CN109022449A - Cucumber CsMLO1 gene and its silencing expression vector establishment method, application - Google Patents
Cucumber CsMLO1 gene and its silencing expression vector establishment method, application Download PDFInfo
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- CN109022449A CN109022449A CN201810824315.6A CN201810824315A CN109022449A CN 109022449 A CN109022449 A CN 109022449A CN 201810824315 A CN201810824315 A CN 201810824315A CN 109022449 A CN109022449 A CN 109022449A
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- C07—ORGANIC CHEMISTRY
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- 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
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- 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
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- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—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 biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—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 biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
Abstract
The invention belongs to molecular biology and field of biotechnology, more particularly to a kind of cucumber CsMLO1 gene and its silencing expression vector establishment method, application, cucumber responds the full length coding region sequence of the CsMLO1 gene in stick spore leaf spot regulatory pathway as shown in sequence table SEQ ID NO.1；Amino acid sequence is as shown in sequence table SEQ ID NO.2.The present invention also provides the construction methods that cucumber CsMLO1 gene specific segment is inserted into silencing expression vector pTRV2, by the plant silencing expression vector of building through Agrobacterium-mediated Transformation to cucumber cotyledons, it obtains transgenosis cucumber plant and carries out Disease Resistance Identification, the results showed that the gene is the negative regulatory factor in cucumber and stick spore leaf spot interaction.The present invention is to provide technological means and theoretical foundation using technique for gene engineering to disclose cucumber CsMLO1 gene functional research comprehensively, have very big application value.
Description
Technical field
The invention belongs to molecular biology and field of biotechnology, and in particular to a kind of cucumber CsMLO1 gene
(Cucsa.207280) and its silencing expression vector establishment method, application.
Background technique
Cucumber (Cucumis sativus) is one of main vegetables crop of northern China facility cultivation, yield and product
Matter is seriously influenced by abiotic stress.Cucumber stick spore leaf spot is a kind of global fungal disease, more by pathomycete
Main stick spore (Corynespora cassiicola) causes；Currently, the research of the disease is concentrated mainly on biological control and chemistry
Prevention and treatment etc. is very few in resistance mechanism research of the cucumber to stick spore leaf spot.
MLO (Mildew Resistance Locus O) albumen is distinctive albumen in plant, research shows that MLO family
Family member is as susceptible gene, and vulnerable to infecting for powdery mildew, and recessive mutation gene mlo is endowed resistance of wide spectrum (Shirasu
et al.,1999；Chen et al.,2006；Kim et al.,2002).Currently, other than the disease-resistant path mlo and powdery mildew
The relationship of other pathogens also receives more and more attention.Bar shaped handle rest fungus (Puccinia striiformis
It f.sp.tritici) is the specialization fungi for causing stripe rust of wheat, main harm wheat leaf portion.Research passes through virus induction gene
Silencing (virus-induced gene silencing) technology, to three of hypersensitive cell death signal path may be participated in
Genes Encoding Transmembrane Proteins TaMLO1, TaMLO2, TaMLO5 carry out gene silencing, and test inoculation stripe rust plant shows different degrees of
Disease resistance, TaMLO1/2/5 may participate in having adjusted to a certain extent small as the negative regulatory factor of apoptosis
The Interaction (Shi Xuexia, 2013) of wheat and strip rust bacteria.Wild-type barley mutated gene mlo3, mlo4, mlo5 and barley leaf are withered
Disease analysis it was found that, Trimutant enhances (Luo Zhen etc. 2009) to the resistance of pathogen.Therefore, thought according to the serial fact,
This ill-resistant protein of MLO may be used as a kind of adjuster, be not only the resistance repressor of powdery mildew, and in other pathogens
Also the defense response mutually inhibited is embodied.
