WO2014101160A1 - Cotton mannose-6-phosphoric acid reductase, gene for encoding same, and application thereof - Google Patents

Cotton mannose-6-phosphoric acid reductase, gene for encoding same, and application thereof Download PDF

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WO2014101160A1
WO2014101160A1 PCT/CN2012/087975 CN2012087975W WO2014101160A1 WO 2014101160 A1 WO2014101160 A1 WO 2014101160A1 CN 2012087975 W CN2012087975 W CN 2012087975W WO 2014101160 A1 WO2014101160 A1 WO 2014101160A1
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plant
gene
seq
expression vector
ghm6pr
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PCT/CN2012/087975
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French (fr)
Chinese (zh)
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王建胜
崔洪志
何云蔚
王婷婷
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创世纪转基因技术有限公司
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Priority to CN201280078031.8A priority Critical patent/CN104884623A/en
Priority to PCT/CN2012/087975 priority patent/WO2014101160A1/en
Publication of WO2014101160A1 publication Critical patent/WO2014101160A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01224—Mannose-6-phosphate 6-reductase (1.1.1.224)
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)

Definitions

  • the present invention relates to plant proteins and their coding genes and applications, and more particularly to a cotton-derived mannose-6-phosphate reductase (M6PR) protein and a gene encoding the same, and in the cultivation of transgenic plants having improved drought resistance Applications.
  • M6PR mannose-6-phosphate reductase
  • 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.
  • a first aspect of the invention provides a coding gene for a mannose-6-phosphate reductase of cotton (herein designated GhM6PR) having the sequence 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-GhM6PR-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 Crd29A-GhM6PR-2300) of GhM6PR.
  • Figure 2 is a plasmid map of the plant expression vector Crd29A-GhM6PR-2300) of GhM6PR.
  • Figure 3 shows the drought resistance of control tobacco and transgenic tobacco (Fig. 3a before drought, Fig. 3b after drought); CK (left): control tobacco; T1P5 (right): transgenic tobacco lines.
  • Figure 4 shows the results of verification of transcriptional levels of drought-tolerant T1 transgenic tobacco plants and drought-tolerant control tobacco plants.
  • M is Marker
  • 1-8 is a drought-tolerant T1 transgenic tobacco plant
  • 9 is a plasmid PCR positive control
  • 10-13 is a drought-tolerant control tobacco plant.
  • BEST MODE FOR CARRYING OUT THE INVENTION The following examples are provided to facilitate a better understanding of the present invention by those skilled in the art. The examples are for illustrative purposes only and are not intended to limit the scope of the invention.
  • Example 1 Cotton SSH library construction under drought stress:
  • African cotton (National Cotton Medium-Term Library, obtained by the China Cotton Research Institute, Uniform No.: ZM-06838) was planted on sterilized vermiculite at 25 ° C, photoperiod 16 h light / 8 h dark (light intensity 2000 - 3000 Lx) Culture under conditions, pouring 1/2MS medium per week (containing 9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM NH4NO3, 0.75 mM MgSO 4 , 1.5 mM CaCl 2 , 50 ⁇ KI, 100 ⁇ 3 ⁇ 3 , 100 MMnSO 4 , 30 MZnSO 4 , 1 ⁇ 2 ⁇ 0 4 , 0.1 ⁇ CoCl 2 , 100 ⁇ Na 2 EDTA, 100 MFeSO 4 ) — times. It was used for experiments when the seedlings were as high as 25-30 cm.
  • the test seedlings were divided into 2 groups, 4 pots per group and 1 pot per pot.
  • the first group was a control group, which was cultured at 25 ° C, photoperiod 16 h light / 8 h dark (light intensity 2000-3000 Lx), and was normally watered.
  • the second group was the drought treatment group, cultured at 25 °C, photoperiod 16h light/8h dark (light intensity 2000-3000 Lx), stopped watering, treated for 10 days, and cut the top of the two seedlings in time after treatment. 3 leaves, quickly frozen with liquid nitrogen, in a -70 ° C refrigerator Save.
  • the cotton leaves of the control group and the drought treatment group were respectively 0.5 g, and the total RNA of cotton leaves was extracted with a plant 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 poly A+ RNA from total RNA.
  • Two tester cDNAs with different adaptors were mixed with excess Driver for the first forward subtractive hybridization.
  • the two products of the first forward subtractive hybridization were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment. .
  • the second inhibitory PCR product of the second subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) and pGEM-T Easy (purchased according to the procedure of pGEM-T Easy kit)
  • the vector was ligated from the Promega 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 ⁇ 1, pGEM-T Easy vector 1 ⁇ l, ⁇ 4 DNA ligase 1 ⁇ l, ligated overnight at 4 °C. Take 10 ⁇ of the reaction product, add 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 add 250 ⁇ LB medium (containing 1% Tryptone).
  • OXOID 0.5% Yeast Extract
  • 1% NaCl purchased from Sinopharm
  • ⁇ / ⁇ 1 ampicillin LB (ibid.) /X-gal/IPTG (X-gal/IPTG purchased from TAKARA) culture plate, incubated at 37 ° C for 18 h.
  • Nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-select TM cDNA Subtraction Kit The kit comes with) PCR amplification of the bacterial solution, 452 positive clones were obtained, and all positive clones were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • sequence was SEQ ID No: 3.
  • Sequence analysis indicated that the protein encoded by the sequence belonged to the mannose-6-phosphate reductase protein.
  • the full-length coding gene corresponding to the sequence of SEQ ID No: 3 was named GhM6PR, and its corresponding protein was named M6PR.
  • AAAAAAACTT ATAATTAGAT TCATCAATCG CCCAAGTTTT CTTCTTCTTC TTTTTTAA
  • GliM6PR GSPI SEQ ID No : 4:
  • G!iM6PR GSF1 SEQ ID o; 5:
  • the first round of PCR amplification was carried out using SEQ ID NO: 4 and the universal 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 ⁇ primers SEQ ID NO: 4 and AUAP 2.0 1 ⁇ 1, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 33 cycles (94 ° C change) Properties 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 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 using SEQ ID NO: 5 and the universal primer AUAP.
  • the specific steps are as follows:
  • PCR reaction system 5 ⁇ ⁇ ⁇ Buffer 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ diluted first round PCR product, 1.0 l Ex Taq, 10 ⁇ primer SEQ ID NO: 5 and AUAP each 2.0 ⁇ 1, and 35 ⁇ ⁇ 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 into LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use.
  • SEQ ID NO: 5 and the universal primer AUAP to carry out bacterial PCR amplification, 5 positive clones were obtained, and 4 positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the cDNA of the gene was obtained. 3' end.
  • GhM6FR GSP3 SEQ ID ot 6:
  • GTCCGATATT TCCGGTCAAT GG GI1M6P GSP4 SEQ- ID No; 7:
  • GhM6PR GSP5 SEQ: ID No : f 8:
  • GTTTCGTTGG ATTCCCCACC GG Experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit was purchased from Invitrogen).
  • 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 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 SEQ ID NO: 8 and primer AUAP.
  • the specific steps are as follows: 50 ⁇ PCR reaction system: 5 ⁇ ⁇ ⁇ Buffer 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ diluted first round PCR product, 1.0 l Ex Taq, 10 ⁇ primer SEQ ID NO: 8 and AUAP each 2.0 ⁇ 1, and 35 ⁇ ⁇ 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 800 bp in the second PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into pGEM-T Easy Vector, and then transformed into JM109 (specific method as above).
