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

A gene and protein technology, applied to the GhPAO gene, its encoded protein and its application in plant resistance to Verticillium wilt, can solve the problems of cloning and functional analysis that are rarely reported, and achieve the effect of improving Verticillium wilt resistance

Inactive Publication Date: 2012-07-11
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 to resistance to plant verticillium wilt
  • GhPAO gene, and encoding protein thereof and application thereof to resistance to plant verticillium wilt
  • GhPAO gene, and encoding protein thereof and application thereof to resistance to plant verticillium wilt

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Cloning of cotton GhPAO gene and construction of eukaryotic expression vector

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

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

[0033] (3) SSH differential fragment acquisition: sequence the differentially expressed fragments before and after the induction of Verticillium dahliae, and then use the e...

Embodiment 2

[0051] Example 2 Conserved domains and homology analysis of the protein encoded by the cotton GhPAO gene

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

Embodiment 3

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

[0058] The specific steps are as follows:

[0059] (1) Cotton seedling culture and Verticillium dahliae inoculation: The seeds of the disease-resistant upland cotton "Jimian 20" are develed with sulfuric acid and soaked overnight at room temperature. The seeds with uniform germination are selected and sown in a nutrient bowl filled with vermiculite . When the cotton seedling grows to the first true leaf, inoculate 10 mL of the spore suspension of Verticillium dahliae (the concentration of the spore suspension is 5×10 7 cfu / mL).

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

[0061] The total RNA from the root tissue of cotton after 0h, 12h, 24h, 48h, 72h stress by Verticillium dahliae was extracted by the aforementioned methods. After reverse transcription, the cotton constitutively expressed gene Ubiquitin was used as an internal reference for real-time q...

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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 coding protein and its application in plant resistance to Verticillium wilt. Background technique [0002] Verticillium wilt is called "cotton cancer". The occurrence of Verticillium wilt causes a serious decline in cotton fiber quality and yield. The annual loss of lint production in the world accounts for about 10%-20% of the lint output, and the year of severe disease can reach 25%-30%. Therefore, Verticillium wilt has become an urgent problem in cotton production. So far, no method has been found to completely prevent cotton verticillium wilt. The use of disease-resistant varieties for the prevention and control of Verticillium wilt is limited by factors such as long breeding cycles and lack of resources for resistance to Verticillium wilt. The successful application of transgenic Bt cotton in cotton production has led to the use of genetic engineering meth...

Claims

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Application Information

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Patent Type & Authority Applications(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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