In conclusion very few about the research to interact between cucumber and stick spore leaf spot.Therefore, cucumber is chosen
CsMLO1 gene 402bp segment is inserted into pTRV2, and building recombinant vector pTRV2-CsMLO1 and pTRV1 are imported plant in equal volume
In body, TRV virus is analyzed by phenotypic evaluation and has been succeeded the mass propagation in cucumber cotyledons, and qRT-PCR technology is combined to detect
Copy number of the CsMLO1 gene in transgenosis cucumber is substantially reduced.To which profound cucumber CsMLO1 gene conduct could be understood
Negative regulatory factor in cucumber and stick spore leaf spot interaction lays the foundation to disclose cucumber CsMLO1 gene function comprehensively.
Summary of the invention
In order to solve the above technical problem, the present invention provides a kind of cucumber CsMLO1 gene and its silencing expression vector establishments
Method, application.
The invention is realized in this way a kind of cucumber CsMLO1 gene is provided, coding region sequence such as SEQ ID NO.1 institute
Show.
There is provided a kind of for expanding the primer of above-mentioned cucumber CsMLO1 gene, nucleotide sequence is as follows:
CsMLO1-F:5 '-ATGGCGGGGGCAGCCGGTGG-3 '
CsMLO1-R:5 '-TTCAACTCTATCAAATGAAA-3 '.
The method expanded using above-mentioned primer pair cucumber CsMLO1 gene is utilized using cucumber leaves as material
The method of RNAprep pure plant total RNA extraction reagent box extracts total serum IgE, and reverse transcription synthesizes cDNA, design such as claim 2
The primer；
Using the cDNA of reverse transcription as template, polymerization chain type PCR reaction is carried out, PCR product is recycled, obtains the piece of 1749bp mesh
Section, purified product is sequenced, sequence cucumber CsMLO1 gene as shown in SEQ ID NO.1 is obtained.
A kind of protein encoded using above-mentioned cucumber CsMLO1 gene, amino acid sequence such as SEQ ID are provided
Shown in NO.2.
The specific regions primer of above-mentioned cucumber CsMLO1 gene is provided, cucumber MLO family gene sequence is compared, is chosen
Specific regions 402bp design primer, nucleotide sequence are as follows:
TRV-CsMLO1-F:5’-gtgagtaaggttaccgaattcGTGGCAGAGGCCCTTCGCAAC-3’
TRV-CsMLO1-R:5’-ggcctcgagacgcgtgagctcTCATTCAACTCTATCAAATGA-3’。
Note: 21bp base gtgagtaaggttaccgaattc before TRV-CsMLO1-F and TRV-CsMLO1-R primer sequence
And ggcctcgagacgcgtgagctc, it is carrier construction needs, what is be artificially added is not belonging to the expression vector of objective gene sequence
Partial sequence.
Polymerization chain type PCR reaction is carried out to the gene-specific region cucumber CsMLO1, PCR product is recycled, obtains 402bp purpose
Purified product is sequenced segment, obtains sequence gene-specific region cucumber CsMLO1 as shown in SEQ ID NO.3.It is pure
The pTRV2 carrier of the glue specific regions recovery product CsMLO1 and linearisation terminal homologous sequence after change, using In-Fusion
The specific regions CsMLO1 are connected into the linear large fragment of pTRV2 by HD Cloning kit, reaction system are as follows: 25 × In- of μ L
Fusion HD Enzyme Premix, the 5 linear large fragments of μ L pTRV2,3 μ L CsMLO1 target fragments, 50 DEG C of reaction 15min；
Connection product is gone in E. coli competent DH5 α, is identified through bacterium colony PCR and sequencing, shakes bacterium, upgrading grain is obtained with mesh
Gene pTRV2-CsMLO1 plant silencing expression vector.
A kind of vegetable transformant of cucumber CsMLO1 gene silencing expression vector that benefit is prepared with the aforedescribed process is provided.
Further, the recipient plant in the vegetable transformant is cucumber " the close thorn in Xintai City ".
Above-mentioned cucumber CsMLO1 gene silencing is provided and is improving cucumber to the application in stick spore leaf spot fungi resistance.
