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Method for quickly identifying chrysanthemum nankingense gene function based on TRV-VIGS technology

A technology of gene function and chrysanthemum brain, applied in the field of genetic engineering, can solve problems that have not been reported, and achieve the effect of low cost, easy operation and realization

Inactive Publication Date: 2017-02-08
NANJING AGRICULTURAL UNIVERSITY
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  • Application Information

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Problems solved by technology

It has been confirmed that the TRV virus vector can be used in Solanaceae plants, such as tobacco, tomato, petunia, and non-Solanaceae Arabidopsis thaliana (Gao et al, 2008). The study of brain gene function has not been reported

Method used

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  • Method for quickly identifying chrysanthemum nankingense gene function based on TRV-VIGS technology
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  • Method for quickly identifying chrysanthemum nankingense gene function based on TRV-VIGS technology

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Embodiment 1

[0059] 1. Cloning of CnPDS gene fragment

[0060] With chrysanthemum brain as material, take 0.3g of leaves, extract the total RNA of leaves with reference to the operation method of the Trizol RNA extraction kit (TaKaRa), and obtain cDNA by reverse transcription according to the M-MLV reverse transcription kit (TaKaRa). For the sequence information of the gene in the chrysanthemum brain library, use primer 5 software to design specific primers to amplify CnPDS;

[0061] Upstream primer CnPDS-EcoRI-F: 5'-TACGTGAATTCTTGCACCAGCAGAAGAAT-3' (SEQ ID NO.2),

[0062] Downstream primer CnPDS-KpnI-R: 5'-TCAGGTACCAGACTCGCCTCAGCAATCAC-3' (SEQ ID NO.3);

[0063] Using leaf cDNA as template, carry out PCR reaction, 50 μL reaction system: 10×PCR Buffer 5.0 μL, CnPDS-EcoRI-F, CnPDS-KpnI-R primers 1.0 μL each (20 μmol L -1 ), dNTP 4.0μL (2.5mmol L -1 ), Pfu DNA Polymerase 0.2 μL, cDNA template 1 μL, ddH 2 O 37.8 μL; reaction program: pre-denaturation at 95°C for 5 min, then melting at 94°...

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Abstract

The invention belongs to the technical field of plant genetic engineering and discloses a method for quickly identifying the chrysanthemum nankingense gene function based on TRV-VIGS technology. TRV2 virus silent expression carrier plasmid containing an endogenous target gene segment is established, the chrysanthemum nankingense is converted instantaneously by a blade injection method, and the silencing of the endogenous target gene of chrysanthemum nankingense is induced so as to identify the function of the endogenous target gene. According to the invention, a system of silencing the endogenous gene of chrysanthemum nankingense by TRV-VIGS so as to identify the gene function is established; and the method has the advantages of high speed, high throughput, easiness in operation and the like and provides a new way of carrying out chrysanthemum nankingense gene function study.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for quickly identifying the function of chrysanthemum brain genes based on TRV-VIGS technology. Background technique [0002] Chrysanthemum (Chrysanthemum morifolium) is native to China. It is one of the top ten traditional famous flowers in my country and one of the four major cut flowers in the world. It has high ornamental and economic value. However, chrysanthemum is an allohexaploid, with a large and highly heterozygous genome, lack of genome information, and the constant genetic transformation mediated by Agrobacterium is highly dependent on genotype and the efficiency of genetic transformation is low, which seriously restricts the process of gene function identification in chrysanthemum. . Chrysanthemum nankingense (Chrysanthemum nankingense) is a diploid wild species closely related to Chrysanthemum. Previous studies have shown that it is...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/83C12N1/21C12Q1/68
CPCC12N9/001C12N15/8203C12N15/8218C12Q1/6895
Inventor 陈素梅王银杰张倩丽陈发棣蒋甲福王海滨宋爱萍
Owner NANJING AGRICULTURAL UNIVERSITY
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