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WO2014082280A1 - 一种棉花锌指蛋白（Czf4）及其编码基因与应用 - Google Patents

一种棉花锌指蛋白（Czf4）及其编码基因与应用 Download PDF

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Publication number
WO2014082280A1
WO2014082280A1 PCT/CN2012/085620 CN2012085620W WO2014082280A1 WO 2014082280 A1 WO2014082280 A1 WO 2014082280A1 CN 2012085620 W CN2012085620 W CN 2012085620W WO 2014082280 A1 WO2014082280 A1 WO 2014082280A1
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plant
seq
gene
expression vector
ghczf4
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French (fr)
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王建胜
王君丹
林余
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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BIOCENTURY TRANSGENE (CHINA) Co Ltd
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Priority to CN201280077196.3A priority Critical patent/CN104837998B/zh
Priority to PCT/CN2012/085620 priority patent/WO2014082280A1/zh
Publication of WO2014082280A1 publication Critical patent/WO2014082280A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the present invention relates to plant proteins and their coding genes and applications, and more particularly to a cotton-derived zinc finger protein (Czf4) and a gene encoding the same, and its use in breeding transgenic plants having improved drought resistance.
  • Czf4 cotton-derived zinc finger protein
  • 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 areas account for about 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 350-40 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the most severe areas in China, and spring droughts frequently reach 10 years.
  • genes and their expression products can be divided into three categories: (1) genes and products involved in signal cascade amplification systems and transcriptional control; (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) Proteins associated with the uptake and transport of water and ions.
  • genes and products involved in signal cascade amplification systems and transcriptional control (2) genes and their expression products that directly contribute to the protection of biofilms and proteins; ) Proteins associated with the uptake and transport of water and ions.
  • the system has a further understanding (Liu Q. 1998.
  • Two transcription factors, DREB1 and DREB2 with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought- and low temperature-responsive gene expression, respectively, in Arabidopsis.
  • the first aspect of the invention provides a gene encoding a zinc finger protein Czf4 of cotton (designated herein as
  • GhCzf4 the sequence of which is SEQ ID NO: 2.
  • a second aspect of the invention provides a recombinant expression vector comprising the gene of the first aspect of the invention, and the nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector; preferably, The vector is the rd29A-Gh Czf4-2300 vector shown in Fig. 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 drought resistance 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 expressing the gene;
  • the plant is tobacco.
  • a fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or plant tissue 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
  • the plant is tobacco.
  • 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 resistance of a plant and for use in plant breeding Use;
  • the plant is tobacco.
  • the seventh aspect of the present invention provides the gene-encoded protein of 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 (rd29A-GhCzf4-2300;> of GhCzf4.
  • Figure 2 is a plasmid map of the plant expression vector Crd29A-GhCzf4-2300 of GhCzf4.
  • Figure 3 shows the drought resistance growth of control tobacco and transgenic tobacco
  • CK left: control tobacco
  • T1C1 middle
  • T1C2 right: transgenic tobacco lines.
  • Figure 4 shows the results of verification of transcriptional levels of T1 transgenic tobacco plants resistant to drought and drought.
  • M is Marker
  • 1-8 is a drought-tolerant T1 transgenic tobacco plant
  • 9-12 is a drought-tolerant T1 transgenic tobacco plant.
  • African cotton (National Cotton Medium Term Bank, obtained by the China Cotton Research Institute, Uniform No.: ZM-06838) was sown on sterilized vermiculite at 25 ° C, photoperiod 16 h / 8 h (light intensity 2000 - 3000 Lx) conditions Under the culture, 1/2MS medium (containing 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 ⁇ , 100 ⁇ 3 ⁇ 3 , 100 MMnSO 4 per week) , 30 ⁇ ZnS0 4 , 1 ⁇ 2 ⁇ 0 4 , 0.1 ⁇ CoCl 2 , 100 ⁇ Na 2 EDTA, 100 MFeSO 4 ). It was used for experiments when the seedlings were as high as 25-30 cm.
  • 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 16 h / 8 h (light intensity 2000 - 3000 Lx), and was normally watered.
  • the second group was the drought treatment group, cultured at 25 °C, photoperiod 16h/8h (light intensity 2000-3000 Lx), stopped watering, treated for 10 days, and cut 1/3 of the top of the two seedlings in time after treatment.
  • the leaves, which were rapidly frozen with liquid nitrogen, were stored in a -70 ° C refrigerator.
  • RNA extraction kit purchased from Invitrogen
  • the absorbance of total RNA at 260 nm and 280 nm was determined by HITACHI's UV spectrophotometer U-2001.
  • the OD 26Q / OD 28Q ratio was 1.8-2.0, indicating a high total RNA purity with 1.0% agarose gel.
  • the total RNA was detected by electrophoresis, and the 28S band was about twice as bright as the 18S band, indicating good RNA integrity.
  • mRNA was isolated using Qiagen's purification of polyA+ RNA from total RNA.
  • Two tester cDNAs with different adaptors were mixed with excess Driver 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 freshly denatured Driver cDNA, and the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.
  • the second PCR product of the 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 specific steps are as follows: The following components were sequentially added using a 200 ⁇ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 ⁇ 1, 2> ⁇ 4 ligase buffer 5 ⁇ l, pGEM-T Easy vector 1 ⁇ l, T4 DNA ligase 1 ⁇ l, ligated overnight at 4 °C. 10 ⁇ of the reaction product was added to 100 ⁇ of competent E.
  • coli JM109 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 seconds, ice bath for 2 min, and additional 250 ⁇ LB medium (containing 1%). Tryptone (purchased 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 min, and inoculated with 200 ⁇ M bacteria in 50 g/ml Ampicillin was cultured on LB (ibid.)/X-gal/IPTG (X-gal/IPTG purchased from TAKARA) plates at 37 ° C for 18 h.