Compared with the prior art, the advantages of the present invention are as follows:
1, the present invention utilizes existing plant gene engineering technology, utilizes gene expression analysis, gene cloning and sequence point
Analysis technology, separation identify cucumber related gene sequence information；
2, what the present invention constructed can directly be lost containing cucumber CsMLO1 gene silencing expression vector to report for the first time
Pass conversion test；
3, cucumber CsMLO1 gene provided by the invention is the encoding gene of a new cucumber response stick spore leaf spot.It is logical
It crosses Agrobacterium_mediated method and silent carrier pTRV2-CsMLO1 and pTRV1 is instantaneously transferred to silencing CsMLO1 after cucumber cotyledons
The expression of gene, its lesion area is reduced compared with the control after loop-carrier spore leaf spot, illustrates which raises cucumber to stick spore leaf
The resistance of pinta bacterium stress, provides new theoretical foundation to the research of the disease-resistant key gene of stick spore leaf spot for cucumber.
Detailed description of the invention
Fig. 1 is the amplification of CsMLO1 coding sequence；
Fig. 2 (a) is recombinant vector pTRV2-CsMLO1 schematic diagram of construction method；
Fig. 2 (b) is that Escherichia coli pTRV2-CsMLO1 detects electrophoresis result；
Fig. 2 (c) is the sequencing result of pTRV2-CsMLO1；
Fig. 3 (a) is the viral spot schematic diagram of cucumber cotyledons presentation after silencing CsMLO1 gene；
Fig. 3 (b) is the expression schematic diagram of silencing cucumber cotyledons CsMLO1 gene；
Compared with Fig. 4 control group is handled with transgenosis cucumber cotyledon pathogen, transgenosis cucumber cotyledon is improved to stick spore leaf
The resistance of pinta bacterium stress.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that specific embodiment described herein is used only for explaining the present invention, and
It is not used in the restriction present invention.
Embodiment 1
The clone of cucumber CsMLO1 gene
1) cDNA sequence of cucumber CsMLO1 gene is as shown in SEQ ID NO.1.Regulate and control cucumber and responds stick spore leaf spot
The amino acid sequence of the protein of CsMLO1 gene coding is as shown in SEQ ID NO.2.
2) cloning process of cucumber CsMLO1 gene is as follows: using cucumber leaves as material, utilizing RNAprep pure plant
The method of total RNA extraction reagent box extracts total serum IgE, and reverse transcription synthesizes cDNA, and design primer expands cucumber CsMLO1 gene, primer
Nucleotide sequence is as follows:
CsMLO1-F:5 '-ATGGCGGGGGCAGCCGGTGG-3 '
CsMLO1-R:5 '-TTCAACTCTATCAAATGAAA-3 '.
Using the cDNA of reverse transcription as template, polymerization chain type PCR reaction is carried out, PCR product is recycled, obtains the piece of 1749bp mesh
Section, with reference to Fig. 1.Purified product is sequenced, sequence cucumber CsMLO1 gene as shown in SEQ ID NO.1 is obtained.
Embodiment 2
Plant silencing expression vector establishment
With reference to Fig. 2 (a), Fig. 2 (b) and Fig. 2 (c), with glue recovery product pTRV2-CsMLO1 after purification, with linearisation end
The special target fragment of CsMLO1, is connected by the pTRV2 carrier for holding homologous sequence using In-Fusion HD Cloning kit
In the linear large fragment of pTRV2, reaction system are as follows: 2 μ L 5 × In-Fusion HD Enzyme Premix, the 5 linear large fragments of μ L,
3 μ L target fragments, 50 DEG C of reaction 15min.Connection product is gone in E. coli competent DH5 α, through bacterium colony PCR and sequencing
Bacterium is shaken in identification, upgrading grain is obtained with target gene pTRV2-CsMLO1 plant silencing expression vector.
Embodiment 3
Instantaneous conversion cucumber cotyledons carry out functional verification, with reference to Fig. 3 (a) and Fig. 3 (b).
