Rice black-streaked dwarf virus (RBSDV) RNA interference vectors, construction method and application

A rice black bar dwarfing and RNA interference technology, applied in the field of genetic engineering, can solve problems such as difficulty and lack of resistance sources

Inactive Publication Date: 2011-02-09
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the lack of ideal resistance sources in traditional breeding methods (the germplasm resources of resistance to black-streaked dwarf virus have not been found in China), it is very difficult to obtain varieties with dual characteristics of disease resistance, high yield and high quality.

Method used

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  • Rice black-streaked dwarf virus (RBSDV) RNA interference vectors, construction method and application
  • Rice black-streaked dwarf virus (RBSDV) RNA interference vectors, construction method and application
  • Rice black-streaked dwarf virus (RBSDV) RNA interference vectors, construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1. Construction of rice black streaked dwarf virus RNA interference vector suitable for Agrobacterium-mediated transformation

[0075] Step 1. Design RNA interference primers for RBSDV S10 fragment

[0076] According to the published sequence of the RBSDV S10 gene (NCBI No.AJ297433) and the presence of multiple cloning sites on the pMCG161 vector, AscI and AvrII can be used to ligate the forward fragment of the target gene to the vector; while SpeI and SacI can be used to The reverse fragment of the S10 partial nucleotide fragment is ligated to the vector. Therefore, the following interference primers are designed:

[0077] SpeI AscI

[0078] SEQ ID NO1: 5'-AG ACTAGT GGCGCGCC TAATGGCTGACATAAGACTC-3’

[0079] SacI AvrII

[0080] SEQ ID NO2: 5’AG GAGCTC CCTAGGG TTGCGTGATGTTGGGTAAAG-3’

[0081] Step 2: Obtain the specific nucleotide sequence of RBSDV S10 partial fragment

[0082] Using the total RNA of rice infected with rice black streaked dwarf virus (RBSDV) collected f...

Embodiment 2

[0091] Example 2: Transformation of Agrobacterium tumefaciens EHA105 by electroporation with viral RNA interference vector

[0092] Since the efficiency of heat shock transformation of the recombinant plasmid pCMBIA1301+ / -B to Agrobacterium EHA105 is very low, the present invention adopts the method of electric shock transformation to obtain the recombinant Agrobacterium containing the target gene. Specific steps are as follows:

[0093] Step 1. Mix 1 μg plasmid (pCMBIA1301+ / -B) with 150 μl Agrobacterium EHA105 competent, add them to the sterilized electric shock cup, and perform electric shock transformation at 2.5KV.

[0094] Step 2. Then add 800μl of liquid culture medium to rinse the electric shock cup and transfer it to EP tube, shake culture at 28°C, 220rpm for 2h.

[0095] Step 3. Take 100-200μl culture medium and apply YM (Kana: 50μg / ml; rifampicin 50μg / ml) plate, and incubate at 28°C for about 18 hours.

[0096] Step 4. Pick a single colony in 5ml YM liquid medium (Kana: 50μg / ...

Embodiment 3

[0099] Example 3 Application of the RNA interference vector of RBSDV-HS10.

[0100] The above-mentioned recombinant Agrobacterium containing the hairpin structure of the target fragment of RBSDV was used to transform the relevant recipient plant-Aichi Asahi rice variety to make it resistant to rice black streaked dwarf virus virus ( Figure 8-14 ).

[0101] The present invention has obtained rice T0 regeneration plants with rice model Aichixu as the recipient, and the number of plants transformed into RBSDV vector pCMBIA1301+ / -B is 122 plants ( Figure 16 ).

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Abstract

The invention relates to rice black-streaked dwarf virus (RBSDV) RNA interference vectors, a construction method and application, and belongs to the technical field of biology. RBSDV RNA interference vectors, namely pMCG161+ / -B and pCMBIA1301+ / -B, are constructed by using a sequence shown in SEQ ID NO:3 as positive-sense and antisense strands. The invention provides a virus gene interference expression vector and a construction method and application thereof for plant disease-resistant genetic engineering, and provides a novel way of thinking for application of transgenic technology and RNA interference in biological resistance breeding. By transferring the vector into rice, antiviral plants are successfully obtained, the expected goal for obtaining new anti-RBSDV materials of rice is achieved by using an RNA interference principle, successful examples for RNA interference-mediated plant resistance breeding and genetic engineering are provided and a gap of RNA interference-mediated RBSDV resistance in the research of the field is filled.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a rice black streaked dwarf virus RNA interference vector suitable for genetic transformation of monocots, a construction method and its application to rice. Background technique [0002] Rice is one of the most important food crops in the world. Rice accounts for 40% of food production and consumption in my country, and it plays an important role in food security and national economy. Since 2000, rice black streaked dwarf virus (RBSDV) caused by rice black streaked dwarf virus (RBSDV) has re-emerged after many years of silence in rice regions such as Jiangsu, Zhejiang, Fujian, and Jiangxi. Approximately 60% of the paddy fields are affected. The incidence rate of plants in general fields is 10-30%, and in severe fields, it can be as high as 40-80%. In 2004, there were 800,000 mu of diseased area in the hybrid rice planting area in south-central Zhejiang Provinc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C12N15/113C12N15/63C12N15/66
Inventor 李莉李红伟王锡锋刘艳吴蓓蕾
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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