  • PCR primers Primer 1 and Primer 2R (Clontech's PCR-select TM cDNA Subtraction Kit kit) were used to carry out PCR amplification of the broth, and 452 positive clones were obtained, and then all the positive clones were sent to the British Shanghai) Trading Co., Ltd. sequencing.
  • the sequence was SEQ ID No: 3, and sequence analysis indicated that the protein encoded by the sequence belonged to the zinc finger protein.
  • the full-length coding gene corresponding to the sequence of SEQ ID No: 3 was named GhCzf4, and its corresponding protein was named Czf4.
  • GhCzf'i GSP1 SEQ ID NO: 4:
  • GhCzf l GSP2 SEQ ID K0: 5:
  • the experimental procedure was performed according to the kit instructions (3' 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: 4 and the 3' primer AUAP (provided with the kit), using mRNA reverse transcribed cDNA as a template. Specific steps are as follows:
  • PCR reaction system 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcribed cDNA, 1.0 ⁇ Ex Taq (purchased from TAKARA), 10 ⁇ primer SEQ ID NO: 4 and AUAP each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (94 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min), 72 °C for 10 min.
  • the resulting PCR product was diluted 50-fold with double distilled water and 2.0 ⁇ L was used as a template, using SEQ ID NOS: 5 and 3'
  • the primer AUAP is used for the second round of PCR amplification.
  • the specific steps are as follows:
  • PCR reaction 5 ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ diluted first round PCR product, 1.0 ⁇ 1 ⁇ ⁇ 3 ⁇ , 10 ⁇ primers SEQ ID NO: 5 and AUAP each 2.0 ⁇ 1, and 35 ⁇ 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 1 min), extension at 72 °C for 10 min.
  • a band of approximately 400 bp in the second PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into pGEM-T Easy Vector, and then transformed into E. coli JM109 (specific method as above).
  • Ten white colonies were randomly picked and inoculated in LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and -80 was stored for use.
  • SEQ ID NO: 5 and 3' primer AUAP to carry out bacterial PCR amplification, 8 positive gram drops were obtained, and 4 positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing. The 3, end of the cDNA of the gene.
  • GhCzf GSP3 SEQ ID NO: 6:
  • GhCzf l GSP4 SEQ ID NO: 7:
  • GhCzf4 GSP5 SEQ ID NO: 8:
  • the first round of PCR amplification was carried out using SEQ ID NO: 7 and 5' universal primer AAP (provided with the kit), and cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 6) was used as a template for the first round of PCR amplification.
  • SEQ ID NO: 7 and 5' universal primer AAP provided with the kit
  • cDNA reverse transcription cDNA reverse transcription primer SEQ ID NO: 6
  • PCR reaction system 5 ⁇ ⁇ Buffer > 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcribed cDNA, 1.0 ⁇ Ex Taq (purchased from TAKARA), 10 ⁇ primers SEQ ID NO: 7 and AAP 2.0 1 ⁇ 1, and 35 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 33 cycles (94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min), 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: 8 and 3 ' primer AUAP.
  • the specific steps are as follows:
  • the GhCzf4 full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11.
  • PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template.
  • 50 ⁇ PCR reaction system 10 ⁇ 5xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA replacement page (Article 26) 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO. 10 and SEQ ID NO. 11 each of 2.0 ⁇ l, and 30 ⁇ 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 1 min), extension at 72 ° C for 10 min.
  • PCR amplification product plus A tail 2.5 times the volume of absolute ethanol was added to the PCR product, placed at -20 ° C for 10 minutes, centrifuged, the supernatant was removed, air-dried, and then dissolved in 21 ⁇ l of double distilled water. Then, 2.5 ⁇ lOxExBuflFer, 0.5 ⁇ 5 mM dATP, 2.5 ⁇ ⁇ Taq were added thereto. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained DNA fragment of about 450 bp was recovered (Omega recovery kit), and ligated to the pGEM T-easy vector, and then transformed into JM109 (method as above).
  • 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 of the ⁇ gene containing the double enhancer was replaced with the Pnos promoter.
  • Primer SEQ ID NO: 12 and SEQ ID NO: 13 were used to amplify Pnos using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and TaKaRa's PrimeSTAR HS DNA polymerase was used.
  • 50 l PCR reaction system 10 ⁇ 5 PS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ ⁇ 12 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 12 and SEQ ID NO: 13 each 2.0 ⁇ 1, and 31 ⁇ l double Steamed water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 33 cycles (94 C denaturation 30 s, 56 ° C annealing 30 s, 72 extension 30 s), 72 ° C extension 10 min.
  • the resulting PCR product was ligated by EcoRI and Bglll (promega, T4 ligase cassette) to pCAMBIA2300 to obtain pCAMBIA2300-1.
  • ATCCAGATCTAGATCCGGTGCAGATTATTTG Tnos was amplified using primers SEQ ID NO: 14 and SEQ ID NO: 15 with pBI121 as a template, and PrimeSTAR HS DNA of TaKaRa was used to polymerize ruthenium.
  • 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 Sacl and EcoRI (promega T4 ligase cassette) to pCAMBIA2300-1 to obtain pCAMBIA2300-2.
  • TCAi3 ⁇ 44J7 uses the primers SEQ ID NO: 16 and SEQ ID NO: 17 to amplify the Arabidopsis thaliana rd29A promoter with Arabidopsis thaliana (Columbia type, available from www.arabidopsis.org) as a template (see Zeng J., et Al. 2002, Preparation of total DNA from "recalcitrant plant taxa", Acta Bot. Sin., 44(6): Method in 694-697 for extraction of Arabidopsis DNA).
