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RNA (Ribose Nucleic Acid) with high TuMV (Turnip Mosaic Virus) resistance and RNAi (RNA interfere) vector for coding same

A technology for expressing vectors and encoding genes, which is applied in the field of RNA and RNAi vectors encoding the RNA, and can solve problems such as ineffective effects and environmental pollution

Inactive Publication Date: 2013-12-25
BEIJING AGRO BIOTECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is impossible to kill all aphids quickly to prevent the spread of the virus by using insecticides. The effect of killing aphids with insecticides is not obvious, and it is easy to cause environmental pollution.

Method used

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  • RNA (Ribose Nucleic Acid) with high TuMV (Turnip Mosaic Virus) resistance and RNAi (RNA interfere) vector for coding same
  • RNA (Ribose Nucleic Acid) with high TuMV (Turnip Mosaic Virus) resistance and RNAi (RNA interfere) vector for coding same
  • RNA (Ribose Nucleic Acid) with high TuMV (Turnip Mosaic Virus) resistance and RNAi (RNA interfere) vector for coding same

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1, the preparation of RNAi carrier

[0057] (1) Preparation of CI Gene Conserved Fragment

[0058] According to the conservation of 118 TuMV strains registered in GenBank, the 882-1247th nucleotide sequence of CI gene with a total of 366 bp conserved fragment was selected as the RNA interference target.

[0059] Extract the total RNA of the TuMV-C4 strain, and reverse transcribe it into cDNA, use the cDNA as a template, and use the following CI366F and CI366R (the underlined parts are restriction sites Xho I, BamH I, EcoR I, Hind III) as primers, PCR amplification with high-fidelity enzyme Fastpfu DNA Polymerase:

[0060] CI366 F 5'-cc ctcgagggatcc TGGAACACCTAGCAAGAAGCACT-3';

[0061] CI366 R 5'-cg gaattcaagctt CGTGCCTGCTTTGCCGTTAC-3'.

[0062] PCR amplification program: 94°C for 5min; 30 cycles of 94°C for 30s, 56°C for 30s, 72°C for 1min; 72°C for 7min.

[0063] The PCR product was purified by agarose electrophoresis and recovered. The obtained PCR...

Embodiment 2

[0079] Example 2, RNA with high resistance to TuMV and functional verification of the RNAi vector encoding the RNA

[0080] (1) Genetic transformation of Arabidopsis and screening of positive seedlings

[0081] The pBBBTu-CI vector was transformed into Agrobacterium GV3101 (pMP90) by electric shock method, and Arabidopsis was infected by Agrobacterium-mediated inflorescence dipping method. Arabidopsis thaliana to be harvested 0 The generation seeds were sown in culture trays filled with nutrient soil, and placed in an artificial climate chamber at 22°C (day) / 18°C (night) with a photoperiod of 16h (light) / 8h (dark). After the seedlings grow two cotyledons, spray water to dilute the glufosinate-ammonium herbicide 500 times by volume (the glufosinate-ammonium herbicide is a commercial herbicide purchased in the market, and the concentration of glufosinate-ammonium is 200mg / L) for screening Positive seedlings.

[0082] After being sprayed 2-3 times with glufosinate-ammonium her...

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Abstract

The invention discloses an RNA (Ribose Nucleic Acid) with high TuMV (Turnip Mosaic Virus) resistance and an RNAi (RNA interfere) vector for coding the same. The RNA with high TuMV resistance has one of the following nucleotide sequences: 1) a nucleotide sequence as shown in SEQ ID No.2 in a sequence table; and 2) RNA sequences having more than 90% homology with the RNA sequence defined by the item 1), specifically more than 95% homology, more specifically more than 96% homology, further more specifically more than 97% homology, further more specifically more than 98% homology and the most specifically more than 99% homology, and having the same functions. The RNA and the RNAi vector for coding the RNA disclosed by the invention can be used for enhancing the resistance of plants to TuMV or glufosinate-ammonium herbicides, and simultaneously, also lay a foundation for breeding high-TuMV-resistance transgenic plants.

Description

technical field [0001] The invention belongs to the field of plant anti-virus genetic engineering, and in particular relates to an RNA highly resistant to TuMV and an RNAi carrier encoding the RNA. Background technique [0002] Turnip Mosaic Virus (TuMV) belongs to Potyviridae and Potyvirus. TuMV has a wide host range and can infect 318 species of plants in 43 families and 156 genera. TuMV is the most serious virus that harms cruciferous crops. Crops infected with the virus are also susceptible to downy mildew and soft rot, causing compound infections and directly affecting yield and commodity value. [0003] Rapeseed is the main oil crop in my country, and turnip mosaic virus is one of the important diseases of rapeseed. In the middle and lower reaches of the Yangtze River in my country, the production of rapeseed due to viral diseases generally reaches 20-30%, and it reaches about 50% in a pandemic year. Infected rapeseed not only reduced yield, but also significantly ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/82C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 姚磊叶艳英曾钢曹鸣庆马荣才
Owner BEIJING AGRO BIOTECH RES CENT
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