  • Ten white colonies were randomly picked and inoculated into LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use.
  • the GhM6PR 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 ⁇ l ⁇ Reaction system 10 ⁇ 5xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 ⁇ l, and 30 ⁇ double steamed 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.
  • the 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 ⁇ ⁇ ⁇ Buffer, 0.5 ⁇ 5 mM dATP, 1.0 ⁇ Ex Taq were added thereto. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained DNA fragment of about 1200 bp was recovered (Omega recovery kit), and ligated to the pGEM T-easy vector (to obtain the GhM6PR-pGEM plasmid), and then transformed into JM109 (method as above).
  • Amino acid sequence of M6PR protein : : SEQ ID NO:
  • 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 inducible rd29A promoter and the terminator Tnos were selected as promoters and terminators of the GhM6PR gene, respectively.
  • Pnos was amplified using the primers SEQ ID NO: 12 and SEQ ID NO: 13 with the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ l ⁇ Reaction system: 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ ⁇ 121 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 12 and P SEQ ID NO: 13 each 2.0 ⁇ 1, and 31 ⁇ 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 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was ligated by EcoRI, Bglll (promega, T4 ligase cassette) to pCAMBIA2300 to obtain pCAMBIA2300-1.
  • ATCCAGATCTAGATCCGGTGCAGA TATTTG Tnos was amplified using primers SEQ ID NO: 14 and SEQ ID NO: 15 with pBI121 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 72 °C for 30 s), extension at 72 °C for 10 min.
  • the resulting PCR product was cleaved by Sacl, EcoRI and ligated into the Cpromega T4 ligase cassette;) to pCAMBIA2300-l to obtain pCAMBIA2300-2.
  • the Arabidopsis thaliana rd29A promoter was amplified using primers SEQ ID NO: 16 and SEQ ID NO: 17 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 694-697 for extracting Arabidopsis DNA). PrimeSTAR HS DNA polymerase from TaKaRa was used.
  • PCR reaction system 10 ⁇ 5 ⁇ PS Buffer 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ Arabidopsis DNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 16 and P SEQ ID NO: 17 each 2.0 ⁇ 1, and 31 11 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 HindIII and Pstl (connection method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3.
  • the full-length sequence of the GhM6PR-encoding gene was amplified with primers SEQ ID NO: 18 and SEQ ID NO: 19 (template was the positive GhM6PR-pGEM plasmid obtained in Example 2) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 ⁇ PCR reaction system: 10 ⁇ 5 ⁇ PS Buffer 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ GhM6PR-pGEM, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 18 and P SEQ ID NO: 19 2.0 ⁇ l, and 31 ⁇ Double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 min, 33 cycles (94 ° C change) Properties 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min.
  • the obtained PCR product was ligated by Pstl and Sacl digestion (the ligation method is the same as above) to pCAMBIA2300-3 to obtain a plant expression vector.
  • 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 inoculation, culture at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin and 50 ⁇ ⁇ / ⁇ 1 streptomycin, and cultured overnight (about 12-16 h) to OD 6 at 28 °C with shaking. . A value of 0.4 forms a seed broth.
  • 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. A mixture of competent cells and rd29A-GhM6PR-2300 plasmid DNA was transferred to an ice-cold electric shock cup (purchased from bio-md) using a pipette, and tapped 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.
  • an ice-cold electric shock cup purchased from bio-md
  • the MicroPulser purchased from bio-rad
  • the MicroPulser is set to "Agr” and the 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-GhM6PR-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 ⁇ PCR 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 ⁇ PCR 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.
  • the seeds of T0P1-T0P20 transgenic tobacco and control tobacco were separately sown on vermiculite, and 15 seeds were seeded per pot at 25 ° C, 14 hours light culture / 10 hours dark culture cycle.
  • 1/2MS was poured once a week, and after 25 days of culture, SEQ ID NO: 10 and SEQ ID NO: 11 were subjected to PCR detection to remove negative plants, and 4-5 seedlings of uniform size were retained per pot for drought experiments, transgenic tobacco, control tobacco Drought for 14 days (without watering), 25 ° C, 14 hours light culture / 10 hours dark culture cycle.
  • T1 transgenic plants plants grown from T0 transgenic plants
  • T1P5 T1P7, T1P15 and T1P17 showed significant drought resistance
  • T1P5 the results of T1P7, T1P15, and T1P17 are similar to T1P5, which is not shown here).
  • Example 7 Verification of M6PR protein expression at the transcriptional level
  • RNA was extracted from the plant RNA extraction kit (invitrogen). Ultraviolet spectrophotometric determination of total RNA at 260 nm and 280 nm The absorbance values were calculated for each RNA concentration.
  • Reverse transcription was carried out according to the method shown by the invitrogen reverse transcription kit Superscript III Reverse Transcriptase (1 total RNA as a template, reverse transcription primer SEQ ID NO: 11). The relative expression of M6PR protein was detected by amplifying GhM6PR by SEQ ID NO: 10 and SEQ ID NO: 20.
  • PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase.
  • 50 ⁇ PCR reaction system 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA, 1.0 ⁇ PrimeSTAR, 10 ⁇ primers SEQ ID NO: 10 and P SEQ ID NO: 20 each 2.0 ⁇ l, and 30 ⁇ Double steamed water.
  • PCR reaction conditions pre-denaturation at 94 °C for 5 min, 29 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.
  • M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-8 is a drought-tolerant T1 transgenic tobacco plant, and 9 is a plasmid PCR positive control (rd29A-GhM6PR). -2300 plasmid), 10-13 is a drought-tolerant control tobacco plant.
  • the band size shown in the figure is consistent with the size of GhM6PR (approximately 900 bp). The results showed that the transcription of GhM6PR in transgenic T1 transgenic tobacco plants was stronger, and the drought-tolerant control tobacco plants did not have GhM6PR transcription.

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Abstract

Provided are a mannose-6-phosphoric acid reductase (M6PR) protein from cotton, a gene for encoding same, and an application thereof in culturing transgenic plants with improved drought-tolerance.

Description

一种棉花甘露糖 -6-磷酸还原酶及其编码基因与应用  Cotton mannose-6-phosphate reductase and its coding gene and application
技术领域 本发明涉及植物蛋白及其编码基因与应用， 特别是涉及一个来源于棉花的甘露糖 -6-磷酸还原酶 (M6PR)蛋白及其编码基因， 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。 FIELD OF THE INVENTION The present invention relates to plant proteins and their coding genes and applications, and more particularly to a cotton-derived mannose-6-phosphate reductase (M6PR) protein and a gene encoding the same, and in the cultivation of transgenic plants having improved drought resistance Applications. BACKGROUND OF THE INVENTION Abiotic stresses, such as drought, salting, extreme temperatures, chemical pollution, and oxygen damage, can cause serious damage to the growth and development of plants and cause significant losses to crop yields. Among them, the impact of drought on crop yields ranks first in many natural adversities, and its harm is equivalent to the sum of other disasters. Many regions are the bottleneck of agricultural development. According to statistics, the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid 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.