1) recombinant plasmid transformed cucumber cotyledons
By pTRV1 and Agrobacterium EHA105 positive colony containing target gene pTRV2-CsMLO1, it is inoculated in liquid YEP
Culture medium (contains 50 μ g mL-1Rif and 50 μ g mL-1Kan), 28 DEG C of 200rpm shake culture 48h make the culture of Agrobacterium bacterium solution extremely
OD600For 0.6-1.0.
Bacterium solution is transferred in sterile centrifuge tube, 5000rpm is centrifuged 10min and collects thallus；Use 10mmolL-1MES+
10mmol·L-1MgCl2+200μmol·L-1The aqueous solution suspension thalline of As is placed at room temperature for 3h until OD600 is 0.4；By thallus
It is 9d cucumber cotyledons that suspension, which injects seedling age with needleless injector,.
Use vegetable material for " the close thorn in Xintai City " cucumber；
2) cucumber gene is instantaneously overexpressed strain identification
A) phenotypic evaluation: injection method is used to be inoculated with recombinant plasmid pTRV2-CsMLO1+ to seedling age for the cucumber cotyledons of 9d
1:1 (v/v) mixed liquor of EHA105 and pTRV1+EHA105 is cultivated under the conditions of 23 DEG C.10d is inoculated in injection TRV:00+
It can be on EHA105 (the 1:1 mixed liquor of pTRV1+EHA105 and pTRV2+EHA105) and TRV:CsMLO1+EHA105 cotyledon
It observes flaviviruses spot, and is showed no viral spot on the cotyledon of Control, illustrate that TRV virus has succeeded in cucumber
Mass propagation in cotyledon.
B) RT-PCR is detected: the further instantaneous silencing expression of analysis CsMLO1 gene, to inject TRV:00+
EHA105 plant cDNA is control, the expression of CsMLO1 gene in the cucumber of detection injection TRV:CsMLO1+EHA105.Note
The expression quantity for penetrating CsMLO1 gene in the cucumber of TRV:CsMLO1 is significantly reduced to 0.03-0.1 times of control；The result shows that
The instantaneous silencing CsMLO1 gene expression of success in transgenosis cucumber cotyledon.
Embodiment 4
The stick spore leaf spot Resistance Identification of instantaneous silencing CsMLO1 gene cucumber
With reference to Fig. 4, in order to further verify CsMLO1 gene negative regulatory factor in cucumber-stick spore leaf spot Interaction
Function, this test carry out the inoculation processing of stick spore leaf spot fungi to the cucumber cotyledons of TRV:CsMLO1 injection 10d to observe its morbidity
Situation.Loop-carrier spore leaf spot fungi 10d injects cucumber of TRV:CsMLO1 compared with Control and injection TRV:00 plant
Ye Wei shows stick spore tikka disease symptoms.Meanwhile the disease index of CsMLO1 silencing plant is 24.3, and Control and injection
The disease index of TRV:00 plant is 52.44 and 58.30 respectively.Therefore, the silencing expression of CsMLO1 gene improves cucumber pair
The resistance of stick spore leaf spot fungi stress.
