GhPAO gene, and encoding protein thereof and application thereof in resistance to plant verticillium wilt

An anti-Verticillium wilt, gene technology, applied in the field of genetic engineering, can solve problems such as cloning and functional analysis, which are rarely reported, and achieve the effect of improving Verticillium wilt resistance

Inactive Publication Date: 2013-03-13
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although a large number of genes related to disease resistance have been cloned from cotton, there are few reports on the cloning and functional analysis of the polyamine oxidase gene PAO in the polyamine metabolic pathway.

Method used

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  • GhPAO gene, and encoding protein thereof and application thereof in resistance to plant verticillium wilt
  • GhPAO gene, and encoding protein thereof and application thereof in resistance to plant verticillium wilt
  • GhPAO gene, and encoding protein thereof and application thereof in resistance to plant verticillium wilt

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The cloning of embodiment 1 cotton GhPAO gene and the construction of eukaryotic expression vector

[0031] (1) Cotton seedling cultivation and Verticillium dahliae inoculation: The seeds of upland cotton "Jimian 20" were velveted with sulfuric acid, dried in the sun, soaked at room temperature overnight, and the seeds that germinated uniformly were selected and sowed in a nutrient bowl filled with vermiculite. When the cotton seedling grows to the flattening of the first true leaf, inoculate 10 mL of Verticillium dahliae spore suspension (the concentration of the spore suspension is 5×10 7 cfu / mL).

[0032] (2) RNA extraction: RNA extraction and reverse transcription use RNA extraction kit and reverse transcription kit (purchased from Beijing Aolibo Biotechnology Co., Ltd.), and operate according to the instructions to obtain double-stranded cDNA (dsDNA) materials.

[0033] (3) Acquisition of SSH differential fragments: Sequence the differentially expressed fragments ...

Embodiment 2

[0051] Example 2 The conserved domain and homology analysis of the protein encoded by the cotton GhPAO gene

[0052] Using the online http: / / www.ncbi.nlm.nih.gov / conserved domain, the conserved region of the GhPAO-encoded protein was analyzed. The results showed that the encoded protein of GhPAO has the conserved sequence of GXGXXG ie GAGVSG combined with NADB-Rossmann (cl09931) family. The GhPAO protein belongs to the amino oxidase (pfam01593) family and has the characteristic sequence (sVIiIGAGvSGLSAAKvLAE) of the riboflavin (flavin) binding site, namely CTTTCTCTCGGTCGTCCGTT. The amino acid sequence (SEQ ID No.2) of GhPAO was blasted and analyzed in the NCBI database, and the results showed that it had high homology with RcPAO (XM-002517737.1) of castor plant and PtPAO (AB200262.1) of poplar, respectively. 84.3% and 82.8%; the homology with the polyamine oxidase AhPAO (AF469064.1) and barley polyamine oxidase (AJ298132.1) is low, reaching 46.8% and 45.4%, the results are sh...

Embodiment 3

[0057] Example 3 Analysis of the expression characteristics of cotton GhPAO gene

[0058] The specific operation steps are as follows:

[0059] (1) Cotton seedling cultivation and Verticillium dahliae inoculation: the seeds of the upland cotton disease-resistant variety "Jimian 20" were develored with sulfuric acid and soaked overnight at room temperature, and the seeds that germinated uniformly were selected and sowed in a nutrient bowl filled with vermiculite . When the cotton seedling grows to the first true leaf flat, inoculate 10mL Verticillium dahliae spore suspension (concentration of spore suspension is 5×10 7 cfu / mL).

[0060] (2) RNA extraction: RNA extraction and cDNA synthesis operations are as described above

[0061] The aforementioned methods were used to extract the total RNA of cotton root tissue after Verticillium dahliae stress for 0h, 12h, 24h, 48h, and 72h, and after reverse transcription, the real-time quantitative PCR reaction was carried out with the...

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Abstract

The invention relates to a GhPAO protein. The GhPAO protein has an amino acid sequence shown as SEQ ID No. 2 or an amino acid sequence which is obtained by replacing, deleting or adding one or more amino acids and has equivalent functions. A cloned GhPAO gene helps to reveal a molecular mechanism of interaction between cotton and Verticillium lecanii, provides an important theoretical basis for studying a metabolic pathway for a plant to resist Verticillium wilt, and provides a more valuable candidate functional gene for improving genetic traits in the field of plant genetic engineering; the verticillium wilt resistance of a transgenic plant obtained from the GhPAO gene is significantly improved, which lays the foundation for further transforming an important agricultural crop so as to improve the disease resistance of the crop; therefore, the GhPAO gene has relatively good application prospect and application value in agricultural production.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the GhPAO gene, its encoded protein and its application in plant resistance to verticillium wilt. Background technique [0002] Verticillium wilt is called "cotton cancer", and the occurrence of Verticillium wilt leads to a serious decline in cotton fiber quality and yield. The annual loss of lint production in the world accounts for about 10%-20% of lint production, and the year of severe disease can reach 25%-30%. Therefore, verticillium wilt has become an urgent problem to be solved in cotton production. So far, there is no way to completely control cotton verticillium wilt. The use of disease-resistant varieties to prevent and control Verticillium wilt is limited by factors such as long breeding cycle and lack of resources for resisting Verticillium wilt. However, the successful application of transgenic Bt cotton in cotton production has made it possible to use genetic e...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/06C12N15/53C12N15/63C12N5/10C12N15/84A01H5/00
Inventor 马峙英默辉娟孙艳香王省芬张艳张桂寅
Owner HEBEI AGRICULTURAL UNIV.
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