Rice stripe virus RNA interference vector, constructing method and application thereof

A rice stripe virus 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: 2012-06-27
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, disease resistance genes are mostly accompanied by inferior genes, and 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 stripe virus RNA interference vector, constructing method and application thereof
  • Rice stripe virus RNA interference vector, constructing method and application thereof
  • Rice stripe virus RNA interference vector, constructing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1, Construction of Rice Stripe Virus RNA Interference Vector Suitable for Agrobacterium-mediated Transformation

[0077] Step 1, design the specific primers of RSV full-length CP gene:

[0078] The following primers were designed according to the reported RSV CP gene sequence (Wei Taiyun, 2003)

[0079] RSV CP1: 5'-AGGTTCAGTCTAGTCATCTGCAC-3'

[0080] RSV CP2: 5'-AGTAGAATGGGTACCAACAAGCCA-3'

[0081]Using the total RNA of rice standard samples (leaves) infected with rice stripe blight collected from Nanjing, Jiangsu as a template, RSVCP1 ((upstream primer)) and RSV CP2 (downstream primer) as primer pairs, amplified by RT-PCR to obtain A specific band of about 1000bp was recovered, and the PCR product was recovered and connected to the pMD18-T vector to obtain the recombinant plasmid pMD18-T-CP, which was identified by PCR and EcoR I digestion, and transformed into Escherichia coli DH5α to obtain a positive clone, and the sequence analysis results It shows that th...

Embodiment 2

[0098] Example 2, Viral RNA interference vector electroporation transformation of Agrobacterium tumefaciens EHA105

[0099] Since the heat shock transformation efficiency of the recombinant plasmid (pCMBIA1301+ / -R) to Agrobacterium EHA105 is very low, the present invention adopts the electric shock transformation method to obtain the recombinant Agrobacterium containing the target gene. Specific steps are as follows:

[0100] Step 1. Mix 1 μg of plasmid (pCMBIA1301+ / -R) 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.

[0101] 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.

[0102] 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.

[0103] Step 4. Pick a single colony and p...

Embodiment 3

[0106] Embodiment 3, application of RSV-HCP interference carrier.

[0107] Apply the above-mentioned recombinant agrobacterium containing the RSV target fragment hairpin structure to transform the relevant recipient plant-Aichi Asahi rice variety, so that it can produce resistance to rice virus disease ( Figure 8-14 ).

[0108] The present invention has obtained rice T0 generation regenerated plants using the rice model variety Aichi Asahi as the recipient, and 173 plants transformed with RSV vector pCMBIA1301+ / -R ( Figure 16 ).

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Abstract

The invention relates to a rice stripe virus RNA interference vector, a constructing method and application thereof, belonging to the technical field of biology. The method establishes the rice stripe virus RNA interference vector pMCG161+ / -R and pCMBIA1301+ / -R by using a sequence shown in SEQ ID NO 3 as sense and antisense strands. The invention provides the vector interfering with virus gene expression and the constructing method and application thereof for genetic engineering of plant disease resistance, and provides a new idea for transgenic technology and application of RNA interference in biological resistance breeding. The vector is transferred into paddy to obtain antivirus plant successfully, the preset target of obtaining the anti RSV paddy new material by using RNA interference principle can be realized, a successful case is provided for RNA interference mediated plant resistance breeding and genetic engineering, and the blank of RNA interference mediated RSV resistance in the research of the field can be filled up.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a rice stripe virus RNA interference vector suitable for genetic transformation of monocot plants, a construction method and its application in rice. 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 30%, and the serious field reaches more than 80%, or even no harvest. The disease broke out successively in 2001, 2004, 2005, 2006 and 2007 in Jiangsu, Anhui, Henan and Shandong. In 2004, the rate of diseased fields in Jiangsu and Henan was more than 75%, and the rate of disea...

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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