SEQUENCE LISTING
<110>Agricultural University Of Shenyang
<120>cucumber CsMLO1 gene and its silencing expression vector establishment method, application
<130> 2018
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1749
<212> DNA
<213>artificial sequence
<400> 1
atggcggggg cagccggtgg caagtcgctg gagcaaacac cgacatgggc cgttgccgtt 60
gtttgctttg ttttgctcgt catctctatt ttcatcgaat atagtctcca tcttatcgga 120
cattggctaa agaagagaca caaacgggcg ttgtttgaag cattagagaa gatcaaatca 180
gagcttatgt tattggggtt tatatcattg ctactaacgg tggggcaagg accaataacg 240
gagatatgta ttccacaaca tgtagctgca acgtggcatc catgtacaaa ggaaagagaa 300
gatgagatga acaaagaggt ggagaaatct gtggaacatt tgggtcttaa tcgccggaga 360
ctccttcatc tcctcggaaa tggtgaaagt ttccggcgga gtttggccgc tgcgggagga 420
gaggataaat gtgccgccaa gggtaaagct tcctttattt cagcagatgg aattcatcaa 480
cttcatatct tcatttttgt gttggctgtt tttcatgttt tgtattgtgt tctaacttat 540
gcgttggcta gagctaagat gaggagttgg aaaacatggg aaaaagagac caaaactgct 600
gaataccaat tctcacatga tccagagagg tttaggtttg caagagacac ctcatttggg 660
agaagacatt tgagcttttg gaccaaaaat cctgccttga tgtggatcgt ttgtttcttc 720
agacaatttg taagatctgt tccaaaagtt gattacttga cattaagaca tgggtttata 780
atggcacatt tagcacctca aagtcataca caatttgatt ttcaaaaata cattaataga 840
tcccttgaag aagacttcaa agttgttgtg ggaatcagcc caccaatttg gttctttgct 900
gttctatttc tcctctcaaa cactcacggt tggagggcgt atctatggct gccattcatc 960
ccactaatca ttttgctgtt gattggaaca aaattgcaag tgatcataac gaaaatggca 1020
ctaagaatac aagaaagagg tgaagtagtg aagggcgtgc cggtggtgga gcctggcgat 1080
gacctctttt ggtttaatcg acctcgcctt attctttatc tcatcaactt tgttctcttt 1140
caaaatgcct tccaagttgc cttctttgct tggacttggt atgagtttgg gttgaattct 1200
tgcttccatg agcatataga agatgtggtg atcagaattt ctatgggggt gcttgtacaa 1260
atcctttgca gttatgttac tcttcctctt tatgcactag tcactcagat gggttcaaca 1320
atgaagccaa ctatattcaa tgagagagtg gcagaggccc ttcgcaattg gtaccactcg 1380
gctcgaaagc acatcaaaca caaccgcggt tcggtcactc caatgtcgag ccgacccgcc 1440
accccgactc acagcatgtc acctgtccac cttctccgac actacaagag tgaagtcgat 1500
agcttccaca cctcaccgag aaggtcaccg ttcgacaccg atcgttggga caacgattcg 1560
ccctctccat ctcgccatgt tgatggttcg tcttcgtcac aaccccacgt tgagatggga 1620
ggttatgaaa aagatcccgt tgaatcaagt tcgtctcaag ttgatccggt tcaaccatct 1680
cgaaaccgca atcaacatga gattcatatt ggaggcccca aagacttttc atttgataga 1740
gttgaatga 1749
<210> 2
<211> 582
<212> PRT
<213>artificial sequence
<400> 2
Met Ala Gly Ala Ala Gly Gly Lys Ser Leu Glu Gln Thr Pro Thr Trp
1 5 10 15
Ala Val Ala Val Val Cys Phe Val Leu Leu Val Ile Ser Ile Phe Ile
20 25 30
Glu Tyr Ser Leu His Leu Ile Gly His Trp Leu Lys Lys Arg His Lys
35 40 45
Arg Ala Leu Phe Glu Ala Leu Glu Lys Ile Lys Ser Glu Leu Met Leu
50 55 60
Leu Gly Phe Ile Ser Leu Leu Leu Thr Val Gly Gln Gly Pro Ile Thr
65 70 75 80
Glu Ile Cys Ile Pro Gln His Val Ala Ala Thr Trp His Pro Cys Thr
85 90 95
Lys Glu Arg Glu Asp Glu Met Asn Lys Glu Val Glu Lys Ser Val Glu
100 105 110
