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Method for improving efficiency of eggplant virus induced gene silencing (VIGS)

A gene silencing and viral gene technology, applied in the field of genetic engineering, can solve the problems of large damage to eggplant seedlings, unfavorable gene function, low silencing efficiency, etc., and achieve the effects of long-lasting traits, significant silencing phenotypes, and overcoming mechanical damage

Inactive Publication Date: 2018-10-09
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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Problems solved by technology

Although VIGS technology has many advantages, it still has certain limitations. For example, this technology can rarely completely silence or inhibit the expression of target genes. Different insert sizes, inoculation methods, and culture environments all affect gene silencing efficiency. larger
In particular, the current way of eggplant VIGS technology to inoculate virus is mostly to inoculate by injection, pressure and penetration into leaves, which has great damage to eggplant seedlings, low silencing efficiency, large amount of preparation of Agrobacterium infection solution required for virus inoculation, and long time-consuming inoculation process. , and it is not conducive to the study of gene function in early development

Method used

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  • Method for improving efficiency of eggplant virus induced gene silencing (VIGS)
  • Method for improving efficiency of eggplant virus induced gene silencing (VIGS)
  • Method for improving efficiency of eggplant virus induced gene silencing (VIGS)

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Experimental program
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Embodiment

[0032] 1. Cloning of SmPDS gene fragment

[0033] Take the leaves of cultivated eggplant Suzhou Niujiao (publicly known and public, purchased in the market), extract the total RNA of the leaves with reference to the operating method of the Plant Total RNA Extraction Kit (TIANGEN), and reverse according to the cDNA Reverse Transcription Kit (Beijing Quanshijin) The cDNA was recorded, and according to the sequence information of the SmPDS gene in the eggplant library, the primer 5 software was used to design specific primers to amplify SmPDS; the upstream primer SmPDS-XbaI-F: 5'-cggtctagaggcactcaactttataaacc-3' (SEQID NO.1), the downstream primer SmPDS - BamHI-R: 5'-cggggatcccttcagttttctgtcaaacc-3' (SEQ ID NO. 2);

[0034] Use leaf cDNA as template to carry out PCR reaction, 50 μL reaction system: 25 μL of 2×PCR SuperMix, 1 μL of upstream and downstream primers (20 μmol / L), 2 μL of cDNA template, 21 μL of ddH2O; reaction program: pre-denaturation at 95°C for 5 minutes, then 94°C...

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Abstract

The invention discloses a method for improving efficiency of eggplant virus induced gene silencing (VIGS), belonging to the technical field of plant genetic engineering. The method comprehensively considers various factors affecting the efficiency of the eggplant VIGS; a recombinant vector is silenced by constructing a TRV2 virus containing a target gene fragment; a vacuum infiltration method is adopted to infect seeds at the stage in germination so as to induce silencing of an endogenous target gene. The silenced plants treated by the method are rapid in phenotype appearance, high in efficiency and long in phenotypic change duration, and the problems of injury, caused by injection and inoculation, to plants, difficulty in injection and inoculation of smaller eggplant cotyledons, and the like can be solved; therefore, an effective method is provided for rapid verification of gene functions.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for improving gene silencing efficiency induced by eggplant virus. Background technique [0002] Virus-induced gene silencing (Virus-induced Gene Silencing, VIGS) is a technology developed by using the defense mechanism of plants against RNA viruses. It is to infect plants with a viral vector carrying a target gene fragment to trigger the silencing of sequence homologous genes in the host, thereby Realize the identification of gene function (Senthil-Kumar & Mysore, 2011). Compared with traditional gene function research methods such as transgenic, gene knockout, and antisense suppression, VIGS technology does not require genetic transformation of plants or acquisition of mutants, and can identify gene functions simply, quickly, instantaneously, efficiently, and specifically. Effective technical methods in the study of plant gene function. With t...

Claims

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Application Information

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IPC IPC(8): C12N15/82
CPCC12N15/8218
Inventor 刘军周晓慧杨艳鲍生有庄勇
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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