WO2014190538A1 - 一个棉花PP2Ac类磷酸酶蛋白PP2Ac-5及其编码基因与应用 - Google Patents

一个棉花PP2Ac类磷酸酶蛋白PP2Ac-5及其编码基因与应用 Download PDF

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WO2014190538A1
WO2014190538A1 PCT/CN2013/076541 CN2013076541W WO2014190538A1 WO 2014190538 A1 WO2014190538 A1 WO 2014190538A1 CN 2013076541 W CN2013076541 W CN 2013076541W WO 2014190538 A1 WO2014190538 A1 WO 2014190538A1
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gene
seq
plant
pp2ac
expression vector
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PCT/CN2013/076541
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French (fr)
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陈文华
孙超
何云蔚
崔洪志
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创世纪转基因技术有限公司
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Priority to PCT/CN2013/076541 priority Critical patent/WO2014190538A1/zh
Priority to CN201380074525.3A priority patent/CN105189536A/zh
Publication of WO2014190538A1 publication Critical patent/WO2014190538A1/zh

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    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)

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  • the present invention relates to a PP2Ac phosphatase protein PP2Ac and a coding gene thereof, and particularly to a cotton-derived phosphatase protein PP2AC-5 and Encoding genes, and their use in cultivating transgenic plants with improved drought tolerance.
  • a PP2Ac phosphatase protein PP2Ac and a coding gene thereof and particularly to a cotton-derived phosphatase protein PP2AC-5 and Encoding genes, and their use in cultivating transgenic plants with improved drought tolerance.
  • PP1A phosphatase is a monomeric polymer in its natural state, composed of a catalytic subunit (PP2Ac) and one or more regulatory subunits A or B.
  • PP2Ac catalytic subunit
  • the first subfamily consisted of OsPP2Ac-l and OsPP2Ac-3. Drought and high salt were induced in rice leaves. The transcription levels of OsPP2Ac-l and OsPP2Ac-3 were increased; the second subfamily consisted of OsPP2Ac-2, OsPP2Ac-4 and OsPP2Ac-5, and Northern blot analysis indicated that these three genes were composed in all rice tissues. Transcription, but the level of transcription under high salt or dry heat stress conditions also changes.
  • Abiotic stresses such as drought, salt, extreme temperature, chemical pollution and oxygen damage, can cause serious damage to plant growth and development, causing great losses to crop yields.
  • the impact of drought on crop yields is in many natural adversities. It ranks first, and its harm is equivalent to the sum of other disasters. It is the bottleneck of agricultural development in many areas. According to statistics, the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid regions account for 52% of the country's land area, and the annual drought-affected area amounts to 200-2.7 million hectares. Cubic meters, due to lack of water, less than 350 to 40 billion kilograms of grain; especially China's major grain-producing areas such as North China, Northeast China and Northwest China are the areas with the most water shortage in China, and the spring drought frequently reaches 10 years.
  • genes and their expression products can be divided into three categories: (1) genes and products involved in signal cascade amplification 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 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.
  • studies on the ability of plants to improve stress tolerance through transgenic techniques, as well as studies on stress-tolerant crops, xerophytes and halophytes have yielded significant results for stress-related genes and signal transduction.
  • the system has a further understanding (Liu Q.1998.
  • DREB 1 and DREB2 Two transcription factors, DREB 1 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; KANGJY.2002. Arabidopsis basic leucine zipper proteins that mediate stress responsive abscisic acid signaling. Plant Cell, 14: 343-357; ABEH.2003.Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) Function as transcriptional activators in abscisic acid signaling. Plant Cell, 15: 63-78. ).
  • the present inventors cloned a PP2Ac phosphatase protein (a DNA sequence encoding a gene encoded herein) by using SSH (Suppression Subtractive Hybridization) in combination with RACE, and found that after insertion into a plant genome, It can significantly improve the drought tolerance of transgenic plants, and these traits can be stably inherited.
  • SSH Selection Subtractive Hybridization
  • the first aspect of the present invention provides a gene encoding a PP2Ac-like phosphatase protein PP2AC-5 of cotton having the sequence of SEQ ID NO: 2.
  • a second aspect of the invention provides a recombinant expression vector comprising the group of the first aspect of the invention Therefore, it is obtained by inserting the gene into an expression vector, wherein a nucleotide sequence of the gene is operably linked to an expression control sequence of the expression vector; preferably, the expression vector is PCAMBIA2300; Preferably, the recombinant expression vector is the rd29A-GhPP2Ac-5-2300 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 drought tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and causing the gene Expression;
  • the plant is tobacco.
  • 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, the recombinant expression vector of the second aspect of the invention, under conditions effective to produce a plant Tissue;
  • 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 tolerance of a plant and for use in plant breeding Use;
  • the plant is tobacco.
  • a seventh aspect of the invention provides the amino acid sequence encoded by the gene of the first aspect of the invention, as set forth in SEQ ID NO: 1.
  • Figure ⁇ is the construction procedure of the plant expression vector Crd29A-GhPP2Ac-5-2300 of GhPP2Ac-5;
  • Figure 2 is a plasmid map of the plant expression vector Crd29A-GhPP2Ac-5-2300 of GhPP2Ac-5;
  • Figure 3 shows the results of drought tolerance simulation experiments of GW ⁇ 24 C -5T Q -transgenic tobacco plants (T Q 1 ; right, T Q 5 ) and non-transgenic tobacco plants (left, CK) as controls.
  • Figure 4 shows the use of reverse transcription PCR for T Q transgenic tobacco plants and non-transgenic control plants.
  • a subtractive library (SSH library;) was constructed by the method of inhibition subtractive hybridization using the method shown in the PCR-selectTM cDNA Subtraction Kit of Clontech.
  • the mRNA of the leaves of the drought-treated cotton seedlings was used as a tester during the experiment, and the mRNA of the leaves of the untreated cotton seedlings was used as a driver.
  • the specific steps are as follows:
  • test seedlings were divided into two groups, each with 4 pots and 1 pot per pot.
  • the first group was the control group, cultured at 25 ° C, lighted, and normally watered.
  • the second group was the drought treatment group, 25 °C, light culture, stop watering, treatment for 10 days. After the treatment, the leaves of the top 1/3 of the two seedlings were cut out in time, and then rapidly frozen with liquid nitrogen at -70 °C. Store in the refrigerator.
