Construction and application of gene silencing vector induced by Chinese wheat yellow mosaic virus

A technology of gene silencing and leaf virus, applied in application, genetic engineering, introduction of foreign genetic material using vectors, etc., can solve problems such as interference in target gene function identification, and achieve the effect of enriching species

Inactive Publication Date: 2018-09-11
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some viral vectors themselves can also induce certain symptoms or physiological changes in plants during growth, which will interfere with the identification of target gene functions.

Method used

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  • Construction and application of gene silencing vector induced by Chinese wheat yellow mosaic virus
  • Construction and application of gene silencing vector induced by Chinese wheat yellow mosaic virus
  • Construction and application of gene silencing vector induced by Chinese wheat yellow mosaic virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Cloning of partial gene fragments of NbPDS: extract the Total RNA of Nicotiana benthamiana, and obtain the cDNA corresponding to Nicotiana benthamiana by reverse transcription, take a small amount of cDNA as a template, and use the primer pair upstream primer PDSN:5'- ACTAGT GAACATATTGAGTCAAAAGGT-3' (the underlined part is the sequence of SpeI) and downstream primer PDSC: 5'- GGTACC GCTTCTGCTGAAGAGCAGATT-3' (the underlined part is the sequence of KpeI) was amplified by PCR, and the obtained target product was a partial fragment of NbPDS, and the length of the amplified sequence was about 300bp. After electrophoresis detection and gel cutting recovery, it was connected to pMD18- Tsimple vector, obtain the pMD18-NbPDS recombinant plasmid, and then transform it into Escherichia coli DH5α, and then pick the verified positive clones for sequencing identification.

[0039] Construction, transformation and screening of the pCB-35S-MCS:NbPDS recombinant vector: The correctly ...

Embodiment 2

[0043] Example 2: CWMV-VIGS vector silencing analysis of Nicotiana benthamiana NbL12, specific steps:

[0044] Cloning of partial gene fragments of NbL12: using primer pair upstream primer L12N:5'- ACTAGT CCTCGCTGACGCCCAGAAACT-3' (the underlined part is the sequence of SpeI) and downstream primer L12C:5'- GGTACC GCCCTAACAGCCTTAATTGTA-3' (the underlined part is the sequence of KpeI) was amplified by PCR, and the size of the target fragment was 300bp. For other construction processes, refer to Example 1.

[0045] Construction, transformation and screening of pCB-35S-MCS:NbL12 recombinant vector: Refer to Case 1 for specific steps.

[0046] CWMV-VIGS: NbL12 infiltration inoculation and cultivation of tobacco: Refer to Case 1 for specific steps.

[0047] Result analysis: observation of the symptoms of Nicotiana benthamiana infiltrated by CWMV-VIGS: NbL12 found that obvious mosaic symptoms appeared on the leaves of the system after 14 days of infection ( Figure 5 ), the trans...

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Abstract

The invention mainly relates to construction of a gene silencing vector induced by Chinese wheat yellow mosaic virus, and application thereof to nicotiana benthamiana. The biological active analysis results show that the constructed CWMV-VIGS can successfully infect the nicotiana benthamiana and can realize the silencing after the effective transcription on relevant genes such as phytoene desaturase and 50S ribosomal protein L12. The types of the VIGS vectors are enriched; in addition, the effective technical measures are provided for the gene function study.

Description

technical field [0001] The invention relates to the field of agricultural applied biotechnology, in particular to a CWMV-VIGS gene silencing carrier and its application in Nicotiana benthamiana. Background technique [0002] Virus-induced gene silencing (VIGS) is a phenomenon of post-transcriptional gene silencing. It is a reverse genetics technique for rapidly identifying plant gene functions and has become a powerful tool for the study of plant gene functions. When the virus carries the cDNA of the target gene and infiltrates plant cells, it will form double-stranded RNA (dsRNA) during the process of replication and expression in plant cells. As the elicitor of gene silencing, dsRNA is first cut into Small fragments of RNA (siRNA), and then siRNA combines with some specific proteins (AGO1) in a single-stranded form in plant cells to form an RNA-induced silencing complex, which specifically binds to homologous RNA in the cytoplasm , causing degradation of homologous RNAs r...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66A01H5/00A01H6/82
CPCC12N15/8218C12N15/8261
Inventor 羊健张恒木彭琪琪陈炫何龙杨锦陈剑平
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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