Cloning Of A Receptor-Like-Protein Gene Gbvdr3and Characterization Of The Promoter Of Gbvdr3in Island Cotton

Verticillium wilt caused by soil-borne pathogens is a kind of serious vascular disease.The pathogen of Verticillium wilt has a wide host range, and it is obvious of the differentiation of the virulence.So far there were no effective way for disease controlling.The main problem for cotton resistance breeding against Verticillium wilt include lack of resistance resources in cotton, the narrow genetic basis of the resistance of Verticillium Wilt, a long breeding period and a single breeding method and so on. With the development of modern molecular biology techniques, transgenic breeding has opened up broad prospects for breeding new disease-resistant varieties. Therefore, cloning resistance genes to Verticillium wilt has become a hotspot in disease resistance research.In this study,a structure domain of receptor like gene about cotton resistance Gbvdr3in Gossypium barbadense was obtained. The full length of Gbvdr3is3412bp,which encodes a protein of1068aa.. The identity of the amino acids between Gbvdr3and Ve1/Ve2is49%. Gbvdr3inserted into the plant over expression vector pCAMBIA2301of promoter CaMV35S controlling and it was transformed into Arabidopsis of ecotype Columbia.5transgenic plants were obtained by antibiotic selection and PCR method.The functional verification of the transgenic Arabidopsis showed varying degrees of resistance. Compared with the wild type, some symptoms were found in transgenic Arabidopsis,but the seeds were shown normal. The conclusion can be drawed that the resistance of Gbvdr3transgenic plants to V991was obviously improved,but the resistance of Gbvdr3transgenic plants to BP2was not obviously improved.In order to the further study of the role of Gbvdr3genes in disease resistance,the promoter region of Gbvdr3in this study was obtained by genome walking, and the sequence was analyzed by PLACE software. The sequence analysis showed the promoter contains disease resistance, stess and light regulation elements.The cloned promoter was fused to the GUS reporter gene and making use of the promoter drives the expression of the GUS gene to identify the expression characteristics of the gene. The recombinant vector was transferred into Arabidopsis by Agrobacterium-mediated transformation method．Transgenic plants have a GUS staining.8transgenic plants were obtained through kanamycin sulfate screening and PCR method. GUS staining revealed the GUS expression was specifically detected in roots, flowers and seeds.The results indicated that the promoter was a tissue-specific promoter. Separation and characterization of the promoter of Gbvdr3promoted the further research and application of the gene.