WO2014169482A1 - Thellungiella halophila molybdenum enzyme cofactor sulfurized enzyme mcsu-2, coding genes of same, and application thereof - Google Patents

Thellungiella halophila molybdenum enzyme cofactor sulfurized enzyme mcsu-2, coding genes of same, and application thereof Download PDF

Info

Publication number
WO2014169482A1
WO2014169482A1 PCT/CN2013/074452 CN2013074452W WO2014169482A1 WO 2014169482 A1 WO2014169482 A1 WO 2014169482A1 CN 2013074452 W CN2013074452 W CN 2013074452W WO 2014169482 A1 WO2014169482 A1 WO 2014169482A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
plant
workers
gene
expression vector
Prior art date
Application number
PCT/CN2013/074452
Other languages
French (fr)
Chinese (zh)
Inventor
陈文华
孙超
崔洪志
Original Assignee
创世纪转基因技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 创世纪转基因技术有限公司 filed Critical 创世纪转基因技术有限公司
Priority to CN201380074524.9A priority Critical patent/CN105008389B/en
Priority to PCT/CN2013/074452 priority patent/WO2014169482A1/en
Publication of WO2014169482A1 publication Critical patent/WO2014169482A1/en

Links

Classifications

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

Definitions

  • the present invention relates to plant proteins and coding genes thereof and applications thereof, and more particularly to a molybdenum coenzyme factor vulcanase MCSt-2 derived from small salt mustard and a gene encoding the same, and the same in the cultivation of transgenic plants with improved drought tolerance application.
  • BACKGROUND OF THE INVENTION Stresses such as temperature, salting and drought can cause serious damage to the growth and development of higher plants, resulting in reduced crop yields, degraded quality, and serious threats to agricultural production and the natural environment. Among them, the impact of drought on crop yields ranks first in many natural adversities, and its harm is equivalent to the sum of other disasters. Many regions are the bottleneck of agricultural development.
  • 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 2 million to 2.7 million hectares.
  • the first aspect of the present invention provides a gene encoding a molybdenum coenzyme vulcanase MC ⁇ /-2 of the small salt mustard (herein named ThMCSU-2, the sequence of which is SEQ ID NO: 2.
  • a second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into an expression vector, preferably, the expression vector is pCAMBIA2300; And the nucleotide sequence of the gene is operably linked to the expression control sequence of the recombinant expression vector; preferably, the recombinant expression vector is -ThMCSU-2-2, vector shown in Figure 2.
  • the third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.
  • a fourth aspect of the present invention provides a method for improving drought tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and causing the gene Expression;
  • the plant is Arabidopsis thaliana.
  • a fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or a plant comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant Tissue;
  • the plant is Arabidopsis thaliana.
  • a sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving drought tolerance of a plant and for use in plant breeding Use;
  • the plant is Arabidopsis thaliana.
  • the seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, the amino acid sequence of which is shown in SEQ ID NO: 1.
  • Fig. 1 is a construction flow of a plant expression vector (35S-r/zMG ⁇ /-2-2300) of ThMCSU-2 (Fig. la-lb).
  • Figure 2 is a plasmid map of the plant expression vector of ThMCSU-2 (5S-ThMCSU-2-7300).
  • Figure 3 shows the results of drought tolerance simulation experiments of r/zMC ⁇ /-2 T2 transgenic Arabidopsis plants (in the figure, T2H3; T2H4) and non-transgenic Arabidopsis plants (Fig., CK1, CK2) as controls.
  • Fig. 3a is an Arabidopsis plant that is normally grown for 20 days
  • Fig. 3b is an Arabidopsis plant that has been treated for 14 days after normal growth for 20 days).
  • FIG. 4 shows T2 transgenic Arabidopsis plants under drought stress and normal growth conditions and control plants ABA containing The amount of change detection results.
  • 1-8 are the lines: T2H1, T2H2, T2H3, T2H4, T2H5, T2H6, CK1, CK2, wherein T2H1, T2H2, T2H3, T2H4, T2H5, T2H6 are transgenic plants, and CK1 and CK2 are control plants.
  • FIG. 5 shows the results of molecular level detection of the ThMCSU-2 gene at the transcriptional level in T2 transgenic Arabidopsis plants and non-transgenic control plants by reverse transcription PCR.
  • M is DNA Ladder Marker (DL2000, TakaRa)
  • 1-4 is a transgenic Arabidopsis T2 plant with insignificant drought tolerance
  • 5-11 is a transgenic Arabidopsis T1 plant with significant drought tolerance (in order: ⁇ 2 ⁇ 1) ⁇ 2 ⁇ 2, ⁇ 2 ⁇ 3, ⁇ 2 ⁇ 4, ⁇ 2 ⁇ 5, ⁇ 2 ⁇ 6, ⁇ 2 ⁇ 7)
  • 12-17 are non-transgenic Arabidopsis plants.
  • the SSH library (subtractive library) was constructed by inhibition subtractive hybridization according to the method described in Clontech's PCR-selectTM cDNA Subtraction Kit kit. During the experiment, the mRNA of the leaves of the small salt mustard seedlings treated with drought during the growth process was used as a sample (Tester), and the mRNA of the leaves of the untreated small salt mustard seedlings was used as a control. Specific steps are as follows:
  • Small salt mustard (T ellungiella halophila, purchased from the Wulanbu and Desert Green Botanical Garden of Halophytes in Bayannao, Inner Mongolia, China) was sown on sterilized vermiculite at 25 ° C, photoperiod 16 hours light / 8 hours Incubate under dark conditions (light intensity 2000-3000 Lx), and pour 1/2 MS medium per week (9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM NH 4 N0 3 , 0.75 mM MgS0 4 , 1.5 mM CaCl 2 , 50 ⁇ KI, 100 ⁇ ⁇ 3 ⁇ 3 , 100 M MnSO 4 , 30 ⁇ ZnS0 4 , 1 ⁇ ⁇ 2 ⁇ 0 4 , 0.1 ⁇ CoCl 2 , 100 ⁇ Na 2 EDTA, 100 M FeSO 4 ). It was used for experiments when the seedlings reached a diameter of 5-6 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, cultured at 25 ° C, photoperiod of 16 hours light / 8 hours dark, and normally watered with 1/2 MS medium.
  • the second group is the arid area In the group, culture at 25 °C, photoperiod of 16 hours light/8 hours dark, stop watering, treat for 10 days, then cut the leaves of the top 1/3 of the seedlings in two groups in time, and then quickly freeze with liquid nitrogen, then - Store in a 70 °C refrigerator.
  • the method according to Clontech's PCR-select TM cDNA Subtraction Kit kit instructions will be shown suppression subtractive hybridization.
  • the Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and 2 ⁇ g of Tester cDNA and 2 ⁇ g of Driver cDNA were used as starting materials for subtractive hybridization.
  • the Tester cDNA and Driver cDNA were digested with Rsa I for 1.5 hours in a 37 ° C water bath, and then the digested Tester cDNA was divided into two equal portions, and the different linkers were ligated, and the Driver cDNA was not ligated.
  • Two tester cDNAs with different adaptors were mixed with excess Driver cDNA for the first forward subtractive hybridization.
  • the two first subtractive hybridization products were mixed and subjected to a second forward subtractive hybridization with the newly denatured Driver cDNA, and then the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.
  • this experiment In order to increase the validity of the Expressed Sequence Tag (EST) (Unigene), avoid the gene-free cleavage site and the obtained sequence in the untranslated region, this experiment simultaneously uses the endonuclease Haelll to tester cDNA according to the above steps.
  • the P Driver cDNA was digested and subjected to two forward subtractive hybridizations and two inhibitory PCR amplifications, and finally the second inhibitory PCR products of the two sets of forward subtractive hybridization cDNA fragments were combined.
  • the second PCR amplification product of the combined forward subtractive hybridization cDNA fragment was purified according to the method described in the product specification of pGEM-T Easy kit (purchased from Promega kit) (purified by QIAquick PCR Purification Kit, purchased from QIAquick PCR Purification Kit) Qiagen) is ligated to the pGEM-T Easy vector.
  • the specific steps are as follows: The following components are sequentially added to the 200 ⁇ l ⁇ tube: The second inhibitory PCR product of the purified combined positive subtractive hybridization cDNA fragment 3 ⁇ 1, 2xT4 DNA ligase buffer 5 ⁇ l, pGEM-T Easy vector 1 ⁇ 1, ⁇ 4 DNA ligase 1 ⁇ l, and ligated overnight at 4 °C.
  • the picked white colony clones were inoculated into LB liquid medium containing 50 ⁇ / ⁇ 1 ampicillin in 96-well cell culture plate (CORNING), and cultured overnight at 37 ° C, and glycerin was added to a final concentration of 20% glycerol. (volume ratio), then stored at -80 ° C for later use.
  • the colony clones were cultured by nested PCR primers Primer 1 and Primer 2R (PCR-selectTM cDNA Subtraction Kit from Clontech) for PCR amplification, and 166 positive clones were obtained, and then all positive clones were obtained. Sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • sequence After removing the redundant DNA from the clone of the colony Gh-B001 in the identified SSH library of the small salt mustard, the sequence is SEQ ID No: 3. Sequence analysis indicated that the encoded amino acid sequence of the sequence was sulfided with the known molybdenum cofactor. The amino acid sequence of the enzyme has high homology.
  • the full-length coding gene corresponding to the clone YLS-1 is named J/zMC ⁇ /-2, and the corresponding protein is named MCSU-2.
  • AAACACTGCA TGAACAGAAA CAAGAGTCCT GGTTTGTGCA GAGAATTAGA AAGCAATATC
  • SEQ ID No: 3 is the coding gene MC ⁇ -2
  • YLS-1GSP1 SEQ ID NO: 4:
  • YLS-1GSP2 SEQ ID NO: 5:
  • YLS-1 GSP3 SEQ ID NO: 6:
  • the kit comes with universal primers:
  • AAP SEQ ID NO: 7:
  • the GGCCACGCGTCGACTAGTAC experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
  • YLS-1GSP1 (SEQ ID NO: 4) was used as a reverse transcription primer, and the mRNA extracted from the leaves of the salt-treated mustard was used as a template for reverse transcription to obtain a cDNA template, and then according to the above 5' RACE kit instructions.
  • the first step of PCR amplification was carried out by adding the tail of the Poly C tail to the template.
  • the primers used were SEQ ID NO: 4 and the universal primer SEQ ID NO: 7 (the kit is self-contained, and I is hypoxanthine). Modified a, c, g or t), the 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 SEQ ID NO: 7 each of 2.0 ⁇ and 35 ⁇ of double distilled water.
  • PCR reaction conditions predenaturation at 94 °C for 5 minutes, 33 cycles (denaturation at 94 °C for 50 seconds, annealing at 54 °C for 50 seconds, extension at 72 °C for 1 minute), extension at 72 °C for 10 minutes.
  • the obtained PCR product was diluted 50-fold with double distilled water, and 2.0 ⁇ L was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 5 and the universal primer SEQ ID NO: 8.
  • 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 ⁇ Ex Taq, 10 ⁇ primers SEQ ID NO: 5 and SEQ ID NO: 8 2.0 ⁇ l, and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 50 seconds, annealing at 54 ° C for 50 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes.
  • a band of about 1 Kbp in the second PCR product (Glue Extraction Kit was purchased from OMEGA) was recovered and ligated into the pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (the same method as above) And screened on LB solid medium containing 50 g/mL ampicillin. Ten white colonies were randomly picked and inoculated in LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 ampicillin. After incubation at 37 °C overnight, glycerol was added to a final concentration of glycerol of 20% (volume ratio), -80 °C. Save spare.
  • the primers SEQ ID NO: 5 and the 3' primer SEQ ID NO: 6 were used for PCR amplification (reaction system and reaction conditions as above), and three positive clones (YJ1-1, YJl-2, YJ1-3) were obtained. , sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. to sequence and sequence, to obtain a 5' end sequence of the cDNA of the gene.
  • SEQ10 GSP1 SEQ ID NO: 11:
  • GSP2 SEQ ID NO: 12: AGAACACATACACCAGTCCCG
  • the experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen). Using SEQ ID NO: 1 1 as a reverse transcription primer, reverse transcription was performed using the mRNA extracted from the leaves of the salt-treated mustard leaves as a template to obtain a cDNA template, and then adding Poly C according to the procedure in the above 5' RACE kit instructions. The first round of PCR amplification was carried out using the tailed product as a template.
  • the primers used were SEQ ID NO: 11 and the universal primer SEQ ID NO: 7 (the kit is self-contained, I is a hypoxanthine modified a, c, g or t), the specific steps are as follows:
  • PCR reaction conditions pre-denaturation at 94 °C for 5 minutes, 33 cycles (94 °C for 45 seconds, 57 °C for 45 seconds, 72 °C for 90 seconds), 72 °C for 10 minutes.
  • the obtained PCR product was diluted 50-fold with double distilled water, and 2.0 ⁇ L was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 12 and the general primer SEQ ID NO: 8.
  • 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 ⁇ primers SEQ ID NO: 12 and SEQ ID NO: 8 2.0 ⁇ and 35 ⁇ double distilled water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation for 45 seconds at 94 ° C, annealing for 45 seconds at 58 ° C, extension of 90 ° at 90 ° C), extension at 72 ° C for 10 minutes.
  • the band of about 1.1K bp in the second PCR product was recovered (the Gel Extraction Kit was purchased from OMEGA), and ligated into the pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (the same method as above) ) and screening on LB solid medium containing 50 g/mL ampicillin. Eight white colonies were randomly picked and inoculated in LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of glycerol of 20% (volume ratio), -80 ° C Save spare.
  • the primers SEQ ID NO: 12 and the 3' primer SEQ ID NO: 13 were used for PCR amplification (reaction system and reaction conditions as above), and three positive clones (Ts-2, Ts-3 and Ts-5) were obtained. , sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. to sequence, obtain a 5' end sequence of the cDNA of the gene.
  • the obtained second round of 5' RACE product clone Ts-5 was sequenced to obtain SEQ ID NO: 14: 1 GGGGGGGGGG ATGGAAGAAT TTCTTAAGGA ATTTGGAGAT TATTATGGAT ACCCAGAAGG 6 1 TCCAAAGAGT ATCAAAGAGA TCCGCGATAC TGAATTCAAG AGATTAGACG AAGGTGTTGT 12 1 ATACTTGGAT CATGCTGGTT CTACTTTGTA TTCTGAGTTG CAGATGGAAG ACATCTTTAA 181 GGACTTTACA AGCAATGTTT ATGGCAATCC TCATAGTCAA AGTGATATCA GTTCAGCGAC
  • SEQ ID NO: 14 The sequence of SEQ ID NO: 14, obtained by the second round of 5' RACE, was spliced with the sequence SEQ ID NO: 10 to obtain SEQ ID NO: 15:
  • a pair of primers were designed according to the sequence of SEQ ID NO: 15 as follows:
  • ThMCSU-2F SEQ ID NO: 16:
  • ThMCSU-2R SEQ ID NO: 17:
  • TTATTCAGTATTTGGATTTACTTC AP SEQ ID NO: 18:
  • ThMCSU-2 The full-length coding sequence of ThMCSU-2 was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.
  • the small salt mustard RNA was extracted, and the primer SEQ ID NO: 18 was used as a reverse transcription primer to obtain a small salt mustard cDNA.
  • PCR reaction conditions 5 ⁇ lO PfuUltra II reaction Buffer 0.5 ⁇ 25 mM dNTP, 2.0 ⁇ cDNA 1.0 ⁇ PfuUltra II Fusion HS DNA Polymerase 10 ⁇ primers SEQ ID NO: 16 and SEQ ID NO: 17 each
  • the PCR amplification product was added with A tail: The PCR product was added with 2.5 volumes of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, and the supernatant was removed, dried, and dissolved in 21 ⁇ l of double distilled water. Add 2.5 ⁇ ⁇ ⁇ Buffer, 0.5 ⁇ 5 mM dATP, 1.0 ⁇ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 2.4 Kbp was recovered (Omega recovery kit), ligated into the pGEM T-easy vector (the ThMCSU-2-pGEM plasmid was obtained), and then transformed into JM109, and 8 white colonies were randomly picked and inoculated to contain 50.
  • the primers SEQ ID NO: 16 and SEQ ID NO: 17 were used for PCR amplification (reaction system and reaction conditions are the same as above), and three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing.
  • the sequence is SEQ ID NO: 2.
  • the amino acid sequence of the encoded protein is SEQ ID NO: 1.
  • Amino acid sequence of MCSU-2 protein SEQ ID NO: 1
  • VVOVVVVDDD V-worker; D-worker £)£) ⁇ V£)Worker:)Worker SSSV:)Worker V£)Worker 89 ⁇ Work V£)Work VDSI S Work SSVDVVS Workman:) Work £)Work 3 Workers and workers V£) £)Works 3£)Works £)Worksman ⁇ 9 ⁇ Work vv :) V workers VIVIDIDV workers SVIDIVVV workers; D D11DV1DVVV ; DID workers; DIDIVV SVVV workers SIVVS workers; ; DID workers; DVV; D workers; DV workers £) £) V; workers vvsmv TOST workers:):) V; D workers K) workers and workers VID workers SVV workers VIDIV; Workers V£) ⁇ ; D ⁇ 11VVVOVVDD £)£)V£)VV£) ⁇
  • LLDLL LLLLLLDLLLL V:) £)£) ⁇ £) £) ⁇ :) ⁇ :) £)£)1V ⁇
  • VVVVV work VIDVID workman D1V workman £)VV£) work;
  • VVVID engineering 3£)£) ⁇ 0VD0D1V10V £) ⁇ ; D ⁇ VID ⁇ £)£)V£) ⁇ T9S ⁇ V£)£)V£m ⁇ OVVVOVOVVO VVVV factory D ⁇ ; D£) ⁇ VVID ⁇ ; D £) ⁇ 3£) ⁇ 6 ⁇ ; DV ⁇ ; VVVVID ⁇ ; D£) ⁇ VS ⁇ £ ) ⁇ 3:) ⁇ :) ⁇ :) ⁇ :) ⁇ :) ⁇ :) £) ⁇ £)£) ⁇ :)£)£) ⁇ ; DV ⁇ ; DIDIVIIS £) ⁇ V ⁇ £)VV ; DV work; D ⁇ VIDIS IXJ 0 IXJ IXJ 0 IXJ IXJ 0 IXJ IXJ Worker VSVDSI :) IQL worker visvssms worker vv; DIDVID DV1DVDV1DO worker si ssvvv:) DSI SIVS worker V £) workman; D
  • LLLLLLVLLLLLLVVLL V 3£)1V£)V£) work £)V£) VVOOVVVVDD work; DVVV work; D work; D work V
  • VV workers Workers V1V Workers : ) £) Workers £) £) V Workers VIDSIV Workers VVS
  • 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 35S promoter containing the double enhancer and the Tnos terminator were selected as promoters and terminators of the ThMCS f/-2 gene, respectively.
  • the construction flow chart is shown in Figure 1.
  • primers SEQ ID NO: 19 and SEQ ID NO: 20 Pnos was amplified using the plant expression vector pBI121 (purchased from Beijing Huaxia Ocean Technology Co., Ltd.) as a template, and TaKaRa's PrimeSTAR HS DNA polymerase was used. 50 ⁇ l ⁇ Reaction system: 10 l 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 1.0 ⁇ ⁇ 121 1.0 ⁇ PrimeSTAR 10 ⁇ primers SEQ ID NO: 19 and SEQ ID NO: 20 each 2.0 ⁇ and 31 ⁇ double steamed water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes.
  • the resulting PCR product was digested with EcoRI and Bglll and ligated into pCAMBIA2300 (Promega, T4 ligase kit) to obtain pCAMBIA2300-1.
  • PrimeSTAR HS DNA polymerase 50 ⁇ PCR reaction system: 10 ⁇ 5 > ⁇ PS Buffer, 3 ⁇ 2.5 mM dNTP 1.0 ⁇ pBI121 1.0 ⁇ PrimeSTAR, 10 ⁇ primer SEQ ID NO: 16 and SEQ ID NO:
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes.
  • the resulting PCR product was digested with Kpnl, EcoRI and ligated into pCAMBIA2300-1 (Promega T4 ligase kit) to obtain pCAMBIA2300-2.
  • SEQ ID NO: 23 and SEQ ID NO: 24 amplify Arabidopsis thaliana 35S with pCAMBIA2300 plasmid as template Promoter.
  • PCR reaction conditions predenaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes.
  • the obtained PCR product was ligated by HindIII and Xbal digestion (connection method is the same as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3
  • SEQ ID NO: 25 and SEQ ID NO: 26 Amplification of the r/zMC ⁇ -2-pGEM plasmid containing the full-length ThMCSU-2 gene cloned in Example 2 as a template, with the designed restriction enzymes at both ends of the amplification The site of the ThMCSU-2 gene. Stratagene's PfuUltra II Fusion HS DNA Polymerase was used.
  • PCR reaction system 5 ⁇ lO PfuUltra II Reaction Buffer 0.5 ⁇ 25 mM dNTP, 2.0 ⁇ ThMCSU-2-pGEM plasmid, 1.0 ⁇ PfuUltra II Fusion HS DNA Polymerase, 10 ⁇ primer SEQ ID NO: 20 and SEQ ID NO: 21 each of 2.0 ⁇ and 37.5 ⁇ of double distilled water.
  • PCR reaction conditions pre-denaturation at 95 °C for 2 minutes, 35 cycles (denaturation at 95 °C for 25 seconds, annealing at 51 °C for 30 seconds, extension at 72 °C for 90 s), extension at 72 °C for 5 minutes.
  • the resulting PCR product was ligated by Xbal and Kpnl (connection method as above) to pCAMBIA2300-3 to obtain a plant expression vector 35S-r/zMC ⁇ 7-2-2300.
  • Agrobacterium LBA4404 (purchased from Biovector Science Lab, Inc) Preparation of Competent Cells: Agrobacterium LBA4404 was plated on LB solid medium containing 50 g/ml rifampicin and 50 g/ml streptomycin 1-2 days in advance Single spot inoculation, culture at 28 ° C for 1 to 2 days. Single colonies were picked and inoculated into 5 ml of LB liquid medium containing 50 ⁇ ⁇ / ⁇ 1 rifampicin and 50 ⁇ ⁇ / ⁇ 1 streptomycin, and cultured overnight (about 12-16 hours) to OD 6 at 28 °C. The K) value is 0.4, and a seed bacterial liquid is formed.
  • Transformation of Agrobacterium Melt LBA4404 competent cells on ice, add 1 ⁇ of the positive 35S-r/zMC ⁇ /-2-2300 plasmid obtained in Example 3 to 40 ⁇ of the competent cells, mix and ice Bath for about 10 minutes. Transfer the mixture of the competent cells and the 35S-r/zMC ⁇ /-2-2300 plasmid after the ice bath to the ice pre-cooled electric shock cup with a pipette, tap to bring the suspension to the bottom, be careful not to have bubble. Place the electric shock cup (purchased from Bio-Rad) on the slide of the electric shock chamber and push the slide to place the electric shock cup on the base electrode of the electric shock chamber.
  • Bio-Rad purchased from Bio-Rad
  • the MicroPuMUer purchased from Bio-Rad
  • the MicroPuMUer is set to "Agr" and the shock is applied once.
  • the electric shock cup is added 200 ⁇ l of ⁇ medium pre-warmed at 28 °C.
  • the suspension was transferred to a 1.5 ml centrifuge tube and incubated at 225 rpm for 1 hour at 28 °C.
  • Plant to be transformed Arabidopsis seeds (Columbia type, from the Arabidopsis Bioresources Center of the Ohio State University (www.arabidopsis.org) sown in peat soil, treated at 4 ° C for 3 days, placed at 23 Germination in a incubator at °C, 16 hours light/8 hours dark. After 7-10 days, transplant to a plastic crucible with a diameter of 7.5 cm containing peat and vermiculite (3:1 by volume). 6 strains were grown in an incubator at 23 ° C, 16 hours light / 8 hours dark. Before transplanting, 40 ml of 1/2MS medium was poured per pot, and the soil moisture was added to the soil moisture in time to improve the growth period. Water 1/2 MS medium.
  • the first inflorescence is cut off after the first inflorescence of most plants is formed. , remove the apical advantage, and promote the simultaneous emergence of multiple secondary inflorescences. Prepare for dip when most inflorescences are about 1-10 cm high (4-8 days after cutting the first inflorescence).
  • Culture of Agrobacterium After removing the bacterial solution of the Agrobacterium-positive transformed clone preserved in Example 4, pick a single colony of Agrobacterium and inoculate it into 10 ml of sterile LB liquid medium (containing 75 mg/1 rifampicin). 100 mg/1 streptomycin and 100 mg/1 kanamycin were shaken overnight at 250 rpm at 28 °C.
  • the obtained bacterial liquid is 1% - 2%
  • Inflorescence dip dyeing The above Agrobacterium-containing dyeing medium was added to a large-mouth container, and 200-300 ml of the Agrobacterium-containing dyeing medium was added to each container having a diameter of 9 cm for dip dyeing. Invert the plants so that the above ground tissues are all immersed in the Agrobacterium suspension for 3-5 seconds and gently agitate. There should be a liquid film on the plant after infiltration. Dip-infected plants are placed in plastic trays, covered with a clean plastic or cling film to moisturize them, and then placed in low light or dark places for the night, taking care to prevent direct sunlight from shining on the plants. Remove the cover approximately 12-24 hours after processing. The plants are cultured normally, and the plants are further grown for 3-5 weeks until the pods are browned and dried. Seeds of the T1 generation were harvested, and the seeds were stored in a centrifuge tube at 4 ° C.
  • Transgenic seed screening Prepare 1/2 MS medium (9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM H 4 N0 3 , 0.75 mM MgSO 4 , 1.5 mM CaCl 2 , 50 ⁇ ⁇ , 100 ⁇ ⁇ 3 ⁇ 3 , 100 ⁇ MnSO 4 , 30 M ZnS0 4 , 1 ⁇ ⁇ 2 ⁇ 0 4 , 0.1 M CoCl 2 , 100 ⁇ Na 2 EDTA, 100 M FeS0 4 ), add 0.8% agar powder, heat in a microwave oven until the agar is completely dissolved.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 °C for 45 seconds, annealing at 51 °C for 45 seconds, extension at 72 °C for 2 minutes), extension at 72 °C for 7 minutes), numbering the plants identified as positive by PCR, normal culture of the plants, and further planting Grow for 3-5 weeks until the pods turn brown and dry. Seeds of T2 were harvested and the seeds were stored in a centrifuge tube at 4 °C.
  • Example 6 Drought Tolerance Simulation Experiment and Functional Identification of Transgenic Arabidopsis T2 Plants Overexpressing ThMCSU-2 The sterilized vermiculite was permeated with 1/2 MS medium.
  • Transgenic Arabidopsis thaliana T2 plants of ThMCSU-2 No. T2H1-T2H6 and control Arabidopsis seeds were planted on vermiculite, 10 seeds per pot, 25 ° C, 10 hours light culture / 14 hours dark culture cycle, 1/2 MS every 7 days, culture for 20 days After that, positive plants were screened using kanamycin, and 4 seedlings of uniform size were retained per pot for drought experiments.
  • T2 transgenic plants showed that the control plants were all wilted, while the T2H1, T2H2, T2H3, T2H4, T2H5, T2H6, T2H7, and T2H8 lines were all 32 in each line. Survival and continued growth showed significant drought tolerance (see Figures 3a and 3b, with T2H3, T2H4 as an example, the results of T2H1, T2H2, T2H5, T2H6, T2H7, T2H8 are similar to T2H3, T2H4, not shown here ).
  • Example 7 Determination of ABA content after drought stress
  • ABA is recognized as a plant hormone associated with stress, which can act as a signaling molecule to regulate the expression of multiple stress-inducing genes, thereby improving the plant's resistance to stress.
  • T2H1, T2H2, T2H3, T2H4, T2H5, T2H6 The kit measures the ABA content (see Figure 4). The results showed that the ABA content of transgenic plants was higher than that of the control (CK1, CK2) under drought treatment and control conditions, which proved that ThMCSU-2 gene can positively regulate the endogenous ABA content of plants.
  • Example 8 Verification of expression of r/zMC ⁇ /-2 gene at the transcriptional level
  • RNA extracted from plant leaves RNA extraction kit (Invitrogen) by 0.05 g each for 10 days of drought.
  • the absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the individual RNA concentrations.
  • Reverse transcription was carried out according to the method shown by Invitrogen Reverse Transcription Kit Superscript III Reverse Transcriptase (2 ⁇ g of total RNA as a template, reverse transcription primer SEQ ID NO: 18).
  • the relative expression of the ⁇ / ⁇ /-2 gene was detected by SEQ ID NO: 16 and SEQ ID NO: ⁇ ⁇ Th ThMCSU-2.
  • PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template.
  • 50 ⁇ l ⁇ Reaction system 10 ⁇ 5 xPS Buffer, 3 ⁇ 2.5 mM dNTP, 2.0 ⁇ cDNA 1.0 ⁇ PrimeSTAR 10 ⁇ primers SEQ ID NO: 16 and P SEQ ID NO: 17 each 2.0 ⁇ and 30 ⁇ double steamed water.
  • PCR reaction conditions pre-denaturation at 94 ° C for 5 minutes, 29 cycles (denaturation at 94 ° C for 45 seconds, annealing at 51 ° C for 45 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.
  • M is DNA Ladder Marker (DL2000, TakaRa)
  • 1-4 is Transgenic Arabidopsis T2 plants with insignificant drought tolerance
  • 5-11 are transgenic Arabidopsis thaliana plants with significant drought tolerance (in order: ⁇ 2 ⁇ 1, ⁇ 2 ⁇ 2, ⁇ 2 ⁇ 3, ⁇ 2 ⁇ 4, ⁇ 2 ⁇ 5, ⁇ 2 ⁇ 6, T1H7)
  • 12 -17 is a non-transgenic Arabidopsis plant.
  • the size of the electrophoresis band of the PCR product shown in the figure is consistent with the size of ThMCSU-2 (about 2.4 Kbp).
  • the results showed that there was no r/zMC ⁇ /-2 transcription in Arabidopsis thaliana, significantly in drought-tolerant transgenic Arabidopsis thaliana T1 plants.
  • ThMCSU-2 The transcription of ThMCSU-2 was stronger, and the drought-tolerant transgenic Arabidopsis thaliana T2 plants were not significantly transcribed.