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 transcriptional activators in abscisic acid signaling. Plant Cell, 15: 63-78. )。 Since the stress tolerance of plants is mostly quantitative, the available germplasm resources are scarce. It is very difficult to improve the stress tolerance of plants by conventional breeding techniques. It is especially difficult to cultivate true stress-tolerant varieties. In recent years, with the deepening of research on the molecular mechanism of plant stress resistance and the rapid development of molecular biology technology, stress resistance research has progressed from physiological level to molecular level, which promotes the development of plant stress resistance genetic engineering. When plants are stressed, they will respond accordingly to reduce or eliminate the damage to plants. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. These 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. In recent years, 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. 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 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甘露糖 -6-磷酸还原酶 (M6PR)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。  However, as far as the current research situation is concerned, due to the complexity of its mechanism, the biochemical and physiological response mechanisms of many plants to stress remain to be further studied. Research on the function and expression regulation of stress-responsive genes will provide an important basis for the study of plant stress-resistance related signaling pathways and signal transmission network systems. SUMMARY OF THE INVENTION The present inventors cloned a gene encoding a mannose-6-phosphate reductase (M6PR) of cotton using SSH (suppression subtractive hybridization) and RACE (rapid amplification of cDNA ends) and determined the gene. Its DNA sequence. It has also been found that the transgenic plants can significantly improve the drought resistance of transgenic plants by introducing them into plants, and these traits can be stably inherited.
本发明第一方面提供棉花的一个甘露糖 -6-磷酸还原酶的编码基因 （本文命名为 GhM6PR), 其序列为 SEQ ID N0: 2。  A first aspect of the invention provides a coding gene for a mannose-6-phosphate reductase of cotton (herein designated GhM6PR) having the sequence 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-GhM6PR-2300载体。  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-GhM6PR-2300 vector shown in Fig. 2.
本发明第三方面提供一种重组细胞， 其含有本发明第一方面所述的基因或者本发 明第二方面所述的重组表达载体； 优选地， 所述重组细胞为重组农杆菌细胞。  A third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.
本发明第四方面提供一种改善植物抗旱性的方法， 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。  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; Preferably, the plant is tobacco.
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。  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 Preferably, the plant is tobacco.
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。  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; Preferably, the plant is tobacco.
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 GhM6PR的植物表达载体 Crd29A-GhM6PR-2300)的构建流程。 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. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a construction flow of a plant expression vector Crd29A-GhM6PR-2300) of GhM6PR.
图 2是 GhM6PR的植物表达载体 Crd29A-GhM6PR-2300)的质粒图。  Figure 2 is a plasmid map of the plant expression vector Crd29A-GhM6PR-2300) of GhM6PR.
图 3是对照烟草和转基因烟草的抗旱性生长情况 （图 3a为干旱前， 图 3b为干旱 后）； CK (左)： 对照烟草； T1P5 (右）： 转基因烟草株系。  Figure 3 shows the drought resistance of control tobacco and transgenic tobacco (Fig. 3a before drought, Fig. 3b after drought); CK (left): control tobacco; T1P5 (right): transgenic tobacco lines.
图 4是耐干旱 T1代转基因烟草植株和不耐干旱对照烟草植株在转录水平上的验 证结果。 M为 Marker, 1-8为耐干旱 T1代转基因烟草植株， 9为质粒 PCR阳性对照， 10-13为不耐干旱对照烟草植株。 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：  Figure 4 shows the results of verification of transcriptional levels of drought-tolerant T1 transgenic tobacco plants and drought-tolerant control tobacco plants. M is Marker, 1-8 is a drought-tolerant T1 transgenic tobacco plant, 9 is a plasmid PCR positive control, and 10-13 is a drought-tolerant control tobacco plant. BEST MODE FOR CARRYING OUT THE INVENTION The following examples are provided to facilitate a better understanding of the present invention by those skilled in the art. The examples are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1. Cotton SSH library construction under drought stress:
具体方法为：  The specific method is:
利用 Clontech公司的 PCR-selectTM cDNA Subtraction Kit 所示的方法通过抑制差 减杂交方法构建差减文库。 在实验过程中以干旱处理的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： The method of PCR-select TM cDNA Subtraction Kit from Clontech shown by using the subtractive hybridization method suppression construct subtraction libraries. The mRNA extracted from the leaves of the drought-treated cotton seedlings was used as a tester during the experiment, and the mRNA extracted from the leaves of the untreated cotton seedlings was used as a driver. The specific steps are as follows:
(1) 供试材料：  (1) Test materials:
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光照 /8h黑暗 （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 mM NH4NO3, 0.75 mMMgSO4， 1.5 mMCaCl2， 50 μΜ KI， 100 μΜΗ3ΒΟ3， 100 MMnSO4， 30 MZnSO4， 1 μΜΝα2Μο04, 0.1 μΜ CoCl2, 100 μΜ Na2EDTA, 100 MFeSO4) — 次。 当苗株高达 25— 30cm时用于实验。 African cotton (National Cotton Medium-Term Library, obtained by the China Cotton Research Institute, Uniform No.: ZM-06838) was planted on sterilized vermiculite at 25 ° C, photoperiod 16 h light / 8 h dark (light intensity 2000 - 3000 Lx) Culture under conditions, pouring 1/2MS medium per week (containing 9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM NH4NO3, 0.75 mM MgSO 4 , 1.5 mM CaCl 2 , 50 μΜ KI, 100 μΜΗ 3 ΒΟ 3 , 100 MMnSO 4 , 30 MZnSO 4 , 1 μΜΝα 2 Μο0 4 , 0.1 μΜ CoCl 2 , 100 μΜ Na 2 EDTA, 100 MFeSO 4 ) — times. It was used for experiments when the seedlings were as high as 25-30 cm.
(2) 材料处理：  (2) Material handling:
将供试幼苗分为 2组， 每组 4盆， 每盆 1株。 第一组为对照组， 在 25°C、 光周期 16h光照 /8h黑暗（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光照 /8h黑暗 （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。 The test seedlings were divided into 2 groups, 4 pots per group and 1 pot per pot. The first group was a control group, which was cultured at 25 ° C, photoperiod 16 h light / 8 h dark (light intensity 2000-3000 Lx), and was normally watered. The second group was the drought treatment group, cultured at 25 °C, photoperiod 16h light/8h dark (light intensity 2000-3000 Lx), stopped watering, treated for 10 days, and cut the top of the two seedlings in time after treatment. 3 leaves, quickly frozen with liquid nitrogen, in a -70 ° C refrigerator Save.
( 3 ) 总 RNA提取：  (3) Total RNA extraction:
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 poly A+ RNA from total RNA)分离 mRNA。 The cotton leaves of the control group and the drought treatment group were respectively 0.5 g, and the total RNA of cotton leaves was extracted with a plant 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 poly A+ RNA from total RNA.
( 4 ) 抑制差减杂交：  (4) Suppression of subtractive hybridization:
按 Clontech公司的 PCR-selectTM cDNA Subtraction Kit试剂盒所示的方法进行抑制 差减杂交。先将 Driver mRNA和 Tester mRNA分别反转录， 得到双链 cDNA, 再以 2 μg Tester cDNA和 2 g Driver cDNA作为起始材料进行差减杂交。 在 37°C水浴下分别将 Tester cDNA和 Driver cDNA用 Rsa I 酶切 1.5 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。 将两种第一次正向差减 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,通过两次抑制 性 PCR扩增差异表达的片段，使其得到富集。 Suppression Subtractive Hybridization performed by PCR-select TM cDNA Clontech's method shown Subtraction Kit kit. The Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and 2 μg 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 for the first forward subtractive hybridization. The two products of the first forward subtractive hybridization were mixed, and a second forward subtractive hybridization was performed with the newly denatured Driver cDNA, and the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment. .