  • RNA extraction kit (Invitrogen). The absorbance of the total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001. The OD260/OD280 ratio was 1.8-2.0, indicating that the total RNA purity was high, using 1.0% agarose gel electrophoresis. To check the integrity of the total RNA, the 28S band is about twice as bright as the 18S band, indicating good RNA integrity. Isolation of mRNAo using Qiagen's purification of polyA+ RNA from total RNA
  • the second PCR product of the combined positive subtractive hybridization cDNA fragment (QIAquick PCR Purification Kit purified from Qiagen) was ligated with the pGEM-T Easy (purchased from Promega kit) vector according to the pGEM-T Easy kit product specification.
  • the specific steps are as follows: Add the following components to the 200 ⁇ PCR tube: Purification of the second PCR product of the positive subtractive hybridization cDNA fragment 3 ⁇ l, ⁇ 4 ligase buffer 5 ⁇ l, pGEM-T Easy vector 1 ⁇ l, T4 DNA ligase 1 ⁇ , ligated overnight at 4 °C. Take 10 ligation reaction products, add to 100 competent E.
  • coli JMI09 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 s, ice bath for 2 min, plus 250 ⁇ L L culture medium (with 1% Tryptone purchased from OXOID, 0.5% Yeast Extract was purchased from OXOID, 1% NaCl was purchased from Sinopharm. Placed in a 37 °C shaker, shaken at 225 r/min for 30 min, and 200 ⁇ L of bacterial solution was planted in 50 g/mL ampicillin ( LB solid plates were purchased from Amresco), 40 ug/mL X-gaK 24 ug/mL IPTG (X-gal/IPTG were purchased from TAKARA), and incubated at 37 ° C for 18 h.
  • PCR primers Primer 1 and P Primer 2R (Clontech's PCR-selectTM cDNA Subtraction Kit kit) were used to carry out PCR amplification of the bacterial cells, and 245 positive clones were obtained, and all positive clones were sent to the British Shanghai) Trading Co., Ltd. sequencing
  • Sequencing result of the positive clone designated Gh-D35 after the redundant DNA was removed, the sequence was SEQ ID No: 3.
  • the long gene was named G/ ⁇ P24c-5, and the corresponding protein was named PP2Ac-5.
  • the GhPP2Ac-5 gene fragment that has been obtained already has a stop codon TGA, and only 5 ' RACE is required.
  • GhPP2Ac-5 GSP 1 SEQ ID NO: 4:
  • GhPP2Ac-5 GSP2 SEQ ID NO: 5:
  • GhPP2Ac-5 GSP3 SEQ ID NO: 6:
  • 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: 5 and 5' universal primer AAP (provided with the kit), and cDNA reverse transcription cDNA (reverse transcription primer SEQ ID NO: 4) was used as a template for the first round of PCR amplification.
  • the specific steps are as follows: Ex Taq was purchased from TAKARA, 50 ⁇ PCR reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ mRNA reverse transcribed cDNA, 1.0 ⁇ l ⁇ ⁇ 10 ⁇ primers SEQ ID NO: 5 and AAP each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions predenaturation at 94 °C for 5 min, 94 denaturation for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
  • the obtained PCR product was diluted 50 times 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 the primer AUAP of SEQ ID NO: 6 3, and the specific steps were as follows: 50 ⁇ l ⁇ Reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ diluted first round PCR product, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 6 and AUAP each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions predenaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min.
  • the second PCR product recovery fragment is about 1000 bp band (Gel Extraction Kit is purchased from OMEGA) and ligated into pGEM-T Easy vector, transformed into E. coli JM109 competent cells (specific method is the same as above), and the transformed bacterial liquid is transformed. Screening was performed on LB solid medium containing 50 g/mL ampicillin.
  • a pair of primers were designed according to the sequence of SEQ ID NO: 17 as follows:
  • GhPP2Ac-5R SEQ ID NO: 8:
  • PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template.
  • 50 ⁇ PCR reaction system 10 ⁇ 5 > ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 ⁇ 1, and 30 ⁇ ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 94 denaturation for 30 s, annealing at 58 °C for 30 s, extension at 72V for 2 min, after 33 cycles, extension at 72 °C for 10 min.
  • PCR amplification product plus A tail The PCR product was added with 2.5 volumes of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, de-cleared, air-dried, and dissolved in 21 ⁇ l of double distilled water. Add 2.5 ⁇ ⁇ Buffer, 0.5 ⁇ l 5 ⁇ dATP, 2.5 ⁇ ⁇ Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 900 bp was recovered (Omega Recovery Kit), ligated into pGEM T-easy vector, transformed into E. coli JM109 competent cells and plated on LB solid medium containing 50 g/mL ampicillin. Screening (method same as above).
  • SEQ ID NO: 7 and SEQ ID NO: 8 were subjected to bacterial cell PCR amplification (reaction system and reaction conditions are the same as above), and four positive clones were obtained, which were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing, and the obtained sequence was SEQ. ID NO: 2.
  • amino acid sequence of the PP2AC-5 protein encoded by the gene is as shown in SEQ ID NO: 1.
  • Amino acid sequence of PP2Ac-5 protein SEQ ID NO: 1
  • Nucleotide sequence of the GhPP2Ac-5 encoding gene SEQ ID NO: 2
  • the plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter of the ⁇ gene containing the double enhancer was replaced with the Pnos promoter to reduce the expression of prion protein in plants. .
  • the inducible promoter rd29A and Tnos terminator were selected as promoters and terminators of the GhPP2Ac-5 gene.
  • Primer SEQ ID NO: 9 and SEQ ID NO: 10 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 ⁇ PCR reaction system: 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ PBI121, 1.0 ⁇ PrimeSTAR 10 ⁇ primers SEQ ID ⁇ : 9 and SEQ ID NO: 10 each 2.0 ⁇ l, and 31 ⁇ double steaming water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min.
  • pCAMBIA2300-l was obtained by EcoRI, Bglll digestion with pCAMBIA2300 (Promega, T4 ligase cassette).
  • Tnos was amplified using primers SEQ ID NO: 11 and SEQ ID NO: 12 with PBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase.
  • PCR reaction conditions pre-variability at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 s, after 33 cycles, extension at 72 °C for 10 min.
  • the pCAMBIA2300-2 was obtained by restriction enzyme digestion with pCAMBIA2300-1 (Promega T4 ligase cassette) by Sacl and EcoRI.