Landscapes

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

Abstract

The present invention relates to a plant protein, coding genes of same, and application thereof, and specifically relates to a molybdenum enzyme cofactor sulfurized enzyme sourced from Thellungiella halophila, ThMCSU-2, the coding genes of same, and an application thereof in cultivating a transgenic plant of increased drought tolerance.

Description

一种小盐芥钼辅酶因子硫化酶 MCSU-2及其编码基因与应用  A small salt mustard molybdenum coenzyme sulfide enzyme MCSU-2 and its coding gene and application
技术领域 本发明涉及植物蛋白及其编码基因与应用， 特别是涉及一个来源于小盐芥的钼 辅酶因子硫化酶 MCSt -2及其编码基因， 以及其在培育耐旱性提高的转基因植物中的 应用。 背景技术 温度、 盐渍和干旱等逆境胁迫会对高等植物的生长发育造成严重危害， 导致作物产 量降低， 品质下降， 严重威胁农业生产和自然环境。 其中干旱对作物产量的影响， 在诸 多自然逆境中占首位， 其危害相当于其它灾害之和， 是许多地区是农业发展的瓶颈。 据 统计， 世界干旱、 半干旱地区占陆地面积的 34%; 我国干旱、 半干旱地区约占国土面积 的 52%， 年受旱面积达 200-270万公顷， 全国灌溉区每年缺水约 30亿立方米， 因缺水而 少收粮食 350-400亿公斤； 特别是我国主要产粮区如华北、 东北和西北， 是我国缺水最 严重的地区， 春旱频繁达到十年九遇。 FIELD OF THE INVENTION The present invention relates to plant proteins and coding genes thereof and applications thereof, and more particularly to a molybdenum coenzyme factor vulcanase MCSt-2 derived from small salt mustard and a gene encoding the same, and the same in the cultivation of transgenic plants with improved drought tolerance application. BACKGROUND OF THE INVENTION Stresses such as temperature, salting and drought can cause serious damage to the growth and development of higher plants, resulting in reduced crop yields, degraded quality, and serious threats to agricultural production and the natural environment. Among them, the impact of drought on crop yields ranks first in many natural adversities, and its harm is equivalent to the sum of other disasters. Many regions are the bottleneck of agricultural development. According to statistics, the world's arid and semi-arid regions account for 34% of the land area; China's arid and semi-arid regions account for 52% of the country's land area, and the annual drought-affected area amounts to 2 million to 2.7 million hectares. Cubic meters, due to lack of water, less than 35-40 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.
植物耐旱性大多属于多基因控制的数量性状， 利用常规育种方法改良作物的抗旱性 受到周期长、 优异种质资源缺乏的限制。 近年来的转录组学、 蛋白组学和基因表达调控 的研究初步揭示了植物干旱胁迫的作用分子机理。 目前， 利用干旱胁迫相关基因提高植 物的抗旱能力， 已经成为植物抗逆分子生物学的研究热点和植物抗逆基因工程重要的研 究方向。  Most of the drought tolerance of plants belongs to the quantitative traits controlled by multiple genes. The use of conventional breeding methods to improve the drought resistance of crops is limited by the long cycle and lack of excellent germplasm resources. Recent studies on transcriptomics, proteomics and gene expression regulation have revealed the molecular mechanism of plant drought stress. At present, the use of drought stress-related genes to improve the drought resistance of plants has become a research hotspot of plant resistance to molecular biology and an important research direction of plant stress resistance genetic engineering.
植物受到逆境胁迫时会产生相应的应答反应， 以降低或消除逆境胁迫给植物带来的 危害。 植物的这种应答反应是一个涉及多基因、 多信号途径及多基因产物的复杂过程。 但就目前的研究状况而言， 由于其机制十分复杂， 许多植物对逆境下的生物化学和生理 学上的响应机制仍有待深入研究。 在抗逆应答基因的功能及表达调控方面的研究将对植 物抗逆相关的信号传递途径之间的联系以及整个信号传递网络系统的研究提供重要的基 础。 发明内容 本发明人利用 SSH (抑制差减杂交） 与 RACE (cDNA末端快速扩增） 相结合的方 法克隆了小盐芥的一个钼辅酶因子硫化酶 （本文命名为 MCSU-2 的编码基因， 并测定 了其 DNA序列。并且发现将其导入受体植物超量表达后，可显著改善转基因植株的耐旱 性， 而且这些性状可稳定遗传。 When plants are stressed by stress, they will respond accordingly to reduce or eliminate the damage caused by stress. This response of plants is a complex process involving multiple genes, multiple signaling pathways, and multiple gene products. However, as far as the current research situation is concerned, due to the complexity of its mechanism, the biochemical and physiological response mechanisms of many plants to stress have yet to be further studied. Studies on the function and expression regulation of stress-responsive genes will provide an important basis for the link between plant stress-resistance-related signaling pathways and the study of the entire signaling network system. SUMMARY OF THE INVENTION The present inventors used SSH (Suppression Subtractive Hybridization) in combination with RACE (Rapid Amplification of cDNA Ends) A molybdenum coenzyme sulfide enzyme of the small salt mustard was cloned (this article was named MCSU-2 encoding gene, and its DNA sequence was determined. It was found that after transduction into the recipient plant, the transgenic plants could be significantly improved. Drought tolerance, and these traits can be stably inherited.
本发明第一方面提供小盐芥的一个钼辅酶因子硫化酶 MC^/-2的编码基因 （本文命 名为 ThMCSU-2 ， 其序列为 SEQ ID NO: 2。  The first aspect of the present invention provides a gene encoding a molybdenum coenzyme vulcanase MC^/-2 of the small salt mustard (herein named ThMCSU-2, the sequence of which is SEQ ID NO: 2.
本发明第二方面提供一种重组表达载体， 其含有本发明第一方面所述的基因， 其是 通过所述基因插入到一种表达载体而获得的，优选地，所述表达载体是 pCAMBIA2300; 并且所述基因的核苷酸序列与所述重组表达载体的表达控制序列可操作地连接；优选地， 所述重组表达载体为附图 2所示的 -ThMCSU-2-2，载体。  A second aspect of the present invention provides a recombinant expression vector comprising the gene of the first aspect of the present invention, which is obtained by inserting the gene into an expression vector, preferably, the expression vector is pCAMBIA2300; And the nucleotide sequence of the gene is operably linked to the expression control sequence of the recombinant expression vector; preferably, the recombinant expression vector is -ThMCSU-2-2, vector shown in Figure 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。  The third aspect of the invention provides a recombinant cell comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention; preferably, the recombinant cell is a recombinant Agrobacterium cell.
本发明第四方面提供一种改善植物耐旱性的方法， 包括： 将本发明第一方面所述的 基因或者本发明第二方面所述的重组表达载体导入植物或植物组织并使所述基因表达； 优选地， 所述植物是拟南芥。  A fourth aspect of the present invention provides a method for improving drought tolerance of a plant, comprising: introducing the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention into a plant or plant tissue and causing the gene Expression; Preferably, the plant is Arabidopsis thaliana.
本发明第五方面提供一种制备转基因植物的方法， 包括： 在有效产生植物的条件下 培养含有本发明第一方面所述的基因或者本发明第二方面所述的重组表达载体的植物或 植物组织； 优选地， 所述植物是拟南芥。  A fifth aspect of the invention provides a method for producing a transgenic plant, comprising: cultivating a plant or a plant comprising the gene of the first aspect of the invention or the recombinant expression vector of the second aspect of the invention under conditions effective to produce a plant Tissue; Preferably, the plant is Arabidopsis thaliana.
本发明第六方面提供本发明第一方面所述的基因、 本发明第二方面所述的重组表达 载体或者本发明第三方面所述的重组细胞用于改善植物耐旱性以及用于植物育种的用途; 优选地， 所述植物是拟南芥。  A sixth aspect of the present invention provides the gene according to the first aspect of the present invention, the recombinant expression vector of the second aspect of the present invention or the recombinant cell of the third aspect of the present invention for improving drought tolerance of a plant and for use in plant breeding Use; Preferably, the plant is Arabidopsis thaliana.
本发明第七方面提供由本发明第一方面所述的基因编码的蛋白质， 其氨基酸序列如 SEQ ID NO: 1所示。 附图说明 图 1是 ThMCSU-2的植物表达载体（35S-r/zMG^/-2-2300)的构建流程（图 la-lb)。 图 2是 ThMCSU-2的植物表达载体 ( 5S-ThMCSU-2-7300 的质粒图。  The seventh aspect of the invention provides the protein encoded by the gene of the first aspect of the invention, the amino acid sequence of which is shown in SEQ ID NO: 1. Brief Description of the Drawings Fig. 1 is a construction flow of a plant expression vector (35S-r/zMG^/-2-2300) of ThMCSU-2 (Fig. la-lb). Figure 2 is a plasmid map of the plant expression vector of ThMCSU-2 (5S-ThMCSU-2-7300).
图 3是 r/zMC^/-2 T2代转基因拟南芥植株 （图中， T2H3 ; T2H4) 和作为对照的非 转基因拟南芥植株（图中， CK1、 CK2) 的耐旱模拟实验结果。 （图 3a为正常生长 20天 的拟南芥植株； 图 3b为正常生长 20天后干旱处理 14天的拟南芥植株）。  Figure 3 shows the results of drought tolerance simulation experiments of r/zMC^/-2 T2 transgenic Arabidopsis plants (in the figure, T2H3; T2H4) and non-transgenic Arabidopsis plants (Fig., CK1, CK2) as controls. (Fig. 3a is an Arabidopsis plant that is normally grown for 20 days; Fig. 3b is an Arabidopsis plant that has been treated for 14 days after normal growth for 20 days).
图 4是干旱胁迫和正常生长条件下的 T2代转基因拟南芥植株及对照植株 ABA含 量变化检测结果。 1-8依次为株系： T2H1、 T2H2、 T2H3、 T2H4、 T2H5、 T2H6、 CK1、 CK2, 其中 T2H1、 T2H2、 T2H3、 T2H4、 T2H5、 T2H6为转基因植株， CK1、 CK2为 对照植株。 Figure 4 shows T2 transgenic Arabidopsis plants under drought stress and normal growth conditions and control plants ABA containing The amount of change detection results. 1-8 are the lines: T2H1, T2H2, T2H3, T2H4, T2H5, T2H6, CK1, CK2, wherein T2H1, T2H2, T2H3, T2H4, T2H5, T2H6 are transgenic plants, and CK1 and CK2 are control plants.
图 5 是利用反转录 PCR 对 T2 代转基因拟南芥植株和非转基因对照植株中 ThMCSU-2 基因在转录水平进行的分子水平检测的结果。 M 为 DNA Ladder Marker ( DL2000, TakaRa) , 1-4为耐旱效果不显著的转基因拟南芥 T2代植株， 5-11为耐旱 效果显著的转基因拟南芥 T1 代植株 （依次为： Τ2Η1、 Τ2Η2、 Τ2Η3、 Τ2Η4、 Τ2Η5、 Τ2Η6、 Τ2Η7) , 12-17为非转基因的拟南芥植株。  Figure 5 shows the results of molecular level detection of the ThMCSU-2 gene at the transcriptional level in T2 transgenic Arabidopsis plants and non-transgenic control plants by reverse transcription PCR. M is DNA Ladder Marker (DL2000, TakaRa), 1-4 is a transgenic Arabidopsis T2 plant with insignificant drought tolerance, and 5-11 is a transgenic Arabidopsis T1 plant with significant drought tolerance (in order: Τ2Η1) Τ2Η2, Τ2Η3, Τ2Η4, Τ2Η5, Τ2Η6, Τ2Η7), 12-17 are non-transgenic Arabidopsis plants.
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。 BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be further described below in conjunction with non-limiting examples.
下面实施例中提到的未注明来源的限制性内切酶均购自 New England Biolabs公司。 实施例 1、 干旱胁迫下小盐芥 SSH文库构建：  The unrecognized restriction enzymes mentioned in the examples below were purchased from New England Biolabs. Example 1. Construction of SSH library of small salt mustard under drought stress:
具体方法为：  The specific method is:
按照 Clontech公司的 PCR-select™ cDNA Subtraction Kit试剂盒说明书所示的方法通 过抑制差减杂交方法构建 SSH文库 （差减文库）。 在实验过程中以生长过程中干旱处理 的小盐芥幼苗的叶片的 mRNA作为样本 ( Tester)，以未处理的小盐芥幼苗的叶片的 mRNA 作为对照 （Driver)。 具体步骤如下：  The SSH library (subtractive library) was constructed by inhibition subtractive hybridization according to the method described in Clontech's PCR-selectTM cDNA Subtraction Kit kit. During the experiment, the mRNA of the leaves of the small salt mustard seedlings treated with drought during the growth process was used as a sample (Tester), and the mRNA of the leaves of the untreated small salt mustard seedlings was used as a control. Specific steps are as follows:
( 1 ) 供试材料：  (1) Test materials:
小盐芥 （ T ellungiella halophila, 购自中国内蒙古巴彦淖尔市乌兰布和沙漠绿色 植物园盐生植物繁育中心）播种到灭菌的蛭石上， 在 25 °C、 光周期 16小时光照 /8小 时黑暗 （光强 2000-3000 Lx) 条件下培养， 每周浇 1/2MS培养基 （9.39 mM KN03， 0.625 mM KH2P04， 10.3 mM NH4N03 , 0.75 mM MgS04， 1.5 mM CaCl2， 50 μΜ KI， 100 μΜ Η3ΒΟ3， 100 M MnSO4， 30 μΜ ZnS04, 1 μΜ Να2Μο04, 0.1 μΜ CoCl2, 100 μΜ Na2EDTA, 100 M FeSO4) —次。 当苗株直径达到 5-6 cm时用于实验。 Small salt mustard (T ellungiella halophila, purchased from the Wulanbu and Desert Green Botanical Garden of Halophytes in Bayannao, Inner Mongolia, China) was sown on sterilized vermiculite at 25 ° C, photoperiod 16 hours light / 8 hours Incubate under dark conditions (light intensity 2000-3000 Lx), and pour 1/2 MS medium per week (9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM NH 4 N0 3 , 0.75 mM MgS0 4 , 1.5 mM CaCl 2 , 50 μΜ KI, 100 μΜ Η 3 ΒΟ 3 , 100 M MnSO 4 , 30 μΜ ZnS0 4 , 1 μΜ Να 2 Μο0 4 , 0.1 μΜ CoCl 2 , 100 μΜ Na 2 EDTA, 100 M FeSO 4 ). It was used for experiments when the seedlings reached a diameter of 5-6 cm.
( 2 ) 材料处理：  (2) Material handling:
将供试幼苗分为 2组， 每组 4盆， 每盆 1株。 第一组为对照组， 在 25 °C、 光周 期 16小时光照 /8小时黑暗条件下培养， 正常用 1/2MS培养基浇灌。 第二组为干旱处 理组， 25 °C、 光周期 16小时光照 /8小时黑暗条件下培养， 停止浇灌， 处理 10天， 然 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, cultured at 25 ° C, photoperiod of 16 hours light / 8 hours dark, and normally watered with 1/2 MS medium. The second group is the arid area In the group, culture at 25 °C, photoperiod of 16 hours light/8 hours dark, stop watering, treat for 10 days, then cut the leaves of the top 1/3 of the seedlings in two groups in time, and then quickly freeze with liquid nitrogen, then - Store in a 70 °C refrigerator.
( 3 ) 总 RNA提取：  (3) Total RNA extraction:
分别取对照组和干旱处理组的小盐芥叶片 0.5 g， 用植物 RNA提取试剂盒（购自 Invitrogen) 提取小盐芥叶片的总 RNA。 用 HITACHI公司的紫外分光光度计 U-2001 测定所得总 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 （从总 RNA中纯化 polyA+ RNA) 分离 mRNA。 0.5 g of small salt mustard leaves of the control group and the drought treatment group were taken, and the total RNA of the leaves of the small salt mustard was extracted with a plant RNA extraction kit (purchased from Invitrogen). The absorbance of the total RNA at 260 nm and 280 nm was measured by HITACHI's UV spectrophotometer U-2001. The OD 260 / OD 280 ratio was 1.8-2.0, indicating that the total RNA purity was higher; 1.0% agarose gel was used. Gel electrophoresis detected the integrity of total RNA. The brightness of the 28S band was approximately twice that of the 18S band, indicating good RNA integrity. mRNA was isolated using Qiagen's Oligotex mRNA Purification Kit (purified polyA+ 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试剂盒说明书所示的方法进行 抑制差减杂交。 先将 Driver mRNA和 Tester mRNA分别反转录， 得到双链 cDNA， 再以 2 μg Tester cDNA和 2 μg Driver cDNA作为起始材料进行差减杂交。 在 37°C水浴下分别 将 Tester cDNA和 Driver cDNA用 Rsa I酶切 1.5小时， 然后将酶切后的 Tester cDNA分 成两等份， 连接上不同的接头， 而 Driver cDNA不连接头。 两种连有不同接头的 Tester cDNA分别与过量的 Driver cDNA混合， 进行第一次正向差减杂交。 将两种第一次差减 杂交的产物混合， 再与新变性的 Driver cDNA进行第二次正向差减杂交， 然后通过两次 抑制性 PCR扩增差异表达的片段， 使其得到富集。 The method according to Clontech's PCR-select TM cDNA Subtraction Kit kit instructions will be shown suppression subtractive hybridization. The Driver mRNA and Tester mRNA were reverse transcribed, respectively, to obtain double-stranded cDNA, and 2 μg of Tester cDNA and 2 μg 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 hours in a 37 ° C water bath, and then the digested Tester cDNA was divided into two equal portions, and the different linkers were ligated, and the Driver cDNA was not ligated. Two tester cDNAs with different adaptors were mixed with excess Driver cDNA for the first forward subtractive hybridization. The two first subtractive hybridization products were mixed and subjected to a second forward subtractive hybridization with the newly denatured Driver cDNA, and then the differentially expressed fragments were amplified by two inhibitory PCRs to obtain enrichment.
为了增加获得表达序列标签 （Expressed Sequence Tag, EST) (Unigene) 的有效性， 避免基因无酶切位点及所获得序列在非翻译区， 本实验同时用内切酶 Haelll按上述步骤 对 Tester cDNA禾 P Driver cDNA进行酶切并先后进行两次正向差减杂交和两次抑制性 PCR扩增， 最后合并两组正向差减杂交 cDNA片段的第二次抑制性 PCR产物。  In order to increase the validity of the Expressed Sequence Tag (EST) (Unigene), avoid the gene-free cleavage site and the obtained sequence in the untranslated region, this experiment simultaneously uses the endonuclease Haelll to tester cDNA according to the above steps. The P Driver cDNA was digested and subjected to two forward subtractive hybridizations and two inhibitory PCR amplifications, and finally the second inhibitory PCR products of the two sets of forward subtractive hybridization cDNA fragments were combined.
( 5 ) 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 subtractive libraries
依照 pGEM-T Easy试剂盒 （购自 Promega的试剂盒）产品说明书所示方法， 将上述 合并的正向差减杂交 cDNA片段的第二次 PCR扩增产物（使用 QIAquick PCR Purification Kit纯化， 购自 Qiagen) 与 pGEM-T Easy载体连接， 其具体步骤如下： 在 200 μ1 ΡΟ 管 中依次加入下列成分： 纯化的合并后的正向差减杂交 cDNA片段的第二次抑制性 PCR产 物 3 μ1、 2xT4 DNA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。 然后取 10 μΐ连接反应产物， 加入到 ΙΟΟ μΙ感受态大肠杆菌 JM109 (购 自 TAKARA)中， 冰浴 30分钟、热休克 60秒、冰浴 2分钟， 另加 250 μΐ LB培养液（ 1% 胰蛋白胨 （Tryptone, 购自 OXOID), 0.5%酵母提取物 （Yeast Extract, 购自 OXOID), l% NaCl (购自国药））后置于 37°C摇床中， 以 225 r/分钟振荡培养 30分钟， 然后从中取 200 μΐ菌液涂布于含 50 μ§/ιη1氨苄青霉素、 40 μ§/ιη1 X-gal ( 5-溴 -4氯 -3-吲哚- β -D-半乳 糖苷）、 24 g/ml IPTG (异丙基 - β -D-硫代吡喃半乳糖苷） （X-gal/IPTG购自 TAKARA) 的 LB (同上） 固体培养平板上， 37°C培育 18小时。 计数培养板中直径 > 1 mm的清晰白 色及蓝色菌落数， 随机挑取 198个白色菌落 (编号： Gh-B001至 Gh-B198)。 将所挑取的 白色菌落克隆分别接种于 96孔细胞培养板（CORNING)中的含 50 μ§/ιη1氨苄青霉素的 LB液体培养基， 37°C培养过夜后加甘油至甘油终浓度为 20% (体积比）， 然后于 -80°C保 存备用。 对所培养的菌落克隆以巢式 PCR 引物 Primer 1和 Primer 2R (来自 Clontech公 司的 PCR-select™ cDNA Subtraction Kit试剂盒）进行菌液 PCR扩增验证， 得到 166个阳 性克隆， 然后将所有阳性克隆在送英潍捷基 （上海） 贸易有限公司测序。 The second PCR amplification product of the combined forward subtractive hybridization cDNA fragment was purified according to the method described in the product specification of pGEM-T Easy kit (purchased from Promega kit) (purified by QIAquick PCR Purification Kit, purchased from QIAquick PCR Purification Kit) Qiagen) is ligated to the pGEM-T Easy vector. The specific steps are as follows: The following components are sequentially added to the 200 μl ΡΟ tube: The second inhibitory PCR product of the purified combined positive subtractive hybridization cDNA fragment 3 μ1, 2xT4 DNA ligase buffer 5 μl, pGEM-T Easy vector 1 μ1, Τ4 DNA ligase 1 μl, and ligated overnight at 4 °C. Then 10 μΐ of the ligation reaction product was added to ΙΟΟ μΙ competent Escherichia coli JM109 (purchased from TAKARA), ice bath for 30 minutes, heat shock for 60 seconds, ice bath for 2 minutes, and 250 μL LB medium (1%) Tryptone (purchased from OXOID), 0.5% yeast extract (Yeast Extract, purchased from OXOID), and 1% NaCl (purchased from Sinopharm) were placed in a 37 ° C shaker and shaken at 225 r/min. For 30 minutes, then take 200 μL of the bacterial solution and apply it to 50 μ § /ιη1 ampicillin, 40 μ § /ιη1 X-gal ( 5-bromo-4-chloro-3-indolyl-β-D-galactoside ), 24 g/ml IPTG (isopropyl-β-D-thiogalactopyranoside) (X-gal/IPTG purchased from TAKARA) on LB (ibid.) solid culture plate, incubated at 37 ° C for 18 hours . Count the number of clear white and blue colonies with a diameter > 1 mm in the culture plate and randomly pick 198 white colonies (number: Gh-B001 to Gh-B198). The picked white colony clones were inoculated into LB liquid medium containing 50 μ§ /ιη1 ampicillin in 96-well cell culture plate (CORNING), and cultured overnight at 37 ° C, and glycerin was added to a final concentration of 20% glycerol. (volume ratio), then stored at -80 ° C for later use. The colony clones were cultured by nested PCR primers Primer 1 and Primer 2R (PCR-selectTM cDNA Subtraction Kit from Clontech) for PCR amplification, and 166 positive clones were obtained, and then all positive clones were obtained. Sent to Yingjie 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 123条表达序 列标签 （Expressed sequence tag, EST) (Unigene)。 经分析有 22个重叠群， 有 101个单 一的序列。 经 BlastN发现其中 53条 EST (Unigene) 在 GenBank 中有同源序列， 21条 EST功能未知或者为假定蛋白，另有 27条 EST未获得同源匹配，推测可能是处于 3 '或 5' 末端非翻译区的较短序列。 实施例 2 小盐芥钼辅酶因子硫化酶编码基因 ThMCSU-2的克隆  After removing the vector and the ambiguous sequence and redundant cDNA from the DNA sequencing results, a total of 123 Expressed sequence tags (EST) (Unigene) were obtained. After analysis, there are 22 contigs with 101 single sequences. It was found by BlastN that 53 ESTs (Unigene) have homologous sequences in GenBank, 21 EST functions are unknown or hypothetical proteins, and 27 ESTs have not obtained homologous matches, presumably at the 3' or 5' end. A shorter sequence of translation regions. Example 2 Cloning of a small salt mustard molybdenum coenzyme-vulcanase-encoding gene ThMCSU-2
将所述鉴定的小盐芥 SSH文库中来自菌落 Gh-B001的克隆子去掉冗余 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与已知钼辅酶因子硫化酶 氨基酸序列有较高同源性，本文将克隆子 YLS-1对应的全长编码基因命名为 J/zMC^/-2， 其对应的蛋白命名为 MCSU-2。  After removing the redundant DNA from the clone of the colony Gh-B001 in the identified SSH library of the small salt mustard, the sequence is SEQ ID No: 3. Sequence analysis indicated that the encoded amino acid sequence of the sequence was sulfided with the known molybdenum cofactor. The amino acid sequence of the enzyme has high homology. In this paper, the full-length coding gene corresponding to the clone YLS-1 is named J/zMC^/-2, and the corresponding protein is named MCSU-2.
SEQ ID No: 3 SEQ ID No: 3
1 CGAGAATGGA TGATTCAGGG TCTGACGGGT GATATTCTTA CCCAAAAGAA GGTGCCTGAA 1 CGAGAATGGA TGATTCAGGG TCTGACGGGT GATATTCTTA CCCAAAAGAA GGTGCCTGAA
6 1 ATGTCTCTTA TTAGGACCTT TATCGACCTT GAGGAAGGAC TATTGTCTGT AGAATCTACT6 1 ATGTCTCTTA TTAGGACCTT TATCGACCTT GAGGAAGGAC TATTGTCTGT AGAATCTACT
12 1 CGCTGCAAAG ACAAGTTGCA CATCAGAATC AAGTCTAATT CATATGATCC AAGGAGCCAT12 1 CGCTGCAAAG ACAAGTTGCA CATCAGAATC AAGTCTAATT CATATGATCC AAGGAGCCAT
18 1 GAGTTCGATA CAGATGCCAA CATACTTGAA AATCATGACG AGGAAACTAG AATCAATGAT18 1 GAGTTCGATA CAGATGCCAA CATACTTGAA AATCATGACG AGGAAACTAG AATCAATGAT
24 1 TGGTTAACCC GTGCCATTGG TCGACAGTGC AAATTGTTAC AGTATTGTAG CTCTAATTCC24 1 TGGTTAACCC GTGCCATTGG TCGACAGTGC AAATTGTTAC AGTATTGTAG CTCTAATTCC
3 0 1 AAACACTGCA TGAACAGAAA CAAGAGTCCT GGTTTGTGCA GAGAATTAGA AAGCAATATC3 0 1 AAACACTGCA TGAACAGAAA CAAGAGTCCT GGTTTGTGCA GAGAATTAGA AAGCAATATC
3 6 1 AACTTCGCTA ATGAAGCTCA GTTCTTGTTG ATATCCGAGG AGAGTGTTGC TGATCTAAAT 421 AGAAGATTAG AAGCAAAAGG CAAGGATTAC AACCGAGCTC TTGAGAAACT CAGTCCATAT3 6 1 AACTTCGCTA ATGAAGCTCA GTTCTTGTTG ATATCCGAGG AGAGTGTTGC TGATCTAAAT 421 AGAAGATTAG AAGCAAAAGG CAAGGATTAC AACCGAGCTC TTGAGAAACT CAGTCCATAT
481 AGATTCAGAC CAAATCTGGT AATATCAGGA GGTGAACCAT ACGCAGAAGA TAAATGGAGA481 AGATTCAGAC CAAATCTGGT AATATCAGGA GGTGAACCAT ACGCAGAAGA TAAATGGAGA
541 ACTCTCAAGA TAGGAGACAG TCATTTCACG TCGTTGGGCG GTTGTAATCG TTGCCAGATG541 ACTCTCAAGA TAGGAGACAG TCATTTCACG TCGTTGGGCG GTTGTAATCG TTGCCAGATG
601 ATAAACATAA GTAATGAAAC TGGTCAAGTG AAGAAATCCA ACGAGCCCTT GACAACTTTA601 ATAAACATAA GTAATGAAAC TGGTCAAGTG AAGAAATCCA ACGAGCCCTT GACAACTTTA
661 GCTTCTTATA GAAGAGTAAA GGGAAAGATC TTGTTTGGAA CTCTTCTGAG ATACGAGATT661 GCTTCTTATA GAAGAGTAAA GGGAAAGATC TTGTTTGGAA CTCTTCTGAG ATACGAGATT
721 GATGCAAAAA CAGAGTCTTG GATTCAAGTT GGGGAAGAAG TAAATCCAAA TACTGAATAA721 GATGCAAAAA CAGAGTCTTG GATTCAAGTT GGGGAAGAAG TAAATCCAAA TACTGAATAA
MC^/-2全长编码基因的克隆 Cloning of MC^/-2 full-length coding gene
根据已经获得的 SEQ ID No: 3序列分析： SEQ ID No: 3为编码基因 MC^ -2的 According to the sequence analysis of SEQ ID No: 3 that has been obtained: SEQ ID No: 3 is the coding gene MC^-2
3端序列。 根据已经获得的 SEQ ID NO: 3序列， 设计如下三条特异性引物， 作为反转录引 物及 5'RACE的 3'端特异性引物。 3-terminal sequence. Based on the sequence of SEQ ID NO: 3 which has been obtained, the following three specific primers were designed as reverse transcription primers and 3'-end specific primers for 5' RACE.
YLS-1GSP1: SEQ ID NO: 4： YLS-1GSP1: SEQ ID NO: 4:
CATTGATTCTAGTTTCCTCGTC CATTGATTCTAGTTTCCTCGTC
YLS-1GSP2: SEQ ID NO: 5： YLS-1GSP2: SEQ ID NO: 5:
ATGGCTCCTTGGATCATATG ATGGCTCCTTGGATCATATG
YLS-1 GSP3: SEQ ID NO: 6： YLS-1 GSP3: SEQ ID NO: 6:
CGAGAATGGA TGATTCAGG  CGAGAATGGA TGATTCAGG
试剂盒自带通用引物：  The kit comes with universal primers:
AAP: SEQ ID NO: 7：  AAP: SEQ ID NO: 7:
GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG
AUAP: SEQ ID NO: 8： AUAP: SEQ ID NO: 8:
GGCCACGCGTCGACTAGTAC 实验步骤按试剂盒说明书操作 （5' RACE System for Rapid Amplification of cDNA Ends试剂盒购自 Invitrogen公司）。  The GGCCACGCGTCGACTAGTAC experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen).
以 YLS-1GSP1 (SEQ ID NO: 4) 为反转录引物， 以干旱处理组小盐芥叶子提取的 mRNA为模板进行反转录， 获得 cDNA模板， 然后按照上述 5' RACE试剂盒说明书中的 步骤加 Poly C尾，以加尾后的产物为模板进行第一轮 PCR扩增，所用引物为 SEQ ID NO: 4与通用引物 SEQ ID NO: 7 (试剂盒自带， I为次黄嘌吟修饰的 a、 c、 g或 t)， 具体步 骤如下：  YLS-1GSP1 (SEQ ID NO: 4) was used as a reverse transcription primer, and the mRNA extracted from the leaves of the salt-treated mustard was used as a template for reverse transcription to obtain a cDNA template, and then according to the above 5' RACE kit instructions. The first step of PCR amplification was carried out by adding the tail of the Poly C tail to the template. The primers used were SEQ ID NO: 4 and the universal primer SEQ ID NO: 7 (the kit is self-contained, and I is hypoxanthine). Modified a, c, g or t), the specific steps are as follows:
50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer、 3 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 SEQ ID NO: 7各 2.0 μΐ以及 35 μΐ的双蒸水。 PCR反应条件： 94 °C预变性 5分钟， 33个循环 （94°C变 性 50秒， 54°C退火 50秒， 72°C延伸 1分钟）， 72°C延伸 10分钟。 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 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 SEQ ID NO: 7 each of 2.0 μΐ and 35 μΐ of double distilled water. PCR reaction conditions: predenaturation at 94 °C for 5 minutes, 33 cycles (denaturation at 94 °C for 50 seconds, annealing at 54 °C for 50 seconds, extension at 72 °C for 1 minute), extension at 72 °C for 10 minutes.
所得的 PCR产物用双蒸水稀释 50倍后取 2.0 μΐ作为模板， 用 SEQ ID NO: 5与通用 引物 SEQ ID NO: 8进行第二轮 PCR扩增， 具体步骤如下：  The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μL was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 5 and the universal primer SEQ ID NO: 8. 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 μΐ Ex Taq、 10 μΜ的引物 SEQ ID NO: 5和 SEQ ID NO: 8各 2.0 μ1， 以 及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5分钟， 33个循环（94°C变性 50秒， 54°C 退火 50秒， 72°C延伸 1分钟）， 72°C延伸 10分钟。 回收第二次 PCR产物中约为 1 Kbp 大小的条带 （ Gel Extraction Kit购自 OMEGA)， 并将其连接到 pGEM-T Easy载体， 然后 转化到大肠杆菌 JM109感受态细胞中（具体方法同上）， 并在含 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个白色菌落分别接种于含有 50 μ§/ιη1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甘油终浓度为 20% (体积比）， -80°C 保存备用。 用引物 SEQ ID NO: 5与 3'端引物 SEQ ID NO: 6进行菌液 PCR扩增 （反应 体系及反应条件同上）， 得到 3个阳性克隆（YJ1-1、 YJl-2、 YJ1-3 ), 送英潍捷基（上海） 贸易有限公司测序测序， 获得该基因的 cDNA的一段 5'端序列。 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 μΐ Ex Taq, 10 μΜ primers SEQ ID NO: 5 and SEQ ID NO: 8 2.0 μl, and 35 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 50 seconds, annealing at 54 ° C for 50 seconds, extension at 72 ° C for 1 minute), extension at 72 ° C for 10 minutes. A band of about 1 Kbp in the second PCR product (Glue Extraction Kit was purchased from OMEGA) was recovered and ligated into the pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (the same method as above) And screened on LB solid medium containing 50 g/mL ampicillin. Ten white colonies were randomly picked and inoculated in LB liquid medium containing 50 μ § /ιη1 ampicillin. After incubation at 37 °C overnight, glycerol was added to a final concentration of glycerol of 20% (volume ratio), -80 °C. Save spare. The primers SEQ ID NO: 5 and the 3' primer SEQ ID NO: 6 were used for PCR amplification (reaction system and reaction conditions as above), and three positive clones (YJ1-1, YJl-2, YJ1-3) were obtained. , sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. to sequence and sequence, to obtain a 5' end sequence of the cDNA of the gene.
所得的 5 'RACE产物克隆子 YJ1 - 1测序获得序列为 SEQ： [D NO: 9：  The obtained 5 'RACE product clone YJ1 - 1 was sequenced to obtain the sequence of SEQ: [D NO: 9:
1 GGGGGGGACT GTTGCTGCTT CAATTGCTGA CATTGACTTT GTAAAAAGAA GAGAAAGCGT 1 GGGGGGGACT GTTGCTGCTT CAATTGCTGA CATTGACTTT GTAAAAAGAA GAGAAAGCGT
6 1 GGAGGATTTT TTTGAGGATG GCTCTGCTTC ATTCTTGAGT ATCGCAGCCA TCCGCCATGG6 1 GGAGGATTTT TTTGAGGATG GCTCTGCTTC ATTCTTGAGT ATCGCAGCCA TCCGCCATGG
12 1 CTTCAAATTA CTCAAGTCCC TTACTACTTC TGCAATTTGG ATGCACACAA CATCACTTTC12 1 CTTCAAATTA CTCAAGTCCC TTACTACTTC TGCAATTTGG ATGCACACAA CATCACTTTC
18 1 CATGTATGTG AAGAAGAAAC TTCAGGCTTT AAGACATGGA AACGGGACTG GTGTATGTGT18 1 CATGTATGTG AAGAAGAAAC TTCAGGCTTT AAGACATGGA AACGGGACTG GTGTATGTGT
24 1 TCTGTATGGC AGTGAAAATC TGAAGTTATC TTCACATAAA TCAGGCCCAA CGGTTACATT24 1 TCTGTATGGC AGTGAAAATC TGAAGTTATC TTCACATAAA TCAGGCCCAA CGGTTACATT
3 0 1 CAACTTGAAG AGACCTGATG GCTCTTGGTT TGGCTACCTG GAGGTGGAAA AGCTCGCTTC3 0 1 CAACTTGAAG AGACCTGATG GCTCTTGGTT TGGCTACCTG GAGGTGGAAA AGCTCGCTTC
3 6 1 TTTATCTGGA ATCCAGTTAC GGACAGGATG CTTTTGCAAT CCTGGCGCAT GTGCAAAGTA3 6 1 TTTATCTGGA ATCCAGTTAC GGACAGGATG CTTTTGCAAT CCTGGCGCAT GTGCAAAGTA
42 1 TCTCGCCTTG TCTCATTCGA ATCTATTGTC TAACGTAGAG GCTGGGCATA TTTGTTGGGA42 1 TCTCGCCTTG TCTCATTCGA ATCTATTGTC TAACGTAGAG GCTGGGCATA TTTGTTGGGA
4 8 1 CGATAATGAT GTGATTAATG GAAAACCAAC TGGAGCTGTT AGAGTTTCGT TTGGTTATAT4 8 1 CGATAATGAT GTGATTAATG GAAAACCAAC TGGAGCTGTT AGAGTTTCGT TTGGTTATAT
54 1 GTCAACCTTT GAAGAGGCCA AGAAATTTCT TGATTTCATC ATAAGTTCAT TTGTTTCACC54 1 GTCAACCTTT GAAGAGGCCA AGAAATTTCT TGATTTCATC ATAAGTTCAT TTGTTTCACC
6 0 1 TTCAAAGAAT ATTGGGAATG GAATTGCCGT CAGTGGAAGG TTTACTCAAC TTCCTAGTGA6 0 1 TTCAAAGAAT ATTGGGAATG GAATTGCCGT CAGTGGAAGG TTTACTCAAC TTCCTAGTGA
66 1 AGAACTTGAA TGTAAAGAAT CCTTTCCAAA CTACTTCCTT AAATCAGTTA CCATATACCC66 1 AGAACTTGAA TGTAAAGAAT CCTTTCCAAA CTACTTCCTT AAATCAGTTA CCATATACCC
72 1 AATCAAGTCG TGTGCTGGAT TTTCTGTGAA TCGTTGGCCA CTTTGCAGAA CAGGCCTGCT72 1 AATCAAGTCG TGTGCTGGAT TTTCTGTGAA TCGTTGGCCA CTTTGCAGAA CAGGCCTGCT
78 1 GCATGATCGA GAATGGATGA TTCAGGGTCT GACGGGTGAT ATTCTTACCC AAAAGAAGGT78 1 GCATGATCGA GAATGGATGA TTCAGGGTCT GACGGGTGAT ATTCTTACCC AAAAGAAGGT
84 1 GCCTGAAATG TCTCTTATTA GGACCTTTAT CGACCTTGAG GAAGGACTAT TGTCTGTAGA84 1 GCCTGAAATG TCTCTTATTA GGACCTTTAT CGACCTTGAG GAAGGACTAT TGTCTGTAGA
90 1 ATCTACTCGC TGCAAAGACA AGTTGCACAT CAGAATCAAG TCTAATTCAT ATGATCCAAG90 1 ATCTACTCGC TGCAAAGACA AGTTGCACAT CAGAATCAAG TCTAATTCAT ATGATCCAAG
96 1 GAGCCAT 将 5 'RACE获得的序列 SEQ ID NO: 9, 与获得的序列 SEQ ID NO: 3拼接， 获 得 SEQ ID NO: 10: 96 1 GAGCCAT The sequence SEQ ID NO: 9 obtained by 5 'RACE was spliced with the obtained sequence SEQ ID NO: 3 to obtain SEQ ID NO: 10:
1 GGGGGGGACT GTTGCTGCTT CAATTGCTGA CATTGACTTT GTAAAAAGAA GAGAAAGCGT 1 GGGGGGGACT GTTGCTGCTT CAATTGCTGA CATTGACTTT GTAAAAAGAA GAGAAAGCGT
61 GGAGGATTTT TTTGAGGATG GCTCTGCTTC ATTCTTGAGT ATCGCAGCCA TCCGCCATGG61 GGAGGATTTT TTTGAGGATG GCTCTGCTTC ATTCTTGAGT ATCGCAGCCA TCCGCCATGG
121 CTTCAAATTA CTCAAGTCCC TTACTACTTC TGCAATTTGG ATGCACACAA CATCACTTTC121 CTTCAAATTA CTCAAGTCCC TTACTACTTC TGCAATTTGG ATGCACACAA CATCACTTTC
181 CATGTATGTG AAGAAGAAAC TTCAGGCTTT AAGACATGGA AACGGGACTG GTGTATGTGT181 CATGTATGTG AAGAAGAAAC TTCAGGCTTT AAGACATGGA AACGGGACTG GTGTATGTGT
241 TCTGTATGGC AGTGAAAATC TGAAGTTATC TTCACATAAA TCAGGCCCAA CGGTTACATT241 TCTGTATGGC AGTGAAAATC TGAAGTTATC TTCACATAAA TCAGGCCCAA CGGTTACATT
301 CAACTTGAAG AGACCTGATG GCTCTTGGTT TGGCTACCTG GAGGTGGAAA AGCTCGCTTC301 CAACTTGAAG AGACCTGATG GCTCTTGGTT TGGCTACCTG GAGGTGGAAA AGCTCGCTTC
361 TTTATCTGGA ATCCAGTTAC GGACAGGATG CTTTTGCAAT CCTGGCGCAT GTGCAAAGTA361 TTTATCTGGA ATCCAGTTAC GGACAGGATG CTTTTGCAAT CCTGGCGCAT GTGCAAAGTA
421 TCTCGCCTTG TCTCATTCGA ATCTATTGTC TAACGTAGAG GCTGGGCATA TTTGTTGGGA421 TCTCGCCTTG TCTCATTCGA ATCTATTGTC TAACGTAGAG GCTGGGCATA TTTGTTGGGA
481 CGATAATGAT GTGATTAATG GAAAACCAAC TGGAGCTGTT AGAGTTTCGT TTGGTTATAT481 CGATAATGAT GTGATTAATG GAAAACCAAC TGGAGCTGTT AGAGTTTCGT TTGGTTATAT
541 GTCAACCTTT GAAGAGGCCA AGAAATTTCT TGATTTCATC ATAAGTTCAT TTGTTTCACC541 GTCAACCTTT GAAGAGGCCA AGAAATTTCT TGATTTCATC ATAAGTTCAT TTGTTTCACC
601 TTCAAAGAAT ATTGGGAATG GAATTGCCGT CAGTGGAAGG TTTACTCAAC TTCCTAGTGA601 TTCAAAGAAT ATTGGGAATG GAATTGCCGT CAGTGGAAGG TTTACTCAAC TTCCTAGTGA
661 AGAACTTGAA TGTAAAGAAT CCTTTCCAAA CTACTTCCTT AAATCAGTTA CCATATACCC661 AGAACTTGAA TGTAAAGAAT CCTTTCCAAA CTACTTCCTT AAATCAGTTA CCATATACCC
721 AATCAAGTCG TGTGCTGGAT TTTCTGTGAA TCGTTGGCCA CTTTGCAGAA CAGGCCTGCT721 AATCAAGTCG TGTGCTGGAT TTTCTGTGAA TCGTTGGCCA CTTTGCAGAA CAGGCCTGCT
781 GCATGATCGA GAATGGATGA TTCAGGGTCT GACGGGTGAT ATTCTTACCC AAAAGAAGGT781 GCATGATCGA GAATGGATGA TTCAGGGTCT GACGGGTGAT ATTCTTACCC AAAAGAAGGT
841 GCCTGAAATG TCTCTTATTA GGACCTTTAT CGACCTTGAG GAAGGACTAT TGTCTGTAGA841 GCCTGAAATG TCTCTTATTA GGACCTTTAT CGACCTTGAG GAAGGACTAT TGTCTGTAGA
901 ATCTACTCGC TGCAAAGACA AGTTGCACAT CAGAATCAAG TCTAATTCAT ATGATCCAAG901 ATCTACTCGC TGCAAAGACA AGTTGCACAT CAGAATCAAG TCTAATTCAT ATGATCCAAG
961 GAGCCATGAG TTCGATACAG ATGCCAACAT ACTTGAAAAT CATGACGAGG AAACTAGAAT961 GAGCCATGAG TTCGATACAG ATGCCAACAT ACTTGAAAAT CATGACGAGG AAACTAGAAT
1021 CAATGATTGG TTAACCCGTG CCATTGGTCG ACAGTGCAAA TTGTTACAGT ATTGTAGCTC1021 CAATGATTGG TTAACCCGTG CCATTGGTCG ACAGTGCAAA TTGTTACAGT ATTGTAGCTC
1081 TAATTCCAAA CACTGCATGA ACAGAAACAA GAGTCCTGGT TTGTGCAGAG AATTAGAAAG1081 TAATTCCAAA CACTGCATGA ACAGAAACAA GAGTCCTGGT TTGTGCAGAG AATTAGAAAG
1141 CAATATCAAC TTCGCTAATG AAGCTCAGTT CTTGTTGATA TCCGAGGAGA GTGTTGCTGA1141 CAATATCAAC TTCGCTAATG AAGCTCAGTT CTTGTTGATA TCCGAGGAGA GTGTTGCTGA
1201 TCTAAATAGA AGATTAGAAG CAAAAGGCAA GGATTACAAC CGAGCTCTTG AGAAACTCAG1201 TCTAAATAGA AGATTAGAAG CAAAAGGCAA GGATTACAAC CGAGCTCTTG AGAAACTCAG
1261 TCCATATAGA TTCAGACCAA ATCTGGTAAT ATCAGGAGGT GAACCATACG CAGAAGATAA1261 TCCATATAGA TTCAGACCAA ATCTGGTAAT ATCAGGAGGT GAACCATACG CAGAAGATAA
1321 ATGGAGAACT CTCAAGATAG GAGACAGTCA TTTCACGTCG TTGGGCGGTT GTAATCGTTG1321 ATGGAGAACT CTCAAGATAG GAGACAGTCA TTTCACGTCG TTGGGCGGTT GTAATCGTTG
1381 CCAGATGATA AACATAAGTA ATGAAACTGG TCAAGTGAAG AAATCCAACG AGCCCTTGAC1381 CCAGATGATA AACATAAGTA ATGAAACTGG TCAAGTGAAG AAATCCAACG AGCCCTTGAC
1441 AACTTTAGCT TCTTATAGAA GAGTAAAGGG AAAGATCTTG TTTGGAACTC TTCTGAGATA1441 AACTTTAGCT TCTTATAGAA GAGTAAAGGG AAAGATCTTG TTTGGAACTC TTCTGAGATA
1501 CGAGATTGAT GCAAAAACAG AGTCTTGGAT TCAAGTTGGG GAAGAAGTAA ATCCAAATAC1501 CGAGATTGAT GCAAAAACAG AGTCTTGGAT TCAAGTTGGG GAAGAAGTAA ATCCAAATAC
1561 TGAATAA 根据 SEQ ID NO: 10序列分析， SEQ ID NO: 10非 ¾MC^7-2的全长序列。 需 进一步进行 5'RACE。设计如下三条特异性引物，作为新一轮的反转录引物及 5，RACE 的 3'端特异性引物。 1561 TGAATAA According to the sequence of SEQ ID NO: 10, the full length sequence of SEQ ID NO: 10 is not MC4-7-2. Further 5' RACE is required. The following three specific primers were designed as a new round of reverse transcription primers and 5, RACE 3'-end specific primers.
SEQ10 GSP1: SEQ ID NO: 11： SEQ10 GSP1: SEQ ID NO: 11:
CAAGTTGAATGTAACCGTTGG SEQ10 GSP2: SEQ ID NO: 12： AGAACACATACACCAGTCCCG CAAGTTGAATGTAACCGTTGG SEQ10 GSP2: SEQ ID NO: 12: AGAACACATACACCAGTCCCG
SEQ1 0 GS P3 : SEQ ID NO : 13 :  SEQ1 0 GS P3 : SEQ ID NO: 13:
ACTGTTGCTGCTTCAATTGCTG  ACTGTTGCTGCTTCAATTGCTG
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）。 以 SEQ ID NO: 1 1为反转录引物， 以干旱处理组小盐芥叶子提取的 mRNA为模 板进行反转录， 获得 cDNA模板， 然后按照上述 5' RACE试剂盒说明书中的步骤加 Poly C尾， 以加尾后的产物为模板进行第一轮 PCR扩增， 所用引物为 SEQ ID NO: 11与通用引物 SEQ ID NO: 7 (试剂盒自带， I为次黄嘌吟修饰的 a、 c、 g或 t) ， 具体步骤如下：  The experimental procedure was performed according to the kit instructions (5' RACE System for Rapid Amplification of cDNA Ends kit purchased from Invitrogen). Using SEQ ID NO: 1 1 as a reverse transcription primer, reverse transcription was performed using the mRNA extracted from the leaves of the salt-treated mustard leaves as a template to obtain a cDNA template, and then adding Poly C according to the procedure in the above 5' RACE kit instructions. The first round of PCR amplification was carried out using the tailed product as a template. The primers used were SEQ ID NO: 11 and the universal primer SEQ ID NO: 7 (the kit is self-contained, I is a hypoxanthine modified a, c, g or t), the specific steps are as follows:
50 μΐ PCR反应体系： 5 μΐ ΙΟ Εχ Buffer、 3 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: 11和 SEQ ID NO: 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: 11 and SEQ ID NO:
7各 2.0 μΐ以及 35 μΐ的双蒸水。 PCR反应条件： 94 °C预变性 5分钟， 33个循环 （94°C变 性 45秒， 57 °C退火 45秒， 72 °C 延伸 90秒）， 72 °C延伸 10分钟。 7 each of 2.0 μΐ and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 °C for 5 minutes, 33 cycles (94 °C for 45 seconds, 57 °C for 45 seconds, 72 °C for 90 seconds), 72 °C for 10 minutes.
所得的 PCR产物用双蒸水稀释 50倍后取 2.0 μΐ作为模板， 用 SEQ ID NO: 12与通 用引物 SEQ ID NO: 8进行第二轮 PCR扩增， 具体步骤如下：  The obtained PCR product was diluted 50-fold with double distilled water, and 2.0 μL was used as a template, and the second round of PCR amplification was carried out using SEQ ID NO: 12 and the general primer SEQ ID NO: 8. 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: 12和 SEQ ID NO: 8各 2.0 μΐ以及 35 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5分钟， 33个循环（94°C 变性 45秒， 58°C 退火 45秒， 72°C延伸 90秒）， 72 °C 延伸 10分钟。 回收第二次 PCR产物中约为 1.1K bp 大小的条带 （ Gel Extraction Kit购自 OMEGA)， 并将其连接到 pGEM-T Easy载体， 然后 转化到大肠杆菌 JM109感受态细胞中 （具体方法同上）， 并在含 50 g/mL氨苄青霉素的 LB固体培养基上进行筛选。随机挑取 8个白色菌落分别接种于含有 50 μ§/ιη1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甘油终浓度为 20% (体积比）， -80°C 保存备用。 用引物 SEQ ID NO: 12与 3'端引物 SEQ ID NO: 13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（Ts-2、 Ts-3和 Ts-5 )， 送英潍捷基（上海） 贸易有限公司测序， 获得该基因的 cDNA的一段 5'端序列。 50 μΐ PCR reaction system: 5 μΐ ΙΟ Εχ Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ diluted first round PCR product, 1.0 l Ex Taq, 10 μΜ primers SEQ ID NO: 12 and SEQ ID NO: 8 2.0 μΐ and 35 μΐ double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation for 45 seconds at 94 ° C, annealing for 45 seconds at 58 ° C, extension of 90 ° at 90 ° C), extension at 72 ° C for 10 minutes. The band of about 1.1K bp in the second PCR product was recovered (the Gel Extraction Kit was purchased from OMEGA), and ligated into the pGEM-T Easy vector, and then transformed into E. coli JM109 competent cells (the same method as above) ) and screening on LB solid medium containing 50 g/mL ampicillin. Eight white colonies were randomly picked and inoculated in LB liquid medium containing 50 μ § /ιη1 ampicillin, and cultured overnight at 37 ° C, glycerol was added to a final concentration of glycerol of 20% (volume ratio), -80 ° C Save spare. The primers SEQ ID NO: 12 and the 3' primer SEQ ID NO: 13 were used for PCR amplification (reaction system and reaction conditions as above), and three positive clones (Ts-2, Ts-3 and Ts-5) were obtained. , sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. to sequence, obtain a 5' end sequence of the cDNA of the gene.
所得的第二轮 5'RACE产物克隆子 Ts-5测序获得序列为 SEQ ID NO: 14： 1 GGGGGGGGGG ATGGAAGAAT TTCTTAAGGA ATTTGGAGAT TATTATGGAT ACCCAGAAGG 6 1 TCCAAAGAGT ATCAAAGAGA TCCGCGATAC TGAATTCAAG AGATTAGACG AAGGTGTTGT 12 1 ATACTTGGAT CATGCTGGTT CTACTTTGTA TTCTGAGTTG CAGATGGAAG ACATCTTTAA 181 GGACTTTACA AGCAATGTTT ATGGCAATCC TCATAGTCAA AGTGATATCA GTTCAGCGACThe obtained second round of 5' RACE product clone Ts-5 was sequenced to obtain SEQ ID NO: 14: 1 GGGGGGGGGG ATGGAAGAAT TTCTTAAGGA ATTTGGAGAT TATTATGGAT ACCCAGAAGG 6 1 TCCAAAGAGT ATCAAAGAGA TCCGCGATAC TGAATTCAAG AGATTAGACG AAGGTGTTGT 12 1 ATACTTGGAT CATGCTGGTT CTACTTTGTA TTCTGAGTTG CAGATGGAAG ACATCTTTAA 181 GGACTTTACA AGCAATGTTT ATGGCAATCC TCATAGTCAA AGTGATATCA GTTCAGCGAC
241 TAGTGATCTT ATAGCGGACG CTCGGCGTCA GGTACTTGAA TACTTCAATG CATCTCCTGA241 TAGTGATCTT ATAGCGGACG CTCGGCGTCA GGTACTTGAA TACTTCAATG CATCTCCTGA
301 AGACTACAGT TGCATATTTA CTTCCGGAGC CACAGCAGCA CTGAAGCTTG TCGGAGAGAC301 AGACTACAGT TGCATATTTA CTTCCGGAGC CACAGCAGCA CTGAAGCTTG TCGGAGAGAC
361 ATTTCCGTGG ACCCACGAGA GCAACTTTTT GTATAGCATG GAAAACCATA ACAGTGTACT361 ATTTCCGTGG ACCCACGAGA GCAACTTTTT GTATAGCATG GAAAACCATA ACAGTGTACT
421 TGGTATTAGG GAATATGCAT TAGGTCAAGG TGCTTCAGCA TGTGCAGTGG ATATTGAAGA421 TGGTATTAGG GAATATGCAT TAGGTCAAGG TGCTTCAGCA TGTGCAGTGG ATATTGAAGA
481 GACAGCTAAC AAGCCCGGTC AGCTAGCAAG TTCAGGACCA TTTATCAAGG TCAAGCATCG481 GACAGCTAAC AAGCCCGGTC AGCTAGCAAG TTCAGGACCA TTTATCAAGG TCAAGCATCG
541 TGCTGTGCAG ATGAGAAACA CTTCTAAACT CCAAAAGGAA GAGTCAAGAG GAGATGCCTA541 TGCTGTGCAG ATGAGAAACA CTTCTAAACT CCAAAAGGAA GAGTCAAGAG GAGATGCCTA
601 TAATTTATTT GCCTTCCCCT CAGAGTGCAA TTTTACCGGC TTGAGGTTCA ACCTAGATCT601 TAATTTATTT GCCTTCCCCT CAGAGTGCAA TTTTACCGGC TTGAGGTTCA ACCTAGATCT
661 GGTGAAGCTG ATTAAAGAAA ATACCGAAAC TACACTGCAA GGCTCTCCCT TTTGCACGAG661 GGTGAAGCTG ATTAAAGAAA ATACCGAAAC TACACTGCAA GGCTCTCCCT TTTGCACGAG
721 CAAGCGGTGG ATGGTCTTGA TTGATGCTGC CAAAGGCTGT GCTACACTAC CACCTAATTT721 CAAGCGGTGG ATGGTCTTGA TTGATGCTGC CAAAGGCTGT GCTACACTAC CACCTAATTT
781 GTCGGAGTAT CCTGCAGATT TTGTTGTTGT GTCATTCTAC AAGTTATTTG GTTATCCTAC781 GTCGGAGTAT CCTGCAGATT TTGTTGTTGT GTCATTCTAC AAGTTATTTG GTTATCCTAC
841 TGGGCTTGGT GCTCTCCTTG TACGAAATGA TGCTGCCAAG TTGCTCAAAA AGACTTACTT841 TGGGCTTGGT GCTCTCCTTG TACGAAATGA TGCTGCCAAG TTGCTCAAAA AGACTTACTT
901 CAGTGGAGGC ACTGTTGCTG CTTCAATTGC TGACATTGAC TTTGTAAAAA GAAGAGAAAG901 CAGTGGAGGC ACTGTTGCTG CTTCAATTGC TGACATTGAC TTTGTAAAAA GAAGAGAAAG
961 CGTGGAGGAT TTTTTTGAGG ATGGCTCTGC TTCATTCTTG AGTATCGCAG CCATCCGCCA961 CGTGGAGGAT TTTTTTGAGG ATGGCTCTGC TTCATTCTTG AGTATCGCAG CCATCCGCCA
1021 TGGCTTCAAA TTACTCAAGT CCCTTACTAC TTCTGCAATT TGGATGCACA CAACATCACT1021 TGGCTTCAAA TTACTCAAGT CCCTTACTAC TTCTGCAATT TGGATGCACA CAACATCACT
1081 TTCCATGTAT GTGAAGAAGA AACTTCAGGC TTTAAGACAT GGAAACGGGA CTGGTGTATG1081 TTCCATGTAT GTGAAGAAGA AACTTCAGGC TTTAAGACAT GGAAACGGGA CTGGTGTATG