( 5 ) cDNA差减文库的构建与初步筛选、 克隆、 鉴定  (5) Construction and preliminary screening, cloning and identification of cDNA subtraction library
依照 pGEM-T Easy试剂盒的程序，将所述正向第二次差减杂交 cDNA片段的第二 次抑制性 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， Τ4 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,另加 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 μ§/ιη1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。 The second inhibitory PCR product of the second subtractive hybridization cDNA fragment (purified using QIAquick PCR Purification Kit, purchased from Qiagen) and pGEM-T Easy (purchased according to the procedure of pGEM-T Easy kit) The vector was ligated from the Promega kit. The specific steps are as follows: The following components were sequentially added using a 200 μΐ PCR tube: The second PCR product of the purified forward subtractive hybridization cDNA fragment 3 μ1, 2><Τ4 ligase buffer 5 Μ1, pGEM-T Easy vector 1 μl, Τ4 DNA ligase 1 μl, ligated overnight at 4 °C. Take 10 μΐ of the reaction product, add 100 μΐ of competent E. coli JM109 (purchased from TAKARA), ice bath for 30 min, heat shock for 60 seconds, ice bath for 2 min, and add 250 μΐ 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 μM bacteria in 50 μm. § /ιη1 ampicillin LB (ibid.) /X-gal/IPTG (X-gal/IPTG purchased from TAKARA) culture plate, incubated at 37 ° C for 18 h. Count the number of clear white and blue colonies > 1 mm in diameter in the culture plate and randomly pick 540 white colonies (number: Gh-D2-001 to Gh-D2-540). All white clones were inoculated into 96-well cell culture plates (CORNING) containing LB liquid medium containing 50 μ § /ιη1 ampicillin, cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C. spare. Nested PCR primers Primer 1 and Primer 2R (Clontech's PCR-select TM cDNA Subtraction Kit The kit comes with) PCR amplification of the bacterial solution, 452 positive clones were obtained, and all positive clones were sent to Yingji Jieji (Shanghai) Trading Co., Ltd. for sequencing.
( 6 ) 差异克隆的 cDNA测序分析：  (6) cDNA sequencing analysis of differential clones:
将 DNA测序结果去除载体和不明确序列及冗余的 cDNA后， 共得到 405个有效 EST(unigene 实施例 2 棉花甘露糖 -6-磷酸还原酶基因 GhM6PR的克隆  After removing the vector and the ambiguous sequence and redundant cDNA from the DNA sequencing results, a total of 405 effective ESTs were obtained (unigene example 2 clone of cotton mannose-6-phosphate reductase gene GhM6PR)
克隆子 Gh-D2-139去掉冗余 DNA后， 序列为 SEQ ID No: 3， 序列分析表明该序 列编码的蛋白属于甘露糖 -6-磷酸还原酶蛋白。 本文将 SEQ ID No: 3序列对应的全长 编码基因命名为 GhM6PR， 其对应的蛋白命名为 M6PR。  After the cloned Gh-D2-139 was removed from the redundant DNA, the sequence was SEQ ID No: 3. Sequence analysis indicated that the protein encoded by the sequence belonged to the mannose-6-phosphate reductase protein. Here, the full-length coding gene corresponding to the sequence of SEQ ID No: 3 was named GhM6PR, and its corresponding protein was named M6PR.
SEQ ID No: 3：  SEQ ID No: 3:
1 ACAAGAAAAC CGTGGCTCAG ATCGTTCTCC GGTGGGGAAT CCAACGAAAC ACGGTAGTCA 1 ACAAGAAAAC CGTGGCTCAG ATCGTTCTCC GGTGGGGAAT CCAACGAAAC ACGGTAGTCA
61 TCCCAAAGAC ATCAAAATTC GAGAGATTGA AAGAGAATTT TGAGGTTTTT GACTTCGAGT61 TCCCAAAGAC ATCAAAATTC GAGAGATTGA AAGAGAATTT TGAGGTTTTT GACTTCGAGT
121 TAGCCAAAGA AGACATGGAC AAAATCAAAG CCATTGACCG GAAATATCGG ACCAATCAAC 181 CTGCCAAGTT TTGGGGCATC GATCTGTATG CTTAGAGTGC GATGCGGGTA TTCTTTGTGT121 TAGCCAAAGA AGACATGGAC AAAATCAAAG CCATTGACCG GAAATATCGG ACCAATCAAC 181 CTGCCAAGTT TTGGGGCATC GATCTGTATG CTTAGAGTGC GATGCGGGTA TTCTTTGTGT
241 GGCGACTTAT GTTTTATGAA GAATAAGAGC AAAAAAAGTC CATGACTTGC A CAACAAAG241 GGCGACTTAT GTTTTATGAA GAATAAGAGC AAAAAAAGTC CATGACTTGC A CAACAAAG
301 AAAAAAACTT ATAATTAGAT TCATCAATCG CCCAAGTTTT CTTCTTCTTC TTTTTTTTAA301 AAAAAAACTT ATAATTAGAT TCATCAATCG CCCAAGTTTT CTTCTTCTTC TTTTTTTTAA
361 ATGT 361 ATGT
GhM6PR全长编码基因的克隆 Cloning of the full-length coding gene of GhM6PR
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。  Based on the sequence of SEQ ID No: 3 that has been obtained, the following two specific primers were designed as the 5'-end specific primer for 3' RACE.
GliM6PR GSPI : SEQ ID No: 4: GliM6PR GSPI : SEQ ID No : 4:
ATCGTTCTCC GGTGGGGAAT CC  ATCGTTCTCC GGTGGGGAAT CC
G!iM6PR GSF1： SEQ ID o; 5:  G!iM6PR GSF1: SEQ ID o; 5:
AT CCAACGAAAC ACGGTAGTCA 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）。  AT CCAACGAAAC ACGGTAGTCA Experimental procedure was performed according to the kit instructions (3 'RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
用 SEQ ID NO: 4与通用弓 I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：  The first round of PCR amplification was carried out using SEQ ID NO: 4 and the universal AUAP (provided with the kit), using mRNA reverse transcribed cDNA as a template. Specific steps are as follows:
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 μΐ 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 性 30 s， 58°C退火 30 s， 72 °C 延伸 lmin)， 72 °C 延伸 10 min。 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer 3 μΐ 2.5 mM dNTP, 2.0 μΐ mRNA reverse transcribed cDNA, 1.0 μΐ Ex Taq (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 4 and AUAP 2.0 1、1, and 35 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 min, 33 cycles (94 ° C change) Properties 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min), extension at 72 °C for 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与通 用引物 AUAP进行第二轮 PCR扩增， 具体步骤如下：  The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μL was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 5 and the universal primer AUAP. The specific steps are as follows:
50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ稀释的第一 轮 PCR产物、 1.0 l Ex Taq、 10 μΜ的引物 SEQ ID NO: 5和 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 °C 保存备用。用 SEQ ID NO: 5与通用引物 AUAP进行菌液 PCR扩增， 得到 5个阳性克 隆， 将其中 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。  50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 l Ex Taq, 10 μΜ primer SEQ ID NO: 5 and AUAP each 2.0 μ1, and 35 ΐ ΐ 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 into LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. Using SEQ ID NO: 5 and the universal primer AUAP to carry out bacterial PCR amplification, 5 positive clones were obtained, and 4 positive clones were sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing, and the cDNA of the gene was obtained. 3' end.
根据已经获得的 GhM6PR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。  Based on the obtained GhM6PR gene fragment, the following three specific primers were designed as the 3'-end specific primer of 5' RACE.