His Leu Gly Leu Asn Arg Arg Arg Leu Leu His Leu Leu Gly Asn Gly
115 120 125
Glu Ser Phe Arg Arg Ser Leu Ala Ala Ala Gly Gly Glu Asp Lys Cys
130 135 140
Ala Ala Lys Gly Lys Ala Ser Phe Ile Ser Ala Asp Gly Ile His Gln
145 150 155 160
Leu His Ile Phe Ile Phe Val Leu Ala Val Phe His Val Leu Tyr Cys
165 170 175
Val Leu Thr Tyr Ala Leu Ala Arg Ala Lys Met Arg Ser Trp Lys Thr
180 185 190
Trp Glu Lys Glu Thr Lys Thr Ala Glu Tyr Gln Phe Ser His Asp Pro
195 200 205
Glu Arg Phe Arg Phe Ala Arg Asp Thr Ser Phe Gly Arg Arg His Leu
210 215 220
Ser Phe Trp Thr Lys Asn Pro Ala Leu Met Trp Ile Val Cys Phe Phe
225 230 235 240
Arg Gln Phe Val Arg Ser Val Pro Lys Val Asp Tyr Leu Thr Leu Arg
245 250 255
His Gly Phe Ile Met Ala His Leu Ala Pro Gln Ser His Thr Gln Phe
260 265 270
Asp Phe Gln Lys Tyr Ile Asn Arg Ser Leu Glu Glu Asp Phe Lys Val
275 280 285
Val Val Gly Ile Ser Pro Pro Ile Trp Phe Phe Ala Val Leu Phe Leu
290 295 300
Leu Ser Asn Thr His Gly Trp Arg Ala Tyr Leu Trp Leu Pro Phe Ile
305 310 315 320
Pro Leu Ile Ile Leu Leu Leu Ile Gly Thr Lys Leu Gln Val Ile Ile
325 330 335
Thr Lys Met Ala Leu Arg Ile Gln Glu Arg Gly Glu Val Val Lys Gly
340 345 350
Val Pro Val Val Glu Pro Gly Asp Asp Leu Phe Trp Phe Asn Arg Pro
355 360 365
Arg Leu Ile Leu Tyr Leu Ile Asn Phe Val Leu Phe Gln Asn Ala Phe
370 375 380
Gln Val Ala Phe Phe Ala Trp Thr Trp Tyr Glu Phe Gly Leu Asn Ser
385 390 395 400
Cys Phe His Glu His Ile Glu Asp Val Val Ile Arg Ile Ser Met Gly
405 410 415
Val Leu Val Gln Ile Leu Cys Ser Tyr Val Thr Leu Pro Leu Tyr Ala
420 425 430
Leu Val Thr Gln Met Gly Ser Thr Met Lys Pro Thr Ile Phe Asn Glu
435 440 445
Arg Val Ala Glu Ala Leu Arg Asn Trp Tyr His Ser Ala Arg Lys His
450 455 460
Ile Lys His Asn Arg Gly Ser Val Thr Pro Met Ser Ser Arg Pro Ala
465 470 475 480
Thr Pro Thr His Ser Met Ser Pro Val His Leu Leu Arg His Tyr Lys
485 490 495
Ser Glu Val Asp Ser Phe His Thr Ser Pro Arg Arg Ser Pro Phe Asp
500 505 510
Thr Asp Arg Trp Asp Asn Asp Ser Pro Ser Pro Ser Arg His Val Asp
515 520 525
Gly Ser Ser Ser Ser Gln Pro His Val Glu Met Gly Gly Tyr Glu Lys
530 535 540
Asp Pro Val Glu Ser Ser Ser Ser Gln Val Asp Pro Val Gln Pro Ser
545 550 555 560
Arg Asn Arg Asn Gln His Glu Ile His Ile Gly Gly Pro Lys Asp Phe
565 570 575
Ser Phe Asp Arg Val Glu
580
<210> 3
<211> 402
<212> DNA
<213>artificial sequence
<400> 3
gtggcagagg cccttcgcaa ttggtaccac tcggctcgaa agcacatcaa acacaaccgc 60
ggttcggtca ctccaatgtc gagccgaccc gccaccccga ctcacagcat gtcacctgtc 120
caccttctcc gacactacaa gagtgaagtc gatagcttcc acacctcacc gagaaggtca 180
ccgttcgaca ccgatcgttg ggacaacgat tcgccctctc catctcgcca tgttgatggt 240
tcgtcttcgt cacaacccca cgttgagatg ggaggttatg aaaaagatcc cgttgaatca 300
agttcgtctc aagttgatcc ggttcaacca tctcgaaacc gcaatcaaca tgagattcat 360
attggaggcc ccaaagactt ttcatttgat agagttgaat ga 402
Claims (10)
1. a kind of cucumber CsMLO1 gene, which is characterized in that its coding region sequence is as shown in SEQ ID NO.1.