  • 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), 72 "C extension for 10 min. Digestion by Pstl, Sacl The resulting PCR product was ligated (ligation method as above) to pCAMBIA2300-3 to obtain a plant expression vector rd29A-GhCzf4-2300.
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 was plated on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin 1-2 days in advance Single spot vaccination, 28 ⁇ culture for 1 to 2 days. A single colony was picked and inoculated into 5 ml of LB liquid medium containing 50 ⁇ / ⁇ 1 rifampicin and 50 ⁇ m ⁇ ⁇ streptomycin, and cultured overnight (about 12-16 h) to OD 6fl under shaking at 28 Torr . A value of 0.4 forms a seed broth.
  • Replacement page 10 min, shake every 3 minutes to allow the bacteria to go to sleep evenly. Centrifuge at 4000 g for 10 min at 4 ° C, discard the supernatant; add a certain amount of ice-cold 10% glycerol to resuspend the cells, centrifuge at 4000 g for 10 min at 4 ° C, collect the precipitate; pre-cool with ice 10 % glycerol was washed 3-4 times repeatedly; then the bacterial pellet was resuspended by adding 10% glycerol pre-cooled with an appropriate amount of ice bath, dispensed at 40 ⁇ /tube, and stored at -70 °C until use.
  • Transformation of Agrobacterium The competent cells were thawed on ice, and 1 ⁇ of the plasmid was added to 40 ⁇ of competent cells, and the mixture was mixed and ice bathed for about 10 minutes. Transfer the mixture of competent cells and DNA to a ice-cold electric shock cup (purchased from bio-md) with a pipette and tap to bring the suspension to the bottom of the electric shock cup, 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 to the base electrode of the electric shock chamber.
  • a ice-cold electric shock cup purchased from bio-md
  • the program of MicroPulser (purchased from bio-rad) is set to "Agr" and the electric shock is applied once.
  • the electric shock cup was immediately taken out and the pre-warmed LB medium at 28 ° C was added. Quickly and gently spread the cells with a pipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C for 225 rpm for 1 h.
  • the leaves of sterile seedlings were cut into 5 mm ⁇ 5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing expression vector rd29A-GhCzf4-2300 in logarithmic growth phase for 10 min, and the bacterial culture was sucked up and co-cultured under dark conditions. 2 days (MS solid medium). Transfer the leaves to a differentiation solid medium (MS+1 mg/1 cytokinin (BA) + 0.1 mg/1 naphthaleneacetic acid (NAA) + 50 mg/1 kanamycin + 500 mg/1 cephalosporin) , incubated with 2000 Lx of light for 16 hours per day for about 45 days.
  • a differentiation solid medium MS+1 mg/1 cytokinin (BA) + 0.1 mg/1 naphthaleneacetic acid (NAA) + 50 mg/1 kanamycin + 500 mg/1 cephalosporin
  • the buds are transferred and transferred to rooting solid medium (MS+50 mg/1 kanamycin + 500 mg/1 cephalosporin) for cultivation. After about a day, after the root system was developed, the seedlings were transferred to MS solid medium supplemented with 500 mg/1 cephalosporin for number storage.
  • rooting solid medium MS+50 mg/1 kanamycin + 500 mg/1 cephalosporin
  • the leaves of the obtained transgenic tobacco plants were cut out, DNA was extracted (the Arabidopsis thaliana DNA extraction method in Example 3), and PCR amplification was carried out using SEQ ID NO: 10 and SEQ ID NO: 11 (50 ⁇ ⁇ reaction system: 5 ⁇ ⁇ ⁇ Buffer 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 10 and SEQ ID NO: 11 each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • SEQ ID NO: 10 and SEQ ID NO: 11 50 ⁇ ⁇ reaction system: 5 ⁇ ⁇ ⁇ Buffer 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 10 and SEQ ID NO: 11 each of 2.0 ⁇ l, and 35 ⁇ of double distilled water.
  • the sterilized vermiculite was soaked in 1/2 MS medium.
  • T0C1—T0C20 transgenic tobacco and control tobacco seeds were planted on vermiculite, 15 seeds per pot, 25 °C, 10 hours light culture/14 hours dark culture cycle.
  • 1/2MS was poured every 5 days. After 25 days of culture, 4-5 seedlings of the same size were kept in each pot for drought test.
  • Transgenic tobacco and control tobacco were dried for 14 days (no watering), 25 °C, 10 Hour light culture / 14 hour dark culture cycle.
  • T1 transgenic plants plants grown from seeds of TO transgenic plants
  • the results of T1C7, T1C9, T1C11, T1C14 are similar to T1C1, T1C2, not shown here).
  • Example 7 Verification of Czf4 protein expression at the transcriptional level
  • Example 6 20 of the 1 ⁇ generation transgenic plants capable of growing normally under drought conditions were randomly selected, and 10 of the 1 ⁇ generation transgenic plants in Example 6 which could not grow normally under drought conditions were randomly selected. The leaves were cut for 0.05 g in 14 days of drought, 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 the invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (1 total RNA as a template, reverse transcription primer SEQ ID NO: 11).
  • Czf4 protein was detected by amplifying GhCzf4 by SEQ ID NO: 10 and SEQ ID NO: 11.
  • the PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ l ⁇ Reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primer SEQ ID NO: 10 and P SEQ ID NO: 11 each 2.0 ⁇ l, and 30 ⁇ Double steamed water.
  • M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-8 is a normal growing plant, and 9-12 is a plant that cannot grow normally.
  • the strip size shown in the figure is the same as the size of GhCzf4.