GhM6FR GSP3: SEQ ID ot 6:  GhM6FR GSP3: SEQ ID ot 6:
GTCCGATATT TCCGGTCAAT GG GI1M6P GSP4: SEQ- ID No; 7:  GTCCGATATT TCCGGTCAAT GG GI1M6P GSP4: SEQ- ID No; 7:
TTGTCCATGT CTTCTTTGGC TA  TTGTCCATGT CTTCTTTGGC TA
GhM6PR GSP5: SEQ: ID No :f 8: GhM6PR GSP5: SEQ: ID No : f 8:
GTTTCGTTGG ATTCCCCACC 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）。  GTTTCGTTGG ATTCCCCACC GG Experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit was purchased from Invitrogen).
用 SEQ ID NO: 7与 5'通用引物 AAP (试剂盒自带）， 以 mRNA逆转录的 cDNA SEQ ID NO: 7 and 5' universal primer AAP (provided with the kit), reverse-transcribed 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： (Reverse transcription primer SEQ ID NO: 6) The first round of PCR amplification was performed for the template. The specific steps are as follows:
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 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 性 30 s， 55 °C退火 30 s， 72 °C 延伸 lmin)， 72 °C 延伸 10 min。  50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer 3 μΐ 2.5 mM dNTP, 2.0 μΐ mRNA reverse transcribed cDNA, 1.0 μΐ Ex Taq (purchased from TAKARA), 10 μΜ primers SEQ ID NO: 7 and AAP 2.0 1、1, and 35 μΐ 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.
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 ΙΟ Εχ Buffer 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ稀释的第一 轮 PCR产物、 1.0 l Ex Taq、 10 μΜ的引物 SEQ ID NO: 8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 1 min)， 72 °C 延伸 10 min。 回收第二次 PCR产物中片段约为 800 bp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 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。 用 SEQ ID NO: 8与引物 AUAP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。 The obtained PCR product was diluted 50 times with double distilled water, and then 2.0 μL was used as a template, and the second round of PCR amplification was carried out with SEQ ID NO: 8 and primer AUAP. The specific steps are as follows: 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 l Ex Taq, 10 μΜ primer SEQ ID NO: 8 and AUAP each 2.0 μ1, and 35 ΐ ΐ 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 800 bp in the second PCR product (Gel Extraction Kit from OMEGA) was recovered and ligated into pGEM-T Easy Vector, and then transformed into JM109 (specific method as above). Ten white colonies were randomly picked and inoculated into LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. Using SEQ ID NO: 8 and primer AUAP for bacterial liquid PCR amplification (reaction system and reaction conditions as above), 7 positive clones were obtained, and 4 clones were selected and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing and sequencing. The 5' end of the cDNA of this gene was obtained.
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 GhM6PR全长 cDNA序列 SEQ ID No: 9：  After the obtained 5' RACE product was cloned and sequenced, it was spliced with the 3' RACE product sequencing result and the SEQ ID No: 3 sequence. The full-length cDNA sequence of GhM6PR was obtained SEQ ID No: 9:
1 GAGATAGCAG ATTTGATTTA CAGAGACCAA AGGGGGGGAA ATGGCGATCA CACTGAACAA 1 GAGATAGCAG ATTTGATTTA CAGAGACCAA AGGGGGGGAA ATGGCGATCA CACTGAACAA
61 TGGATTTAAG ATGCCAGTGG TTGGTTTAGG TGTTTGGCGT ATGGATGGAA AAGACGTTAG61 TGGATTTAAG ATGCCAGTGG TTGGTTTAGG TGTTTGGCGT ATGGATGGAA AAGACGTTAG
121 AGACCTCATC ATCAACTCCA TCAAGCTTGG TTATCGTCAT TTCGATTGTG CTGCTGACTA121 AGACCTCATC ATCAACTCCA TCAAGCTTGG TTATCGTCAT TTCGATTGTG CTGCTGACTA
181 CAAGAATGAA GCAGAAGTTG GTGAGGCATT GTCTGAAGCA TTCAAAACCG GGCTTGTTAA181 CAAGAATGAA GCAGAAGTTG GTGAGGCATT GTCTGAAGCA TTCAAAACCG GGCTTGTTAA
241 GAGAGAGGAC CTCTTTATTA CAACCAAGCT TTGGAATTCT GATCACGGGC ATGTTCTTGA241 GAGAGAGGAC CTCTTTATTA CAACCAAGCT TTGGAATTCT GATCACGGGC ATGTTCTTGA
301 GGCCTGTAAA GACAGTCTTA AGAAGCTTCA GTTGGATTAT CTGGATTTGT ATCTAGTTCA301 GGCCTGTAAA GACAGTCTTA AGAAGCTTCA GTTGGATTAT CTGGATTTGT ATCTAGTTCA
361 CTTTCCCGTT GCTGTAAAGC ACACTGGAGT CGGTCAAACG GGCAGTCCTT TGGACAAGGA361 CTTTCCCGTT GCTGTAAAGC ACACTGGAGT CGGTCAAACG GGCAGTCCTT TGGACAAGGA
421 TGGAGTCCTG GACATAGACA CCACTATATC GTTGGAGACT ACCTGGCACG CGATGGAAGA421 TGGAGTCCTG GACATAGACA CCACTATATC GTTGGAGACT ACCTGGCACG CGATGGAAGA
481 CTTGGTTTCC AAGGGTTTAG TTCGTAGCAT TGGGATCAGC AACTACGACA TCTTTTTAAC481 CTTGGTTTCC AAGGGTTTAG TTCGTAGCAT TGGGATCAGC AACTACGACA TCTTTTTAAC
541 AAGAGATTGC CTAGCTTACT CCAAGGTGAA GCCTGCTGTA AACCAGA AG AGACACATCC541 AAGAGATTGC CTAGCTTACT CCAAGGTGAA GCCTGCTGTA AACCAGA AG AGACACATCC
601 TTACTTCCAA CGTGATTGTC TTGTAAAATT TTGCCAGAAG CACGGAATCT GTGTTACTGC601 TTACTTCCAA CGTGATTGTC TTGTAAAATT TTGCCAGAAG CACGGAATCT GTGTTACTGC
661 TCACACTCCA TTAGGAGGTG CTGCTGCTAA TGCAGAATGG TTTGGAACAG TGTCATGTTT661 TCACACTCCA TTAGGAGGTG CTGCTGCTAA TGCAGAATGG TTTGGAACAG TGTCATGTTT
721 GGATGATCCT GTCCTTAAGG CGCTAGCTGA GAAGTACAAG AAAACCGTGG CTCAGATCGT721 GGATGATCCT GTCCTTAAGG CGCTAGCTGA GAAGTACAAG AAAACCGTGG CTCAGATCGT
781 TCTCCGGTGG GGAATCCAAC GAAACACGGT AGTCATCCCA AAGACA CAA AATTCGAGAG781 TCTCCGGTGG GGAATCCAAC GAAACACGGT AGTCATCCCA AAGACA CAA AATTCGAGAG
841 ATTGAAAGAG AATTTTGAGG TTTTTGACTT CGAGTTAGCC AAAGAAGACA TGGACAAAAT841 ATTGAAAGAG AATTTTGAGG TTTTTGACTT CGAGTTAGCC AAAGAAGACA TGGACAAAAT
901 CAAAGCCATT GACCGGAAAT ATCGGACCAA TCAACCTGCC AAGTTTTGGG GCATCGATCT901 CAAAGCCATT GACCGGAAAT ATCGGACCAA TCAACCTGCC AAGTTTTGGG GCATCGATCT
961 GTATGCTTAG AGTGCGATGC GGGTATTCTT TGTGTGGCGA CTTATGTTTT ATGAAGAATA961 GTATGCTTAG AGTGCGATGC GGGTATTCTT TGTGTGGCGA CTTATGTTTT ATGAAGAATA
1021 AGAGCAAAAA AAGTCCATGA CTTGCATCAA CAAAGAAAAA AACTTATAAT TAGATTCATC1021 AGAGCAAAAA AAGTCCATGA CTTGCATCAA CAAAGAAAAA AACTTATAAT TAGATTCATC
1081 AATCGCCCAA GTTTTCTTCT CTTCTTTTT TTTAAATGTA CCTAAGTTTC TTGTATGTTA1081 AATCGCCCAA GTTTTCTTCT CTTCTTTTT TTTAAATGTA CCTAAGTTTC TTGTATGTTA
1141 TGACATTTAC GTTTATCTGT ATGGATTGAG AATAATTTTT CTTTAAAAAA AAAAAAAAAA1141 TGACATTTAC GTTTATCTGT ATGGATTGAG AATAATTTTT CTTTAAAAAA AAAAAAAAAA
1201 AAAAAAA 根据 SEQ ID NO:9序列设计一对引物如下: SEQ ID No: 10: 1201 AAAAAAA A pair of primers were designed according to the sequence of SEQ ID NO: 9 as follows: SEQ ID No: 10:
ATGGCGATCACACTGAACAATGG ATGGCGATCACACTGAACAATGG
SEQ ID No: I I : SEQ ID No: I I :
CTAAGCATAC AGATCGATGC CCC 通过 SEQ ID NO: 10和 SEQ ID NO: 11来克隆 GhM6PR全长编码基因。  CTAAGCATAC AGATCGATGC CCC The GhM6PR full-length coding gene was cloned by SEQ ID NO: 10 and SEQ ID NO: 11.