  • SEQ ID NO: 13 and SEQ ID NO: 14 to Arabidopsis thaliana (Columbia type, purchased from Www.arabidopsis.org) DNA is used as a template to amplify the Arabidopsis thaliana rd29A promoter (see Zeng J., et L. 2002, Preparation of total DNA from "recalcit rant plant taxa", Acta Bot. Sin., 44(6) : Method 694-697 for extracting Arabidopsis DNA). PrimeSTAR HS DNA polymerase using TaKaRa.
  • PCR reaction system 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ Arabidopsis DNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 13 and SEQ ID NO: 14 each 2.0 ⁇ l, and 31 ⁇ of double distilled water.
  • PCR reaction conditions predenaturation at 94 °C for 5 min, 94 denaturation for 30 s, annealing at 58 °C for 30 s, 72V for 30 s, 33 cycles, and 72 °C for 10 min.
  • the pCAMBIA2300-3 was obtained by digesting with HindIII and Pstl (connection method is the same as above) pCAMBIA2300-2.
  • GhPP2Ac-5 (template was GhPP2Ac-5 obtained in Example 2) was amplified using primers SEQ ID NO: 15 and SEQ ID NO: 16, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ PCR reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ GhPP2Ac-5-pGEM, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 15 and SEQ ID NO: 16 each 2.0 ⁇ l, And 31 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, denaturation at 94 V for 30 s, annealing at 58 °C for 30 s, extension at 72 V for 2 min, after 33 cycles, extension at 72 °C for 10 min.
  • the plant expression vector rd29A-GhPP2Ac-5-2300 was obtained by cleavage of Pstl and Sacl (connection method as above) pCAMBIA2300-3 (Fig. 2).
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Competent preparation: Agrobacterium LBA4404 was spotted on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin 1-2 days in advance Inoculate, incubate at 28 °C for 1 to 2 days. Pick a single colony inoculated in 5 ml containing 50 ⁇ ⁇ / ⁇ 1 rifampicin In LB liquid medium with 50 ⁇ ⁇ / ⁇ 1 streptomycin, shake culture at 28 °C overnight (about 12-16 h) to an OD600 value of 0.4 to form a seed broth.
  • Transformation of Agrobacterium Melt LBA4404 competent cells on ice, add 1 ⁇ of the plasmid to 40 ⁇ of the competent cells, mix and ice bath for about 10 min. Transfer the mixture of competent cells after ice bath and rd29A-GhPP2Ac-5-2300 plasmid DNA into a pre-cooled electric shock cup with a micropipette and tap to bring the suspension to the bottom, taking care not to have air bubbles. Place the electric shock cup (purchased from bio-rad) on the slide of the electric shock room and push the slide to place the electric shock cup on the base electrode of the electric shock chamber.
  • bio-rad electric shock cup
  • the program of the 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 mix the cells with a micropipette.
  • the suspension was transferred to a 1.5 ml centrifuge tube and incubated at 28 ° C, 225 rpm for 1 h.
  • the leaves of the sterile seedlings were cut into 5 mm ⁇ 5 mm leaf discs, and the leaf discs were inoculated with Agrobacterium containing the expression vector rd29A-GhPP2Ac-5-2300 in the logarithmic growth phase for 10 min, and the bacterial liquid was sucked in the dark condition. Co-culture for 2 days (MS medium).
  • the obtained transgenic tobacco leaves were extracted, and DNA (the Arabidopsis thaliana DNA extraction method in Example 3) was used, and SEQ ID NO: 7 and SEQ ID NO: 8 (50 ⁇ PCR reaction system: 5 ⁇ ⁇ ⁇ Buffer, 3 ⁇ ) 2.5 mM dNTP, 2.0 ⁇ DNA, 1.0 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 9 and SEQ ID NO: 10 each 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions 94 ° C pre- Denaturation for 5 min, 94 denaturation for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min, after 33 cycles, extension at 72 °C for 10 min), PCR identification, preservation of positive plants for number TQ1-T 0 20.
  • T Q 1-T Q 20 and control tobacco tissue culture seedlings were transplanted to vermiculite, respectively, at 25 ° C, 10 hours light culture / 14 hours dark culture cycle, 1/2 MS every 5 days, 15 days after strong seedling culture Drought stress experiments were carried out, transgenic tobacco, control tobacco drought for 14 days (without watering), 25 ° C, 10 hours light culture / 14 hours dark culture cycle.
  • T Q transgenic plants showed that the control plants were wilting, while Tol, ⁇ 5, ⁇ 8, ⁇ 13, ⁇ 0 14, ⁇ 0 16, To 17 plants were able to grow normally, especially Tol and ⁇ 5 showed obvious Drought tolerance (see figure).
  • Example 7 verified G at the transcriptional level / ⁇ expression P24c-5 genes are taken to control tobacco, drought effect is not significant in transgenic tobacco T Q generation plants drought significant effect of transgenic tobacco T Q-generation plants (grow well The leaves were dried at a dose of 0.05 g for 14 days, and total RNA was extracted using a plant RNA extraction kit (Invitrogen).
  • RNA concentration values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the respective RNA concentrations.
  • Reverse transcription was carried out according to the method shown by Invitrogen reverse transcription assay L1 box Superscript III Reverse Transcriptase (2 ⁇ ⁇ total RNA as a template, reverse transcription primer SEQ ID NO: 8).
  • the relative expression of the PP2Ac-5 protein was examined by amplifying GhPP2Ac-5 by SEQ ID NO: 7 and SEQ ID NO: 8.
  • the PCR reaction was carried out using the reverse-transcribed cDNA of TaKaRa as a template using PrimeSTAR HS DNA polymerase of TaKaRa.
  • PCR reaction system 10 ⁇ 5 ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 7 and SEQ ID NO: 8 each 2.0 ⁇ 1, and 30 ⁇ Double steamed water.
  • PCR reaction conditions predenaturation at 94 °C for 5 min, 94 denaturation for 30 s, annealing at 58 °C for 30 s, 72V for 1 min, 29 cycles, 72 °C for 10 min.
  • M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-6 is a transgenic tobacco T Q plant with significant drought tolerance, and 7-12 is drought tolerant. Transgenic tobacco To plants with insignificant effects, 13-15 were control tobacco.
  • the strip size shown in the figure is the same as the size of G/ ⁇ 3 ⁇ 44c-5.
  • the results showed that the transgenic tobacco T Q plants with strong drought tolerance had stronger transcription of the foreign gene G/ ⁇ 3 ⁇ 44c-5, and the drought-tolerant effect was not significant.
