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TuMV (Turnip Mosaic Virus) high-resistance RNA (Ribonucleic Acid) and an RNAi carrier for encoding RNA

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: 2013-12-18
BEIJING AGRO BIOTECH RES CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] TuMV is mainly transmitted by aphids in a non-persistent manner. It is reported that at least 89 species of aphids can be transmitted, and it is impossible to kill all aphids quickly to prevent the spread of the virus by using insecticides. The effect of aphids on preventing and controlling viral diseases is not obvious, and it is easy to cause environmental pollution

Method used

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  • TuMV (Turnip Mosaic Virus) high-resistance RNA (Ribonucleic Acid) and an RNAi carrier for encoding RNA
  • TuMV (Turnip Mosaic Virus) high-resistance RNA (Ribonucleic Acid) and an RNAi carrier for encoding RNA
  • TuMV (Turnip Mosaic Virus) high-resistance RNA (Ribonucleic Acid) and an RNAi carrier for encoding RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1, the preparation of RNAi carrier

[0051] (1) Preparation of HC-Pro gene conservative fragment

[0052] 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 HC-ProF and HC-Pro R (the underlined part is the enzyme cutting site EcoRI, XbaI, KpnI, ClaI) as primers , PCR amplification with high-fidelity enzyme Fastpfu DNA Polymerase:

[0053] HC-Pro F 5'-cg gaattctctaga GTTAGCAAGTTACAGGGTGAC-3';

[0054] HC-Pro R 5'-gg ggtaccatcgat GACGTGATATCCAGTTGACAGT-3'.

[0055] PCR amplification program: 94°C for 5min; 35 cycles of 94°C for 45s, 52°C for 30s, 72°C for 1min; 72°C for 7min.

[0056] The PCR product was purified by agarose electrophoresis and recovered. The obtained PCR product was sequenced to prove that the nucleic acid sequence was correct, and its nucleic acid sequence had the nucleic acid sequence shown in SEQ ID No. 1 in the sequence listing, and its encoded RNA had t...

Embodiment 2

[0072] Example 2, RNA with high resistance to TuMV and functional verification of the RNAi vector encoding the RNA

[0073] (1) Genetic transformation of Arabidopsis and screening of positive seedlings

[0074] The pBBBTu-HC-Pro 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 the glufosinate-ammonium herbicide diluted 500 times by volume with water (the glufosinate-ammonium herbicide is a commercial herbicide purchased in the market, and the concentration of glufosinate-ammonium is 200mg / L) , Screen positive seedlings.

[0075] After spraying 2-3 times of glufosinate-ammonium herbicid...

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Abstract

The invention claims a TuMV (Turnip Mosaic Virus) high-resistance RNA (Ribonucleic Acid) and an RNAi carrier for encoding the RNA. The TuMV high-resistance RNA consists of one of the following nucleotide sequences: 1) nucleotide sequence indicated by SEQ ID No.2 in a sequence table; 2) limited RNA sequence with more than 90% of homology and same functions, wherein concretely the homology is more than 95%, then concretely more than 96%, then concretely more than 97%, then concretely more than 98% and then concretely more than 99%. The RNA and the RNAi carrier for encoding the RNA are used for enhancing the resistance of the plant to TuMV or glufosinate-ammonium and meanwhile can be used for laying a foundation for cultivating the TuMV high-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 the family Potyviridae and the genus Potyvirus. The host range of TuMV is very wide, and it can infect more than 318 species of plants in 43 families and 156 genera. TuMV is the most harmful viral disease in 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 most important oil crop in my country. Turnip mosaic virus is one of the important diseases of rapeseed. In recent years, the occurrence of viral diseases in the main rapeseed producing areas in my country has become increasingly popular and aggravated. According to the survey, the incidence rate of diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/40C12N15/63C12N5/10C12N1/15C12N1/19C12N1/21A01H5/00
Inventor 姚磊叶艳英曾钢曹鸣庆马荣才
Owner BEIJING AGRO BIOTECH RES CENT
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