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶， 以棉花的 cDNA为模板进行 PCR 反应。 50 μ1 ΡΟ 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: 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。  The PCR reaction was carried out using TaKaRa's PrimeSTAR HS DNA polymerase and cotton cDNA as a template. 50 μl ΡΟ Reaction system: 10 μΐ 5xPS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 10 and SEQ ID NO: 11 each 2.0 μl, and 30 μΐ double steamed 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 Ι ΟχΕχ Buffer, 0.5 μΐ 5 mM的 dATP、 1.0 μΐ Ex Taq。反应条件： 70°C反应 30分钟。将得到的约 1200bp 的 DNA片段回收 （Omega回收试剂盒）， 并将其连接至 pGEM T-easy载体上 （得到 GhM6PR-pGEM质粒） ，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 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。 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 6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 SEQ ID NO: 2， 其编码的蛋白质的氨基酸序列为 SEQ ID NO: 1。  The 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 μl of double distilled water. Then, 2.5 μΐ Ι ΟχΕχ Buffer, 0.5 μΐ 5 mM dATP, 1.0 μΐ Ex Taq were added thereto. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. The obtained DNA fragment of about 1200 bp was recovered (Omega recovery kit), and ligated to the pGEM T-easy vector (to obtain the GhM6PR-pGEM plasmid), and then transformed into JM109 (method as above). Ten white colonies were randomly picked and inoculated into LB liquid medium containing 50 g/ml ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of 20%, and stored at -80 ° C until use. PCR amplification of SEQ ID NO: 10 and SEQ ID NO: 11 (reaction system and reaction conditions as above), 6 positive clones were obtained, and 4 positive clones were selected and sent to Yingji Jieji (Shanghai). The company was sequenced and the sequence is SEQ ID NO: 2, and the amino acid sequence of the encoded protein is SEQ ID NO: 1.
M6PR蛋白的氨基酸序列: ： SEQ ID NO:  Amino acid sequence of M6PR protein: : SEQ ID NO:
1 MAITLNNGFK MPWGLGVWR  1 MAITLNNGFK MPWGLGVWR
21 MDGKDVRDLI INS IKLGYRH  21 MDGKDVRDLI INS IKLGYRH
41 FDCAADYKNE AEVGEALSEA  41 FDCAADYKNE AEVGEALSEA
61 FKTGLVKRED LFITTKLWNS  61 FKTGLVKRED LFITTKLWNS
81 DHGHVLEACK DSLKKLQLDY  81 DHGHVLEACK DSLKKLQLDY
101 LDLYLVHFPV AVKHTGVGQT  101 LDLYLVHFPV AVKHTGVGQT
121 GSPLDKDGVL DIDTTISLET  121 GSPLDKDGVL DIDTTISLET
141 TWHAMEDLVS KGLVRS IGIS  141 TWHAMEDLVS KGLVRS IGIS
161 NYDI FLTRDC LAYSKVKPAV  161 NYDI FLTRDC LAYSKVKPAV
181 NQIETHPYFQ RDCLVKFCQK  181 NQIETHPYFQ RDCLVKFCQK
201 HGICVTAHTP LGGAAANAEW  201 HGICVTAHTP LGGAAANAEW
221 FGTVSCLDDP VLKALAEKYK 241 KTVAQIVLRW GIQRNTWI P 221 FGTVSCLDDP VLKALAEKYK 241 KTVAQIVLRW GIQRNTWI P
261 KTSKFERLKE NFEVFDFELA  261 KTSKFERLKE NFEVFDFELA
281 KEDMDKIKAI DRKYRTNQPA  281 KEDMDKIKAI DRKYRTNQPA
301 KFWGIDLYA*  301 KFWGIDLYA*
Figure imgf000010_0001
实施例 3 GhM6PR基因植物表达载体构建
Figure imgf000010_0001
Example 3 Construction of GhM6PR Gene Plant Expression Vector
选择植物双元表达载体 pCAMBIA2300 (购自北京鼎国昌盛生物技术有限责任公 司） 作为植物表达载体， 用 Pnos启动子替换 ΝΡΤΠ基因含双增强子的 35S启动子， 以降低 ΝΡΤΠ蛋白在植物中的表达。 选择诱导型的 rd29A启动子及终止子 Tnos分别 作为 GhM6PR基因的启动子和终止子。  The plant binary expression vector pCAMBIA2300 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) was selected as a plant expression vector, and the 35S promoter containing the double enhancer of the ΝΡΤΠ gene was replaced with the Pnos promoter to reduce the expression of prion protein in plants. . The inducible rd29A promoter and the terminator Tnos were selected as promoters and terminators of the GhM6PR gene, respectively.
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， 采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μ1 ΡΟ 反应体系： 10 μΐ 5 xPS Buffer、 3 μΐ 2.5 mM的 dNTP、 1.0 μΐ ρΒΙ121 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 12禾 P SEQ ID NO: 13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， 56°C退火 30 s， 72 °C 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 Pnos was amplified using the primers SEQ ID NO: 12 and SEQ ID NO: 13 with the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μl ΡΟ Reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ ρΒΙ121 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 12 and P SEQ ID NO: 13 each 2.0 μ1, and 31 μΐ 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 30 s), extension at 72 °C for 10 min. The resulting PCR product was ligated by EcoRI, Bglll (promega, T4 ligase cassette) to pCAMBIA2300 to obtain pCAMBIA2300-1. SEQ ID NO: 12
GCACGAATTC ggcgggaaac gacaatctga  GCACGAATTC ggcgggaaac gacaatctga
SEQ ID NO: 13：  SEQ ID NO: 13:
ATCCAGATCTAGATCCGGTGCAGA TATTTG 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聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5><PS Buffer、 3 μ1 2.5 mM 的 dNTP、 1.0 μΐ pBI121、 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 14禾 P SEQ ID ^«): 15各2.0 ^、 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 Cpromega T4 连接酶盒;)到 pCAMBIA2300-l 获得 pCAMBIA2300-2。  ATCCAGATCTAGATCCGGTGCAGA TATTTG Tnos was amplified using primers SEQ ID NO: 14 and SEQ ID NO: 15 with pBI121 as a template, using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5><PS Buffer, 3 μl 2.5 mM dNTP, 1.0 μΐ pBI121, 1.0 μΐ PrimeSTAR, 10 μΜ primer SEQ ID NO: 14 and P SEQ ID ^«): 15 each 2.0 ^ , and 31 μΐ 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 cleaved by Sacl, EcoRI and ligated into the Cpromega T4 ligase cassette;) to pCAMBIA2300-l to obtain pCAMBIA2300-2.
