耐盐性状良好的转基因 1\代植株中随机选取 8棵 （依次属 于上述 T\F2-4、 T\F2-9、 T\F2_13与 T\F2_15四个耐盐株系， 每个株系 2株） ， 实施例 6中对照植株随机选取 4棵， 各剪取 150 mM NaCl处理 14天的叶片 0. 05 g， 用植物 RNA提取试剂盒 （Invitrogen ) 提取总 RNA。 紫外分光光度测定总 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 ( 1 总 RNA作为模板， 反转录引物为 SEQ ID NO : 14 ) 。
通过 SEQ ID NO : 23 和 SEQ ID NO : 24 引物扩增 A tactin-2 (http : //www. uniprot. org/uniprot/Q96292 ) ， 检测看家基因 Actin-2蛋白的 相对表达情况， 作为内参。 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 l PCR反应体系： 10 μ 1 5 X PS Buf f er， 3 μ 1 2. 5 mM的 dNTP， 2. 0 μ 1 cDNA, 1. 0 μ 1 PrimeSTAR HS DNA聚合酶、 10 μ M的引物 SEQ ID NO : 23和 SEQ ID NO : 24各 2. 0 μ 1， 以及 30 μ 1的双 蒸水。 PCR反应条件： 94°C预变性 5 min， 32个循环 （94°C 变性 30 s， 58 °C 退火 30 s， 72 °C 延伸 lmin ) ， 72 °C 延伸 10 min。
SEQ ID NO : 23：
GCTGATGATATTCAACCAATCGTG
SEQ ID NO : 24：
CTCTGCTGTTGTGGTGAACATG
通过 SEQ ID NO : 13禾 P SEQ ID NO : 14引物扩增 GhZPT5-l, 检测 ZPT5-1 蛋白相对表达情况。
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 y l PCR反应体系： 10 μ 1 5 X PS Buffer, 3 μ 1 2. 5 mM的 dNTP， 2. 0 μ 1 cDNA, 1· 0 μ 1 PrimeSTAR HS DNA聚合酶、 10 μ M的引物 SEQ ID NO : 13 禾 P SEQ ID NO : 14各 2. 0 μ 1， 以及 30 μ 1的双蒸水。 PCR反应条件： 94°C预 变性 5 min， 32个循环（94°C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 lmin)， 72 °C 延伸 10 min。
产物电泳结果如图 4所示，图中所示条带大小与阳性对照的大小一致。 图 中所示上下两个部分条带分别属于 GhZPT5-l与 A tactin-2, 以 A tac tin-2 ^ 为内参。 1-4为不耐盐的非转基因对照拟南芥植株， 5-12为耐盐的 T1代转基因 拟南芥植株（依次属于上述 T\F2-4、 T\F2-9、 T\F2_13与 T\F2_15四个耐盐株系， 每个株系 2株） ， 13为 35S-GhZPT5-l-2300质粒 PCR阳性对照， 14-17为转基 因不耐盐拟南芥植株（分属 2个株系， 各检测 2株）， 18为无模板的空白对照。 结果表明， 不耐盐非转基因对照拟南芥植株中无 GhZPT5-l 的转录，耐盐转基 因拟南芥 1\代植株中 GhZPT5-l 的转录水平较高， 不耐盐转基因拟南芥植株 GhZPT5-l的转录水平很低。

Claims

权 利 要 求 书
1. 棉花的一个锌指蛋白， 其序列为 SEQ ID NO: 1。
2. 编码权利要求 1的锌指蛋白的基因， 其序列为 SEQ ID NO: 2。
3. 一种重组表达载体， 其是通过将权利要求 2所述的基因插入到一种表达 载体而获得的，并且所述基因的核苷酸序列与所述表达载体的表达控制序列可操 作地连接； 优选地， 所述表达载体是 pCAMBIA2300。
4. 权利要求 3所述的载体， 其为图 2所示的 35S-GhZPT5-l-2300载体。
5. —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。
6. 一种改善植物耐盐性的方法， 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。
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。
8. 权利要求 7所述的方法， 其中所述植物是拟南芥。
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 5所述的重组细胞用于改善植物耐盐性以及用于植物育种的用途。
10. 权利要求 9所述的用途， 其中所述植物是拟南芥。
PCT/CN2013/000989 2013-08-22 2013-08-22 一种棉花锌指蛋白zpt5-1及其编码基因与应用 WO2015024144A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201380074330.9A CN105102472B (zh) 2013-08-22 2013-08-22 一种棉花锌指蛋白zpt5-1及其编码基因与应用
PCT/CN2013/000989 WO2015024144A1 (zh) 2013-08-22 2013-08-22 一种棉花锌指蛋白zpt5-1及其编码基因与应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2013/000989 WO2015024144A1 (zh) 2013-08-22 2013-08-22 一种棉花锌指蛋白zpt5-1及其编码基因与应用

Publications (1)

Publication Number Publication Date
WO2015024144A1 true WO2015024144A1 (zh) 2015-02-26

Family

ID=52482902

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/000989 WO2015024144A1 (zh) 2013-08-22 2013-08-22 一种棉花锌指蛋白zpt5-1及其编码基因与应用

Country Status (2)