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Abstract

提供了一种来源于棉花的锌指蛋白（Czf4）及其编码基因，以及其在培育抗旱性提高的转基因植物中的应用。

Description

一种棉花锌指蛋白 （Czf4) 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 （Czf4) 及其编码基因， 以及其在培育抗旱性提高的转基因植物中的应用。 背景技术 非生物胁迫,如干旱、盐渍、 极端温度、 化学污染和氧损伤等能够对植物的生长发 育造成严重的危害，对作物产量造成极大损失。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。
由于植物的耐胁迫性大多属于数量性状,现有可利用的种质资源匮乏，采用常规育 种技术改良植物胁迫耐性的难度相当大,培育出真正的耐胁迫品种就尤为困难。近年来, 随着对植物抗逆分子机理研究的不断深入和分子生物学技术的迅猛发展， 抗逆研究已 经从生理水平深入到分子水平,促进了植物抗逆基因工程的发展。当植物在受到胁迫时 会产生相应的应答反应,来降低或消除给植株带来的危害。植物的这种应答反应是一个 涉及多基因、 多信号途径、 多基因产物的复杂过程。 这些基因及其表达产物可以分为 3 类： （1 ) 参与信号级联放大系统和转录控制的基因及产物； （2) 直接对保护生物 膜和蛋白质起作用的基因及其表达产物； （3 ) 与水和离子的摄入和转运相关的蛋白 质。 近年来,通过转基因技术提高植物对胁迫耐受能力的研究，以及对胁迫具有耐受能 力的农作物、旱生植物和盐生植物的研究都取得了显著的成果，对胁迫相关基因和信号 转导系统也有了更进一步的了解 (Liu Q. 1998. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain, separate two cellular signal transduction pathways in drought- and low temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell, 10: 1391-1406； KANG JY. 2002. Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling. Plant Cell, 14: 343-357； ABE H. 2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as l transcriptional activators in abscisic acid signaling. Plant Cell, 15: 63-78. )。
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及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 方法克隆出了棉花的一个锌指蛋白（本文命名为 Czf4)的编码基因， 并测定了其 DNA 序列。 并且发现通过转基因将其导入植株后， 可明显改善转基因植株的抗旱性， 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 Czf4 的编码基因 （本文命名为
GhCzf4)， 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 载体为附图 2所示的 rd29A-Gh Czf4-2300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抗旱性的方法， 包括： 将本发明第一方面所述 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， 所述植物是烟草。
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， 所述植物是烟草。
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抗旱性以及用于植物育种 的用途； 优选地， 所述植物是烟草。
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 N0: 1所示。 附图说明 图 1是 GhCzf4的植物表达载体 (rd29A-GhCzf4-2300;>的构建流程。 图 2是 GhCzf4的植物表达载体 Crd29A-GhCzf4-2300)的质粒图。
图 3是对照烟草和转基因烟草的抗旱性生长情况； CK (左)：对照烟草； T1C1 (中） 和 T1C2 (右）： 转基因烟草株系。
图 4是耐干旱和不耐干旱 T1代转基因烟草植株在转录水平上的验证结果。 M为 Marker, 1-8为耐干旱的 T1代转基因烟草植株， 9一 12为不耐干旱的 T1代转基因烟 草植株。
具体实施方式 提供以下实施例， 以方便本领域技术人员更好地理解本发明。 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。 实施例 1. 干旱胁迫下棉花 SSH文库构建：
具体方法为：
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棉花幼苗的叶片中提取的 mRNA 作为样本 （tester), 以未处理的棉花幼苗的叶片中提取的 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 种到灭过菌的蛭石上， 在 25°C、 光周期 16h/8h (光强 2000— 3000 Lx) 条件下培养， 每周浇 1/2MS培养基 （含有 9.39 mMKN03， 0.625 mM KH2P04， 10.3 mMNH4N03, 0.75 mMMgSO4， 1.5 mMCaCl2， 50 μΜΚΙ, 100 μΜΗ3ΒΟ3， 100 MMnSO4， 30 μΜ ZnS04， 1 μΜΝα2Μο04, 0.1 μΜ CoCl2, 100 μΜ Na2EDTA, 100 MFeSO4) —次。 当 苗株高达 25— 30cm时用于实验。
(2) 材料处理：
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 16h/8h (光强 2000— 3000 Lx) 培养， 正常浇灌。 第二组为干旱处理组， 25°C、 光周 期 16h/8h (光强 2000— 3000 Lx) 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。
(3) 总 RNA提取：
分别取对照组和干旱处理组的棉花叶片 0.5 g， 用植物 RNA提取试剂盒 （购自 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， OD26Q/OD28Q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 polyA+ RNA from total RNA)分离 mRNA。
( 4 ) 抑制差减杂交：
