Functional Analysis Of Resistant-related Genes AtBRG1 Of Arabidopsis Thaliana Against Botrytis Cinerea

The host of Botrytis cinerea is widespread, which can infect more than 200 kinds of plants, concluding fruits, vegetables and so on, and this caused serious economic losses to agriculture. The genome of Arabidopsis thaliana is the smallest, approximately 130 Mb, and only five pairs of chromosomes in somatic cell, so it is the model plant for the study of interaction between plant and pathogen. In the recent years, many disease-resistant gene (R gene) of plant and the resistant related gene were obtained from Arabidopsis thaliana. In the earlier period, we obtained BRG1 (Botrytis cinerea-resistant related gene) of Arabidopsis thaliana Col-0 ecotype by DDRT-PCR and RACE technology. In order to understand the function of BRG1 gene and its role in the disease-resistant way, prokaryotic expression technology was used to confirmed BRG1 protein can inhibit the germination of Botrytis cinerea spore. Compared Arabidopsis thaliana Col-0 wide type with brg1 (T-DNA insertion mutant of BRG1 gene), the role of the BRG1 gene in Arabidopsis thaliana resisting Botrytis cinerea and abiotic stress such as cold stress and drought stress was determined, The complementation and overexpression experiment were performed to validate the function of BRG1 gene.Constructing the prokaryotic expression vector of the BRG1 gene, The protein expressed in E. coli showed inhibit germination of the Botrytis cinerea spore in some degree.The T-DNA insertion mutant brg1 of BRG1 gene was obtained from Arabidopsis thaliana SALK Library and was identified by PCR, Southern Blotting, and RT-PCR technology. Homozygous mutant brg1 was obtained and it was determined that the BRG1 gene is partly knocked out. Mutants brg1 and wild-type Col-0 plants were inoculated with Botrytis cinerea. The results showed Col-0 was immune and mutants brg1 were highly susceptible to Botrytis cinerea. A series of abiotic stress on the brg1 mutants were carried out, compared with wild-type, it turned out to be that BRG1 mediates responses not only to biotic but also abiotic stress such as cold stress and drought stress. RT-PCR were used to examine the expression of some gene of disease-resistant way and the defense enzyme in brg1 mutant. It showed mutant of BRG1 gene can affect the expression of disease-resistant gene and defensive enzyme gene.The wild type BRG1 gene of was obtained by PCR and inserted into the XbaI and kpnI restriction sites of the plant expression vector pCAMBIA1300. The pCAMBIA1300 vectors harboring BRG1 were transformed into brg1 mutants and WT by an Agrobacterium-mediated transformation. The expression of BRG1 in transgenic lines, brg1 mutants and WT were analyzed by semi-quantitative RT-PCR. The results showed that the expression levels of BOSA in transgenic lines were significantly higher than those in brg1 mutants plants. Inoculation of wild-type Col-0 plants, brg1 mutants, and transgenic lines with B. cinerea was performed. 48 h later, Col-0 ecotype appeared immunity, and brg1 mutants appeared obvious disease symptoms, and while transgenic lines appeared significantly smaller size of lesions. while BRG1-complement lines appeared HR response, overexpression lines was immune. So we concluded the BRG1 gene have the influential role in the Arabidopsis thaliana anti-Botrytis cinerea process.