  • the transgenic tobacco T Q generation plants had no transcription or transcription of the foreign gene GhPP2Ac-2. Weak, no signal of transcription of the foreign gene GhPP2Ac-5 was found in the control tobacco.

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Abstract

一个来源于棉花的PP2Ac类磷酸酶蛋白PP2AC-5及其编码基因，以及其在培育耐旱性提高的转基因植物中的应用。

Description

一个棉花 PP2Ac类磷酸酶蛋白 PP2AC-5及其编码基因与应用 技术领域 本发明涉及 PP2Ac类磷酸酶蛋白 PP2Ac及其编码基因与应用， 特别是涉及一 个来源于棉花的磷酸酶蛋白 PP2AC-5及其编码基因， 以及其在培育耐旱性提高的 转基因植物中的应用。 技术背景 在生物体内激酶的磷酸化作用和磷酸酶的脱磷酸作用相互协同，保证了生命 过程的有序进行。据估计，真核生物中约 97%的蛋白质去磷酸化是由丝 /苏氨酸类 的磷酸酶完成的， 丝 /苏氨酸类磷酸酶一般又可分为 PP1、 PP2A、 PP2B和 PP2C等 类型。 其中， PP2A类磷酸酶在天然状态下是单体聚合物， 由催化亚基 （PP2Ac) 以及一个或多个调控亚基 A或 B组成。 在植物中对 PP2Ac家族的报道最早来自拟 南芥， 在拟南芥中有 5个^ 24c基因。 随后， 在水稻基因组中发现也有 5个^ 24c 基因， 这 5个基因被划分为 2个亚家族， 第一个亚家族由 OsPP2Ac-l和 OsPP2Ac-3 组成， 干旱、 高盐都会诱导水稻叶片中的 OsPP2Ac-l和 OsPP2Ac-3的转录水平提 高； 第二个亚家族由 OsPP2Ac-2、 OsPP2Ac-4和 OsPP2Ac-5组成， Northern blot分 析表明， 这 3个^ 2^基因在所有水稻组织中组成型转录， 但在高盐或干热胁迫 条件的转录水平也会发生变化。
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 300亿立方米， 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,DREB 1 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； KANGJY.2002. Arabidopsis basic leucine zipper proteins that mediate stress responsive abscisic acid signaling. Plant Cell, 14: 343- 357； ABEH.2003.Arabidopsis AtMYC2 (bHLH) and AtMYB2(MYB) function as 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 方面的研究将对抗逆相关的信号传递途径之间的联系以及整个信号传递网络系 统的机理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相结合的方法克隆了棉花的 —个 PP2Ac类磷酸酶蛋白 （本文命名为 的编码基因的 DNA序列。 并 发现将其插入植物基因组后， 可明显改善转基因植株的耐旱性， 而且这些性状可 稳定遗传。
本发明第一方面提供棉花的一个 PP2Ac类磷酸酶蛋白 PP2AC-5的编码基因， 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， 其中所述基因的核苷 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； 优选地， 所述重组表达载体为 附图 2 所示的 rd29A-GhPP2Ac-5-2300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旱性的方法， 包括： 将本发明第一方面 所述的基因或者本发明第二方面所述的重组表达载体导入植物或植物组织并使 所述基因表达； 优选地， 所述植物是烟草。
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的基因、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耐旱性以及用于 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由本发明第一方面所述的基因所编码的氨基酸序列，如 SEQ ID NO: 1所示。 附图说明 图 ί是 GhPP2Ac-5的植物表达载体 Crd29A-GhPP2Ac-5-2300;>的构建流程。 图 2是 GhPP2Ac-5的植物表达载体 Crd29A-GhPP2Ac-5-2300;>的质粒图。 图 3是 GW^24C-5TQ代转基因烟草植株（图中， TQ1 ; 右， TQ5 )和作为对照的 非转基因烟草植株 （图左， CK) 的耐旱模拟实验结果。
图 4是利用反转录 PCR对 TQ代转基因烟草植株和非转基因对照植株中
G/^P24c-5基因在转录水平上的分子检测的验证结果。 M为 Marker, 1-6为耐旱效 果显著的转基因烟草 TQ代植株 ,7-12为耐旱效果不显著的转基因烟草 TQ代植株, 13-15为非转基因对照烟草植株。 具体实施方式 下面结合非限制性实施例对本发明进行进一步说明, 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说明书所示的方法 通过抑制差减杂交方法构建差减文库 (SSH文库；)。在实验过程中以干旱处理的棉 花幼苗的叶子的 mRNA作为样本（tester), 以未处理的棉花幼苗的叶子的 mRNA 作为对照 （driver)。 具体步骤简述如下：
( 1 ) 供试材料：
冀棉 14 (国家棉花中期库，获取单位中国棉花研究所，统一编号： ZM-30270) 播种到灭过菌的蛭石上， 在 25°C、 光周期 16h/8h条件下培养， 每周浇 1/2MS培 养基（9.39 mM KN03， 0.625 mM KH2P04, 10.3 mM H4N03, 0.75 mM MgSO4， 1.5 mM CaCl2, 50 μ M KI, 100 μ M H3B03, 100 M MnSO4, 30 M ZnSO4, 1 M Na2Mo04, 0.1 M CoCl2, 100 μ M Na2EDTA, 100 M FeSO4, 其余为水） 一次。 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、 光照培养， 正常浇灌。 第二组为干旱处理组， 25°C、 光照培养， 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子各 0.5g， 用植物 RNA提取试剂盒 (Invitrogen) 提取棉花的总 RNA。 用 HITACHI公司的紫外分光光度计 U-2001 测定所得总 RNA在 260 nm和 280 nm的吸光度值， OD260/OD280比值为 1.8-2.0， 表明总 RNA纯度较高， 用 1.0%的琼脂糖凝胶电泳检测总 RNA的完整性， 28S 条带的亮度约为 18S条带的 2倍， 表明 RNA的完整性良好。 使用 Qiagen 公司 的 Oligotex mRNA纯化试剂盒 (purification of polyA+ RNA from total RNA)分离 mRNAo