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。 应用 Clontech的 PCR-Select cDNA Subtraction Kits 差减杂交试剂盒， 先将 Driver mRNA和 Tester mRNA分别反转录， 得到双链 cDNA, 再以 2 Tester cDNA和 2 ig 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 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 与新鲜变性的 Driver cDNA进行第二次正向差减杂交,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试剂盒的程序，将所述正向差减杂交 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， 2><Τ4连接酶缓冲液 5 μ1， pGEM-T Easy载体 1 μ1， T4 DNA连接酶 1 μ1， 于 4°C连接过夜。取 10 μΐ连接反应产 物，加入到 100 μΐ感受态大肠杆菌 JM109(购自 TAKARA)中,冰浴 30 min、 热休克 60 秒、 冰浴 2 min,另力口 250 μΐ LB培养液（含有 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 rpm振荡培养 30 min,取 200 μΐ 菌液接种于含 50 g/ml 氨苄青霉素的 LB (同上） /X-gal/IPTG ( X-gal/IPTG购自 TAKARA)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 540个白色菌落 (编号： Gh-D2-001至 Gh-D2-540)。 将所有白色克隆接种于含有 50 μ§/ιη1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-selectTM cDNA Subtraction Kit 试剂盒自带） 进行菌液 PCR 扩增， 得到 452个阳性克隆,然后将所有阳性克隆送英潍 捷基 （上海） 贸易有限公司测序。 ( 6) 差异克隆的 cDNA测序分析：
将 DNA测序结果去除载体和不明确序列及冗余的 cDNA后， 共得到 405个有效 EST(unigene)。 实施例 2 棉花锌指蛋白基因 GhCzf4的克隆
克隆子 Gh-D2-112去掉冗余 DNA后， 序列为 SEQ ID No: 3 , 序列分析表明该序 列编码的蛋白属于锌指蛋白。 本文将 SEQ ID No: 3序列对应的全长编码基因命名为 GhCzf4， 其对应的蛋白命名为 Czf4。
SEQ ID No: 3
丄 AC&TCTGAAT TTACAITTGA GSAGTTCCGS AGTAGGACGC CGGGAATTCA A TTGACAC c _ TTATGCAGTT GCi¾AGCGAGC TGAGAATGAC TGTCCGGTCT GCCTGTCTGA GTTTGAGCCT 丄：:丄 AAATCC— :5Α'ΞΑ？ AACCGA'TT GTCTTGCGGC CA CTCTTTC ACAAGGTGTG C TGGAAAAA 丄 TGGT GAACT A TGAAAGT TACTTGCCCT CTT1GCAGGA CT'CTGCTGC GCCGGAAG¾A 4 ^ GAGAGTTGC ACTTGGGATA AGAATATTAA GAAGCATGAT GAGTATGAGG AAATTIC-AAT
301 GT
GhCzf4全长编码基因的克隆
根据已经获得的 SEQ ID No: 3序列， 设计如下两条特异性引物， 作为 3'RACE 的 5'端特异性引物。
GhCzf'i GSP1: SEQ ID NO: 4：
GGAGTTCCGG AGTAGGACGC
GhCzf l GSP2: SEQ ID K0: 5：
G CG& AATTCA ATTTGACACT
实验步骤按试剂盒说明书操作 （3' RACE System for Rapid Amplification of cDNA Ends试剂盒购自 invitrogen公司）。
用 SEQ ID NO: 4与 3'端引物 AUAP (试剂盒自带）， 以 mRNA逆转录的 cDNA为 模板进行第一轮 PCR扩增。 具体步骤如下：
50 μΐ PCR反应体系： 5 μΐ ΙΟ^Εχ Buffer、 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ mRNA反转 录的 cDNA、 1.0 μ〗 Ex Taq (购自 TAKARA)、 10 μΜ的引物 SEQ ID NO: 4和 AUAP 各 2.0 μ1、以及 35 μΐ双蒸水。 PCR反应条件： 94°C预变性 5 min, 33个循环（94°C 变 件 30 s， 58。C退火 30 s, 72 °C 延伸 lmin)， 72 °C 延伸 10 min。
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 3'
替换页 （细则第 26条） 端引物 AUAP进行第二轮 PCR扩增， 具体步骤如下：
50 μΐ PCR反应休系： 5 μΐ ΙΟχΕχ Buffer, 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ稀释的第一 轮 PCR产物、 1.0 μ1 Εχ Τ3ς、 10 μΜ的引物 SEQ ID NO: 5和 AUAP各 2.0 μ1、 以及 35 μΐ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 lmin)， 72 °C 延伸 10 min。 回收第二次 PCR产物中片段约为 400bp的条带（Gel Extraction Kit购自 OMEGA), 并将其连接于 pGEM-T Easy Vector, 然后转化到大肠杆菌 JM109(具体方法同上)。 随机挑取 10个白色菌落接种于含有 50 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 保存备用。用 SEQ ID NO: 5与 3'端引物 AUAP进行菌液 PCR扩增， 得到 8个阳性克 降， 将其屮 4个阳性克隆送至英潍捷基（上海）贸易有限公司测序测序，获得该基因的 cDNA的 3，端。
根据已经获得的 GhCzf4基因片段，设计如下三条特异性引物，作为 5'RACE的 3' 端特异性引物。
GhCzf GSP3: SEQ ID NO: 6：
TAGCAACTCT CTTCTTCCGG
GhCzf l GSP4: SEQ ID NO: 7：
CAGCAGCAGA GTCCTGCAAA GA
GhCzf4 GSP5: SEQ ID NO: 8：
GCACACCTTG TGAAAGASAT GG 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: 7与 5'通用引物 AAP (试剂盒自带）， 以 mRNA逆转录的 cDNA (反转录引物 SEQ ID NO: 6) 为模板进行第一轮 PCR扩增， 具体步骤如下：
50 μΐ PCR反应体系： 5 μΐ ΙΟχΕχ Buffer > 3 μΐ 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: 7和 AAP各 2.0 μ1、 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 33个循环（94°C 变 件 30 s， 55 °C退火 30 s, 72 °C 延伸 lmin)， 72 °C 延伸 10 min。