SEQ ID NO; !4：：  SEQ ID NO; !4::
AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID NO: I S: AAGGAGCTCGAATTTCCCCGATCGTTCAAA SEQ ID NO: IS :
TCAGAATTCCCAGTGAATTCCCGATCTAGTA 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 应体系： 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禾 P 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。  TCAGAATTCCCAGTGAATTCCCGATCTAGTA The Arabidopsis thaliana rd29A promoter was amplified using primers SEQ ID NO: 16 and SEQ ID NO: 17 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 694-697 for extracting Arabidopsis DNA). PrimeSTAR HS DNA polymerase from TaKaRa was used. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ Arabidopsis DNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 16 and P SEQ ID NO: 17 each 2.0 μ1, and 31 11 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 HindIII and Pstl (connection method as above) to pCAMBIA2300-2 to obtain pCAMBIA2300-3.
SEQ ID NO: 16：  SEQ ID NO: 16:
ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO; 17；  ACTAAGCTTCCTTCTTGACATCATTCAATTTTA SEQ ID NO; 17;
TGACTGCAGTCCAAAGATTTTTTTCTTTC^CAATAG 用引物 SEQ ID NO: 18和 SEQ ID NO: 19扩增 GhM6PR编码基因的全长序列（模 板是实施例 2所获得阳性 GhM6PR-pGEM质粒），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 GhM6PR-pGEM、 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 18禾 P SEQ ID NO: 19 各 2.0 μ1、 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
Figure imgf000012_0001
TGACTGCAGTCCAAAGATTTTTTTCTTTC^CAATAG The full-length sequence of the GhM6PR-encoding gene was amplified with primers SEQ ID NO: 18 and SEQ ID NO: 19 (template was the positive GhM6PR-pGEM plasmid obtained in Example 2) using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5×PS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ GhM6PR-pGEM, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 18 and P SEQ ID NO: 19 2.0 μl, and 31 μΐ Double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 min, 33 cycles (94 ° C change) Properties 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 2 min), extension at 72 °C for 10 min. The obtained PCR product was ligated by Pstl and Sacl digestion (the ligation method is the same as above) to pCAMBIA2300-3 to obtain a plant expression vector.
Figure imgf000012_0001
SEQ ID NO: 1.8：  SEQ ID NO: 1.8:
TGACTGCAG ATGGCGATCACACTGAACAATGG SEQ ID NO: 19：  TGACTGCAG ATGGCGATCACACTGAACAATGG SEQ ID NO: 19:
AAGGAGCTC CTAAGCATAC AGATCGATGC CCC 实施例 4 rd29A-GhM6PR-2300表达载体转化农杆菌  AAGGAGCTC CTAAGCATAC AGATCGATGC CCC Example 4 rd29A-GhM6PR-2300 Expression Vector Transformation Agrobacterium
农杆菌 LBA4404 (购自 Biovector Science Lab,Inc) 感受态细胞的制备： 提前 1-2 天将农杆菌 LBA4404在含 50 g/ml利福平和 50 g/ml链霉素的 LB固体培养基上划 单斑接种， 28 °C培养 1至 2天。挑取单菌落接种于 5 ml含 50 μ§/ιη1利福平和 50 μ§/ιη1 链霉素的 LB液体培养基中， 28°C下摇动培养过夜 (约 12-16 h)至 OD6。。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§/ιη1利福平和 50 μ§/ιη1链霉素的 LB液体培养基中， 28°C摇动培养 2-2.5 h至 OD6QQ=0.8。 冰浴菌液 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。 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 inoculation, culture at 28 °C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 μ § /ιη1 rifampicin and 50 μ § /ιη1 streptomycin, and cultured overnight (about 12-16 h) to OD 6 at 28 °C with shaking. . A value of 0.4 forms a seed broth. 5 ml of activated bacterial solution (1:20 ratio) was inoculated into 100 ml of LB liquid medium containing 50 μ § /ιη1 rifampicin and 50 μ § /ιη1 streptomycin, and cultured at 28 ° C with shaking 2 2.5 h to OD 6QQ = 0.8. The ice bath liquid was shaken for 10 min every 3 min to allow the bacteria to enter the dormant state uniformly. 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 μΐ/tube, and stored at -70 °C until use.
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 rd29A-GhM6PR-2300质粒 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 μ§/ιη1链霉素、 50 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 利用农杆菌介导的转化法获得转基因烟草 Transformation of Agrobacterium: The competent cells were thawed on ice, and 1 μΐ of the plasmid was added to 40 μΐ of competent cells, and the mixture was mixed and ice bathed for about 10 minutes. A mixture of competent cells and rd29A-GhM6PR-2300 plasmid DNA was transferred to an ice-cold electric shock cup (purchased from bio-md) using a pipette, and tapped 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. When using a 0.1cm size electric shock cup, the MicroPulser (purchased from bio-rad) program is set to "Agr" and the 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. 100-200 μL of bacterial solution is applied to the corresponding resistant screening medium plate (LB solid medium containing 50 μ § /ιη1 rifampicin, 50 μ § /ιη1 streptomycin, 50 g/ml card Natamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Obtaining Transgenic Tobacco by Agrobacterium-mediated Transformation
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、 50μΜΚΙ、 100μΜΗ3ΒΟ3、 100 MMnSO4、 30 MZnSO4、 1 μΜΝα2Μο04 0.1 MCoCl2、 100 μΜΝα2ΕϋΤΑ 100 MFeSO4、 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-GhM6PR-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。 To soak tobacco seeds with 75% alcohol (National Tobacco Medium Term Bank, obtained by the Institute of Tobacco, Chinese Academy of Agricultural Sciences, library number I5A00660) 30 s, then wash twice with sterile double distilled water. Soak it in 0.1% liters of mercury for 8 min, then use Sterilize twice in steamed water to complete surface sterilization. Surface-sterilized tobacco seeds were placed in MS solid medium (containing 18.78 mM KN0 3 1.25 mM KH 2 P0 4 , 20.6 mM H 4 N0 3 1.5 mM MgS0 4 , 3.0 mM CaCl 2 , 50 μΜΚΙ, 100 μΜΗ 3 ΒΟ 3 , 100 MMnSO 4 , 30 MZnSO 4 , 1 μΜΝα 2 Μο0 4 0.1 MCoCl 2 , 100 μΜΝα 2 ΕϋΤΑ 100 MFeSO 4 , 7.4 g/1 agar, 30 g/1 sucrose) were prepared by germination under sterile conditions to prepare sterile seedlings. 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-GhM6PR-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. After the buds are grown, they 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.