1141 TGTTCT 1141 TGTTCT
将第二轮 5' RACE获得的序列 SEQ ID NO: 14, 与序列 SEQ ID NO: 10拼接， 获得 SEQ ID NO: 15：  The sequence of SEQ ID NO: 14, obtained by the second round of 5' RACE, was spliced with the sequence SEQ ID NO: 10 to obtain SEQ ID NO: 15:
1 GGGGGGGGGG ATGGAAGAAT TTCTTAAGGA ATTTGGAGAT TATTATGGAT ACCCAGAAGG 1 GGGGGGGGGG ATGGAAGAAT TTCTTAAGGA ATTTGGAGAT TATTATGGAT ACCCAGAAGG
61 TCCAAAGAGT ATCAAAGAGA TCCGCGATAC TGAATTCAAG AGATTAGACG AAGGTGTTGT61 TCCAAAGAGT ATCAAAGAGA TCCGCGATAC TGAATTCAAG AGATTAGACG AAGGTGTTGT
121 ATACTTGGAT CATGCTGGTT CTACTTTGTA TTCTGAGTTG CAGATGGAAG ACATCTTTAA121 ATACTTGGAT CATGCTGGTT CTACTTTGTA TTCTGAGTTG CAGATGGAAG ACATCTTTAA
181 GGACTTTACA AGCAATGTTT ATGGCAATCC TCATAGTCAA AGTGATATCA GTTCAGCGAC181 GGACTTTACA AGCAATGTTT ATGGCAATCC TCATAGTCAA AGTGATATCA GTTCAGCGAC
241 TAGTGATCTT ATAGCGGACG CTCGGCGTCA GGTACTTGAA TACTTCAATG CATCTCCTGA241 TAGTGATCTT ATAGCGGACG CTCGGCGTCA GGTACTTGAA TACTTCAATG CATCTCCTGA
301 AGACTACAGT TGCATATTTA CTTCCGGAGC CACAGCAGCA CTGAAGCTTG TCGGAGAGAC301 AGACTACAGT TGCATATTTA CTTCCGGAGC CACAGCAGCA CTGAAGCTTG TCGGAGAGAC
361 ATTTCCGTGG ACCCACGAGA GCAACTTTTT GTATAGCATG GAAAACCATA ACAGTGTACT361 ATTTCCGTGG ACCCACGAGA GCAACTTTTT GTATAGCATG GAAAACCATA ACAGTGTACT
421 TGGTATTAGG GAATATGCAT TAGGTCAAGG TGCTTCAGCA TGTGCAGTGG ATATTGAAGA421 TGGTATTAGG GAATATGCAT TAGGTCAAGG TGCTTCAGCA TGTGCAGTGG ATATTGAAGA
481 GACAGCTAAC AAGCCCGGTC AGCTAGCAAG TTCAGGACCA TTTATCAAGG TCAAGCATCG481 GACAGCTAAC AAGCCCGGTC AGCTAGCAAG TTCAGGACCA TTTATCAAGG TCAAGCATCG
541 TGCTGTGCAG ATGAGAAACA CTTCTAAACT CCAAAAGGAA GAGTCAAGAG GAGATGCCTA541 TGCTGTGCAG ATGAGAAACA CTTCTAAACT CCAAAAGGAA GAGTCAAGAG GAGATGCCTA
601 TAATTTATTT GCCTTCCCCT CAGAGTGCAA TTTTACCGGC TTGAGGTTCA ACCTAGATCT601 TAATTTATTT GCCTTCCCCT CAGAGTGCAA TTTTACCGGC TTGAGGTTCA ACCTAGATCT
661 GGTGAAGCTG ATTAAAGAAA ATACCGAAAC TACACTGCAA GGCTCTCCCT TTTGCACGAG661 GGTGAAGCTG ATTAAAGAAA ATACCGAAAC TACACTGCAA GGCTCTCCCT TTTGCACGAG
721 CAAGCGGTGG ATGGTCTTGA TTGATGCTGC CAAAGGCTGT GCTACACTAC CACCTAATTT721 CAAGCGGTGG ATGGTCTTGA TTGATGCTGC CAAAGGCTGT GCTACACTAC CACCTAATTT
781 GTCGGAGTAT CCTGCAGATT TTGTTGTTGT GTCATTCTAC AAGTTATTTG GTTATCCTAC781 GTCGGAGTAT CCTGCAGATT TTGTTGTTGT GTCATTCTAC AAGTTATTTG GTTATCCTAC
841 TGGGCTTGGT GCTCTCCTTG TACGAAATGA TGCTGCCAAG TTGCTCAAAA AGACTTACTT841 TGGGCTTGGT GCTCTCCTTG TACGAAATGA TGCTGCCAAG TTGCTCAAAA AGACTTACTT
901 CAGTGGAGGC ACTGTTGCTG CTTCAATTGC TGACATTGAC TTTGTAAAAA GAAGAGAAAG901 CAGTGGAGGC ACTGTTGCTG CTTCAATTGC TGACATTGAC TTTGTAAAAA GAAGAGAAAG
961 CGTGGAGGAT TTTTTTGAGG ATGGCTCTGC TTCATTCTTG AGTATCGCAG CCATCCGCCA 102 1 TGGCTTCAAA TTACTCAAGT CCCTTACTAC TTCTGCAATT TGGATGCACA CAACATCACT961 CGTGGAGGAT TTTTTTGAGG ATGGCTCTGC TTCATTCTTG AGTATCGCAG CCATCCGCCA 102 1 TGGCTTCAAA TTACTCAAGT CCCTTACTAC TTCTGCAATT TGGATGCACA CAACATCACT
10 8 1 TTCCATGTAT GTGAAGAAGA AACTTCAGGC TTTAAGACAT GGAAACGGGA CTGGTGTATG10 8 1 TTCCATGTAT GTGAAGAAGA AACTTCAGGC TTTAAGACAT GGAAACGGGA CTGGTGTATG
114 1 TGTTCTGTAT GGCAGTGAAA ATCTGAAGTT ATCTTCACAT AAATCAGGCC CAACGGTTAC114 1 TGTTCTGTAT GGCAGTGAAA ATCTGAAGTT ATCTTCACAT AAATCAGGCC CAACGGTTAC
12 0 1 ATTCAACTTG AAGAGACCTG ATGGCTCTTG GTTTGGCTAC CTGGAGGTGG AAAAGCTCGC12 0 1 ATTCAACTTG AAGAGACCTG ATGGCTCTTG GTTTGGCTAC CTGGAGGTGG AAAAGCTCGC
126 1 TTCTTTATCT GGAATCCAGT TACGGACAGG ATGCTTTTGC AATCCTGGCG CATGTGCAAA126 1 TTCTTTATCT GGAATCCAGT TACGGACAGG ATGCTTTTGC AATCCTGGCG CATGTGCAAA
132 1 GTATCTCGCC TTGTCTCATT CGAATCTATT GTCTAACGTA GAGGCTGGGC ATATTTGTTG132 1 GTATCTCGCC TTGTCTCATT CGAATCTATT GTCTAACGTA GAGGCTGGGC ATATTTGTTG
13 8 1 GGACGATAAT GATGTGATTA ATGGAAAACC AACTGGAGCT GTTAGAGTTT CGTTTGGTTA13 8 1 GGACGATAAT GATGTGATTA ATGGAAAACC AACTGGAGCT GTTAGAGTTT CGTTTGGTTA
144 1 TATGTCAACC TTTGAAGAGG CCAAGAAATT TCTTGATTTC ATCATAAGTT CATTTGTTTC144 1 TATGTCAACC TTTGAAGAGG CCAAGAAATT TCTTGATTTC ATCATAAGTT CATTTGTTTC
15 0 1 ACCTTCAAAG AATATTGGGA ATGGAATTGC CGTCAGTGGA AGGTTTACTC AACTTCCTAG15 0 1 ACCTTCAAAG AATATTGGGA ATGGAATTGC CGTCAGTGGA AGGTTTACTC AACTTCCTAG
156 1 TGAAGAACTT GAATGTAAAG AATCCTTTCC AAACTACTTC CTTAAATCAG TTACCATATA156 1 TGAAGAACTT GAATGTAAAG AATCCTTTCC AAACTACTTC CTTAAATCAG TTACCATATA
162 1 CCCAATCAAG TCGTGTGCTG GATTTTCTGT GAATCGTTGG CCACTTTGCA GAACAGGCCT162 1 CCCAATCAAG TCGTGTGCTG GATTTTCTGT GAATCGTTGG CCACTTTGCA GAACAGGCCT
16 8 1 GCTGCATGAT CGAGAATGGA TGATTCAGGG TCTGACGGGT GATATTCTTA CCCAAAAGAA16 8 1 GCTGCATGAT CGAGAATGGA TGATTCAGGG TCTGACGGGT GATATTCTTA CCCAAAAGAA
174 1 GGTGCCTGAA ATGTCTCTTA TTAGGACCTT TATCGACCTT GAGGAAGGAC TATTGTCTGT174 1 GGTGCCTGAA ATGTCTCTTA TTAGGACCTT TATCGACCTT GAGGAAGGAC TATTGTCTGT
18 0 1 AGAATCTACT CGCTGCAAAG ACAAGTTGCA CATCAGAATC AAGTCTAATT CATATGATCC18 0 1 AGAATCTACT CGCTGCAAAG ACAAGTTGCA CATCAGAATC AAGTCTAATT CATATGATCC
186 1 AAGGAGCCAT GAGTTCGATA CAGATGCCAA CATACTTGAA AATCATGACG AGGAAACTAG186 1 AAGGAGCCAT GAGTTCGATA CAGATGCCAA CATACTTGAA AATCATGACG AGGAAACTAG
192 1 AATCAATGAT TGGTTAACCC GTGCCATTGG TCGACAGTGC AAATTGTTAC AGTATTGTAG192 1 AATCAATGAT TGGTTAACCC GTGCCATTGG TCGACAGTGC AAATTGTTAC AGTATTGTAG
198 1 CTCTAATTCC AAACACTGCA TGAACAGAAA CAAGAGTCCT GGTTTGTGCA GAGAATTAGA198 1 CTCTAATTCC AAACACTGCA TGAACAGAAA CAAGAGTCCT GGTTTGTGCA GAGAATTAGA
2 04 1 AAGCAATATC AACTTCGCTA ATGAAGCTCA GTTCTTGTTG ATATCCGAGG AGAGTGTTGC2 04 1 AAGCAATATC AACTTCGCTA ATGAAGCTCA GTTCTTGTTG ATATCCGAGG AGAGTGTTGC
2 10 1 TGATCTAAAT AGAAGATTAG AAGCAAAAGG CAAGGATTAC AACCGAGCTC TTGAGAAACT2 10 1 TGATCTAAAT AGAAGATTAG AAGCAAAAGG CAAGGATTAC AACCGAGCTC TTGAGAAACT
2 16 1 CAGTCCATAT AGATTCAGAC CAAATCTGGT AATATCAGGA GGTGAACCAT ACGCAGAAGA2 16 1 CAGTCCATAT AGATTCAGAC CAAATCTGGT AATATCAGGA GGTGAACCAT ACGCAGAAGA
222 1 TAAATGGAGA ACTCTCAAGA TAGGAGACAG TCATTTCACG TCGTTGGGCG GTTGTAATCG222 1 TAAATGGAGA ACTCTCAAGA TAGGAGACAG TCATTTCACG TCGTTGGGCG GTTGTAATCG
22 8 1 TTGCCAGATG ATAAACATAA GTAATGAAAC TGGTCAAGTG AAGAAATCCA ACGAGCCCTT22 8 1 TTGCCAGATG ATAAACATAA GTAATGAAAC TGGTCAAGTG AAGAAATCCA ACGAGCCCTT
234 1 GACAACTTTA GCTTCTTATA GAAGAGTAAA GGGAAAGATC TTGTTTGGAA CTCTTCTGAG234 1 GACAACTTTA GCTTCTTATA GAAGAGTAAA GGGAAAGATC TTGTTTGGAA CTCTTCTGAG
24 0 1 ATACGAGATT GATGCAAAAA CAGAGTCTTG GATTCAAGTT GGGGAAGAAG TAAATCCAAA24 0 1 ATACGAGATT GATGCAAAAA CAGAGTCTTG GATTCAAGTT GGGGAAGAAG TAAATCCAAA
246 1 TACTGAATAA 246 1 TACTGAATAA
根据 SEQ ID NO: 15序列设计一对引物如下：  A pair of primers were designed according to the sequence of SEQ ID NO: 15 as follows:
ThMCSU-2F: SEQ ID NO: 16： ThMCSU-2F: SEQ ID NO: 16:
ATGGAAGAATTTCTTAAGGAA ATGGAAGAATTTCTTAAGGAA
ThMCSU-2R: SEQ ID NO: 17： ThMCSU-2R : SEQ ID NO: 17:
TTATTCAGTATTTGGATTTACTTC AP: SEQ ID NO: 18： TTATTCAGTATTTGGATTTACTTC AP: SEQ ID NO: 18:
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT  GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT
通过 SEQ ID NO: 11和 SEQ ID NO: 12来克隆 ThMCSU-2全长编码序列。  The full-length coding sequence of ThMCSU-2 was cloned by SEQ ID NO: 11 and SEQ ID NO: 12.
提取小盐芥 RNA， 以引物 SEQ ID NO: 18为反转录引物， 获取小盐芥 cDNA。 采用 Stratagene的 PfuUltra II Fusion HS DNA Polymerase, 以小盐芥的 cDNA为模板进 行 PCR反应。 50 μΐ PCR反应体系： 5 μΐ lO PfuUltra II reaction Buffer 0.5 μΐ 25 mM 的 dNTP、 2.0 μΐ cDNA 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ的引物 SEQ ID NO: 16和 SEQIDNO: 17各 2.0 μΐ以及 37.5 μΐ的双蒸水。 PCR反应条件：The small salt mustard RNA was extracted, and the primer SEQ ID NO: 18 was used as a reverse transcription primer to obtain a small salt mustard cDNA. Using Stratagene's PfuUltra II Fusion HS DNA Polymerase, using the cDNA of the small salt mustard as a template The PCR reaction was carried out. 50 μΐ PCR reaction system: 5 μΐ lO PfuUltra II reaction Buffer 0.5 μΐ 25 mM dNTP, 2.0 μΐ cDNA 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase 10 μΜ primers SEQ ID NO: 16 and SEQ ID NO: 17 each 2.0 μΐ and 37.5 μΐ Double steamed water. PCR reaction conditions:
95°C预变性 2分钟， 35个循环 （95°C 变性 25秒， 50°C退火 30秒， 72°C 延伸 2分 钟）， 72 °C延伸 5分钟。 Pre-denaturation at 95 °C for 2 minutes, 35 cycles (denaturation for 25 seconds at 95 °C, annealing for 30 seconds at 50 °C, extension of 2 minutes at 72 °C), extension at 72 °C for 5 minutes.
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 ΙΟ Εχ Buffer, 0.5 μΐ 5 mM的 dATP, 1.0 μΐ Ex Taq。 反应条件： 70°C反应 30分钟。 将得到约 2.4 Kbp的 DNA片段 回收 （Omega回收试剂盒） ， 连接至 pGEM T-easy载体上 （得到 ThMCSU-2-pGEM 质粒） ， 然后转化 JM109, 随机挑取 8个白色菌落分别接种于含有 50 g/ml氨苄青霉 素的 LB液体培养基中培养， 37°C培养过夜后加甘油至终浓度 20% (体积比） ， -80°C 保存备用。 用引物 SEQIDNO: 16与 SEQ ID NO: 17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， 送至英潍捷基 （上海） 贸易有限公司测序， 序列为 SEQIDNO: 2， 其编码的蛋白的氨基酸序列为 SEQIDNO: 1。  The PCR amplification product was added with A tail: The PCR product was added with 2.5 volumes of absolute ethanol, placed at -20 ° C for 10 minutes, centrifuged, and the supernatant was removed, dried, and dissolved in 21 μl of double distilled water. Add 2.5 μΐ Εχ Εχ Buffer, 0.5 μΐ 5 mM dATP, 1.0 μΐ Ex Taq. Reaction conditions: The reaction was carried out at 70 ° C for 30 minutes. A DNA fragment of about 2.4 Kbp was recovered (Omega recovery kit), ligated into the pGEM T-easy vector (the ThMCSU-2-pGEM plasmid was obtained), and then transformed into JM109, and 8 white colonies were randomly picked and inoculated to contain 50. G/ml ampicillin was cultured in LB liquid medium, cultured at 37 ° C overnight, and glycerin was added to a final concentration of 20% (volume ratio), and stored at -80 ° C until use. The primers SEQ ID NO: 16 and SEQ ID NO: 17 were used for PCR amplification (reaction system and reaction conditions are the same as above), and three positive clones were obtained and sent to Yingjie Jieji (Shanghai) Trading Co., Ltd. for sequencing. The sequence is SEQ ID NO: 2. The amino acid sequence of the encoded protein is SEQ ID NO: 1.
MCSU-2蛋白的氨基酸序列： SEQ ID NO: 1  Amino acid sequence of MCSU-2 protein: SEQ ID NO: 1
1 MEEFLKEFGD YYGYPEGPKS IKEIRDTEFK RLDEGVVYLD HAGSTLYSEL QMEDIFKDFT 1 MEEFLKEFGD YYGYPEGPKS IKEIRDTEFK RLDEGVVYLD HAGSTLYSEL QMEDIFKDFT
61 SNVYGNPHSQ SDISSATSDL IADARRQVLE YFNASPEDYS CIFTSGATAA LKLVGETFPW61 SNVYGNPHSQ SDISSATSDL IADARRQVLE YFNASPEDYS CIFTSGATAA LKLVGETFPW
121 THESNFLYSM ENHNSVLGIR EYALGQGASA CAVDIEETAN KPGQLASSGP FIKVKHRAVQ121 THESNFLYSM ENHNSVLGIR EYALGQGASA CAVDIEETAN KPGQLASSGP FIKVKHRAVQ
181 MRNTSKLQKE ESRGDAYNLF AFPSECNFTG LRFNLDLVKL IKENTETTLQ GSPFCTSKRW181 MRNTSKLQKE ESRGDAYNLF AFPSECNFTG LRFNLDLVKL IKENTETTLQ GSPFCTSKRW
241 MVLIDAAKGC ATLPPNLSEY PADFVVVSFY KLFGYPTGLG ALLVRNDAAK LLKKTYFSGG241 MVLIDAAKGC ATLPPNLSEY PADFVVVSFY KLFGYPTGLG ALLVRNDAAK LLKKTYFSGG
301 TVAASIADID FVKRRESVED FFEDGSASFL SIAAIRHGFK LLKSLTTSAI WMHTTSLSMY301 TVAASIADID FVKRRESVED FFEDGSASFL SIAAIRHGFK LLKSLTTSAI WMHTTSLSMY
361 VKKKLQALRH GNGTGVCVLY GSENLKLSSH KSGPTVTFNL KRPDGSWFGY LEVEKLASLS361 VKKKLQALRH GNGTGVCVLY GSENLKLSSH KSGPTVTFNL KRPDGSWFGY LEVEKLASLS
421 GIQLRTGCFC NPGACAKYLA LSHSNLLSNV EAGHICWDDN DVINGKPTGA VRVSFGYMST421 GIQLRTGCFC NPGACAKYLA LSHSNLLSNV EAGHICWDDN DVINGKPTGA VRVSFGYMST
481 FEEAKKFLDF IISSFVSPSK NIGNGIAVSG RFTQLPSEEL ECKESFPNYF LKSVTIYPIK481 FEEAKKFLDF IISSFVSPSK NIGNGIAVSG RFTQLPSEEL ECKESFPNYF LKSVTIYPIK
541 SCAGFSVNRW PLCRTGLLHD REWMIQGLTG DILTQKKVPE MSLIRTFIDL EEGLLSVEST541 SCAGFSVNRW PLCRTGLLHD REWMIQGLTG DILTQKKVPE MSLIRTFIDL EEGLLSVEST
601 RCKDKLHIRI KSNSYDPRSH EFDTDANILE NHDEETRIND WLTRAIGRQC KLLQYCSSNS601 RCKDKLHIRI KSNSYDPRSH EFDTDANILE NHDEETRIND WLTRAIGRQC KLLQYCSSNS
661 KHCMNRNKSP GLCRELESNI NFANEAQFLL ISEESVADLN RRLEAKGKDY NRALEKLSPY661 KHCMNRNKSP GLCRELESNI NFANEAQFLL ISEESVADLN RRLEAKGKDY NRALEKLSPY
721 RFRPNLVISG GEPYAEDKWR TLKIGDSHFT SLGGCNRCQM INISNETGQV KKSNEPLTTL721 RFRPNLVISG GEPYAEDKWR TLKIGDSHFT SLGGCNRCQM INISNETGQV KKSNEPLTTL
781 ASYRRVKGKI LFGTLLRYEI DAKTESWIQV GEEVNPNTE 编码基因 ThMCSU-2的核苷酸序列： SEQ ID NO: 2 781 ASYRRVKGKI LFGTLLRYEI DAKTESWIQV GEEVNPNTE The nucleotide sequence of the coding gene ThMCSU-2: SEQ ID NO: 2
1 ATGGAAGAAT TTCTTAAGGA ATTTGGAGAT TATTATGGAT ACCCAGAAGG TCCAAAGAGT1 ATGGAAGAAT TTCTTAAGGA ATTTGGAGAT TATTATGGAT ACCCAGAAGG TCCAAAGAGT
61 ATCAAAGAGA TCCGCGATAC TGAATTCAAG AGATTAGACG AAGGTGTTGT ATACTTGGAT61 ATCAAAGAGA TCCGCGATAC TGAATTCAAG AGATTAGACG AAGGTGTTGT ATACTTGGAT
121 CATGCTGGTT CTACTTTGTA TTCTGAGTTG CAGATGGAAG ACATCTTTAA GGACTTTACA V工工工; DVVIDVS 工工 33:)£)V∞V VDD1VVV0VV ； DVVVS工 VV工 V3VVV工 V TQZZ121 CATGCTGGTT CTACTTTGTA TTCTGAGTTG CAGATGGAAG ACATCTTTAA GGACTTTACA V workers; DVVIDVS workers 33:) £) V∞V VDD1VVV0VV; DVVVS VV V3VVV V TQZZ
SlVSV Sl工 ∞工 VV工 £)工工 £) SIDVID工工工 VID工 工 工：)工：) V τζζζSlVSV Sl Work Completion VV Work £) Workman £) SIDVID Workman VID Workman:) Work:) V τζζζ
VSVSSIVVV工 VOVVOVDODV 工 工 £)£) VOOVD1V1VV 工 £)£)工3工 VVVID DVOVD11VOV 工 VIVIDIDISVID 工; 工工 ：)工:) £)V∞3VV DV11VOOVVD OOVVVVDOVV OV11VOVVOV τοτζ 工 VVV工; 工 3£)工工 £)1£)V£)V 0 IXJ IXJO IXJ IXJ IXJ IXJO 工：) 工 V V工; 工工; DVVVSVSSIVVV VOVVOVDODV Workers £) £) VOOVD1V1VV Jobs £) £) Workers 3 VVVID DVOVD11VOV Workers VIVIDIDISVID Workers; Workers:) Workers:) £) V∞3VV DV11VOOVVD OOVVVVDOVV OV11VOVVOV τοτζ VVV Workers; Workers 3 £) £)1£)V£)V 0 IXJ IXJO IXJ IXJ IXJ IXJO Work:) VV workers; workers; DVV
； DIVIVVIDSVV V0V11VV0V0 VDSISI工工 £)£) 工：) DISVSVV:) VVVS DVVS工 VD01DVDVVV Τ86Τ; DIVIVVIDSVV V0V11VV0V0 VDSISI workers £)£) Work:) DISVSVV:) VVVS DVVS VD01DVDVVV Τ86Τ
； DID工工 VV工; D工; D £)V工 £)工工 ； DV工工 £)工工 VVV DSISVDVS:)工 £)£)工工 V SIS τζβτ 工 VSIVVIDIVV SV工：) ravsivD工 VV VV011DV1VD VIVSID工工 £)V£) Τ98Τ 工 DD1V01V1VD 工工 VV工; D工 SVV ：)工 工 V3£)l工 TOST 工; DV工; 工 £)工3工 £)工工 V工 DVOOVVOOVO 工工; DiDvsmv工 工工; DIDVSSV工工 V工工; D工; D工 £)1V ; DID workers VV workers; D workers; D £) V workers £) workers; DV workers £) workers VVV DSISVDVS:) work £) £) workers V SIS τζβτ work VSIVVIDIVV SV workers:) ravsivD workers VV VV011DV1VD VIVSID workman £)V£) Τ98Τ DD1V01V1VD Worker VV work; D work SVV:)Worker V3£)l work TOST work; DV work; work £) work 3 work £) work V work DVOOVVOOVO work DiDvsmv workers; DIDVSSV workers V workers; D workers; D workers £) 1V