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: 8与 3 ' 端引物 AUAP进行第二轮 PCR扩增， 具体步骤如下：
50 μΐ PCR反应体系： 5 μΐ ΙΟχΕχ Buffer、 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ稀释的第一
6
替换页 （细则第 26条) 轮 PCR产物、 1.0 l Ex Taq、 10 μΜ的引物 SEQ ID NO: 8和 AUAP各 2.0 μ1、 以及 35 μΐ的双蒸水。 PCR反应条件： 94Ό预变性 5 min， 33个循环 （94°C 变性 30 s， 58°C 退火 30 s， i C 延伸 1 min)， 72 °C 延伸 10 min。 回收第二次 PCR产物中片段约为 400bp的条带（Gel Extraction Kit购自 OMEGA), 并将其连接于 pGEM-T Easy Vector, 然后转化到 JM109(具体方法同上)。随机挑取 10个白色菌落接种于含有 50 μδ/ιη1氨苄 青霉素的 LB 液体培养基中，37Ό培养过夜后加甘油至终浓度 20%， -80°C保存备用。 用 SEQ ID NO: 8与 3'端引物 AUAP进行菌液 PCR扩增（反应体系及反应条件同上）, 得到 9个阳性克隆,选取其中 4个克隆送至英潍捷基 （上海） 贸易有限公司测序测序, 获得该基因的 cDNA的 5'端。
所得的 5'RACE产物克隆测序后，将其与 3'RACE产物测序结果以及 SEQ ID No:
3序列进行拼接。 获得 GhCzf4全长 cDNA序列 SEQ ID No: 9：
SEQ ID NO: 9:
1 AGCTAAAACT TAGATfCATA TACAAGAAAT GGGTCTCTCA AG CTGCCAT CTCCGTCAGA 61 AGGAATGCTA GTAT^ CT TGGTAAACAC AGCTT ATCC &TATCTGTSA TTAAAGGCAT
121 ACTTCGATCC ATCCTTCACG TTGTCGGTAT CCATCTC CG TCGTTATCAC CATCCCCGGA
181 TTCCA CGAA AATGAATCAT TTGATTTCCA GTTCAGTACA TCTGAATTTT ACATTGAGGA
241 GTTCCGGAGr AGGACGCCGG CAATTCAATT GACACT TA TGCAG TGCA AGCGAGCTGA
301 GAA GACTGT CCGGTCTGCC TGTCTGAGTT GAGCCTAAA TCGGAGAT A ACCGA TGTC
361 TTGCGGCCA C CTT CAC¾ AGGTGTGCTT GGAAAAATGG TTGAATTATT TGAAAG TAC
421 TTGCCCTCTT TGCAGGACTC TGCTGCTGCC GGAAGAAGAG AGTTGCTACT TGGGA AAGA
481 ATA TAAGAA GCATGASGAG TATGAGGAAA TTTGAA^GTA CAGCGTGTTT ATGGTATA'TG
541 GG ATAT?J¾A TTAAATGTAR IGAGCATTGG CCTAGTGCTA AGCT&CCCGC A&GTTTTAAC
601 TTSATGTGG GATGTGCT&A TGGATGTACA GT TTCftAA GTTTCTAATG ATAtGCGA
661 ATCTCAAGTC TfTTCCATT CTAAAAAAAA ΆΑΆΑΑΆΑΑΆΆ 根据 SEQ ID NO:9序列设计一对引物如下： GhCzf4: SEQ ID K0: 10：
ATGGGTC CT CAAG CTGCC A C
GhCzf : SEQ ID NO: 11：
TTATCCCAAG AGCAACTCT CTTCTT 通过 SEQ ID NO: 10和 SEQ ID NO: 11来克隆 GhCzf4全长编码基因。
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶， 以棉花的 cDNA为模板进行 PCR 反应。 50 μΐ PCR反应体系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ cDNA 替换页 （细则第 26条) 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO.10和 SEQ ID NO.11各 2.0 μ1、 以及 30 μΐ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 lmin)， 72 °C 延伸 10 min。
PCR扩增产物加 A尾： PCR产物中加入 2.5倍体积的无水乙醇， -20°C放置 10分 钟，离心，去上清，晾干，然后用 21 μΐ双蒸水溶解。然后向其中加入 2.5 μΐ lOxExBuflFer, 0.5 μΐ 5 mM的 dATP、 2.5 μΐ ΙΟχΕχ Taq。 反应条件： 70°C反应 30分钟。 将得到的约 450bp的 DNA片段回收 （Omega回收试剂盒）， 并将其连接至 pGEM T-easy载体上, 然后转化 JM109(方法同上)。随机挑取 10个白色菌落接种于含有 50 μ§/η1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。用 SEQ ID NO: 10与 SEQ ID NO: 11进行菌液 PCR扩增 （反应体系及反应条件同上） ， 得到 7个阳性克隆，选取其中 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 SEQIDNO: 2， 其编码的蛋白质的氨基酸序列为 SEQIDNO: 1。
Czf 蛋 f 的 a ¾酸序列： SEQ ID NO: 1
丄 MGLSSLPSPS EGMLGIILVN
2： TALSISVIKG ILSSILHWG
4_ IHLSSLSPSP DSIEKESFDF1
61 QFS SEFYIE EFRSRTPAIQ
3_ FDTLCSCKRA ENDCPVCLSE
101 FEPKSEINRL SCGHLFHKVC
12 _ LEKWLNYLKV CPLCRTLLL
141. PEEESCYLG*
Gl zf4編码 ¾ ½核 酸序列： SEQID O: 2
丄 ATC'GGTCTCT CAAGTCTGCC ATCTCCGTCA GAAGCAATGC TATGTATAAT CTTGGTAAAC
61 ACAGC TTAT CC¾ ATC G AAT AAAGGC ATACTTCGAT CCATCCTTCA CGTTGTCGGT
121 AT CATG'TCT CGTCG TATC ACCATCCCCG GA TCCATCG AAAATGAATC ATTTGATTTC 丄 81 CAGTTCAGTA CATCTGAATT TACAT GAG GAGTTCCGGA G AGGACGCC GGCAATTCAA
：41 TT GACACTT TATC4CAGTTG CAAGCGAGCT GAGAA GACT GTCCGGTCTG CCTGTCTGAG
3 U 1 TTTGAGCCTA A&TCGGAGAT TAACCGAfTG TCTTGCGGCC A CTCTTTCA CAAGGTGTGC
