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Method for mediating virus-induced gene silencing by TRV vectors in forsythia suspensa leaf

A carrier, forsythia technology, applied in the field of genetic engineering, can solve the problems of restricting the molecular biology research of forsythia, and there is no efficient gene function verification method.

Active Publication Date: 2018-12-07
BEIJING FORESTRY UNIVERSITY
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  • Claims
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Problems solved by technology

With the popularization and application of high-throughput sequencing technology, a large number of gene expression sequence tags can be obtained, but there is no efficient and stable method for gene function verification, which limits the research on the molecular biology of forsythia

Method used

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  • Method for mediating virus-induced gene silencing by TRV vectors in forsythia suspensa leaf
  • Method for mediating virus-induced gene silencing by TRV vectors in forsythia suspensa leaf
  • Method for mediating virus-induced gene silencing by TRV vectors in forsythia suspensa leaf

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Embodiment 1

[0028] 1. Acquisition of experimental materials: Potted forsythia cutting seedlings were taken, re-cut and placed in an artificial climate chamber for cultivation, and set aside. Culture conditions: light 300μmol·m -2 ·s -1 , 16h, 22°C, dark 8h, 18°C, humidity 60%.

[0029] 2. Recombinant vector construction: The amplified Forsythia phytoene dehydrogenase (hereinafter referred to as: FsPDS) gene fragment was connected to the TRV virus-induced gene silencing vector pTRV2 by the method of genetic engineering to obtain the recombinant pTRV- FsPDS. Specific steps are as follows:

[0030] Preparation of cDNA: Extract the total mRNA of Forsythia leaves according to the Trizol method, perform reverse transcription of the total mRNA according to the operation instructions of the Tiangen Reverse Transcription Kit, obtain Forsythia cDNA, dilute 20 times, and store at -20°C for later use.

[0031] Amplification of Forsythia PDS gene fragment (FsPDS):

[0032] PCR primers were design...

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Abstract

The invention belongs to the technical field of genetic engineering, and concretely discloses a method for mediating virus-induced gene silencing by TRV vectors in a forsythia suspensa leaf. The method includes the following steps: transforming the TRV2 vector and the TRV1 vector containing specific target gene fragments into an agrobacterium competent state; performing screening to obtain a positive strain, and performing proliferation culture to obtain two monoclonal cells containing the different vectors; centrifuging the two monoclonal cells, resuspending the two monoclonal cells until OD600 is equal to 0.9-1.1 by using an infection solution, and then mixing the two monoclonal cells in equal volume to obtain a mixed infection solution; and injecting the mixed infection solution into the forsythia suspensa to silence the target genes. The method deeply deeply explores key factors that can improve a success rate of the infection and screens out an optimal infection solution composition formula and an OD value of the infection solution containing the cells during the infection. The method of the invention is used to infect the forsythia suspensa leaf, and so the success rate of the forsythia suspensa leaf VIGS infection can be more than 90%.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for virus-induced gene silencing mediated by TRV vectors of forsythia leaves. Background technique [0002] Virus-induced gene silencing (VIGS) is a type of RNA interference and belongs to post-transcriptional gene silencing. This technology is to synthesize dsRNA under the action of RNA-guided RNA polymerase (RDRP) after the recombinant virus vector carrying the target gene fragment enters the plant nucleus, and Dice enzyme acts on the dsRNA to further make it produce 21-25 nucleotides. The antisense strand of the siRNA is then activated by combining with the RNA-induced silencing complex (RISC), and the activated silencing complex can specifically bind to the single-stranded mRNA of the target gene, resulting in its specificity Degradation, the target gene cannot perform the messenger function to continue to transmit the genetic information of ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00A01H6/00
CPCC12N9/001C12N15/8218C12Y103/99
Inventor 潘会堂申建双司未佳张启翔程堂仁王佳
Owner BEIJING FORESTRY UNIVERSITY
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