VVOVVVVDDD V工工; D工工 工 £)£)∞V£)工:)工 SSSV:)工工 V£)工 Τ89Τ 工 V£)工 VDSI S 工 SSVDVVS 工工：) 工 £)工3工工工工 V£) £)工3£)工£)工∞工 τζ9τ 工 vv :) V工 VIVIDIDV工工 SVIDIVVV工工; D D11DV1DVVV ； DID工工工; DIDIVV SVVV工 SIVVS 工工; 工 SV工; DID工工; DVV ； D工; DV工工工 £)£)V ； 工工 vvsmv TOST 工工:)：) V ； D工工工 £)工工工 VID 工工 SVV工 VIDIV ； D工工工 V£)工工; D工 11VVVOVVDD £)£)V£)VV£)工工工 \^\ VVOVVVVDDD V-worker; D-worker £)£)∞V£)Worker:)Worker SSSV:)Worker V£)Worker 89ΤWork V£)Work VDSI S Work SSVDVVS Workman:) Work £)Work 3 Workers and workers V£) £)Works 3£)Works £)Worksman τζ9τ Work vv :) V workers VIVIDIDV workers SVIDIVVV workers; D D11DV1DVVV ; DID workers; DIDIVV SVVV workers SIVVS workers; ; DID workers; DVV; D workers; DV workers £) £) V; workers vvsmv TOST workers:):) V; D workers K) workers and workers VID workers SVV workers VIDIV; Workers V£)工工; D工11VVVOVVDD £)£)V£)VV£)工工工\^\
； DIDVVID工 工 V工工 £)£)工工工∞ 工工工 £)V£)V工工 £) 工：) SVSSI VV DDVVVVOOIV V工工 V£)工 £)1V£) τδετ 工 VV工 V∞V£)£) £)工工 £)工工工 V工 V ：) £)£)£) I £)£)V£) 工工 V工; DIVVSID 工工 VID工; D工 £)工工 τζζτDIDVVID 工工工工£)£)工工工工工工£)V£)V工工£) 工:) SVSSI VV DDVVVVOOIV V工工V£)工£)1V£) τδετ VV V∞V£)£) £)Workers £)Workers V V:) £)£)£) I £)£)V£) Work V; DIVVSID Work VID; D Work £) Worker τζζτ
33∞工：)工 VIS ； D£)工工工工; D£)工 V ssvDvsrav工 工 工 VV£)£) ΐ9ζτ33 Completion:) VIS; D£) 工工工; D£)工 V ssvDvsrav 工工 VV£)£) ΐ9ζτ
LLDLL LLLLLLDLLLL ：) V工:) £)£)工工工 £) £)工工：)工:) £)£)1V τοζτLLDLL LLLLLLDLLLL :) V:) £)£)工工 £) £)工工:)工:) £)£)1V τοζτ
£)工工; DVVID工工 V ：) V工工 £)∞νν3 33£)£)V:)工 VVV 工 VIDVID工工; D1V 工工 £)VV£)工; D工 V VVVS工 τ^ττ 工 V工 £)工3工工 £)工 工 £)工£)£)工：） VOOODVVVOO 工 工工工 3£)£)V:)工工：) VV VSVVSVVS工 £) τδοτ 工 V工 SIVIDID工工 工：) 工 工工 工; D工工 ：) V工:) V工工 3：)：) 工 SVVID工; DV工工 τζοτ£)工工; DVVID工工V :) V工工£)∞νν3 33£)£)V:)VVV work VIDVID workman; D1V workman £)VV£) work; D work V VVVS work τ^ Ττ工V工£)工3工工£)工工£)工£)£)工:) VOOODVVVOO 工工工3£)£)V:)工工:) VV VSVVSVVS工£) τδοτ 工 V SIVIDID workers:) workers and workers; D workers:) V workers:) V workers 3:):) workers SVVID workers; DV workers τζοτ
VVVID工工; 3£)£)工 0VD0D1V10V £)工工; D工工 VID工工 £)£)V£)工工工工工工 T9S 工 V£)£)V£m∞ OVVVOVOVVO VVVVV工 Ώ工工工 ； DVS工工 工 ； D£)工工 VVID工工; D £)工3£)工工 το6 工工; DV工工; VVVVID工; D£)工工 VS工 ννν∞ν工 £)工工3：)工：)工:) £) 工 £)£)工工:) £)£)£)工 ； DV工; DIDIVIIS £)工工工 V工工 £)VV ； DV工; D工工 VIDIS IXJ 0 IXJ IXJ 0 IXJ IXJ 0 IXJ IXJ 工工 VSVDSI :) IQL 工 visvssms 工工工 vv工; DIDVID DV1DVDV1DO 工 si ssvvv:) DSI SIVS工工 V£)工工; D工 £)£)ΐν IZL svravDSi工工 工:) 331 工 VVDSI VDV工 ：) VVV∞3V1V VVVSVVV工工 V Τ99 工; 工; VID工工 £)£)V£)工工 3£)∞:)V工工工工 VVDOIOVOVD 1 3：)：)工工 το9VVVID engineering; 3£)£)工0VD0D1V10V £)工工; D工工VID工工£)£)V£)工工工工工T9S 工V£)£)V£m∞ OVVVOVOVVO VVVVV factory D工工工工; D£)工工VVID工工; D £)工3£)工工το6 工工; DV工工; VVVVID工; D£)工工VS工ννν∞ν工 £ )工工3:)工:)工:) £)工£)£)工工:) £)£)£)工; DV工; DIDIVIIS £)工工V工工£)VV ; DV work; D工工VIDIS IXJ 0 IXJ IXJ 0 IXJ IXJ 0 IXJ IXJ Worker VSVDSI :) IQL worker visvssms worker vv; DIDVID DV1DVDV1DO worker si ssvvv:) DSI SIVS worker V £) workman; D work £) £ ) ΐν IZL svravDSi workers :) 331 workers VVDSI VDV workers :) VVV ∞ 3V1V VVVSVVV workers V Τ 99 workers; workers; VID workers £) £) V £) workers 3 £) ∞:) V workers VVDOIOVOVD 1 3:):)Worker το9
LLLLLLVLLLLLLVVLL V工:) 3£)1V£)V£) 工 £)V£) VVOOVVVVDD 工; DVVV工; D工工; D 工 V LLLLLLVLLLLLLVVLL V:) 3£)1V£)V£) work £)V£) VVOOVVVVDD work; DVVV work; D work; D work V
EmvDSvv:)工 SSVVID工 V工工工 v vssv:)工工 工:) £)V ：)工 VV EmvDSvv:) SSVVID Worker V Worker v vssv:) Workers:) £)V :) Worker VV
：) VV工:) 工工 V1V 工工: )£)工 工 £)£)V工 工 VIDSIV工 VVS :) VV workers :) Workers V1V Workers : ) £) Workers £) £) V Workers VIDSIV Workers VVS
£)£)V工工 V工 £)£)工 工; DV工 SISVIDV V1VDDVVVVO SIVIDSV工 工工工工工; DVVIDS VOVODVDDDV Τ9Ζ £)£)V工工 V工 £)£)工工; DV工 SISVIDV V1VDDVVVVO SIVIDSV 工工工工; DVVIDS VOVODVDDDV Τ9Ζ
£)£)工∞3工工工 V £)工工:) £)VV£)工：） VDOVDOVDVD ：) £)ν£)∞:)1工：） V工工工 V工 V3£)工 τοε 工 工：) VSV SIVVID工工; DV工 VVS工工; DV工 £)£) 工∞£)∞工3 £)£)工∞3工工V £)工工:) £)VV£)工:) VDOVDOVDVD :) £)ν£)∞:)1工:) V 工工V工V3£) Τοε 工工 :) VSV SIVVID 工工; DV 工 VVS 工工; DV工£)£) 工∞£) Completion 3
工工; 工 SV工 ：) ν∞£)ν3工工 £) VD1V1V010V VVIDISVIVID工 工工工 SIVVIDSV Τ8Τ Workman; Work SV Work :) ν∞£)ν3工工 £) VD1V1V010V VVIDISVIVID Engineering Worker SIVVIDSV Τ8Τ
ZSM7.0/CT0ZN3/X3d 234 1 GCTTCTTATA GAAGAGTAAA GGGAAAGATC TTGTTTGGAA CTCTTCTGAG ATACGAGATT 24 0 1 GATGCAAAAA CAGAGTCTTG GATTCAAGTT GGGGAAGAAG TAAATCCAAA TACTGAATAA 实施例 3 r/zMC^/-2基因植物表达载体构建 ZSM7.0/CT0ZN3/X3d 234 1 GCTTCTTATA GAAGAGTAAA GGGAAAGATC TTGTTTGGAA CTCTTCTGAG ATACGAGATT 24 0 1 GATGCAAAAA CAGAGTCTTG GATTCAAGTT GGGGAAGAAG TAAATCCAAA TACTGAATAA Example 3 Construction of r/zMC^/-2 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。 选择含双增强子的 35S启动子及 Tnos终止子分别作为 ThMCS f/- 2基因的启动子和终止子。 构建流程图如图 1所示。  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 35S promoter containing the double enhancer and the Tnos terminator were selected as promoters and terminators of the ThMCS f/-2 gene, respectively. The construction flow chart is shown in Figure 1.
使用引物 SEQ ID NO: 19和 SEQ ID NO: 20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 l 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: 19禾口 SEQ ID NO: 20各 2.0 μΐ以及 31 μΐ 的双蒸水。 PCR反应条件： 94°C预变性 5分钟， 33个循环 （ 94°C 变性 30秒， 56°C 退火 30秒， 72°C延伸 30秒）， 72°C延伸 10分钟。通过 EcoRI、 Bglll酶切后将所得 PCR 产物连接到 pCAMBIA2300 ( Promega, T4连接酶试剂盒） 获得 pCAMBIA2300-l。  Using primers SEQ ID NO: 19 and SEQ ID NO: 20, Pnos was amplified 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 μl ΡΟ Reaction system: 10 l 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 1.0 μΐ ρΒΙ121 1.0 μΐ PrimeSTAR 10 μΜ primers SEQ ID NO: 19 and SEQ ID NO: 20 each 2.0 μΐ and 31 μΐ double steamed water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 56 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was digested with EcoRI and Bglll and ligated into pCAMBIA2300 (Promega, T4 ligase kit) to obtain pCAMBIA2300-1.
SEQ ID NO: 19 ： SEQ ID NO: 19:
GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 20：  GCACGAATTCATACAAATGGACGAACGGAT SEQ ID NO: 20:
ATCCAGATCTAGATCCGGTGCAGATTATTTG SEQ ID NO: 21禾 P SEQ ID NO: 22以 pBI121为模板扩增 Tnos, 采用 TaKaRa的 ATCCAGATCTAGATCCGGTGCAGATTATTTG SEQ ID NO: 21 and P SEQ ID NO: 22 amplifies Tnos with pBI121 as a template, using TaKaRa
PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5 ><PS Buffer、 3 μΐ 2.5 mM的 dNTP 1.0 μΐ pBI121 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 16禾口 SEQ ID NO:PrimeSTAR HS DNA polymerase. 50 μΐ PCR reaction system: 10 μΐ 5 ><PS Buffer, 3 μΐ 2.5 mM dNTP 1.0 μΐ pBI121 1.0 μΐ PrimeSTAR, 10 μΜ primer SEQ ID NO: 16 and SEQ ID NO:
17各 2.0 μΐ以及 31 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5分钟， 33个循环（94°C 变性 30秒， 58 °C退火 30秒， 72°C延伸 30秒）， 72°C延伸 10分钟。 通过 Kpnl、 EcoRI 酶切后将所得 PCR产物连接到 pCAMBIA2300-l (Promega T4连接酶试剂盒） 获得 pCAMBIA2300-2。 17 each of 2.0 μΐ and 31 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The resulting PCR product was digested with Kpnl, EcoRI and ligated into pCAMBIA2300-1 (Promega T4 ligase kit) to obtain pCAMBIA2300-2.
SEQ ID NO: 21： SEQ ID NO: 21:
CGGGG7MCCGAATTTCCCCGATCGTTCAAA  CGGGG7MCCGAATTTCCCCGATCGTTCAAA
SEQ ID NO: 22：  SEQ ID NO: 22:
TCKGAA JJCCC AGTGAATTCCCGATCT AGT A TCKGAA JJCCC AGTGAATTCCCGATCT AGT A
SEQ ID NO: 23和 SEQ ID NO: 24以 pCAMBIA2300质粒为模板扩增拟南芥 35S 启动子。采用 TaKaRa的 PrimeSTAR HS DNA聚合酶。 50 μΐ PCR反应体系： 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM的 dNTP、 1.0 μΐ稀释 50倍的 pCAMBIA2300质粒、 1.0 μΐ PrimeSTAR、 10 μΜ的引物 SEQ ID NO: 23禾 P SEQ ID NO: 24各 2.0 μΐ以及 31 μΐ的双蒸水。 PCR 反应条件： 94°C预变性 5分钟， 33个循环（94°C变性 30秒， 50°C退火 30秒， 72°C延 伸 30秒）， 72°C延伸 10分钟。 通过 HindIII、 Xbal酶切后将所得 PCR产物连接到 （连 接方法同上） pCAMBIA2300-2获得 pCAMBIA2300-3 SEQ ID NO: 23 and SEQ ID NO: 24 amplify Arabidopsis thaliana 35S with pCAMBIA2300 plasmid as template Promoter. PrimeSTAR HS DNA polymerase using TaKaRa. 50 μΐ PCR reaction system: 10 μΐ 5 xPS Buffer 3 μΐ 2.5 mM dNTP, 1.0 μΐ diluted 50-fold pCAMBIA2300 plasmid, 1.0 μΐ PrimeSTAR, 10 μΜ primer SEQ ID NO: 23 and P SEQ ID NO: 24 each 2.0 μΐ And 31 μΐ of double distilled water. PCR reaction conditions: predenaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 ° C for 30 seconds, annealing at 50 ° C for 30 seconds, extension at 72 ° C for 30 seconds), extension at 72 ° C for 10 minutes. The obtained PCR product was ligated by HindIII and Xbal digestion (connection method is the same as above) pCAMBIA2300-2 to obtain pCAMBIA2300-3
SEQ ID NO: ： 23： SEQ ID NO: : 23:
ACT^GCrJATGGTGGAGCACGACACTCT  ACT^GCrJATGGTGGAGCACGACACTCT
SEQ ID NO: 24：  SEQ ID NO: 24:
GC r ¾ AG AG AT AG ATTTGT AG AG AG AG AC  GC r 3⁄4 AG AG AT AG ATTTGT AG AG AG AG AC
SEQ ID NO: 25和 SEQ ID NO: 26扩增实施例 2中所克隆的含有全长 ThMCSU-2 基因的 r/zMC^ -2-pGEM质粒为模板， 扩增两端带有所设计酶切位点的 ThMCSU-2基 因。 采用 Stratagene的 PfuUltra II Fusion HS DNA Polymerase。 50 μΐ PCR反应体系： 5 μΐ lO PfuUltra II Reaction Buffer 0.5 μΐ 25 mM的 dNTP、2.0 μΐ ThMCSU-2-pGEM质粒、 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase、 10 μΜ的引物 SEQ ID NO: 20禾口 SEQ ID NO: 21各 2.0 μΐ以及 37.5 μΐ的双蒸水。 PCR反应条件： 95 °C预变性 2分钟， 35个循 环（95 °C变性 25秒， 51 °C退火 30秒， 72°C延伸 90s) ， 72°C延伸 5分钟。 通过 Xbal、 Kpnl酶切后将所得 PCR产物连接 （连接方法同上） 到 pCAMBIA2300-3， 获得植物表 达载体 35S-r/zMC^7-2-2300。 SEQ ID NO: 25：  SEQ ID NO: 25 and SEQ ID NO: 26 Amplification of the r/zMC^-2-pGEM plasmid containing the full-length ThMCSU-2 gene cloned in Example 2 as a template, with the designed restriction enzymes at both ends of the amplification The site of the ThMCSU-2 gene. Stratagene's PfuUltra II Fusion HS DNA Polymerase was used. 50 μΐ PCR reaction system: 5 μΐ lO PfuUltra II Reaction Buffer 0.5 μΐ 25 mM dNTP, 2.0 μΐ ThMCSU-2-pGEM plasmid, 1.0 μΐ PfuUltra II Fusion HS DNA Polymerase, 10 μΜ primer SEQ ID NO: 20 and SEQ ID NO: 21 each of 2.0 μΐ and 37.5 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 95 °C for 2 minutes, 35 cycles (denaturation at 95 °C for 25 seconds, annealing at 51 °C for 30 seconds, extension at 72 °C for 90 s), extension at 72 °C for 5 minutes. The resulting PCR product was ligated by Xbal and Kpnl (connection method as above) to pCAMBIA2300-3 to obtain a plant expression vector 35S-r/zMC^7-2-2300. SEQ ID NO: 25:
AArC7¾&4ATGGGAGATAGATTCATGATG  AArC73⁄4&4ATGGGAGATAGATTCATGATG
SEQ ID NO: 26：  SEQ ID NO: 26:
GCGG7¾CCCATAAATGATTTCGCATACAC 实施例 4 35S-ThMCSU-2-2300表达载体转化农杆菌  GCGG73⁄4CCCATAAATGATTTCGCATACAC Example 4 Transformation of Agrobacterium by 35S-ThMCSU-2-2300 Expression Vector
农杆菌 LBA4404 (购自 Biovector Science Lab, Inc) 感受态细胞的制备： 提前 1-2 天将农杆菌 LBA4404在含 50 g/ml利福平和 50 g/ml链霉素的 LB固体培养基上划单斑 接种， 28°C培养 1至 2天。挑取单菌落接种于 5 ml含 50 μ§/ιη1利福平和 50 μ§/ιη1链霉素 的 LB液体培养基中， 28°C下摇动培养过夜 （约 12-16小时） 至 OD6(K)值为 0.4， 形成种 子菌液。取 5 ml培养活化后的种子菌液（1 :20的比例）接种于 100 ml同样浓度抗生素的 LB液体培养基中， 28°C摇动培养 2-2.5小时至 OD6(K)=0.8。 冰浴菌液 10分钟， 每隔 3分 钟摇匀一次， 使细菌均匀进入休眠状态。 于 4°C下 4000 g离心 10分钟， 弃上清液； 加入 l ml冰预冷的 10% (体积比） 甘油重悬浮菌体， 4°C下 4000 g离心 10分钟， 收集沉淀； 用冰预冷的 10% (体积比） 甘油重复洗 3-4次； 加入适量冰预冷的 10% (体积比） 甘油 重新悬浮细菌沉淀， 即制得 LBA4404感受态细胞， 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 hours) to OD 6 at 28 °C. The K) value is 0.4, and a seed bacterial liquid is formed. 5 ml of culture-activated seed broth (1:20 ratio) was inoculated to 100 ml of the same concentration of antibiotics. In LB liquid medium, incubate at 28 ° C for 2 to 2.5 hours to OD 6 (K) = 0.8. The ice bath liquid was shaken for 10 minutes every 3 minutes to allow the bacteria to enter the dormant state evenly. Centrifuge at 4000 g for 10 minutes at 4 ° C, discard the supernatant; resuspend the cells by adding 1 ml of ice pre-cooled 10% (by volume) glycerol, centrifuge at 4000 g for 10 minutes at 4 ° C, collect the precipitate; 10% (by volume) of pre-cooled glycerin was washed 3-4 times repeatedly; 10% (by volume) of glycerol was added to resuspend the bacterial pellet to prepare LBA4404 competent cells, which were prepared at 40 μΐ/tube. Dispense, store at -70 ° C for use.
转化农杆菌： 在冰上融化 LBA4404感受态细胞， 向 40 μΐ的所述感受态细胞中加入 1 μΐ实施例 3中所得的阳性 35S-r/zMC^/-2-2300质粒， 混匀后冰浴约 10分钟。 将冰浴 后的所述感受态细胞和 35S-r/zMC^/-2-2300质粒的混合物用移液枪转移到冰预冷的电击 杯中， 轻敲使悬浮液到达底部， 注意不要有气泡。 将电击杯（购自 Bio-Rad)放到电击室 的滑道上， 推动滑道将电击杯放至电击室基座电极处。 使用 0.1 cm 规格的电击杯， MicroPuMUer (购自 Bio-Rad) 的程序设置为 "Agr"， 电击一次。 立即取出电击杯， 加入 28°C预热的 200 μ1 ίΒ培养基。快速而轻柔的用移液枪将感受态细胞打匀。将悬浮液转入 1.5 ml的离心管， 在 28°C下 225 rpm培养 1小时。 取 100-200 μΐ的菌液涂布于相应的抗 性筛选培养基平板上 （LB固体培养基， 含 50 μ§/ιη1利福平、 50 μ§/ιη1链霉素、 50 μ§/ιη1 卡那霉素）， 28°C培养。 筛选阳性转化克隆， 并将其菌液于 -70°C保存备用。 实施例 5 利用农杆菌介导的转化法获得转基因拟南芥 Transformation of Agrobacterium: Melt LBA4404 competent cells on ice, add 1 μΐ of the positive 35S-r/zMC^/-2-2300 plasmid obtained in Example 3 to 40 μΐ of the competent cells, mix and ice Bath for about 10 minutes. Transfer the mixture of the competent cells and the 35S-r/zMC^/-2-2300 plasmid after the ice bath to the ice pre-cooled electric shock cup with a pipette, tap to bring the suspension to the bottom, be careful not to have bubble. Place the electric shock cup (purchased from Bio-Rad) on the slide of the electric shock chamber and push the slide to place the electric shock cup on the base electrode of the electric shock chamber. Using a 0.1 cm size electric shock cup, the MicroPuMUer (purchased from Bio-Rad) program is set to "Agr" and the shock is applied once. Immediately remove the electric shock cup and add 200 μl of 预 medium pre-warmed at 28 °C. Quickly and gently mix the competent cells with a pipette. The suspension was transferred to a 1.5 ml centrifuge tube and incubated at 225 rpm for 1 hour at 28 °C. 100-200 μL of bacterial solution was applied to the corresponding resistant selection medium plate (LB solid medium containing 50 μ § /ιη1 rifampicin, 50 μ § /ιη1 streptomycin, 50 μ § /ιη1 Kanamycin), cultured at 28 °C. Positive transformed clones were screened and their bacterial stocks were stored at -70 °C until use. Example 5 Obtaining transgenic Arabidopsis thaliana using Agrobacterium-mediated transformation
