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Method for cultivating anti-TYLCV (Tomato Yellow Leaf Curl Virus) tomato plant by using RNAi technology

A kind of tomato technology, applied in the field of breeding TYLCV-resistant tomato, genetic transformation of crops (tomato) and selection of new transgenic varieties

Inactive Publication Date: 2012-07-04
JIANGSU ACAD OF AGRI SCI
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Problems solved by technology

Tomato cultivars resistant to TYLCV can only be obtained by crossing tomato cultivars with wild species, but this requires extensive and long-term breeding and identification work (Ji Y, Scott J, Hanson P, Graham E, Maxwell D. Sources of resistance, inheritance, and location of genetic loci conferring resistance to members of the tomato-infecting begomoviruses. Tomato Yellow Leaf Curl Virus Disease, 2007: 343-362)

Method used

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  • Method for cultivating anti-TYLCV (Tomato Yellow Leaf Curl Virus) tomato plant by using RNAi technology
  • Method for cultivating anti-TYLCV (Tomato Yellow Leaf Curl Virus) tomato plant by using RNAi technology
  • Method for cultivating anti-TYLCV (Tomato Yellow Leaf Curl Virus) tomato plant by using RNAi technology

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Experimental program
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Embodiment Construction

[0046] 1. Construction of V2 and C1 gene RNAi vectors of TYLCV

[0047] 1.1 Extraction of total plant DNA

[0048] Use the CTAB method to extract DNA from tomato plant diseased leaf samples with typical symptoms of TYLCV (curling of top new leaves and ear protrusions). The specific steps are as follows: Take 1g of fresh leaves, grind them into powder with liquid nitrogen, transfer them to a 1.5 ml centrifuge tube, add 600 μl preheated CTAB lysate (Table 1), vortex to mix, 65 ℃ water bath for 1 hour, add 600 μl chloroform-isoamyl alcohol (24:1), invert 50 times to mix, centrifuge at 10000 rpm for 15 minutes, take the supernatant in a new 1.5 ml centrifuge tube, add an equal volume of pre-cooled isopropanol, mix well, place at -20°C for 30 minutes, centrifuge at 12000rpm for 15 minutes, discard the supernatant, add 1ml 75% alcohol to wash the DNA precipitate, discard the alcohol, air dry, add 100μl TE to dissolve DNA was stored at 4°C for later use.

[0049]

[0050] Table 1 ...

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Abstract

The invention relates to a method for cultivating an anti-TYLCV (Tomato Yellow Leaf Curl Virus) tomato plant by using an RNAi (RNA interference) technology, belonging to the field of biotechnology. According to the method, the conserved sequences in genes of V2 and C1 of TYLCV are inosculated, an RNAi carrier with inverted repeat sequences is constructed, the aseptic seedling cotyledons or hypocotyl explants of the tomato are transformed through an agrobacterium-mediated transformation method, the transgenic tomato expresses resistance to the TYLCV after the inoculation of the agrobacterium infectious clone injection of the TYLCV and the TYLCV-carrying Bemisia tabaci virus, and no obvious disease symptoms are observed. According to the method, the anti-TYLCV transgenic tomato plant can be cultivated by using the RNAi technology, so that the technical support for the control of the TYLCV is provided.

Description

Technical field: [0001] The invention relates to a method for cultivating TYLCV-resistant tomato by using RNAi technology, which is specially used for genetic transformation of crops (tomato) and selection of new transgenic varieties, and belongs to the field of biotechnology. [0002] Background technique: [0003] Tomato Yellow Leaf Curl Virus (TYLCV) belongs to the genus Begomovirus of the family Geminiviridae. Most Begomoviruses contain 2 closed circular single-stranded genomes with a size of 2.5-3.0Kb, namely DNA-A and DNA-B components; a few contain only one DNA-A component, and their genome structure is equivalent to that of two-component viruses DNA-A. DNA-A encodes 6 ORFs, which are the V1 gene (encoding capsid protein, CP) located on the viral chain, the V2 gene (encoding movement protein, MP) and the C1 gene located on the complementary chain (encoding Replication initiator protein, Rep), C2 gene (encodes transcriptional activator protein, TrAP), C3 gene (enc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 陈天子余文贵张保龙朱成松赵统敏杨郁文
Owner JIANGSU ACAD OF AGRI SCI
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