3 i TTGGAAAAAT GG TGAA TA TTTGAAAG T ACTTGCCC C TT GCAGGAC TCTGCTGCTG
421 CCGGAAGAAG AGAG TGCTA CTTGGGATAA 实施例 3 GhCzf4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，
8
替换页 （细则第 26条) 以降低 ΝΡΤΠ蛋白在植物中的表达。 选择诱导型的 rd29A启动子及终止子 Tnos分别 作为 GhCzf4基因的启动子和终止子。
用引物 SEQ ID NO: 12和 SEQ ID NO: 13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, 釆用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 l PCR反应体系： 10 μΐ 5 PS Buffer、 3 μΐ 2.5 mM的 dNTP、 1.0 μΐ ρΒΙ12 1.0 μΐ PrimeSTAR, 10 μΜ的引物 SEQ ID NO: 12和 SEQ ID NO: 13各 2.0 μ1、 以及 31 μ1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 延伸 30 s)， 72 °C 延伸 10 min。 通过 EcoRI、 Bglll酶切将所得的 PCR产物 连接 (promega, T4 连接酶盒)到 pCAMBIA2300获得 pCAMBIA2300-l。
SEQ ID NO ; 12 ：
GCACGAATTCATAC ATCGACG CGGAT SEQ ID NO: 13:
ATCCAGATCTAGATCCGGTGCAGATTATTTG 用引物 SEQ ID NO: 14和 SEQ ID NO: 15以 pBI121为模板扩增 Tnos，采用 TaKaRa 的 PrimeSTAR HS DNA聚合嗨。 50 μΐ PCR反应体系： 10 μΐ 5xPS 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和 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 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 Sacl、 EcoRI酶切将所得的 PCR产物连接 (promega T4 连接酶盒)到 pCAMBIA2300-l获得 pCAMBIA2300-2。
SEQ ID W : 14：
AAG6Mi¾^TG TTTCCCCGATCGTTC A
SEQ ID 0 : 15:
TCAi¾4J7(iCCAGTGAATTCCCGATCTAGTA 用引物 SEQ ID NO: 16 和 SEQ ID NO: 17 以拟南芥 （哥伦比亚型 ， 购自 www.arabidopsis.org) DNA为模板扩增拟南芥 rd29A启动子（参考 Zeng J., et al. 2002, Preparation of total DNA from "recalcitrant plant taxa", Acta Bot. Sin., 44(6): 694-697 中 的方法提取拟南芥 DNA)。采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反
9
替换页 （细则第 26条） 应休系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM 的 dNTP、 1.0 μΐ 拟南芥 DNA、 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 16和 SEQ ID NO: 17各 2.0 μ1、 以及 31 μ1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 HindIII、 Pstl酶切将所得的 PCR产物连 接 （连接方法同上） 到 pCAMBIA2300-2获得 pCAMBIA2300-3。
SEQ ID KO : 16：
ACTAAGCTTCCTTCTTGACATCATTC TTnA
SEQ ID NO : 17 :
TGAfTmi^rCCAAAGATTTTTTTGTTTCCMTAG 用引物 SEQ ID NO: 18和 SEQ ID NO: 19扩增 GhCzf4编码基因的全长序列 （模 板是实施例 2所获得 GhCzf4全长编码基因），采用 TaKaRa的 PrimeSTA HS DNA聚 合酶。 50 μΐ PCR 反应休系： 10 μΐ 5xPS Buffer、 3 μΐ 2.5 mM 的 dNTP、 1.0 μΐ GhCzf4-pGEM、 1.0 μΐ PrimeSTAR, 10 μΜ的引物 SEQ ID NO: 18和 SEQ ID NO: 19 各 2.0 μΙ、 以及 31 μΐ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。 通过 Pstl、 Sacl酶切 将所得的 PCR产物连接 （连接方法同上） 到 pCAMBIA2300-3， 获得植物表达载体 rd29A- GhCzf4-2300。
SEQ ID NO : 18：
IQkCTGCAG ATGGGTCTCT CAAGTCTGCC ATC
SEQ ID : 19：
kMjGAGCTC TATCCCAAG TAGCAACTCT CTTCTT 实施例 4 rd29A-GhCzf4-2300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： 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 Ό培养 1至 2天。挑取单菌落接种于 5 ml含 50 μ§/η 1利福平和 50 μ^ιηΐ 链霉素的 LB液体培养基中， 28Ό下摇动培养过夜 (约 12-16 h)至 OD6fl。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 g/ml利福平和 50 g/ml链霉素的 LB液体培养基中， 28Ό摇动培养 2-2.5 h至 OD6。。=0.8。 冰浴菌液
10
替换页 （细则第 26条） 10 min, 每隔 3 min摇匀一次， 令所述细菌均匀进入休眠状态。 于 4°C下 4000 g离心 10 min, 弃上清液； 加入一定量冰预冷的 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； 然后加入适量冰浴预冷的 10% 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。
转化农杆菌： 在冰上融化所述的感受态细胞， 往 40 μΐ的感受态细胞中加入 1 μΐ 的质粒， 混匀后冰浴约 10 min。 将感受态细胞和 DNA的混合物用移液枪转移到冰预 冷的电击杯 （购自 bio-md) 中， 轻敲使悬浮液到达电击杯底部， 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。 使用 0.1cm规格的电击杯的时候， MicroPulser (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 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 225 rpm摇动培养 1 h。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 g/ml链霉素、 50 g/ml卡那霉素）， 28°C培养。 实施例 5 利用农杆菌介导的转化法获得转基因烟草