待转化植株培养： 拟南芥种子 （哥伦比亚型， 来自美国俄亥俄州立大学的拟南芥生 物资源中心（www.arabidopsis.org)播种在泥炭土中，经 4°C低温处理 3天后，置于 23 °C、 16小时光照 /8小时黑暗的培养箱中发芽。 7-10天后移栽到装有泥炭土和蛭石 (体积比 3: 1 ) 的口径为 7.5 cm的塑料钵中， 每钵栽种 6株， 置于 23 °C， 16小时光照 /8小时黑暗的培养 箱中生长。 移栽前每钵浇 1/2MS培养基 40 ml, 移栽后视土壤湿度及时补充水分。 在生 长期间适当浇灌 1/2MS培养基。 按需要每 3-4周一次（或者时间更长）。 为了在每个植株 上得到较多的花芽， 当大多数植株第一个花序形成后剪去第一个花序， 解除顶端优势， 促使多个次生花序的同步出现。 当大多数花序约 1-10 cm高 （剪去第一个花序后 4-8天） 时准备浸染。  Plant to be transformed: Arabidopsis seeds (Columbia type, from the Arabidopsis Bioresources Center of the Ohio State University (www.arabidopsis.org) sown in peat soil, treated at 4 ° C for 3 days, placed at 23 Germination in a incubator at °C, 16 hours light/8 hours dark. After 7-10 days, transplant to a plastic crucible with a diameter of 7.5 cm containing peat and vermiculite (3:1 by volume). 6 strains were grown in an incubator at 23 ° C, 16 hours light / 8 hours dark. Before transplanting, 40 ml of 1/2MS medium was poured per pot, and the soil moisture was added to the soil moisture in time to improve the growth period. Water 1/2 MS medium. Every 3-4 weeks (or longer) as needed. In order to get more flower buds on each plant, the first inflorescence is cut off after the first inflorescence of most plants is formed. , remove the apical advantage, and promote the simultaneous emergence of multiple secondary inflorescences. Prepare for dip when most inflorescences are about 1-10 cm high (4-8 days after cutting the first inflorescence).
农杆菌的培养： 取出实施例 4中保种的农杆菌阳性转化克隆的菌液活化后， 挑取农 杆菌单菌落接种到 10 ml无菌 LB液体培养基中 （含 75 mg/1利福平、 100 mg/1链霉素和 100 mg/1卡那霉素）， 28°C恒温下 250转 /分钟振摇过夜培养。再将所得到的菌液按 1%-2% 的比例接种到 200 ml同样含上述抗生素的 LB液体培养基中， 28°C恒温振摇使农杆菌的 浓度达到 OD6(K)=1.8， 然后在室温下 3000转 /分钟离心 15分钟， 弃去上清液后用浸染培 养基 （该浸染培养基含有 5.0%的蔗糖和 0.05% ( 500 μ1/1) 的 Silwet L-77) 重新悬浮农杆 菌， 悬浮至 OD6QQ约 0.80。 Culture of Agrobacterium: After removing the bacterial solution of the Agrobacterium-positive transformed clone preserved in Example 4, pick a single colony of Agrobacterium and inoculate it into 10 ml of sterile LB liquid medium (containing 75 mg/1 rifampicin). 100 mg/1 streptomycin and 100 mg/1 kanamycin were shaken overnight at 250 rpm at 28 °C. Then the obtained bacterial liquid is 1% - 2% The ratio was inoculated into 200 ml of LB liquid medium containing the above antibiotics, shaking at a constant temperature of 28 ° C to bring the concentration of Agrobacterium to OD 6 (K) = 1.8, and then centrifuging at 3000 rpm for 15 minutes at room temperature, discarding After removing the supernatant, the Agrobacterium was resuspended with a dip-dye medium containing 5.0% sucrose and 0.05% (500 μl/1) of Silwet L-77, and suspended to an OD 6QQ of about 0.80.
花序的浸染： 将上述含农杆菌的浸染培养基加入大口容器中， 每个口径 9 cm的容器 中加入 200-300 ml所述含农杆菌的浸染培养基用于浸染。 将植株倒置， 使地上组织全部 浸没在农杆菌悬浮液中 3-5 秒， 并要轻轻搅动。 浸润后植株上应该有一层液体膜。 浸染 过的植株放在塑料盘中， 用干净的塑料或保鲜膜覆盖以保湿， 然后放置在弱光或暗处过 夜， 注意小心防止阳光直射植株。 处理后约 12-24 小时去掉覆盖。 正常培养植株， 植株 进一步生长 3-5周， 直至角果变褐变干。 收获 T1代种子， 并将所述种子用离心管在 4°C 下干燥贮存。  Inflorescence dip dyeing: The above Agrobacterium-containing dyeing medium was added to a large-mouth container, and 200-300 ml of the Agrobacterium-containing dyeing medium was added to each container having a diameter of 9 cm for dip dyeing. Invert the plants so that the above ground tissues are all immersed in the Agrobacterium suspension for 3-5 seconds and gently agitate. There should be a liquid film on the plant after infiltration. Dip-infected plants are placed in plastic trays, covered with a clean plastic or cling film to moisturize them, and then placed in low light or dark places for the night, taking care to prevent direct sunlight from shining on the plants. Remove the cover approximately 12-24 hours after processing. The plants are cultured normally, and the plants are further grown for 3-5 weeks until the pods are browned and dried. Seeds of the T1 generation were harvested, and the seeds were stored in a centrifuge tube at 4 ° C.
转基因种子筛选： 配制 1/2MS培养基水溶液 （9.39 mM KN03, 0.625 mM KH2P04， 10.3 mM H4N03, 0.75 mM MgSO4， 1.5 mM CaCl2, 50 μΜ ΚΙ， 100 μΜ Η3ΒΟ3， 100 μΜ MnSO4，30 M ZnS04， 1 μΜ Να2Μο04, 0.1 M CoCl2， 100 μΜ Na2EDTA, 100 M FeS04)， 加入 0.8 %琼脂粉， 用微波炉加热至琼脂完全溶化， 待冷却到 50°C左右， 加入所需量的终 浓度为 50 mg 1的卡那霉素， 摇匀后每培养皿中倒入 25 ml， 置实验台冷却凝固后即可播 种。 把称量好的种子倒在一张普通复印纸上， 用手指轻敲复印纸， 将种子均匀地播种在 琼脂胶上， 盖上培养皿盖， 置 4 °C冰箱冷处理 72小时后， 移至 23 °C、 16小时光照 /8小 时黑暗的培养箱中发芽， 定期统计种子发芽和幼苗生长情况， 将抗性幼苗及时移栽到营 养土中。 移栽后视土壤湿度及时补充水分。 在生长期间适当浇灌 1/2MS培养基。 取生长 20天的拟南芥叶片 0.1 g，提取 DNA，用 SEQ ID NO: 16：和 SEQ ID NO: 17扩增 ThMCSU-2: ( 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: 16和 SEQ ID NO: 17各 2.0 μΐ以及 35 μΐ的双蒸水。 PCR反 应条件： 94°C预变性 5分钟， 33个循环 （94°C变性 45秒， 51 °C退火 45秒， 72°C延伸 2 分钟）， 72°C延伸 7分钟）， 将 PCR鉴定为阳性的植株进行编号， 正常培养植株， 植株进 一步生长 3-5周， 直至角果变褐变干。 收获 T2带种子， 并将种子用离心管在 4°C下干燥 贮存。 实施例 6 过表达 ThMCSU-2的转基因拟南芥 T2代植株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。 将 ThMCSU-2的转基因拟南芥 T2代植株编 号 T2H1-T2H6及对照拟南芥种子分别播种在蛭石上， 每盆播种 10颗种子， 25°C、 10小 时光培养 /14小时暗培养循环， 每 7天浇一次 1/2MS, 培养 20天之后， 使用卡那霉素筛 选阳性植株， 每盆保留大小较一致的 4棵苗， 用于干旱实验。 转基因拟南芥、 对照拟南 芥干旱 14天 （不浇水）， 25°C、 10小时光培养 /14小时暗培养循环。 T2代转基因植株的 抗旱性鉴定表明， 对照植株都萎蔫严重， 而 T2H1、 T2H2、 T2H3、 T2H4、 T2H5、 T2H6、 T2H7、 T2H8六个株系共 32每株系各 4棵） 拟南芥中 27能够存活并继续生长显现出显 著的耐旱性（参见图 3a和 3b， 以 T2H3、 T2H4为例， T2H1、 T2H2、 T2H5、 T2H6、 T2H7、 T2H8 的结果与 T2H3、 T2H4类似， 在此未示出）。 实施例 7 干旱胁迫后 ABA含量的测定 Transgenic seed screening: Prepare 1/2 MS medium (9.39 mM KN0 3 , 0.625 mM KH 2 P0 4 , 10.3 mM H 4 N0 3 , 0.75 mM MgSO 4 , 1.5 mM CaCl 2 , 50 μΜ ΚΙ, 100 μΜ Η 3 ΒΟ 3 , 100 μΜ MnSO 4 , 30 M ZnS0 4 , 1 μΜ Να 2 Μο0 4 , 0.1 M CoCl 2 , 100 μΜ Na 2 EDTA, 100 M FeS0 4 ), add 0.8% agar powder, heat in a microwave oven until the agar is completely dissolved. To be cooled to about 50 ° C, add the required amount of kanamycin at a final concentration of 50 mg 1 , shake well, pour 25 ml into each dish, and set the bench to cool and solidify. Pour the weighed seeds onto a piece of plain copy paper, tap the copy paper with your fingers, evenly sow the seeds on the agar gel, cover the Petri dish, and cool the chamber at 4 °C for 72 hours, then move to Germinating in a 23 ° C, 16 hour light / 8 hour dark incubator, regular seed germination and seedling growth, and timely transplanting the resistant seedlings into the nutrient soil. After transplanting, the soil moisture is added to the water in time. 1/2 MS medium was appropriately watered during the growth period. 0.1 g of leaves of Arabidopsis thaliana grown for 20 days, DNA was extracted, and ThMCSU-2 was amplified with SEQ ID NO: 16: and SEQ ID NO: 17 (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: 16 and SEQ ID NO: 17 each of 2.0 μΐ and 35 μΐ of double distilled water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 33 cycles (denaturation at 94 °C for 45 seconds, annealing at 51 °C for 45 seconds, extension at 72 °C for 2 minutes), extension at 72 °C for 7 minutes), numbering the plants identified as positive by PCR, normal culture of the plants, and further planting Grow for 3-5 weeks until the pods turn brown and dry. Seeds of T2 were harvested and the seeds were stored in a centrifuge tube at 4 °C. Example 6 Drought Tolerance Simulation Experiment and Functional Identification of Transgenic Arabidopsis T2 Plants Overexpressing ThMCSU-2 The sterilized vermiculite was permeated with 1/2 MS medium. Transgenic Arabidopsis thaliana T2 plants of ThMCSU-2 No. T2H1-T2H6 and control Arabidopsis seeds were planted on vermiculite, 10 seeds per pot, 25 ° C, 10 hours light culture / 14 hours dark culture cycle, 1/2 MS every 7 days, culture for 20 days After that, positive plants were screened using kanamycin, and 4 seedlings of uniform size were retained per pot for drought experiments. Transgenic Arabidopsis thaliana, control Arabidopsis thaliana drought for 14 days (no watering), 25 ° C, 10 hours light culture / 14 hours dark culture cycle. The drought resistance of the T2 transgenic plants showed that the control plants were all wilted, while the T2H1, T2H2, T2H3, T2H4, T2H5, T2H6, T2H7, and T2H8 lines were all 32 in each line. Survival and continued growth showed significant drought tolerance (see Figures 3a and 3b, with T2H3, T2H4 as an example, the results of T2H1, T2H2, T2H5, T2H6, T2H7, T2H8 are similar to T2H3, T2H4, not shown here ). Example 7 Determination of ABA content after drought stress
ABA是公认的与逆境胁迫相关的一种植物激素， 可以作为信号分子调控多个逆境诱 导基因的表达， 从而提高植物的抗逆能力。 我们取干旱胁迫 10天和正常生长条件下的转 基因植株 （T2H1、 T2H2、 T2H3、 T2H4、 T2H5、 T2H6 ) 及对照植株 （CK1、 CK2 ) 叶 片各 0.2 g， 用中国农业大学作物化控研究中心制备的试剂盒测定 ABA含量 （见图 4)。 实验结果表明，无论干旱处理还是对照条件下，转基因植株的 ABA含量均高于对照 ( CK1、 CK2), 证明 ThMCSU-2基因可以正调控植物内源的 ABA含量。 实施例 8 在转录水平上验证 r/zMC^/-2基因的表达  ABA is recognized as a plant hormone associated with stress, which can act as a signaling molecule to regulate the expression of multiple stress-inducing genes, thereby improving the plant's resistance to stress. We took 0.2 g of transgenic plants (T2H1, T2H2, T2H3, T2H4, T2H5, T2H6) and control plants (CK1, CK2) under drought stress for 10 days and normal growth conditions, and prepared with the Crop Control Research Center of China Agricultural University. The kit measures the ABA content (see Figure 4). The results showed that the ABA content of transgenic plants was higher than that of the control (CK1, CK2) under drought treatment and control conditions, which proved that ThMCSU-2 gene can positively regulate the endogenous ABA content of plants. Example 8 Verification of expression of r/zMC^/-2 gene at the transcriptional level
分别取对照拟南芥植株、显著耐旱转基因拟南芥 T2代植株 (分别属于 T2H1、T2H2、 T2H3、 T2H4、 T2H5、 T2H6六个株系）和不显著耐旱转基因拟南芥 T1代植株的干旱 10 天的叶片各 0.05 g， 用植物 RNA提取试剂盒 （Invitrogen) 提取的总 RNA。 用 HITACHI 公司的紫外分光光度计 U-2001测定所得总 RNA在 260 nm和 280 nm的吸光度值， 计算 各个 RNA浓度。 依照 Invitrogen反转录试剂盒 Superscript III Reverse Transcriptase所示 方法进行反转录（2 μg总 RNA作为模板，反转录引物 SEQ ID NO: 18)。通过 SEQ ID NO: 16和 SEQ ID NO: \Ί ψ增 ThMCSU-2， 检测 Γ/ζΜΟ^/-2基因相对表达情况。  Control Arabidopsis thaliana plants, significantly drought-tolerant transgenic Arabidopsis thaliana T2 plants (two strains belonging to T2H1, T2H2, T2H3, T2H4, T2H5, T2H6, respectively) and T4 plants with no drought-tolerant transgenic Arabidopsis thaliana Total RNA extracted from plant leaves RNA extraction kit (Invitrogen) by 0.05 g each for 10 days of drought. The absorbance values of total RNA at 260 nm and 280 nm were measured using a HITACHI UV spectrophotometer U-2001 to calculate the individual RNA concentrations. Reverse transcription was carried out according to the method shown by Invitrogen Reverse Transcription Kit Superscript III Reverse Transcriptase (2 μg of total RNA as a template, reverse transcription primer SEQ ID NO: 18). The relative expression of the Γ/ζΜΟ^/-2 gene was detected by SEQ ID NO: 16 and SEQ ID NO: Ί ψ Th ThMCSU-2.
采用 TaKaRa的 PrimeSTAR HS DNA聚合酶，以反转录的 cDNA为模板进行 PCR 反应。 50 μ1 ΡΟ 反应体系： 10 μΐ 5 xPS Buffer、 3 μΐ 2.5 mM的 dNTP、 2.0 μΐ cDNA 1.0 μΐ PrimeSTAR 10 μΜ的引物 SEQ ID NO: 16禾 P SEQ ID NO: 17各 2.0 μΐ以及 30 μΐ的双蒸水。 PCR反应条件： 94°C预变性 5分钟， 29个循环 （94°C变性 45秒， 51 °C退火 45秒， 72°C 延伸 2分钟） ， 72°C延伸 10分钟。  PCR was performed using TaKaRa's PrimeSTAR HS DNA polymerase with reverse transcribed cDNA as a template. 50 μl ΡΟ Reaction system: 10 μΐ 5 xPS Buffer, 3 μΐ 2.5 mM dNTP, 2.0 μΐ cDNA 1.0 μΐ PrimeSTAR 10 μΜ primers SEQ ID NO: 16 and P SEQ ID NO: 17 each 2.0 μΐ and 30 μΐ double steamed water. PCR reaction conditions: pre-denaturation at 94 ° C for 5 minutes, 29 cycles (denaturation at 94 ° C for 45 seconds, annealing at 51 ° C for 45 seconds, extension at 72 ° C for 2 minutes), extension at 72 ° C for 10 minutes.
产物电泳结果如图 5所示： M为 DNA Ladder Marker ( DL2000, TakaRa), 1-4为 耐旱效果不显著的转基因拟南芥 T2代植株， 5-11为耐旱效果显著的转基因拟南芥 Τ2 代植株 （依次为： Τ2Η1、 Τ2Η2、 Τ2Η3、 Τ2Η4、 Τ2Η5、 Τ2Η6、 T1H7 ) , 12-17为非转 基因的拟南芥植株。 The product electrophoresis results are shown in Figure 5: M is DNA Ladder Marker (DL2000, TakaRa), 1-4 is Transgenic Arabidopsis T2 plants with insignificant drought tolerance, 5-11 are transgenic Arabidopsis thaliana plants with significant drought tolerance (in order: Τ2Η1, Τ2Η2, Τ2Η3, Τ2Η4, Τ2Η5, Τ2Η6, T1H7), 12 -17 is a non-transgenic Arabidopsis plant.
图中所示 PCR产物电泳条带大小与 ThMCSU-2的大小一致（约 2.4 Kbp ) 。 结果 表明， 对照拟南芥没有 r/zMC^/-2转录， 显著耐旱转基因拟南芥 T1代植株中 The size of the electrophoresis band of the PCR product shown in the figure is consistent with the size of ThMCSU-2 (about 2.4 Kbp). The results showed that there was no r/zMC^/-2 transcription in Arabidopsis thaliana, significantly in drought-tolerant transgenic Arabidopsis thaliana T1 plants.
ThMCSU-2的转录较强， 不显著耐旱转基因拟南芥 T2代植株 ThMCSU-2转录很弱。 The transcription of ThMCSU-2 was stronger, and the drought-tolerant transgenic Arabidopsis thaliana T2 plants were not significantly transcribed.