(4) 抑制差减杂交： 为了增加获得表达序列标签 （Expressed sequence tag, EST) (unigene)的有效 性， 避免基因无酶切位点及所获得序列在非翻译区。 本实验室用 Rsal， Haelll 分别对双链 cDNA (按照 PCR-selectTM cDNA Subtraction Kit试剂盒说明书记载的 方案， 由 mRNA逆转录获得） 进行消化， 做两组抑制差减， 其他步骤及方法按 Clontech公司的 PCR-select™ cDNA Subtraction Kit试剂盒说明书所示的方法进 行， 最后合并两组正向差减杂交 cDNA片段的第二次 PCR产物。
( 5 ) 差减文库的构建与初步筛选、 克隆及鉴定
合并的正向差减杂交 cDNA 片段的第二次 PCR 产物 （QIAquick PCR Purification Kit纯化， 购自 Qiagen) 与 pGEM-T Easy (购自 Promega试剂盒)载体 连接， 依照 pGEM-T Easy试剂盒产品说明书所示方法， 具体步骤如下： 向 200 μΐ PCR管中依次加入下列成分： 纯化的正向差减杂交 cDNA片段的第二次 PCR产 物 3 μ1， Τ4连接酶缓冲液 5 μ1， pGEM-T Easy载体 1 μ1， T4 DNA连接酶 1 μΐ ， 于 4°C连接过夜。取 10 连接反应产物，加入到 100 感受态大肠杆菌 JMI09(购 自 TAKARA)中，冰浴 30 min、热休克 60 s、冰浴 2 min,另加 250 μL L 培养液（含 1% Tryptone购自 OXOID, 0.5% Yeast Extract购自 OXOID, 1% NaCl购自国药） 置 37°C摇床中， 以 225 r/min振荡培养 30 min,取 200 μL菌液种植于含 50 g/mL 氨苄青霉素 (购自 Amresco)、 40 ug/mL X-gaK 24 ug/mL IPTG (X-gal/IPTG均购 自 TAKARA) 的 LB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 360 个白色菌落（编号： Gh-D001 至 Gh-D360)。 将所有白色菌落挑于含有 50 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禾 P 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 245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 DNA测序结果去除载体和不明确序列及冗余的 cDNA后， 共得到 121个 EST(unigene), 分别编号为 Gh-Dl-121。 经 BlastN发现其中 69 条 unigene在 GenBank 中有同源序列 （蛋白同源性 50%以上）， 31条 EST功能未知或者为假 定蛋白， 另有 21条未获得同源匹配， 推测可能是处于 3 '、 5 ' 末端非翻译区的较 短序列。 实施例 2 棉花磷酸酶蛋白基因 的克隆
编号为 Gh-D35的阳性克隆的测序结果去掉冗余 DNA后， 序列为 SEQ ID No: 3， 序列分析表明该序列的编码的氨基酸序列属于磷酸酶蛋白， 本文将 SEQ ID No: 3编码的全长基因命名为 G/^P24c-5， 对应的蛋白命名为 PP2Ac-5。 SEQ ID No: 3
1 AACTTCCAGT AGCAAATGGA TGGGGAGAAA AGCACAAGCT TTTCATTCAG TGGAAGCACA
61 TTGAAGCTAA GGCTATTGCA TACTATCTTC ACGGATTAAT CCTCGAAGAA GGGAACACAT
121 CTGCTGGCAT AGCTGCAGCT GCTCTGCAAG CAGCAGAAGA GTATCTCAAA GAAAGCAAAA 181 TGGCCTGCGA TTCTTTCCAT GTGACACTCC CTTCGTCACG TAATCCACCT CCCTGGGGAG 241 CCAGCAAGTA TCTTGCTGAG AGGATTCCAA AAGATATATC AAGCAAAACA ATCAACTGGG 301 ATTCACAAAA ACATGAAATG ATTAAGCATG TAGCTCCAGC ATTGCCTGAC TTTGTGTTGT 361 CCCTAAAACC TGATGAGTAT CAGCTTCCTT CGACAGATCC CTCCTGGAAT GATTTACATG 421 CTCAAAACGT GGTTCCTACC AAGTGAAGCC TCAAGGGCCA GAATGTCAGA TGTGTATCAA 481 ATAATTATAA TATATTACGA ATGTAAAACA CCATTTGGAA TAAATCAAAG CTGTTGAATG 541 TCTTCCAATA TTCATCCACA
GhPP2Ac-5全长基因的克隆
已经获得的 GhPP2Ac-5 基因片段， 已经有终止密码子 TGA,只需要做 5 ' RACE。
根据已经获得的 Gh-D35基因片段， 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。
GhPP2Ac-5 GSP 1： SEQ ID NO: 4：
CATCTGACAT TCTGGCCCTT G
GhPP2Ac-5 GSP2: SEQ ID NO: 5：
CTTTGAGATA CTCTTCTGCT G
GhPP2Ac-5 GSP3： SEQ ID NO: 6：
CTTCTTCGAG GATTAATCCG
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: 5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:4 )为模板进行第一轮 PCR扩增，具体步骤如下： Ex Taq购自 TAKARA, 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 μ1 Εχ Τα 10 μΜ的引物 SEQ ID NO: 5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, 94 变性 30 s，55 °C退火 30 s, 72°C 延伸 2min， 33个循环后， 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: 6 3' 端引物 AUAP进行第二轮 PCR扩增， 具体步骤如下： 50 μ1 ΡΟ 反应体系： 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM的 dNTP, 2.0 μΐ稀释的第一轮 PCR产物， 1.0 μΐ Ex Taq、 10 μΜ的引物 SEQ ID NO: 6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， 94 °C 变性 30 s， 58 °C退火 30 s， 72 °C 延伸 2 min， 33个循环后， 72°C 延伸 10 min。第二次 PCR产物回收片段约为 1000bp条带（Gel Extraction Kit购自 OMEGA)连接于 pGEM-T Easy载体，转化到大肠杆菌 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 g/mL氨苄青霉素 的 LB固体培养基上进行筛选。 随机挑取 10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: 6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 3 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 5'端。 所得的 5'RACE产物克隆测序后， 与 Gh-D35阳性克隆测序结果拼接， 获得
SEQ ID NO: 17：
1 CTTTTTAACT GTTTTTAGTT TCTTGTTTCA CTTGTCTTTC TAGTTGCTTC AAACAGCTGA
61 GGTGTATATA CATATATAAT GGAGATTCTA ATTAGATGTT TTCAAATTAT TGCATGAGAG
