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Plant antiviral gene engineering method mediated by dsRNA

A genetic engineering and anti-virus technology, applied in the field of genetic engineering, can solve problems such as difficult to obtain plants, low frequency of resistance of transgenic plants, and difficulty in achieving immunity

Inactive Publication Date: 2003-08-20
ZHEJIANG UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

The obtained transgenic plants have achieved certain effects in preventing or reducing virus infection, but unfortunately it is difficult to obtain plants that are immune to related viruses
[0005] The existing anti-viral genetic engineering methods in my country are still in the stage of anti-viral genetic engineering mediated by traditional viral gene translation products or non-translated sequences. The resistance frequency of the obtained transgenic plants is not high, and it is difficult to achieve immunity.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Method of ToMV MP full-length dsRNA-mediated plant anti-virus genetic engineering 1 Construction of ToMV MP full-length gene dsRNA plant expression vector pBIN438 1.1 RT-PCR amplification of ToMV MP full-length gene and sequence determination

[0022] Design specific primers according to the sequence of the ToMV MP gene (Accession No. AJ006991), its sequence is: 5' primer: 5' CC GGATCCA TGGCTCTAGTTGTTAAAG-3' (underlined BamH I restriction site) 3' primer: 5'-TTAATACGAATCAGAATCCGCG-3'

[0023] Immunocapture PCR (IC-PCR) method was used to amplify the gene, and the operation steps were as follows: Add 200 μL of ToMV antiserum diluted 200 times (diluted with ELISA coating buffer) to a 0.5ml PCR tube, and place it at 37°C for 2 hours , pour out the coating solution, wash with PBST 3 times, each time for 3 minutes, add 200 μL of ToMV diseased leaf grinding juice and centrifuge supernatant (0.2g fresh leaves + 2ml diseased leaf grinding, 3000rpm centrifugation for 2...

Embodiment 2

[0065] In order to analyze the expression of the target gene at the transcriptional level in transgenic tobacco, five transgenic tobacco plants that were positive in PCR and Southern hybridization and one non-transgenic common tobacco were randomly selected as controls, and [α- 32 P]dCTP-labeled ToMV MP gene was used as a probe, and Northern blot analysis was performed according to conventional methods. The results showed that the five transgenic plants had specific RNA hybridization bands, indicating that the target genes of the five transgenic plants had been normally transcribed, while there was no specific transcription product in the non-transgenic tobacco control. Example 2 Method of CMV-Fny partial Rep gene DsRNA-mediated plant anti-virus genetic engineering 1 Construction of CMV-Fny partial Rep gene HairpinRNA plant expression vector pBIN438 1.1 RT-PCR amplification of CMV-Fny partial Rep gene and sequence determination

[0066] Design specific primers according to the...

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Abstract

The present invention relates to one kind of dsRNA mediated plant antiviral gene engineering method. The cDNA of whole length of partial nucleotide sequence of some gene in target virus genome is first obtained via RT-PCR and inserted to two ends of pBlue SK containing intron to constitute the reverse duplicated gene segment; and the reverse duplicated gene segment containing intron is then inserted to plant expression vector pBIN438 and the recombinant vector is introduced into agrobacterium strain via triparental cross process to obtain agrobacterium mediated plant antiviral vector. Via agrobacterium mediated process, the vector is transferred to tobacco leaf. The donor virus and relevant virus resistance test on the obtained transgenic plant shows that the present invention makes it possible to obtain corresponding plant virus immunizing plant through the transferring.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular, the invention relates to a method of plant anti-virus genetic engineering, more specifically, the present invention relates to a method of dsRNA-mediated plant anti-virus genetic engineering. Background technique [0002] As a kind of main pathogens infecting plants, plant viruses cause diseases of crops, fruit trees, flowers, pastures, and medicinal plants all over the world, resulting in a decline in yield and quality, and even destructive damage. According to incomplete statistics, the annual output loss caused by plant virus diseases in the world accounts for about 10% of the total grain output. Effectively controlling the harm of viruses is an urgent task for agricultural production. [0003] To date, there are few chemical agents that are effective in removing viruses from virus-infected plants. Some chemical agents (such as purine and pyrimidine analogues) can sl...

Claims

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Application Information

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IPC IPC(8): A01H1/00C12N15/63C12N15/70C12N15/74C12N15/82
Inventor 周雪平牛颜冰陶小荣张凯李桂新吴建祥
Owner ZHEJIANG UNIV
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