2. the primer for expanding cucumber CsMLO1 gene as described in claim 1, which is characterized in that its nucleotide sequence is such as
Under:
CsMLO1-F:5 '-ATGGCGGGGGCAGCCGGTGG-3 '
CsMLO1-R:5 '-TTCAACTCTATCAAATGAAA-3 '.
3. the method expanded using primer pair cucumber CsMLO1 gene as claimed in claim 2, which is characterized in that with Huang
Melon blade is material, extracts total serum IgE, reverse transcription synthesis using the method for RNAprep pure plant total RNA extraction reagent box
CDNA designs primer as claimed in claim 2；
Using the cDNA of reverse transcription as template, polymerization chain type PCR reaction is carried out, PCR product is recycled, obtains 1749bp target fragment,
Purified product is sequenced, sequence cucumber CsMLO1 gene as shown in SEQ ID NO.1 is obtained.
4. the protein that cucumber CsMLO1 gene as described in claim 1 encodes, which is characterized in that its amino acid sequence
As shown in SEQ ID NO.2.
5. the specific regions primer of cucumber CsMLO1 gene as described in claim 1, which is characterized in that compare cucumber MLO
Family gene sequence chooses specific regions 402bp design primer, and nucleotide sequence is as follows:
TRV-CsMLO1-F:5’-gtgagtaaggttaccgaattcGTGGCAGAGGCCCTTCGCAAC-3’
TRV-CsMLO1-R:5’-ggcctcgagacgcgtgagctcTCATTCAACTCTATCAAATGA-3’。
6. utilizing the method for the gene-specific region primer pair cucumber CsMLO1 as claimed in claim 5 amplification, which is characterized in that
Polymerization chain type PCR reaction is carried out, PCR product is recycled, obtains 402bp target fragment, purified product is sequenced, cucumber is obtained
TRV-CsMLO1 genetic fragment, as shown in SEQ ID NO.3 in sequence table.
7. the construction method of the silent carrier of cucumber CsMLO1 gene, which is characterized in that the TRV- obtained using claim 6
CsMLO1 genetic fragment, using the pTRV2 carrier of linearisation terminal homologous sequence, using In-Fusion HD Cloning kit
TRV-CsMLO1 genetic fragment is connected into pTRV2 carrier, reaction system are as follows: 2 μ L 5 × In-Fusion HD Enzyme
Premix, 5 μ L pTRV2 carriers, 3 μ L TRV-CsMLO1 genetic fragments, 50 DEG C of reaction 15min；Connection product is gone into large intestine
It in bacillus competence DH5 α, is identified through bacterium colony PCR and sequencing, shakes bacterium, upgrading grain is obtained with target gene pTRV2-CsMLO1
Plant silencing expression vector.
8. a kind of plant of cucumber CsMLO1 gene silencing expression vector prepared by the method containing using method of claim 7 turns
Change body.
9. vegetable transformant as claimed in claim 8, which is characterized in that the recipient plant in the vegetable transformant is Huang
Melon " the close thorn in Xintai City ".
10. cucumber CsMLO1 gene as described in claim 1 is in negative regulation cucumber to the application in stick spore leaf spot fungi resistance.
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