Isolation Of NBS-LRR Resistance Gene Analogs And Characterization Of Gbarvin From Gossypium Barbadense L.

Verticillium wilt has become one of the most severe diseases of cotton in China for years. Morover, the resistance to verticillium wilt is diffcult to be popularized in cultivated cotton species Gossypium hirsutum L. by cross breeding from Gossypium barbadense L., which has high resistance to. So far, more than 50 genes resistant to different pathogens, including bacteria, fungi, nematodes and viruses, have been cloned in plant. All of these plant disease resistance (R) genes have the conserved NBS and LRR domain, although their overall sequence homology is low. In this thesis, the NBS-LRR genes and resistance gene analogs (RGAs) are isolated from the genome of Gossypium barbadense L. XH21 by polymerase chain reaction (PCR)-based approach with degenerate oligonucleotide primers designed from these conserved domains. The characteristic, organization and evolution of these novel disease resistance genes in Gossypium barbadense L. genomes are identified, which are significant for understanding of plant-pathogen interactions and development of novel approaches to effective control of plant pathogens in agriculture.The study of the Gossypium barbadense L. XH21 main genes expression induced by Verticillium dahliae Kleb , is significant not only for understanding the interaction mechanism between fungi with host but also for applying genes to molecular breeding in Gossypium hirsutum L.. Servel pathogenesis-related (PR) proteins will be produced in plant-pathogen interactions, including PR-4 proteins. One class of PR-4 (classe I) contains two domains, one is chitin-binding domain, and another is catalytic domain, which have many biological function and the antifungal activity is an important one of them. Class I proteins contain an N-terminal cysteine-rich chitin-binding domain, a Gly or Pro -rich linker region, and a C-terminal catalytic domain. Barwin-like protein isolated from barly belongs to this class, having ribouclease activity besides antifungal activity.To date, Ve 1 and Ve2 isolated from tomato, are resistant to Verticillium dahliae, and both of them contain the conserved LRR domain. Gbarvin, which encodes 211 amino acids, has a chitin-binding domain in N-terminal and a barwin domain in C-terminal. In this research, it was isolated from the cDNA of cultivar XH21 (Gossypium barbadense L.), which was induced by Verticillium dahlia., by a polymerase chain reaction (PCR)-based approach with degenerate oligonucleotide primers designed from the conserved motifs in Vel and Ve2. It was expressed in E. coli (BL21) and was showed resistant to Verticillium dahliae.It was proposed that Gbarvin has ribouclease activity due to its nucleotide binding site. The second structure of Gbarvin is similar to part of those of Vel and Ve2. It indicates that Gbarvin adopts the same mechanism for antifungal acitivity as Vel and Ve2. The residues involved in nucleotide binding form an active site according to its tertiary structure.RNA interference (RNAi) results of the Gbarvin in Arabidopsis thaliana (Columbia-24) by using the pCPCbCLV system, which made from Gminivirus. Expression vector was constructed by Gbarvin cDNA and pCAMBIA1300 with CaMV 35S promoter, infected Arabidopsis thaliana (ecotype...