CN109022449B - Cucumber CsMLO1 gene and construction method and application of silent expression vector thereof - Google Patents
Cucumber CsMLO1 gene and construction method and application of silent expression vector thereof Download PDFInfo
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Abstract
The invention belongs to the field of molecular biology and biotechnology, and particularly relates to a cucumber CsMLO1 gene and a construction method and application of a silent expression vector thereof, wherein the sequence of the full-length coding region of the CsMLO1 gene in a cucumber response corynespora leaf spot regulation and control pathway is shown as a sequence table SEQ ID NO. 1; the amino acid sequence is shown in a sequence table SEQ ID NO. 2. The invention also provides a construction method for inserting the specific segment of the cucumber CsMLO1 gene into the silent expression vector pTRV2, the constructed plant silent expression vector is transformed into cucumber cotyledons through agrobacterium, a transgenic cucumber plant is obtained, and the disease resistance identification is carried out, and the result shows that the gene is a negative regulatory factor in the interaction of cucumber and corynespora leaf spot. The invention provides a technical means and a theoretical basis for comprehensively disclosing the research of the functions of the CsMLO1 gene of the cucumber by utilizing a gene engineering technology, and has great application value.
Description
Technical Field
The invention belongs to the field of molecular biology and biotechnology, and particularly relates to a cucumber CsMLO1 gene (Cucsa.207280) and a construction method and application of a silencing expression vector thereof.
Background
Cucumber (Cucumis sativus) is one of the main vegetable crops cultivated in northern facilities in China, and the yield and the quality of the cucumber are seriously influenced by abiotic stress. Corynespora cassiicola is a worldwide fungal disease caused by the pathogenic fungus Corynespora cassiicola; at present, the research on the diseases mainly focuses on the aspects of biological control, chemical control and the like, and the research on the resistance mechanism of the cucumber to the corynespora leaf spot is very little.
MLO (Mildew Resistance Locus O) protein is a unique protein in plants, and studies have shown that MLO family members, as susceptible genes, are susceptible to infection by powdery mildew, whereas the recessive mutant MLO gene is conferred broad-spectrum Resistance (Shirasu et al, 1999; Chen et al, 2006; Kim et al, 2002). At present, the relationship between the mlo disease-resistant pathway and other pathogenic bacteria besides powdery mildew is receiving more and more attention. Puccinia striiformis f.sp.tritici is a specialized fungus that causes stripe rust of wheat, mainly damaging wheat leaves. Research shows that three transmembrane protein genes TaMLO1, TaMLO2 and TaMLO5 which possibly participate in allergic cell death signal pathways are subjected to gene silencing through a virus-induced gene silencing technology, inoculated stripe rust plants show different degrees of disease resistance, and TaMLO1/2/5 possibly participate in regulating the interaction process of wheat and stripe rust to a certain extent as a negative regulator of programmed cell death (Schleria schrenki, 2013). Comparison of the wild-type barley mutant genes mlo3, mlo4, mlo5 with barley leaf blight analysis revealed enhanced resistance of the three mutants to pathogenic bacteria (Rozhen et al 2009). Therefore, it is thought from a series of facts that the disease-resistant protein MLO may act as a regulator, and not only as a resistance inhibitor against powdery mildew, but also in other pathogenic bacteria, exhibits a mutually inhibitory defense response.
In conclusion, very little research has been done on the interaction between cucumber and leaf spot of corynespora. Therefore, a cucumber CsMLO1 gene 402bp fragment is selected to be inserted into pTRV2, the constructed recombinant vectors pTRV2-CsMLO1 and pTRV1 with equal volume are introduced into a plant body, TRV viruses are successfully propagated in cucumber leaves in large quantity through phenotype identification and analysis, and the copy number of the CsMLO1 gene in transgenic cucumber is obviously reduced by combining qRT-PCR technology detection. Therefore, the CsMLO1 gene of the cucumber can be deeply known as a negative regulatory factor in the interaction of the cucumber and the leaf spot of the corynespora, and a foundation is laid for comprehensively disclosing the function of the CsMLO1 gene of the cucumber.
Disclosure of Invention
In order to solve the technical problems, the invention provides a cucumber CsMLO1 gene and a construction method and application of a silencing expression vector thereof.
The invention is realized in such a way, and provides a cucumber CsMLO1 gene, wherein the sequence of a coding region of the cucumber CsMLO1 gene is shown as SEQ ID NO. 1.
