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Cotton GbVe gene, protein coded thereby and use thereof in vegetable verticillium wilt resistance

A gene coding and protein technology, applied in the field of genetic engineering, to achieve the effect of improving the resistance of Verticillium wilt

Inactive Publication Date: 2010-12-29
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the research work on cotton Verticillium wilt resistance genes has been carried out. Most of the genes screened for transformation are antifungal genes with broad-spectrum resistance, and there are few genes specifically resistant to Verticillium dahliae. There is no related report about Verticillium dahliae resistance gene Ve in cotton

Method used

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  • Cotton GbVe gene, protein coded thereby and use thereof in vegetable verticillium wilt resistance
  • Cotton GbVe gene, protein coded thereby and use thereof in vegetable verticillium wilt resistance
  • Cotton GbVe gene, protein coded thereby and use thereof in vegetable verticillium wilt resistance

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Embodiment 1

[0026] The cloning of embodiment 1 cotton GbVe gene

[0027] Include steps:

[0028] 1) Amplification of the middle fragment of the GbVe gene;

[0029] 2) 3′ chromosome walking amplification of GbVe gene;

[0030] 3) 5′ chromosome walking amplification of GbVe gene;

[0031] 4) gDNA and cDNA amplification of the full sequence of the GbVe gene.

[0032] Specifically, step 1) selects the Plant Genomic DNA Kit (Plant Genomic DNA Kit) and the Plant Genomic DNA Extraction Kit provided by Tiangen Biochemical Co., Ltd., and extracts the young cotton from the sea-island cotton "Pima 90-53" resistant to Verticillium wilt. gDNA and total RNA were extracted from leaf and root tissues respectively, and purified for subsequent chromosome walking amplification and cDNA synthesis. The specific method is as follows: According to the tomato Ve gene sequence published on GenBank, BLAST comparison was performed on NCBI, according to According to the comparison results, specific primers were ...

Embodiment 2

[0056] Example 2 Homology analysis of the amino acid sequence of the protein encoded by the cotton GbVe gene

[0057] The amino acid sequence (SEQ ID No.2) of the protein encoded by the cotton GbVe gene was blasted and analyzed in the NCBI database, and it was found that it is related to the receptor-like disease resistance gene (LRR-TM), which is important in responding to pathogen infection in various plants. There is a certain degree of similarity, but the homology is low. The identity of GbVe gene with StVe and SlVe2 is less than 55%, but they both belong to the important receptor resistance gene (LRR-TM) for plant response to pathogenic bacteria infestation, and contain signal peptide, transmembrane domain, LRR repeat unit, N Conserved domains such as terminal mature regions. Using the DNAman analysis software, the amino acid sequence encoded by the isolated and cloned GbVe gene and the important receptor disease-resistant gene on GenBank in response to pathogenic bacter...

Embodiment 3

[0060] Example 3 Application of Cotton GbVe Gene in Improving Arabidopsis Verticillium Wilt Resistance

[0061] 1. Construction and transformation of cotton GbVe gene plant expression vector

[0062] The above constructed pMDGbVe and eukaryotic gene expression promoter 35s and terminator were constructed as a fusion expression gene, which was connected to the EcoRI / HindIII restriction site of the binary expression vector pCambia1300 (commercially available) (the structure is as follows: figure 2 shown), the recombinant plasmid pCamGbVe was obtained, which was identified as two target fragments ( image 3 ), indicating that the GbVe gene was inserted into pCambia1300 with the correct reading frame. pCamGbVe was transformed into Agrobacterium GV3101. Transgenic plants resistant to hygromycin (hptII) were obtained by transforming Arabidopsis thaliana according to the method of infecting Arabidopsis catkins.

[0063] 2. PCR detection of transgenic GbVe plants

[0064] The you...

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Abstract

The invention relates to cotton GbVe gene and a protein coded thereby. In the invention, a Ve gene associated with verticillium wilt resistance is obtained by cloning in a gossypium barbadense variety having high verticillium wilt resistance; a 5'-terminal GbVe gene fragment and a 3'-terminal GbVe gene fragment, which are obtained by amplification, are spliced; a primer capable of amplifying the full-length GbVe gene is designed and synthesized according to a gene sequence; the sequence of the full-length GbVe gene is obtained by the conventional colony sequencing method according to an RT-PCR technique; and the nucleotide sequence of the full-length GbVe gene is represented by SEQ ID No.1. In the invention; a new and important candidate gen is provided for vegetable verticillium wilt resistant gene project; and the obtained full-length GbVe gene and a vegetable expression vector constructed by the full-length GbVe gene lay a foundation for further transforming important crops for improving the verticillium wilt resistance of transgenic crops and have great economic benefits and application values.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the cotton GbVe gene, its encoded protein and its application in plant resistance to verticillium wilt. Background technique [0002] Cotton Verticillium wilt is called "Cotton Cancer". It has become one of the major diseases affecting cotton yield and fiber quality worldwide. Verticillium wilt is a soil-borne vascular disease, and its germs can remain latent in the soil for a long time. Can not find the way of thoroughly controlling cotton verticillium wilt so far. The use of cotton Verticillium wilt-resistant varieties for Verticillium wilt control has not been widely used due to the limitations of long breeding cycle and lack of Verticillium wilt-resistant resources. Therefore, the use of genetic engineering technology to isolate genes related to plant resistance to Verticillium wilt and to breed new varieties resistant to Verticillium wilt through genetic transformation ...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10A01H5/00
Inventor 马峙英王省芬张艳杨硕张桂寅吴立强李志坤
Owner HEBEI AGRICULTURAL UNIV.
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