Claims

权 利 要 求 书 Claim
1. 小盐芥的一个钼辅酶因子硫化酶， 其氨基酸序列如 SEQ ID NO : 1所示。 A molybdenum coenzyme factor vulcanase of small salt mustard having an amino acid sequence as shown in SEQ ID NO: 1.
2. 编码权利要求 1的钼辅酶因子硫化酶的基因， 其序列为 SEQ ID NO : 2。  2. A gene encoding the molybdenum coenzyme sulfase of claim 1, the sequence of which is SEQ ID NO: 2.
3. 一种重组表达载体，其是通过将权利要求 2的基因插入到一种表达载体而获得 的， 并且所述基因的核苷酸序列与所述表达载体的表达控制序列可操作地连接， 优 选地， 所述表达载体是 pCAMBIA2300。  A recombinant expression vector obtained by inserting the gene of claim 2 into an expression vector, and 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.
4. 权利要求 3所述的重组表达载体， 其为附图 2所示的 2 -ThMCSU-2-220Q载 体。  4. The recombinant expression vector of claim 3 which is the 2-ThMCSU-2-220Q vector shown in Figure 2.
5. 一种重组细胞， 其含有权利要求 2的基因或者权利要求 3或 4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 2 or the recombinant expression vector of claim 3 or 4; preferably, the recombinant cell is a recombinant Agrobacterium cell.
6. 一种改善植物耐旱性的方法， 包括： 将权利要求 2的基因或者权利要求 3或 4的重组表达载体导入植物或植物组织并使所述基因表达； 优选地， 所述植物是拟南 芥。  A method for improving drought tolerance of a plant, comprising: introducing the gene of claim 2 or the recombinant expression vector of claim 3 or 4 into a plant or plant tissue and expressing the gene; preferably, the plant is intended to be Southern mustard.
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的基因或者权利要求 3或 4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 2 or the recombinant expression vector of claim 3 or 4 under conditions effective to produce a plant.
8. 权利要求 7的方法， 其中所述植物是拟南芥。  8. The method of claim 7, wherein the plant is Arabidopsis thaliana.
9. 权利要求 2的基因、 权利要求 3或 4的重组表达载体或者权利要求 4所述的 重组细胞用于改善植物耐旱性以及用于植物育种的用途。  9. The gene of claim 2, the recombinant expression vector of claim 3 or 4 or the recombinant cell of claim 4 for improving drought tolerance of a plant and for use in plant breeding.
10. 权利要求 9所述的用途， 其中所述植物是拟南芥。  10. The use of claim 9, wherein the plant is Arabidopsis thaliana.
PCT/CN2013/074452 2013-04-19 2013-04-19 Thellungiella halophila molybdenum enzyme cofactor sulfurized enzyme mcsu-2, coding genes of same, and application thereof WO2014169482A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201380074524.9A CN105008389B (en) 2013-04-19 2013-04-19 A kind of small salt mustard molybdenum cofactors vulcanization enzyme MCSU-2 and its encoding gene and application
PCT/CN2013/074452 WO2014169482A1 (en) 2013-04-19 2013-04-19 Thellungiella halophila molybdenum enzyme cofactor sulfurized enzyme mcsu-2, coding genes of same, and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2013/074452 WO2014169482A1 (en) 2013-04-19 2013-04-19 Thellungiella halophila molybdenum enzyme cofactor sulfurized enzyme mcsu-2, coding genes of same, and application thereof

Publications (1)

Publication Number Publication Date
WO2014169482A1 true WO2014169482A1 (en) 2014-10-23

Family

ID=51730713

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2013/074452 WO2014169482A1 (en) 2013-04-19 2013-04-19 Thellungiella halophila molybdenum enzyme cofactor sulfurized enzyme mcsu-2, coding genes of same, and application thereof

Country Status (2)

Country Link
CN (1) CN105008389B (en)
WO (1) WO2014169482A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004092349A2 (en) * 2003-04-15 2004-10-28 Basf Plant Science Gmbh Plant cells and plants with increased tolerance to environmental stress
CN1553950A (en) * 2001-09-06 2004-12-08 ��������ɣ�Ǵ�ѧ���»� Method for increasing stress tolerance in plants

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1553950A (en) * 2001-09-06 2004-12-08 ��������ɣ�Ǵ�ѧ���»� Method for increasing stress tolerance in plants
WO2004092349A2 (en) * 2003-04-15 2004-10-28 Basf Plant Science Gmbh Plant cells and plants with increased tolerance to environmental stress

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 11 June 2010 (2010-06-11), "Arabidopsis lyrata subsp. Lyrata ABA3/ATABA3/LOS5/SIR3 mRNA", accession no. M_002890125.1 *
DATABASE PROTEIN 7 September 2000 (2000-09-07), "Similar to molybdopterin cofactor sulfurase [Arabidopsis thaliana", accession no. AG10824.1 *
YANG, RUOLIN ET AL.: "The Reference Genome of the Halophytic Plant Eutrema salsugineum.", FRONTIERS IN PLANT SCIENCE, vol. 4, 21 March 2013 (2013-03-21), pages 1 - 14 *

Also Published As

Publication number Publication date
CN105008389B (en) 2018-11-30
CN105008389A (en) 2015-10-28

Similar Documents

Publication Publication Date Title
WO2013185258A1 (en) Hkt protein of cotton and coding gene and application thereof
WO2014172852A1 (en) Myb transcription factor myb1-1 of cotton and coding gene, and use thereof
WO2015042742A1 (en) Calcineurin b-like protein cbl-3 from cotton, and coding gene and use thereof
WO2015042740A1 (en) Thellungiella halophila calcineurin b-like protein cbl-4, coding gene of same, and application thereof
WO2014169472A1 (en) Cotton leucine zipper protein bzip-3, coding genes of same, and application thereof
WO2014205597A1 (en) Transporter hkt2 with high affinity for potassium ions and which is derived from cotton and the coding gene and use thereof
WO2014172847A1 (en) Myb transcription factor myb1-2 of thellungiella salsuginea and coding gene, and use thereof
WO2014172826A1 (en) Tonoplast pyrophosphatase vp1 from thellungiella halophila, and coding gene and application thereof
WO2015042738A1 (en) Thellungiella halophila homeotic-leucine zipper protein hdbzip-3, coding gene of same, and application thereof
WO2015042749A1 (en) Thellungiella halophila dehydrin protein dh5, coding gene of same, and application thereof
WO2014107863A1 (en) Cotton homeotic-leucine zipper protein hdbzip-1 and coding gene and use thereof
WO2014169482A1 (en) Thellungiella halophila molybdenum enzyme cofactor sulfurized enzyme mcsu-2, coding genes of same, and application thereof
WO2015058323A1 (en) Bruguiear gymnorrhiza betaine aldehyde dehydrogenase (badh), coding gene thereof, and application of coding gene
WO2014172825A1 (en) Tonoplast sodium-hydrogen anti-protein nhx2 of thellungiella halophila and coding gene and use thereof
WO2015058322A1 (en) Bruguiera gymnorrhiza molybdenum coenzyme factor sulfurylase mcsu and coding gene and use thereof
WO2014082280A1 (en) Cotton zinc finger protein (czf4) and coding gene and application thereof
WO2014205598A1 (en) High-affinity potassium ion transport protein hkt1 derived from thellungiella halophila, and coding gene and use thereof
WO2015058321A1 (en) Avicennia marina monooxygenase cmo and coding gene and use thereof
WO2015042745A1 (en) Thellungiella halophila dehydrin protein dh2, coding gene of same, and application thereof
WO2015042741A1 (en) Thellungiella halophila leucine zipper protein bzip-6, coding gene of same, and application thereof
WO2015024145A1 (en) Zinc finger protein zpt5-3 from cotton, and coding gene and uses thereof
WO2014172842A1 (en) Cysteine protease cysp1-2 of cotton and coding gene, and use thereof
WO2014101153A1 (en) Cotton isopentenyl transferase ipt2, gene for encoding same, and application thereof
WO2014082285A1 (en) Cotton zinc finger protein (czf5) and coding gene and use thereof
WO2015039272A1 (en) Trehalose synthase tps-2 of cotton and coding gene and use thereof

Legal Events

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

Ref document number: 13882067

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13882067

Country of ref document: EP

Kind code of ref document: A1