Provides a primer for amplifying the cucumber CsMLO1 gene, and the nucleotide sequence of the primer is as follows:
CsMLO1-F：5’-ATGGCGGGGGCAGCCGGTGG-3’
CsMLO1-R：5’-TTCAACTCTATCAAATGAAA-3’。
the method for amplifying the CsMLO1 gene of cucumber by using the primer comprises the steps of taking cucumber leaves as materials, extracting total RNA by using a method of an RNAprep pure plant total RNA extraction kit, carrying out reverse transcription to synthesize cDNA, and designing the primer according to claim 2;
and (3) carrying out polymerization chain PCR reaction by taking the reverse transcribed cDNA as a template, recovering a PCR product to obtain a 1749bp target fragment, and sequencing the purified product to obtain the cucumber CsMLO1 gene with the sequence shown as SEQ ID NO. 1.
Provides a protein obtained by utilizing the CsMLO1 gene code of the cucumber, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.
Providing specific region primers of the cucumber CsMLO1 gene, comparing the sequences of cucumber MLO family genes, and selecting specific region 402bp design primers, wherein the nucleotide sequences are as follows:
TRV-CsMLO1-F:5’-gtgagtaaggttaccgaattcGTGGCAGAGGCCCTTCGCAAC-3’
TRV-CsMLO1-R:5’-ggcctcgagacgcgtgagctcTCATTCAACTCTATCAAATGA-3’。
note: the first 21bp bases gtgagtaaggttaccgaattc and ggcctcgagacgcgtgagctc of the primer sequences TRV-CsMLO1-F and TRV-CsMLO1-R are partial sequences of an expression vector which is required for constructing the vector and is not a target gene sequence and added artificially.
And (3) carrying out polymerization chain PCR reaction on the specific region of the cucumber CsMLO1 gene, recovering a PCR product to obtain a 402bp target fragment, and sequencing the purified product to obtain the specific region of the cucumber CsMLO1 gene with the sequence shown as SEQ ID NO. 3. The purified gel recovery product CsMLO1 specific region and linearized end homologous sequence pTRV2 vector were used to ligate CsMLO1 specific region into pTRV2 linear large fragment using In-Fusion HD Cloning kit, the reaction system was: 2. mu.L of 5 XIn-Fusion HD Enzyme Premix, 5. mu.L of pTRV2 linear large fragment, 3. mu.L of CsMLO1 target fragment, reacted at 50 ℃ for 15 min; transferring the ligation product into escherichia coli competence DH5 alpha, and obtaining the plant silencing expression vector with the target gene pTRV2-CsMLO1 through colony PCR and sequencing identification, bacteria shaking and quality improvement.
Provides a plant transformant of the cucumber CsMLO1 gene silencing expression vector prepared by the method.
Further, the recipient plant in the plant transformant is cucumber "Xintai Mici".
Provides the application of the cucumber CsMLO1 gene silencing in improving the resistance of cucumbers to alternaria clavata.
Compared with the prior art, the invention has the advantages that:
1. the related gene sequence information of the cucumber is separated and identified by utilizing the existing plant genetic engineering technology and utilizing the gene expression analysis, gene cloning and sequence analysis technologies;
2. the silencing expression vector containing the cucumber CsMLO1 gene constructed by the invention is reported for the first time, and can be directly used for genetic transformation tests;
3. the cucumber CsMLO1 gene provided by the invention is a novel coding gene for responding to corynespora leaf spot of cucumber. After the silencing vectors pTRV2-CsMLO1 and pTRV1 are transiently transferred into cucumber cotyledons by an agrobacterium-mediated transformation method, the expression of the silencing CsMLO1 gene is silenced, and the disease spot area of the inoculated corynespora leaf spot disease is reduced compared with that of a control, so that the resistance of the cucumber to the stress of the corynespora leaf spot disease is improved, and a new theoretical basis is provided for the research of the cucumber on the disease-resistant key gene of the corynespora leaf spot disease.