121 AAGAACTTGT CAGTTGAGAG GAACTAAATT GGAAGGAAAT GGGGTGCCTG TTATCGACGC
181 CAGAGGTTGC AGGAGGAGCT CGAAGAAGGC CGAGAAATAT TGGTGAAGTT GTAGTTTTTG
241 TTCCTGGACT GAGAATTCCT AGGCCCCTTG ATTTCGCACA GCCACTCGGT GATGGACTAT
301 CCAAGAGCTT GGTTGAACGT CTATCGGCAT TGAGAACACG TATAGAGGTC ATGGCAGGTC
361 AAGAAGCACC TATGACTACT AAACCGAGGA GAACAGCCAC ACAACACGGA GGTTCAACGT
421 TGACTGATTT GCAGCAGGCT CTCGAGGATT ATCTGCCGGT CTTGTTGGGG CTAGTTGAAA
481 ATGGTAACTG GCTAAAACAT AACTTGCGGT TCTGTTGGAT AAATCAGGAA GATGATGTGG
541 AGGAAACTAC CATGTCGGAT TCTTGGTATG AGGTACTATC AGTGTTGCAC TTGATGGCAG
601 TTCTATTGTT ATCACAGGCC AATTTATTGC TTCTTCCAAA CAAATTTACC AGTGGTTATC
661 AGTCTACAGC ATTGGAAGAT TGCAAACGAG CATCCATCGA CATTTTCTTA AAGGCAGCTG
721 GATATCTGGA CTTCGCTGTG CAGAAAGTTC TCCCTCAATT GCCCTTGGAA CTTAGGAGTG
781 ATTTGCCACT AGATCTGTCA GAGGGAGTGC TTAAAGCCCT TTGTTTGCAA GCATTAGGCC
841 AGGGTGTTGA GATTCAACTG GGAATGGCAA TTGATAGCAT TAAGGCCACT CTGGCCGTGA
901 AAAGGAGGCT AGCATGTGAG ATGGTTAAAT ACTGGCATCA GGCTGAGGAA TACATAAAGG 961 AACTTCCAGT AGCAAATGGA TGGGGAGAAA AGCACAAGCT TTTCATTCAG TGGAAGCACA
1021 TTGAAGCTAA GGCTATTGCA TACTATCTTC ACGGATTAAT CCTCGAAGAA GGGAACACAT
1081 CTGCTGGCAT AGCTGCAGCT GCTCTGCAAG CAGCAGAAGA GTATCTCAAA GAAAGCAAAA
1141 TGGCCTGCGA TTCTTTCCAT GTGACACTCC CTTCGTCACG TAATCCACCT CCCTGGGGAG
1201 CCAGCAAGTA TCTTGCTGAG AGGATTCCAA AAGATATATC AAGCAAAACA ATCAACTGGG
1261 ATTCACAAAA ACATGAAATG ATTAAGCATG TAGCTCCAGC ATTGCCTGAC TTTGTGTTGT
1321 CCCTAAAACC TGATGAGTAT CAGCTTCCTT CGACAGATCC CTCCTGGAAT GATTTACATG
1381 CTCAAAACGT GGTTCCTACC AAGTGAAGCC TCAAGGGCCA GAATGTCAGA TGTGTATCAA
1441 ATAATTATAA TATATTACGA ATGTAAAACA CCATTTGGAA TAAATCAAAG CTGTTGAATG
1501 TCTTCCAATA TTCATCCACA
根据 SEQ ID NO: 17序列设计一对引物如下:
GhPP2Ac-5F-. SEQ ID NO: 7：
ATGGGGTGCCTGTTATCGAC
GhPP2Ac-5R: SEQ ID NO: 8：
TCACTTGGTAGGAACCACGTTTTG 通过 SEQ ID NO:7和 SEQ ID NO: 8来克隆 GhPP2Ac-5全长。
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 5 ><PS Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 7和 SEQ ID NO: 8各 2.0 μ1， 以及 30 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 94 变性 30 s， 58 °C 退火 30 s， 72V 延伸 2min， 33个循环后， 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 μΐ双蒸水溶解。 加入 2.5 μΐ ΙΟχΕχ Buffer, 0.5 μ1 5 ιηΜ的 dATP ， 2.5 μΐ ΙΟχΕχ Taq。 反应条件： 70°C反应 30分钟。 将得到约 900 bp的 DNA片段回收 （Omega回收试剂盒）， 连接至 pGEM T-easy载体上，转 化到大肠杆菌 JM109感受态细胞中并在含 50 g/mL氨苄青霉素的 LB固体培养 基上进行筛选 (方法同上)。 随机挑取 10个白色菌落于含有 50 μ§/ιη∑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: 7与 SEQ ID NO: 8进行菌液 PCR扩增 （反应体系及反应条件 同上） ， 得到 4个阳性克隆,送至英潍捷基 （上海） 贸易有限公司测序,所得序列 为 SEQ ID NO: 2。 根据该核苷酸序列， 可推论该基因编码的 PP2AC-5蛋白氨基 酸序列如 SEQ ID NO: l所示。 PP2Ac-5蛋白的氨基酸序列： SEQ ID NO: 1
1 MGCLLSTPEV AGGARRRPRN
2 1 IGEVWFVPG LRI PRPLDFA
4 1 QPLGDGLSKS LVERLSALRT
61 RI EVMAGQEA P TTKPRRTA
81 TQHGGSTLTD LQQALEDYLP
101 VLLGLVENGN WLKHNLRFCW
12 1 INQEDDVEET T SDSWYEVL
14 1 SVLHLMAVLL LSQANLLLLP
161 NKFTSGYQST ALEDCKRAS I
181 DIFLKAAGYL DFAVQKVLPQ
2 01 LPLELRSDLP LDLSEGVLKA
22 1 LCLQALGQGV EIQLG AIDS
24 1 I KATLAVKRR LACE VKYWH
261 QAEEYIKELP VANGWGEKHK
2 81 LFIQWKHIEA KAIAYYLHGL
3 01 ILEEGNTSAG IAAAALQAAE
32 1 EYLKESKMAC DSFHVTLPSS
34 1 RNPPPWGASK YLAERI PKDI
361 SSKTINWDSQ KHE IKHVAP
3 81 ALPDFVLSLK PDEYQLPSTD
4 01 PSWNDLHAQN WPTK*
GhPP2Ac-5 编码基因的核苷酸序列： SEQ ID NO: 2
1 ATGGGGTGCC TGTTATCGAC GCCAGAGGTT GCAGGAGGAG CTCGAAGAAG GCCGAGAAAT
61 ATTGGTGAAG TTGTAGTTTT TGTTCCTGGA CTGAGAATTC CTAGGCCCCT TGATTTCGCA
121 CAGCCACTCG GTGATGGACT ATCCAAGAGC TTGGTTGAAC GTCTATCGGC ATTGAGAACA
181 CGTATAGAGG TCATGGCAGG TCAAGAAGCA CCTATGACTA CTAAACCGAG GAGAACAGCC
241 ACACAACACG GAGGTTCAAC GTTGACTGAT TTGCAGCAGG CTCTCGAGGA TTATCTGCCG