Country Link
CN (1) CN105102472B (zh)
WO (1) WO2015024144A1 (zh)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003007699A2 (en) * 2001-06-22 2003-01-30 Syngenta Participations Ag Transcription factors of cereals
CN102181454A (zh) * 2011-04-25 2011-09-14 吉林农业大学 大豆锌指蛋白基因sctf-1及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009114733A2 (en) * 2008-03-13 2009-09-17 Ceres, Inc. Nucleotide sequences and corresponding polypeptides conferring modulated growth rate and biomass in plants grown in saline and oxidative conditions
CN1818065A (zh) * 2005-10-14 2006-08-16 山东农业大学 棉花GhZFP1基因序列及其克隆与应用
JP5758376B2 (ja) * 2009-04-08 2015-08-05 シャンハイ インスティチューツ フォー バイオロジカル サイエンセズ， シーエーエスＳｈａｎｇｈａｉ Ｉｎｓｔｉｔｕｔｅｓ Ｆｏｒ Ｂｉｏｌｏｇｉｃａｌ Ｓｃｉｅｎｃｅｓ， Ｃａｓ イネのジンクフィンガータンパク質転写因子ｄｓｔならびに渇水および塩の耐性を調節するためのその使用

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003007699A2 (en) * 2001-06-22 2003-01-30 Syngenta Participations Ag Transcription factors of cereals
CN102181454A (zh) * 2011-04-25 2011-09-14 吉林农业大学 大豆锌指蛋白基因sctf-1及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
TIAN, LUMING ET AL.: "Advances of Plant Zinc Finger Proteins Involved in Abiotic Stress", BIOTECHNOLOGY BULLETIN, 26 December 2005 (2005-12-26), pages 12 - 16 *
WANG, DONG ET AL.: "Cloning and Characterization of cDNA Encoding Cotton STZ-like Protein", JOURNAL OF FUDAN UNIVERSITY ( NATURAL SCIENCE, vol. 41, no. 01, 25 February 2002 (2002-02-25), pages 42 - 46 *
YOUNG, N.D. ET AL.: "Zinc finger protein", EMBL: G7L0M5_MEDTR, 25 January 2012 (2012-01-25) *

Also Published As

Publication number Publication date
CN105102472A (zh) 2015-11-25
CN105102472B (zh) 2018-05-29

Similar Documents

Publication Publication Date Title
CN101942449A (zh) 提高植物非生物胁迫耐受性和/或生物量的方法及用该方法所产生的植物
WO2013185258A1 (zh) 棉花hkt蛋白及其编码基因与应用
WO2015042742A1 (zh) 一种棉花钙调磷酸酶b类似蛋白cbl-3及其编码基因与应用
WO2015024143A1 (zh) 一种棉花锌指蛋白zat10-1及其编码基因与应用
WO2015024142A1 (zh) 一种棉花锌指蛋白azf2-1及其编码基因与应用
WO2014205597A1 (zh) 一种棉花高亲和钾离子转运蛋白hkt2及其编码基因与应用
WO2015042734A1 (zh) 一种木榄液泡焦磷酸酶vp2及其编码基因与应用
WO2014172826A1 (zh) 一种小盐芥液泡膜焦磷酸酶vp1及其编码基因与应用
WO2015024145A1 (zh) 一种棉花锌指蛋白zpt5-3及其编码基因与应用
WO2015024147A1 (zh) 一种棉花锌指蛋白zpt5-5及其编码基因与应用
WO2015042740A1 (zh) 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
WO2014063270A1 (zh) 棉花离子通道类蛋白及其编码基因和应用
WO2014205598A1 (zh) 一种小盐芥高亲和钾离子转运蛋白hkt1及其编码基因与应用
WO2015024144A1 (zh) 一种棉花锌指蛋白zpt5-1及其编码基因与应用
WO2015058322A1 (zh) 一种木榄钼辅因子硫化酶mcsu及其编码基因与应用
WO2014172825A1 (zh) 一种小盐芥液泡膜钠氢反向转运蛋白nhx2及其编码基因与应用
WO2015058323A1 (zh) 一种木榄甜菜碱脱氢酶badh及其编码基因与应用
WO2015024146A1 (zh) 一种棉花锌指蛋白zpt5-4及其编码基因与应用
WO2014205595A1 (zh) 一种棉花ATP水解酶ATPase-1及其编码基因与应用
WO2015042738A1 (zh) 一种小盐芥同源异型-亮氨酸拉链蛋白HDbZIP-3及其编码基因与应用
WO2015042749A1 (zh) 一种小盐芥脱水素蛋白dh5及其编码基因与应用
WO2014107863A1 (zh) 一种棉花同源异型-亮氨酸拉链蛋白HDbZIP-1及其编码基因与应用
WO2014205599A1 (zh) 一种小盐芥液泡膜钠氢反向转运蛋白nhx3及其编码基因与应用
WO2015042731A1 (zh) 一种秋茄钠氢转运蛋白nha1及其编码基因与应用
WO2015042733A1 (zh) 一种木榄蛋白激酶cipk1及其编码基因与应用

Legal Events

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

Ref document number: 201380074330.9

Country of ref document: CN

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

Ref document number: 13891801

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13891801

Country of ref document: EP

Kind code of ref document: A1