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Method for cultivating plant capable of preventing rice black-streaked dwarf virus infestation

A technology of plants and fragments, applied in botany equipment and methods, biochemical equipment and methods, plant gene improvement, etc., can solve the problems of aggravation of rice and corn diseases, achieve broad prospects, specific targets, and no disease resistance effect Ecological Risk Effects

Inactive Publication Date: 2013-02-06
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to changes in climate and farming systems in recent years, rice and corn diseases caused by rice black-streaked dwarf virus have increased significantly, and the incidence rate in some rice-producing areas can reach 90%.

Method used

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  • Method for cultivating plant capable of preventing rice black-streaked dwarf virus infestation
  • Method for cultivating plant capable of preventing rice black-streaked dwarf virus infestation
  • Method for cultivating plant capable of preventing rice black-streaked dwarf virus infestation

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1, the discovery of target sequence (S7-1-Hp)

[0044] 1. Extraction of double-stranded RNA

[0045] Take 0.3g of rice (Huajing No. 6) leaves infected with rice black-streaked dwarfvirus (Rice black-streaked dwarfvirus), add liquid nitrogen and grind it into powder, put it into a 2mL centrifuge tube; add 700μL 2×STE buffer, 100μL SDS solution (10%), 900 μL of water-saturated phenol, vortexed for 30 minutes; the vortexed sample was centrifuged at 10000 g for 15 minutes, the supernatant was taken, and ethanol was added to a final concentration of 16.5%, then 3% CF11 cellulose powder was added and shaken for 30 minutes Minutes; centrifuge at 12000g for 3-5 minutes at 4°C, discard the supernatant, add 0.5ml of 1×STE buffer solution containing 16.5% ethanol to the pellet, shake for 5 minutes, then centrifuge at 12000g for 3-5 minutes at 4°C, discard the supernatant Wash the precipitate three times in this way; add 0.25mL 1×STE buffer to the thoroughly rinsed prec...

Embodiment 2

[0057] Embodiment 2, cultivating resistant rice black-streaked dwarf virus-infected plants

[0058] 1. Construction of recombinant plasmid pSK-hairpin (hairpin intermediate vector)

[0059] 1. Synthesize the double-stranded DNA (S7-1-Hp) shown in sequence 2 of the sequence listing.

[0060]2. Using the double-stranded DNA shown in Sequence 2 as a template, perform PCR amplification with a primer pair consisting of Hp left F (701) and Hp left R (702), to obtain PCR amplification product A.

[0061] Hp left F(701): 5'- GTC GAC ATGGATAGACCTGCTCGAGAAC-3';

[0062] Hp left R (702): 5'-gccg AAGCTT TTCTCAACGTCATTTTTGATTAC-3'.

[0063] The Sal I recognition sequence is underlined in Hp left F(701). In Hp left R (702), the HindIII recognition sequence is underlined.

[0064] 3. Using the double-stranded DNA shown in Sequence 2 as a template, perform PCR amplification with a primer pair consisting of Hp right F (703) and Hp right R (704), to obtain PCR amplification product B. ...

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Abstract

The invention discloses a method for cultivating a plant capable of preventing a rice black-streaked dwarf virus infestation. The invention provides a double-strand DNA segment which is a gene segment shown as a formula (I). A forward sequence(SEQ) is shown as a sequence 2 in a sequence table and a reverse sequence(SEQ) is reversely complemented with the forward sequence(SEQ). X is a spacer sequence between the forward sequence(SEQ) and the reverse sequence(SEQ), and X is neither complemented with the forward sequence(SEQ) nor complemented with the reverse sequence(SEQ). The double-strand DNA segment is introduced into a targeted plant so that a transgenic plant having stronger capability to prevent the rice black-streaked dwarf virus infestation than the targeted plant can be obtained. According to the invention, a resistance material can be obtained within a short period of time. Meanwhile, a gene silencing strategy is employed so that targeting is more focused and disease resistance effect is good with no ecological risks. The method of the invention provides an effective way for prevention of the rice black-streaked dwarf virus and has a promising prospect with regard to prevention of serious diseases. The forward sequence(SEQ)-X-the reverse sequence(SEQ) (I).

Description

technical field [0001] The invention relates to a method for cultivating plants resistant to rice black-streaked dwarf virus infection. Background technique [0002] Rice black-streaked dwarfvirus (RBSDV) can infect wheat, rice, corn and other gramineous plants through the spread of the vector insect SBPH, especially rice and corn. According to previous crop disease history data, rice black-streaked dwarf virus is not a major disease that causes economic losses of major crops. However, due to changes in climate and farming systems in recent years, the rice and corn diseases caused by rice black-streaked dwarf virus have obviously increased, and the disease incidence rate can reach 90% in some rice-producing areas. The production experience shows that indica rice varieties show certain resistance, which varies greatly with different varieties, while japonica rice is generally susceptible, and generally speaking, resistant varieties are scarce. [0003] Current studies have ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/63C12N5/10C12N15/83C12N15/33
Inventor 范在丰贾蒙骜李永强雷蕾
Owner CHINA AGRI UNIV
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