用 75%酒精浸泡烟草种子 （国家烟草中期库， 获取单位： 中国农科院烟草所， 库 编号 I5A00660) 30 s, 然后用灭菌双蒸水洗两次。 再用 0.1%升汞浸泡 8 min， 然后用 灭菌双蒸水洗两次， 完成表面灭菌。 将表面灭菌的烟草种子置于 MS固体培养基 （含 有 18.78 mM KN03 1.25 mM KH2P04、 20.6 mM H4N03 1.5 mM MgS04、 3.0 mM CaCl2、 50 μΜ ΚΙ、 100 μΜ Η3ΒΟ3、 100 M MnSO4、 30 M ZnSO4、 1 μΜ Να2Μο04 0.1 M CoCl2、 100 μΜ Να2ΕϋΤΑ 100 M FeSO4、 7.4 g/1琼脂， 30 g/1 蔗糖） 上于无 菌条件下发芽， 制备无菌苗。 取无菌苗叶片剪成 5 mmx5 mm大小的叶盘， 用处于对 数生长期的含表达载体 rd29A-GhCzf4-2300的农杆菌浸染叶盘 10 min， 吸干菌液， 在 黑暗条件下共培养 2天 （MS固体培养基）。 将叶片转到分化固体培养基 （MS+1 mg/1 细胞分裂素 （BA) +0.1 mg/1萘乙酸 （NAA) +50 mg/1卡那霉素 +500 mg/1头孢霉素） 上， 每天用 2000 Lx的光照 16h， 培养 45天左右， 待芽长大后切下转移到生根固体培 养基（MS+50 mg/1卡那霉素 +500 mg/1头孢霉素） 中培养 30天左右， 待根系发达后将 小苗转入仅加有 500 mg/1头孢霉素的 MS固体培养基上进行编号保存。
剪取获得的转基因烟草植株的叶片， 提取 DNA (同实施例 3 中拟南芥 DNA提 取方法）， 用 SEQ ID NO: 10和 SEQ ID NO: 11进行 PCR扩增鉴定（50 μΙ ΡΟ 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: 10和 SEQ ID NO: 11各 2.0 μ1、 以及 35 μΐ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 2 min)， 72 °C 延伸 10 min)， 将 PCR鉴定为阳性植株编号为 T0C1-T0C20并保存。 实施例 6 过表达 GhCzf4转基因烟草 T1代植株的抗旱模拟实验
灭过菌的蛭石用 1/2MS培养基浸透。 T0C1— T0C20转基因烟草及对照烟草的种 子分别播种在蛭石上，每盆播种 15颗种子， 25 °C、 10小时光培养 /14小时暗培养循环。 每 5天浇一次 1/2MS，培养 25天之后，每盆保留大小较一致的 4-5棵苗用于干旱实验， 转基因烟草、对照烟草干旱 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 （TO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 T1C1、 T1C2、 T1C7、 T1C9、 T1C11、 T1C14、 六个株系共 30棵 烟草中有 20棵能够正常生长， 表现出明显的抗旱性 （见图 3， 以 T1C1、 T1C2为例， T1C7、 T1C9、 T1C11、 T1C14的结果与 T1C1、 T1C2类似， 在此未示出）。 实施例 7 在转录水平上验证 Czf4蛋白表达
实施例 6中 20棵能够在干旱条件下正常生长的 1\代转基因植株中随机选取 8棵， 实施例 6中 10棵不能在干旱条件下正常生长的 1\代转基因植株中随机选取 4棵， 各 剪取干旱 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 SEQ ID NO: 11 )。通过 SEQ ID NO: 10和 SEQ ID NO: 11扩增 GhCzf4， 检测 Czf4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 μ1 ΡΟ 反应体系： 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 10禾 P SEQ ID NO: 11各 2.0 μ1， 以及 30 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 29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： M为 DNA Ladder Marker ( DL2000,购自深圳瑞真生 物技术有限公司）， 1-8为正常生长植株， 9-12为不能正常生长植株。 图中所示条带大 小与 GhCzf4的大小一致。结果表明正常生长植株中 GhCzf4的转录较强， 不能正常生 长植株中没有 GhCzf4转录或转录很弱。 本领域技术人员可以理解的是本发明也适用于特定描述之外的变化和调整， 并且 本发明包括所有的这种变化和调整。发明人要求落入下文权利要求范围和精神内的修 改和改变的权利。

Claims

权 利 要 求 书
1. 棉花的一个锌指蛋白的编码基因， 被命名为 GhCzf4， 其核苷酸序列如 SEQ ID NO: 2所示。
2. 一种重组表达载体，其含有权利要求 1所述的基因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。
3. 权利要求 2所述的载体， 其为附图 2所示的 rd29A-GhCzf4-2300载体。
4. 一种重组细胞，其含有权利要求 1所述的基因或者权利要求 2或 3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. 一种改善植物抗旱性的方法， 包括： 将权利要求 1所述的基因或者权利要求 2 或 3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 是烟草。
6. 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 1所述的基因或者权利要求 2或 3所述的重组表达载体的植物或植物组织。
7. 权利要求 6所述的方法， 其中所述植物是烟草。
8. 权利要求 1所述的基因、权利要求 2或 3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抗旱性以及用于植物育种的用途。
9. 权利要求 8所述的用途， 其中所述植物是烟草。
10. 权利要求 1所述的基因编码的蛋白， 其序列如 SEQ ID NO: 1所示。
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CN110791523A (zh) * 2019-12-13 2020-02-14 南京农业大学 一种棉花抗旱相关基因GhRCHY1及其应用
CN110791523B (zh) * 2019-12-13 2022-05-10 南京农业大学 一种棉花抗旱相关基因GhRCHY1及其应用

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