Method for constructing gene of raining dual resistances of potato on PVX virus and PVY virus

A potato and double-resistance technology, applied in botany equipment and methods, biochemical equipment and methods, genetic engineering, etc., to achieve high double-resistant plant rate, high resistance performance, and solve the effect of shrinking flower leaf type degradation

Inactive Publication Date: 2007-12-26
山西省农业科学院作物遗传研究所
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Problems solved by technology

[0005] The present invention provides a method for constructing a gene for improving the double resistance of potatoes to PVX virus and

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  • Method for constructing gene of raining dual resistances of potato on PVX virus and PVY virus
  • Method for constructing gene of raining dual resistances of potato on PVX virus and PVY virus

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[0021] Step 1: Obtaining the full sequence cp of the PVX coat protein gene and the full sequence NIb of the PVY replicase gene

[0022] 1. Acquisition of the complete sequence cp of the PVX coat protein gene

[0023] 1) According to the nucleotide sequence of the PVX coat protein gene complete sequence cp (accession: AF528555),

[0024] Design PCR primers:

[0025] Forward primer: 5'-ATGTCAGCACCAGCTAGCAC-3';

[0026] Reverse primer: 5'-TTATGGTGGTGGTAGAGTGA-3'.

[0027] Primers were synthesized by Sanbo Company.

[0028] 2) Synthesis of the first strand of the full-length DNA sequence of the PVX coat protein gene cp: extract the total RNA of tobacco leaves infected with PVX virus, take 2 μL, then add 2 μL of 10 μM reverse primer, 2 μL of 10 mM dNTP, ddH 2 O 12 μL. 65 ℃ water bath for 5 minutes, ice bath for 2 minutes. Then add the following components to the tube: 5 μL of 5×RT buffer, 1 μL (5u) of RNasin, 1 μL (100u) of M-MLVRT enzyme, mix well, and proceed to the reactio...

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Abstract

This invention relates to a method for constructing fusion gene for improving both PVX resistance and PVY resistance of potato. The method comprises: (1) extracting total RNA from PVX-infected and PVY-infected plants, and performing RT-PCR to obtain coat protein (cp) gene of PVX, and replicase gene Nib of PVY; (2) performing PCR to amplify conserved sequences of cp gene and Nib gene; (3) digesting the conserved sequences, and ligating to obtain fusion gene cn; (4) constructing inverted repeat sequence with an intron, a forward cn gene sequence and a backward cn gene sequence to obtain double-resistance gene RNAiCN; (5) connecting the flanking sequences of RNAiCN with promoter and terminator. When the fusion gene is transferred into potato, both PVX resistance and PVY resistance of the potato are improved.

Description

technical field [0001] The invention relates to a method for constructing a gene, in particular to a method for constructing a gene for improving the double resistance of potatoes to PVX virus and PVY virus. Background technique [0002] Potato is an important food and vegetable crop. During the cultivation process, due to the invasion of viruses, the normal physiological functions of the plants are destroyed, resulting in plant growth decline, plant dwarfing, leaf shrinkage, yellow and green mosaic spots on the leaves, and even Leaf veins are necrotic, the entire compound leaf falls off, and the resulting potato pieces are small and poor in character, which seriously affects the yield and quality. This is the phenomenon of potato degeneration that is usually said. There are more than 30 types of viruses that can infect potatoes, and 16 are named after potatoes (Xie Lianhui et al., 1999). In my country, the main viruses infecting potato are potato virus X (PVX), potato viru...

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Application Information

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IPC IPC(8): C12N15/29C12N15/62
Inventor 白云凤张维锋王国英
Owner 山西省农业科学院作物遗传研究所
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