Drawings
FIG. 1 shows the result of amplification of the coding region sequence of the CsMLO1 gene;
FIG. 2(a) is a schematic diagram of a construction method of recombinant vector pTRV2-CsMLO 1;
FIG. 2(b) shows the result of electrophoresis in E.coli pTRV2-CsMLO 1;
FIG. 2(c) is the sequencing result of pTRV2-CsMLO 1;
FIG. 3(a) is a schematic diagram showing virus spots of cucumber cotyledons after silencing of CsMLO1 gene;
FIG. 3(b) is a schematic diagram of the expression of the silenced cucumber cotyledon CsMLO1 gene;
FIG. 4 comparison of control group and transgenic cucumber cotyledon pathogen treatment, transgenic cucumber cotyledon has improved resistance to clavibacter clavulans stress.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
Cloning of cucumber CsMLO1 Gene
1) The cDNA sequence of the cucumber CsMLO1 gene is shown in SEQ ID NO. 1. The amino acid sequence of the protein for regulating and controlling cucumber response corynespora leaf spot CsMLO1 gene code is shown in SEQ ID NO. 2.
2) The cloning method of the cucumber CsMLO1 gene comprises the following steps: the method comprises the steps of taking cucumber leaves as a material, extracting total RNA by using a method of an RNAprep pure plant total RNA extraction kit, synthesizing cDNA through reverse transcription, designing a primer to amplify the cucumber CsMLO1 gene, wherein the nucleotide sequence of the primer is as follows:
CsMLO1-F：5’-ATGGCGGGGGCAGCCGGTGG-3’
CsMLO1-R：5’-TTCAACTCTATCAAATGAAA-3’。
taking the reverse transcribed cDNA as a template, performing a polymerization chain PCR reaction, and recovering a PCR product to obtain a 1749bp target fragment, which is shown in FIG. 1. Sequencing the purified product to obtain the cucumber CsMLO1 gene with the sequence shown in SEQ ID NO. 1.
Example 2
Construction of plant silencing expression vector
Referring to FIGS. 2(a), 2(b) and 2(c), the purified gel recovery product pTRV2-CsMLO1, pTRV2 vector with linearized terminal homologous sequence, CsMLO1 specific target fragment was ligated into pTRV2 linear large fragment using In-Fusion HD Cloning kit, the reaction system was: mu.L of 5 XIn-Fusion HD Enzyme Premix, 5. mu.L of the linear large fragment, 3. mu.L of the target fragment, and reacted at 50 ℃ for 15 min. Transferring the ligation product into escherichia coli competence DH5 alpha, and obtaining the plant silencing expression vector with the target gene pTRV2-CsMLO1 through colony PCR and sequencing identification, bacteria shaking and quality improvement.
Example 3
The transiently transformed cucumber cotyledons were functionally verified, with reference to FIGS. 3(a) and 3 (b).
1) Recombinant plasmid transformed cucumber cotyledon
pTRV1 and Agrobacterium EHA105 positive clone containing the gene of interest pTRV2-CsMLO1 were inoculated into liquid YEP medium (containing 50. mu.g mL)-1Rif and 50. mu.g mL-1Kan), culturing for 48h at 28 ℃ and 200rpm with shaking to ensure that the agrobacterium liquid is cultured to OD600Is 0.6-1.0.
Transferring the bacterial liquid into a sterile centrifugal tube, centrifuging at 5000rpm for 10min, and collecting thalli; using 10 mmol. L-1MES+10mmol·L-1MgCl2+200μmol·L-1Suspending thallus with As water solution until OD600 is 0.4, and standing at room temperature for 3 h; the thallus suspension is injected into cucumber cotyledons with the seedling age of 9d by a needle-free syringe.
Adopting a plant material of 'Xintai Mici' cucumber;
2) identification of cucumber gene transient over-expression strain
a) And (3) phenotype identification: cucumber cotyledons with the age of 9d are inoculated with a 1:1(v/v) mixed solution of recombinant plasmids pTRV2-CsMLO1+ EHA105 and pTRV1+ EHA105 by an injection method and cultured at the temperature of 23 ℃. When the strain is inoculated for 10d, yellow virus spots can be observed on both the TRV:00+ EHA105 (1: 1 mixed solution of pTRV1+ EHA105 and pTRV2+ EHA 105) and TRV: CsMLO1+ EHA105 cotyledons, and no virus spots are observed on the Control cotyledons, which indicates that the TRV virus has been successfully propagated in the cucumber leaf.
b) And (3) RT-PCR detection: the transient silencing expression level of the CsMLO1 gene is further analyzed, and the expression level of the CsMLO1 gene in the cucumber injected with the TRV: CsMLO1+ EHA105 is detected by taking the plant cDNA injected with the TRV:00+ EHA105 as a control. CsMLO1 gene expression level of cucumber injected with TRV CsMLO1 is obviously reduced to 0.03-0.1 times of that of a control; the result shows that the CsMLO1 gene expression is successfully and transiently silenced in the transgenic cucumber seed leaves.
