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Cucumis sativus CsMLO1 gene, construction method and application of silencing expression vector thereof

A cucumber and gene technology, applied in the fields of molecular biology and biology, can solve the problems of little research on the interaction between cucumber and Corynebacterium leaf spot, and achieve the effects of improving resistance and reducing the area of ​​disease spots

Active Publication Date: 2018-12-18
SHENYANG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In summary, little research has been done on the interaction between cucumber and Corynespora leaf spot

Method used

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  • Cucumis sativus CsMLO1 gene, construction method and application of silencing expression vector thereof
  • Cucumis sativus CsMLO1 gene, construction method and application of silencing expression vector thereof
  • Cucumis sativus CsMLO1 gene, construction method and application of silencing expression vector thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Cloning of Cucumber CsMLO1 Gene

[0035] 1) The cDNA sequence of cucumber CsMLO1 gene is shown in SEQ ID NO.1. The amino acid sequence of the protein encoded by the CsMLO1 gene that regulates cucumber response to Corynesporium leaf spot is shown in SEQ ID NO.2.

[0036] 2) The cloning method of the cucumber CsMLO1 gene is as follows: use the cucumber leaves as the material, use the RNAprep pure plant total RNA extraction kit to extract the total RNA, reverse transcribe to synthesize cDNA, design primers to amplify the cucumber CsMLO1 gene, and the nucleotide sequence of the primers as follows:

[0037] CsMLO1-F: 5'-ATGGCGGGGGCAGCCGGTGG-3'

[0038] CsMLO1-R: 5'-TTCAACTCTATCAAATGAAA-3'.

[0039] Using the reverse-transcribed cDNA as a template, carry out polymerization chain PCR reaction, recover the PCR product, and obtain the 1749bp target fragment, refer to figure 1 . The purified product was sequenced to obtain the cucumber CsMLO1 gene whose sequence is shown in ...

Embodiment 2

[0041] Plant silencing expression vector construction

[0042]Referring to Figure 2(a), Figure 2(b) and Figure 2(c), the product pTRV2-CsMLO1 was recovered from the purified gel, and the pTRV2 vector with homologous sequences at the linearized end was used to clone CsMLO1 using the In-Fusion HD Cloning kit. The specific target fragment was ligated into the large linear fragment of pTRV2. The reaction system was: 2 μL 5×In-Fusion HD Enzyme Premix, 5 μL large linear fragment, 3 μL target fragment, and reacted at 50°C for 15 minutes. The ligation product was transferred to Escherichia coli competent DH5α, identified by colony PCR and sequencing, the bacteria were shaken, and the plasmid was extracted to obtain a plant silencing expression vector carrying the target gene pTRV2-CsMLO1.

Embodiment 3

[0044] Transient transformation of cucumber cotyledon for functional verification, refer to Figure 3(a) and Figure 3(b).

[0045] 1) Transformation of cucumber cotyledon with recombinant plasmid

[0046] Inoculate pTRV1 and Agrobacterium EHA105 positive clones containing the target gene pTRV2-CsMLO1 in liquid YEP medium (containing 50 μg mL -1 Rif and 50 μg mL -1 Kan), 28 ℃ 200rpm shaking culture for 48h, so that the Agrobacterium liquid culture to OD 600 0.6-1.0.

[0047] Transfer the bacterial solution into a sterile centrifuge tube and centrifuge at 5000rpm for 10min to collect the bacterial cells; use 10mmol·L -1 MES+10mmol·L -1 MgCl 2 +200μmol·L -1 Suspend the bacteria in the aqueous solution of As until the OD600 is 0.4, and place at room temperature for 3 hours; inject the bacteria suspension into the cotyledon of cucumber seedlings with a needle-free syringe of 9 days old.

[0048] The plant material is "Xintai Mici" cucumber;

[0049] 2) Identification of cucu...

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Abstract

Belonging to the field of molecular biology and biotechnology, the invention specifically relates to a cucumis sativus CsMLO1 gene, a construction method and application of a silencing expression vector of the gene. In a cucumis sativus responsive corynespora leaf spot regulation approach, a full-length coding region sequence of the CsMLO1 gene is shown as SEQ ID NO.1 in the sequence table, and the amino acid sequence is shown as SEQ ID NO.2 in the sequence table. The invention also provides a construction method of inserting a cucumis sativus CsMLO1 gene specific fragment into the silencing expression vector pTRV2, the constructed plant silencing expression vector is transformed to cucumis sativus cotyledons by Agrobacterium tumefaciens so as to obtain transgenic cucumis sativus plants, and disease resistance identification is carried out. The result shows that the gene is a negative regulatory factor in the interaction between cucumis sativus and corynespora leaf spot. The inventionprovides technical means and theoretical basis for fully revealing the functional study of cucumis sativus CsMLO1 gene by means of genetic engineering technology, and has great application value.

Description

technical field [0001] The invention belongs to the field of molecular biology and biotechnology, and specifically relates to a cucumber CsMLO1 gene (Cucsa.207280) and a method for constructing a silent expression vector and application thereof. Background technique [0002] Cucumber (Cucumis sativus) is one of the main vegetable crops in protected cultivation in northern my country, and its yield and quality are seriously affected by abiotic stress. Corynespora cassiicola leaf spot of cucumber is a worldwide fungal disease caused by the pathogenic fungus Corynespora cassiicola; at present, the research on this disease mainly focuses on biological control and chemical control. There are few studies on the resistance mechanism of leaf spot disease. [0003] MLO (Mildew Resistance Locus O) protein is a unique protein in plants. Its research shows that members of the MLO family are susceptible to powdery mildew infection, while the recessive mutation gene mlo is endowed with b...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/11C12N15/10C12N15/66C07K14/415C12N15/82A01H5/00A01H6/34
CPCC07K14/415C12N15/10C12N15/66C12N15/8282
Inventor 范海延于广超崔娜于洋王翔宇孟祥南陈秋敏赵珺玥杨云
Owner SHENYANG AGRI UNIV
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