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Anti-verticillium wilt protein from sea island cotton as well as coding gene and application thereof

A coding and gene technology, applied in the field of genetic engineering, can solve the problems of disease susceptibility or disease resistance, poor disease resistance of Verticillium wilt, and achieve the effect of improved disease resistance and high application value

Active Publication Date: 2014-10-08
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Upland cotton is characterized by wide adaptability, high yield, long fiber, and good quality, but its resistance to Verticillium dahliae is poor, and most of them are susceptible or disease-resistant varieties at present.

Method used

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  • Anti-verticillium wilt protein from sea island cotton as well as coding gene and application thereof
  • Anti-verticillium wilt protein from sea island cotton as well as coding gene and application thereof
  • Anti-verticillium wilt protein from sea island cotton as well as coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Embodiment 1, the acquisition of GbAt11 gene

[0072] 1. Verticillium dahliae V 991 Induced Sea Island Cotton Leaves

[0073] Verticillium dahliae V 991 Cultivate in Cha's medium for 15 days, filter with gauze, count the filtrate with a counting plate under a microscope, and then adjust the spore concentration to 1×10 6 / mL. Take the young leaves of sea island cotton 7124 and soak them in 1×10 6 / mLV991 spore suspension, record the time when the leaves were soaked, and take out the leaves at 0h, 3h, 6h, 12h and 24h after soaking, wrap them in tinfoil, freeze them with liquid nitrogen, and store them in a -80°C refrigerator stand-by.

[0074] 2. Extraction of sea island cotton RNA

[0075] Use the EASYspin Plant RNA Rapid Extraction Kit to extract sea island cotton RNA:

[0076] (1) Mix 500 μl lysis solution RLT (beta-mercaptoethanol has been added) and 50 μl PLANTaid in a 1.5 ml centrifuge tube.

[0077] (2) Grind the sample in liquid nitrogen, transfer about 50...

Embodiment 2

[0099] Example 2, the acquisition and functional analysis of GbAt11 transgenic cotton

[0100] 1. The acquisition and identification of GbAt11 transgenic cotton

[0101] 1. Acquisition of recombinant expression vector pSPT-02-GbAt11

[0102] Design primers At11-Sal1 and At11-KpnI according to the GbAt11cDNA sequence (sequence 2), the sequences are as follows:

[0103] At11-Sal1: 5'-CG GTC GAC ATGTCGATCG CGTTGGAACG-3' (underlined is the Sal I enzyme recognition site, and the subsequent sequence is the 1st to 20th position of sequence 2);

[0104] At11-KpnI:5'-CG GGTACC GTT ATA TTC ACG TAC ATC AGC C-3' (the underlined mark is the Kpn I enzyme recognition site, and the following sequence is the reverse complementary sequence from the 744th to the 765th position of sequence 2).

[0105] Using the recombinant plasmid pMD18-GbAt11 obtained in Example 1 as a template, PCR amplification was performed with primers At11-Sal1 and At11-KpnI.

[0106] After the reaction, the obtai...

Embodiment 3

[0191] Example 3, Obtaining and Functional Analysis of GbAt11 Transgenic Arabidopsis

[0192] 1. Acquisition and identification of GbAt11 transgenic Arabidopsis

[0193] 1. Acquisition of recombinant expression vector super1300-MYC-GbAt11

[0194] Design primers At11-Sal1 and At11-KpnI according to the GbAt11cDNA sequence (sequence 2), the sequences are as follows:

[0195] At11-Sal1: 5'-CG GTC GAC ATGTCGATCG CGTTGGAACG-3' (underlined is the Sal I enzyme recognition site, and the subsequent sequence is the 1st to 20th position of sequence 2);

[0196] At11-KpnI:5'-CG GGTACC GTT ATA TTC ACG TAC ATC AGC C-3' (the underlined mark is the Kpn I enzyme recognition site, and the following sequence is the reverse complementary sequence from the 744th to the 765th position of sequence 2).

[0197] Using the recombinant plasmid pMD18-GbAt11 obtained in Example 1 as a template, PCR amplification was performed with primers At11-Sal1 and At11-KpnI.

[0198] After the reaction, the...

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Abstract

The invention discloses anti-verticillium wilt protein from sea island cotton as well as a coding gene and application thereof. The protein provided by the invention is following (a) or (b): (a), protein consisting of an amino acid sequence as shown by sequence 1 in a sequence table; (b), protein, obtained by substituting and / or deleting and / or adding with one or more amino acid residues of the amino acid sequence of the protein limited by (a), and associated with plant verticillium wilt disease. Experiment proves that GbAt11 transgenic cotton and GbAt11 transgenic arabidopsis have resistance which is remarkably superior to that of a non-transgenic wild type on verticillium, field verticillium wilt nursery evaluation proves that the anti-verticillium wilt phenotype of most transgenic strains is superior to that of the wild type, and yield is found to be improved after transgenosis at the same time. Therefore, the GbAt11 gene provided by the invention has important action on plant anti-verticillium wilt disease, and also has a very high application value for cultivating a novel plant variety with high verticillium wilt disease resistance.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a Verticillium wilt resistance protein derived from sea-island cotton, its coding gene and its application. Background technique [0002] Cotton belongs to the genus Malvaceae, and there are four main cultivars: Asian cotton (coarse staple cotton), African cotton, upland cotton (fine staple cotton), and Sea Island cotton (long staple cotton). According to the statistics of the International Cotton Council, the total output of upland cotton and sea island cotton accounts for 95% and 2% of the world's cotton output respectively. [0003] Upland cotton is called American cotton because it was first planted in the American continent. It is the most important variety among the four major cotton cultivars in the world. Upland cotton is characterized by wide adaptability, high yield, long fiber and good quality, but its resistance to Verticillium dahliae is poor, and most of them are s...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8282
Inventor 齐俊生巩志忠陈智忠赵佳
Owner CHINA AGRI UNIV
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