301 GTCTTGTTGG GGCTAGTTGA AAATGGTAAC TGGCTAAAAC ATAACTTGCG GTTCTGTTGG
361 ATAAATCAGG AAGATGATGT GGAGGAAACT ACCATGTCGG ATTCTTGGTA TGAGGTACTA
421 TCAGTGTTGC ACTTGATGGC AGTTCTATTG TTATCACAGG CCAATTTATT GCTTCTTCCA
481 AACAAATTTA CCAGTGGTTA TCAGTCTACA GCATTGGAAG ATTGCAAACG AGCATCCATC
541 GACATTTTCT TAAAGGCAGC TGGATATCTG GACTTCGCTG TGCAGAAAGT TCTCCCTCAA
601 TTGCCCTTGG AACTTAGGAG TGATTTGCCA CTAGATCTGT CAGAGGGAGT GCTTAAAGCC
661 CTTTGTTTGC AAGCATTAGG CCAGGGTGTT GAGATTCAAC TGGGAATGGC AATTGATAGC
721 ATTAAGGCCA CTCTGGCCGT GAAAAGGAGG CTAGCATGTG AGATGGTTAA ATACTGGCAT
781 CAGGCTGAGG AATACATAAA GGAACTTCCA GTAGCAAATG GATGGGGAGA AAAGCACAAG
841 CTTTTCATTC AGTGGAAGCA CATTGAAGCT AAGGCTATTG CATACTATCT TCACGGATTA
901 ATCCTCGAAG AAGGGAACAC ATCTGCTGGC ATAGCTGCAG CTGCTCTGCA AGCAGCAGAA
961 GAGTATCTCA AAGAAAGCAA AATGGCCTGC GATTCTTTCC ATGTGACACT CCCTTCGTCA
1021 CGTAATCCAC CTCCCTGGGG AGCCAGCAAG TATCTTGCTG AGAGGATTCC AAAAGATATA
1081 TCAAGCAAAA CAATCAACTG GGATTCACAA AAACATGAAA TGATTAAGCA TGTAGCTCCA
1141 GCATTGCCTG ACTTTGTGTT GTCCCTAAAA CCTGATGAGT ATCAGCTTCC TTCGACAGAT 1201 CCCTCCTGGA ATGATTTACA TGCTCAAAAC GTGGTTCCTA CCAAGTGA 实施例 3 GhPP2Ac-5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 ΡΤΠ基因含双增强子的 35S 启动子， 以降低 ΡΤΠ蛋白在植物中的表达。 选择诱导型启动子 rd29A及 Tnos 终止子作为 GhPP2Ac-5基因的启动子和终止子。
用引物 SEQ ID NO: 9和 SEQ ID NO: 10以植物表达载体 PBI121 (购自北京 华夏远洋科技有限公司）为模板扩增 Pnos,采用 TaKaRa的 PrimeSTAR HS DNA 聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM的 dNTP， 1.0 μΐ PBI121 , 1.0 ^ PrimeSTAR 10 μΜ的引物 SEQ ID ΝΟ:9和 SEQ ID NO: 10各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 94 °C 变性 30 s， 56°C退火 30 s， 72 °C 延伸 30 s， 33个循环后， 72°C 延伸 10 min。 通过 EcoRI、 Bglll酶切连接到 pCAMBIA2300 (Promega, T4 连接酶盒)获得 pCAMBIA2300-l。
SEQ ID NO: 9 ：
GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 10：
ATCCAGATCTAGATCCGGTGCAGATTATTTG
用引物 SEQ ID NO: 11和 SEQ ID NO: 12以 PBI121为模板扩增 Tnos, 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反应体系： 10 μΐ 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: 11和 SEQ ID NO: 12各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变 性 5 min， 94 °C 变性 30 s， 58°C退火 30 s， 72 °C 延伸 30 s， 33个循环后， 72°C 延伸 10 min。 通过 Sacl、 EcoRI酶切连接到 pCAMBIA2300-1 (Promega T4 连接 酶盒)获得 pCAMBIA2300-2
SEQ ID NO: 11：
AAGG^GCJCGAATTTCCCCGATCGTTCAAA
SEQ ID NO: 12：
TCKGAA r7UCC AGTGAATTCCCGATCT AGT A
用引物 SEQ ID NO: 13和 SEQ ID NO: 14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 L. 2002, Preparation of total DNA from"recalcit rant 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反应体系： 10 ^ 5 xPS 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: 13和 SEQ ID NO: 14各 2.0 μ1， 以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 94 变性 30 s， 58 °C退火 30 s， 72V 延伸 30 s， 33个循环后， 72°C 延伸 10 min。通过 HindIII、 Pstl酶切连接到 （连接方法同上） pCAMBIA2300-2获得 pCAMBIA2300-3
SEQ ID NO: 13：
ACT^GCJrCCTTCTTGACATCATTCAATTTTA
SEQ ID NO: 14：
TGACJGC4GTCCAAAGATTTTTTTCTTTCCAATAG
用引物 SEQ ID NO: 15和 SEQ ID NO: 16扩增 GhPP2Ac-5 (模板是实施例 2 所获得 GhPP2Ac-5 )， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR 反应体系： 10 ^ 5 xPS Buffer， 3 μΐ 2.5 mM的 dNTP， 1.0 μΐ GhPP2Ac-5-pGEM, 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 15和 SEQ ID NO: 16各 2.0 μ1， 以 及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min， 94 V 变性 30 s， 58 °C退 火 30 s， 72V 延伸 2min， 33个循环后， 72°C 延伸 10 min。 通过 Pstl、 Sacl酶 切连接到 （连接方法同上） pCAMBIA2300-3， 获得植物表达载体 rd29A- GhPP2Ac-5-2300 (图 2)。
SEQ ID NO: 15：
TGACJGC4GATGGGGTGCCTGTTATCGAC
SEQ ID NO: 16：
AAGG^GCJCTCACTTGGTAGGAACCACGTTTTG 实施例 4 rd29A-GhPP2Ac-5-2300表达载体转化农杆菌