Example 4
Resistance identification of corynespora leaf spot of cucumber transiently silenced with CsMLO1 gene
Referring to FIG. 4, in order to further verify the function of the CsMLO1 gene as a negative regulator in the cucumber-Corynespora clavuligeri interaction process, the present experiment performed Corynespora clavuligeri inoculation treatment on cucumber cotyledons injected with 10d of TRV: CsMLO1 to observe the onset of the disease. Compared with the Control and the TRV:00 plant, the inoculated corynebacterium sp.10 d has no corynebacterium sp.leaf spot disease symptom in the cucumber cotyledon injected with the TRV: CsMLO 1. Meanwhile, the disease index of CsMLO1 silent plants was 24.3, while the disease index of Control and TRV 00 injected plants were 52.44 and 58.30, respectively. Therefore, the silent expression of the CsMLO1 gene improves the resistance of the cucumber to the stress of the corynebacterium clavatum.
SEQUENCE LISTING
<110> Shenyang agriculture university
<120> cucumber CsMLO1 gene and construction method and application of silent expression vector thereof
<130> 2018
<160> 3
<170> PatentIn version 3.3
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<211> 1749
<212> DNA
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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
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Met Ala Gly Ala Ala Gly Gly Lys Ser Leu Glu Gln Thr Pro Thr Trp
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Glu Ile Cys Ile Pro Gln His Val Ala Ala Thr Trp His Pro Cys Thr
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His Leu Gly Leu Asn Arg Arg Arg Leu Leu His Leu Leu Gly Asn Gly
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Glu Ser Phe Arg Arg Ser Leu Ala Ala Ala Gly Gly Glu Asp Lys Cys
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Ala Ala Lys Gly Lys Ala Ser Phe Ile Ser Ala Asp Gly Ile His Gln
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Leu His Ile Phe Ile Phe Val Leu Ala Val Phe His Val Leu Tyr Cys
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Val Leu Thr Tyr Ala Leu Ala Arg Ala Lys Met Arg Ser Trp Lys Thr
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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 (1)
1. An application of a cucumber CsMLO1 gene in negative regulation of cucumber resistance to corynebacterium clavatum is characterized in that a coding region sequence of the cucumber CsMLO1 gene is shown as SEQ ID NO.1, the corynebacterium clavatum is a pathogenic fungus, namely, corynebacterium polyspora, and the Latin article name is Corynespora casicicola.
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| CN201810824315.6A CN109022449B (en) | 2018-07-25 | 2018-07-25 | Cucumber CsMLO1 gene and construction method and application of silent expression vector thereof |
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| CN109022449A CN109022449A (en) | 2018-12-18 |
| CN109022449B true CN109022449B (en) | 2021-03-23 |
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| CN109576303B (en) * | 2018-12-29 | 2021-09-07 | 中国农业科学院郑州果树研究所 | Cucumber green mottle mosaic virus induced gene silencing vector and construction method and application thereof |
| CN111690679B (en) * | 2020-06-03 | 2021-12-28 | 华中农业大学 | Recombinant expression vector for cultivating cucumber male sterile line and construction method and application thereof |
| CN112746073A (en) * | 2020-12-09 | 2021-05-04 | 华中农业大学 | Method for obtaining cucumber seed material with high powdery mildew resistance through polygene editing |
| CN113025632B (en) * | 2021-01-15 | 2023-10-20 | 沈阳农业大学 | Cucumber lignin biosynthesis key enzyme gene CsCSE and application thereof |
| CN113528538B (en) * | 2021-08-16 | 2022-04-19 | 东北农业大学 | Cucumber CsSTK gene, protein, expression vector and application |
| CN113980975B (en) * | 2021-11-08 | 2022-10-28 | 湖南农业大学 | Application of CsCS gene in inhibition of cucumber lateral shoot germination |
| CN114438105B (en) * | 2022-03-24 | 2023-11-17 | 云南中烟工业有限责任公司 | Tobacco NtMLO6-1 gene and knockout method and application thereof |
| CN115960927B (en) * | 2022-07-01 | 2024-03-22 | 沈阳农业大学 | Cucumber powdery mildew stress related gene CsRab11A and application thereof |
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