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 SEQIDNO: 10和 SEQIDNO: 11进行 PCR扩增鉴定（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 SEQIDNO: 10和 SEQIDNO: 11各 2.0 μ1、 以及 35 μΐ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， 72 °C 延伸 2min)， 72 °C 延伸 10 min), 将 PCR鉴定为阳性植株编号为 T0P1-T0P20并保存。 实施例 6 过表达 GhM6PR转基因烟草 T1代植株的抗旱模拟实验  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 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer 3 μΐ 2.5 mM dNTP, 2.0 μΐ DNA, 1.0 μΐ Ex Taq, 10 μΜ primers SEQ ID NO: 10 and SEQ ID NO: 11 each of 2.0 μl, and 35 μΐ 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 identified as positive plants numbered T0P1-T0P20 and stored. Example 6 Overexpression of GhM6PR Transgenic Tobacco T1 Generation Drought Resistance Simulation Experiment
灭过菌的蛭石用 1/2MS培养基浸透。 T0P1— T0P20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、 14小时光培养 /10小时暗培养循环。 每周浇一次 1/2MS, 培养 25天之后， SEQIDNO: 10和 SEQIDNO: 11做 PCR检测， 去除阴性植株，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、 14小时光培养 /10小时暗培养循环。 T1代转基因植株（T0 代转基因植株的种子长成的植株）的抗旱性鉴定表明，对照植株都萎蔫严重，而 T1P5、 T1P7、 T1P15、 T1P17表现出明显的抗旱性 （见图 3a和 3b， 以 T1P5为例， T1P7、 T1P15、 T1P17的结果与 T1P5类似， 在此未示出）。 实施例 7 在转录水平上验证 M6PR蛋白表达  The sterilized vermiculite was soaked in 1/2 MS medium. The seeds of T0P1-T0P20 transgenic tobacco and control tobacco were separately sown on vermiculite, and 15 seeds were seeded per pot at 25 ° C, 14 hours light culture / 10 hours dark culture cycle. 1/2MS was poured once a week, and after 25 days of culture, SEQ ID NO: 10 and SEQ ID NO: 11 were subjected to PCR detection to remove negative plants, and 4-5 seedlings of uniform size were retained per pot for drought experiments, transgenic tobacco, control tobacco Drought for 14 days (without watering), 25 ° C, 14 hours light culture / 10 hours dark culture cycle. The drought resistance of T1 transgenic plants (plants grown from T0 transgenic plants) showed that the control plants were wilting, while T1P5, T1P7, T1P15 and T1P17 showed significant drought resistance (see Figures 3a and 3b). For example, T1P5, the results of T1P7, T1P15, and T1P17 are similar to T1P5, which is not shown here). Example 7 Verification of M6PR protein expression at the transcriptional level
实施例 6中抗旱性好的 T1代转基因植株中随机选取 8棵 （分别属于上述 4个抗 旱株系），实施例 6中对照植株选取 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反转录试剂盒 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: 20扩增 GhM6PR， 检测 M6PR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 μΐ PCR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: 20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为耐干旱 T1代转基 因烟草植株， 9为质粒 PCR阳性对照 （rd29A-GhM6PR-2300质粒）， 10-13为不耐干 旱对照烟草植株。 图中所示条带大小与 GhM6PR的大小一致 （约为 900bp)。 结果表 明， 耐干旱 T1代转基因烟草植株中 GhM6PR的转录较强， 不耐干旱对照烟草植株没 有 GhM6PR转录。 Among the T1 transgenic plants with good drought resistance in Example 6, 8 were randomly selected (four drought-tolerant strains respectively), and 4 of the control plants in Example 6 were selected, and 0.05 g of leaves were cut for 14 days. Total RNA was extracted from the plant RNA extraction kit (invitrogen). Ultraviolet spectrophotometric determination of total RNA at 260 nm and 280 nm The absorbance values were calculated for each RNA concentration. Reverse transcription was carried out according to the method shown by the invitrogen reverse transcription kit Superscript III Reverse Transcriptase (1 total RNA as a template, reverse transcription primer SEQ ID NO: 11). The relative expression of M6PR protein was detected by amplifying GhM6PR by SEQ ID NO: 10 and SEQ ID NO: 20. The PCR reaction was carried out using reverse-transcribed cDNA as a template using TaKaRa's PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA, 1.0 μΐ PrimeSTAR, 10 μΜ primers SEQ ID NO: 10 and P SEQ ID NO: 20 each 2.0 μl, and 30 μΐ Double steamed water. PCR reaction conditions: pre-denaturation at 94 °C for 5 min, 29 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: M is DNA Ladder Marker (DL2000, purchased from Shenzhen Ruizhen Biotechnology Co., Ltd.), 1-8 is a drought-tolerant T1 transgenic tobacco plant, and 9 is a plasmid PCR positive control (rd29A-GhM6PR). -2300 plasmid), 10-13 is a drought-tolerant control tobacco plant. The band size shown in the figure is consistent with the size of GhM6PR (approximately 900 bp). The results showed that the transcription of GhM6PR in transgenic T1 transgenic tobacco plants was stronger, and the drought-tolerant control tobacco plants did not have GhM6PR transcription.
SEQ ID NO: 20: SEQ ID NO: 20:
GGCAGGTTGA TTGGTCCGAT A  GGCAGGTTGA TTGGTCCGAT A

Claims

权 利 要 求 书 Claim
1. 棉花的一个甘露糖 -6-磷酸还原酶的编码基因， 被命名为 GhM6PR， 其核苷酸 序列如 SEQ ID NO: 2所示。 1. A gene encoding a mannose-6-phosphate reductase of cotton, designated GhM6PR, having a nucleotide sequence as shown in SEQ ID NO: 2.
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。  2. A recombinant expression vector comprising the gene of claim 1 and the nucleotide sequence of the gene operably linked to an expression control sequence of the expression vector.
3. 权利要求 2所述的载体， 其为附图 2所示的 rd29A-GhM6PR-2300载体。 3. The vector of claim 2 which is the rd29A-GhM6PR-2300 vector shown in Figure 2.
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。 A recombinant cell comprising the gene of claim 1 or the recombinant expression vector of claim 2 or 3; preferably, the recombinant cell is a recombinant Agrobacterium cell.
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。  A method for improving drought resistance of a plant, comprising: introducing the gene of claim 1 or the recombinant expression vector of claim 2 or 3 into a plant or plant tissue and expressing the gene; preferably, the The plant is tobacco.
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。  A method for producing a transgenic plant, comprising: cultivating a plant or plant tissue comprising the gene of claim 1 or the recombinant expression vector of claim 2 or 3 under conditions effective to produce a plant.
7. 权利要求 6所述的方法， 其中所述植物是烟草。  7. The method of claim 6 wherein the plant is tobacco.
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。  The gene of claim 1, the recombinant expression vector of claim 2 or 3, or the recombinant cell of claim 4 for use in improving drought resistance of a plant and for use in plant breeding.
9. 权利要求 8所述的用途， 其中所述植物是烟草。  9. The use of claim 8 wherein the plant is tobacco.
10. 权利要求 1所述的基因编码的蛋白， 其氨基酸序列如 SEQ ID NO: 1所示。  The gene-encoded protein according to claim 1, which has an amino acid sequence as shown in SEQ ID NO: 1.
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Citations (2)

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US6416985B1 (en) * 1996-10-15 2002-07-09 Board Of Trustees Of Michigan State University DNA encoding mannose 6-phosphate reductase and recombinants produced therefrom
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WO2008034648A1 (en) * 2006-04-05 2008-03-27 Metanomics Gmbh Process for the production of a fine chemical

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