农杆菌 LBA4404 (购自 Biovector Science Lab, Inc) 感受态制备： 提前 1-2 天将农杆菌 LBA4404在含 50 g/ml利福平和 50 g/ml链霉素的 LB固体培养基 上划单斑接种， 28 °C培养 1至 2天。 挑取单菌落接种于 5 ml含 50 μ§/ιη1利福平 和 50 μ§/ιη1 链霉素的 LB 液体培养基中， 28 °C下摇动培养过夜 (约 12-16 h)至 OD600值为 0.4， 形成种子菌液。 取 5 ml活化后的菌液 （1 :20的比例） 接种于 100 ml同样浓度抗生素的 LB液体培养基中，28°C摇动培养 2-2.5 h至 OD6(K)=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 10%甘油重悬浮菌体, 4°C下 4000 g离 心 10 min， 收集沉淀； 用 10%甘油重复洗 3-4次； 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 LBA4404感受态细胞，往 40 μΐ的所述感受态细胞 中加入 1 μΐ 的质粒， 混匀后冰浴约 10 min。 将冰浴后的感受态细胞和 rd29A- GhPP2Ac-5-2300质粒 DNA的混合物用微量移液器转移到预冷的电击杯中，轻敲 使悬浮液到达底部， 注意不要有气泡。 将电击杯 （购自 bio-rad) 放到电击室的 滑道上，推动滑道将电击杯放至电击室基座电极处。使用 0.1cm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 μΐ的菌液涂布与 相应的抗性筛选培养基平板上 （LB固体培养基， 含 50 g/ml利福平、 50 μ§/ιη1 链霉素、 50 μ§/ιη1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, 用灭菌双蒸水洗两次。 再用 0.1%升汞浸泡 8 min， 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 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 Η3Β03， 100 M MnS04， 30 M ZnS04， 1 μΜ Να2Μο04, 0.1 M CoCl2， 100 μΜ Na2EDTA, 100 μΜ FeS04， 7.4 g/L琼脂， 蔗糖 30 g/L， 其余为水） 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-GhPP2Ac-5-2300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培养基）。 将叶片 转到分化培养基 （MS+1 mg/L细胞分裂素 （BA) +0.1 mg/L萘乙酸 （NAA) +50 mg/L卡那霉素 +500 mg/L头孢霉素） 上， 光照条件下培养 45天左右， 待芽长大 后切下转移到生根培养基 （MS+50 mg/L卡那霉素 +500 mg/L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/L头孢霉素的 MS培养基上 进行编号保存。
取获得的转基因烟草叶片， 提取 DNA (同实施例 3中拟南芥 DNA提取方 法），用 SEQ ID NO: 7和 SEQ ID NO: 8 ( 50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM的 dNTP， 2.0 μΐ DNA, 1.0 μΐ Ex Taq, 10 μΜ的引物 SEQ ID NO: 9 禾口 SEQ ID NO: 10各 2.0 μ1， 以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5 min, 94 变性 30 s， 58°C退火 30 s， 72 °C 延伸 2 min， 33个循环后， 72°C 延 伸 10 min)， PCR鉴定， 保存阳性植株进行编号 TQ1-T020。
实施例 6 过表达 GhPP2Ac-5转基因烟草 TQ的耐旱模拟实验及功能鉴定 将灭过菌的蛭石用 1/2MS培养基浸透。 TQ1-TQ20及对照烟草组培苗分别移 栽至蛭石上， 25°C、 10小时光培养 /14小时暗培养循环， 每 5天浇一次 1/2MS, 壮苗培养 15天之后进行干旱胁迫实验， 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。 TQ代转基因植株的抗旱性鉴定 表明， 对照植株都萎蔫严重， 而 Tol、 Το5、 Το8、 Το13、 Τ014, Τ016, To 17七个 植株能够正常生长， 尤其 Tol、 Το5表现出明显的耐旱性 （参见图）。 实施例 7 在转录水平上验证 G/^P24c-5基因的表达 分别取对照烟草、 耐旱效果不显著的转基因烟草 TQ代植株、 耐旱效果显著 的转基因烟草 TQ代植株 （生长状况良好） 干旱 14天的叶子 0.05g， 用植物 RNA 提取试剂盒 (Invitrogen)提取总 RNA。用 HITACHI公司的紫外分光光度计 U-2001 测定所得总 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 (2μβ总 RNA作为模板，反转录引物 SEQ ID NO: 8)。通过 SEQ ID NO:7和 SEQ ID NO: 8扩增 GhPP2Ac-5， 检测 PP2Ac-5蛋白相对表达情况。 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶，以上述反转录的 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, 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 7和 SEQ ID NO: 8各 2.0 μ1， 以及 30 μΐ 的双蒸水。 PCR反应条件： 94°C预变性 5 min， 94 变性 30 s， 58 °C退火 30 s， 72V 延伸 lmin， 29个循环后， 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）， 1-6为耐 旱效果显著的转基因烟草 TQ代植株， 7-12为耐旱效果不显著的转基因烟草 To代 植株， 13-15为对照烟草。 图中所示条带大小与 G/^ ¾4c-5的大小一致。 结果表 明耐旱效果显著的转基因烟草 TQ代植株对外源基因 G/^ ¾4c-5的转录较强， 耐 旱效果不显著的转基因烟草 TQ代植株中外源基因 GhPP2Ac-2没有转录或转录很 弱， 对照烟草中没有发现外源基因 GhPP2Ac-5转录的信号。

Claims

权 利 要 求 书
1. 棉花的一个 PP2Ac类磷酸酶蛋白， 其序列为 SEQ ID N0: 1。
2. 编码权利要求 1的 PP2Ac类磷酸酶蛋白的基因，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，其中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。
4. 权利要求 2所述的载体，其为附图 2所示的 rd29A-GhPP2Ac-5-2300载体。
5. 一种重组细胞， 其含有权利要求 2所述的基因或者权利要求 3或 4所述 的重组表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。
6. 一种改善植物耐旱性的方法， 包括： 将权利要求 2所述的基因或者权利 要求 3或 4所述的重组表达载体导入植物或植物组织并使所述基因表达;优选地， 所述植物是烟草。
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， 其中所述植物是烟草。
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旱性以及用于植物育种的用途。
10. 权利要求 9所述的用途， 其中所述植物是烟草。
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