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RNA (Ribonucleic Acid) interference carriers of RSV (Rice Stripe Virus) and RBSDV (Rice Black-Streaked Dwarf Virus) as well as construction method and application thereof

A technology of RNA interference and interference vector, applied in the field of genetic engineering, can solve the problems of difficulty and lack of anti-origin

Inactive Publication Date: 2012-05-23
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 an ideal source of resistance in the traditional breeding method (the germplasm resources of resistance to black-streaked dwarf virus have not been found in China), and the disease-resistant genes are mostly accompanied by inferior genes, it is difficult to obtain varieties with dual characteristics of disease resistance, high yield and high quality. very big

Method used

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  • RNA (Ribonucleic Acid) interference carriers of RSV (Rice Stripe Virus) and RBSDV (Rice Black-Streaked Dwarf Virus) as well as construction method and application thereof
  • RNA (Ribonucleic Acid) interference carriers of RSV (Rice Stripe Virus) and RBSDV (Rice Black-Streaked Dwarf Virus) as well as construction method and application thereof
  • RNA (Ribonucleic Acid) interference carriers of RSV (Rice Stripe Virus) and RBSDV (Rice Black-Streaked Dwarf Virus) as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1, construction of bivalent marker-free gene RNA interference vector against RSV and RBSDV

[0084] Step 1: Design overlapping primers for RSV-HCP and RBSDV-HS10

[0085] According to the published nucleotide sequences of RSV CP gene and RBSDV S10, overlapping PCR primers SEQ ID NO 3 and SEQ ID NO 4 were designed.

[0086] SEQ ID NO 3: 5'-AGTCTTATGTCAGCCATTAACAGCCATCTTAACACCCAG-3'

[0087] SEQ ID NO 4: 5'-CTGGTGTTA AGATGGCTGTTAATGGCTGACATAAGACTC-3'

[0088] Step 2: Obtaining of tandem RSV and RBSDV-specific nucleotide sequences

[0089] Using the total RNA of rice infected with rice stripe virus (RSV) collected from Nanjing, Jiangsu as a template, and using SEQ ID NO.2 (upstream primer) and SEQ ID NO.3 (downstream primer) as a primer pair, RSV was amplified by RT-PCR -HCP, to obtain PCR product I, the target fragment is about 250bp. The PCR product I was recovered, ligated with the pMD18-T vector, transformed into Escherichia coli DH 5α to obtain the positiv...

Embodiment 2

[0102] Example 2, Viral RNA interference vector electroporation transformation of Agrobacterium tumefaciens EHA105

[0103] Since the efficiency of heat shock transformation of the recombinant plasmid pCMBIA1301+ / -D 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:

[0104] Step 1. Mix 1 μg of plasmid (pCMBIA1301+ / -D) and 150 μl of Agrobacterium EHA105 competently, add them into a sterilized electric shock cup, and perform electric shock transformation at a voltage of 2.5KV.

[0105] Step 2, then add 800 μl of liquid medium to rinse the electric shock cup and transfer to EP tube, shake at 28°C and 220rpm for 2 hours.

[0106] Step 3. Take 100-200 μl of the culture solution and apply it on a YM (Kana: 50 μg / ml; rifampicin 50 μg / ml) plate, and incubate at 28° C. for about 18 hours.

[0107] Step 4. Pick a single colony ...

Embodiment 3

[0110] Example 3. Application of virus gene interference virus gene expression vector.

[0111] Apply the above-mentioned recombinant Agrobacterium containing the purpose structure to transform the relevant recipient plant-Aichi Asahi rice variety, so that it can produce resistance to rice virus disease ( Figure 10-16 ).

[0112] The present invention has obtained rice T0 regenerated plants using the rice model variety Aichi Asahi as the recipient, among which 267 plants were transformed with the bivalent vector pCMBIA1301+ / -D, and were screened for disease resistance in the field in Zhengzhou, Henan Province, an area where viral diseases frequently occur Evaluation( Figure 18 ).

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Abstract

The invention relates to RNA (Ribonucleic Acid) interference carriers of RSV (Rice Stripe Virus) and RBSDV (Rice Black-Streaked Dwarf Virus) as well as a construction method and application thereof, belonging to the technical field of biology. The invention establishes RNA interference carriers pMCG161+ / -D and pCMBIA1301+ / -D of the RSV by using sequences shown as SEQ ID NO1. The invention provides an interference virus gene expression carrier for plant disease-resistant gene engineering as well as a construction method and application thereof and provides a new concept for the application of a transgenic technology and RNA interference in biological resistant breeding. By transplanting the carrier to rice, a virus-resistant plant is successfully acquired, the expected target of obtaining a RSV and RBSDV-resistant rice new material according to the principle of RNA interference is realized, a successful example is provided for plant resistant breeding and gene engineering of RNA interference mediums and the blank of researches on the RSV and RBSDV resistance of the RNA interference mediums in the field is made up.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to the construction of an RNA interference vector suitable for genetic transformation of monocot plants and resistant to two viral diseases and its application in rice. It further relates to the expression vector of rice stripe virus (RSV) coat protein (CP) gene and rice black-streaked dwarf virus (RBSDV) S10 partial nucleotide sequence, and its genetic transformation of gramineous rice and obtaining resistant plants method. Background technique [0002] Rice is one of the most important food crops in the world. In my country, rice accounts for 40% of food production and consumption, and plays a pivotal role in food security and national economy. Rice stripe blight caused by rice stripe virus (RSV) is the most important rice virus disease in japonica rice growing areas in my country. In the popular year, the yield of the general field is reduced by 20% to 3...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/63C12N15/113C12N15/66A01H5/00
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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