The Effects Of Copper Sulfate Induction On Copper Resistance And Expression Of Copper Resistance Related Genes Of Xanthomonas Axonopodis Pv. Citri

Citrus canker is caused by Xanthomonas axonopodis pv. citri (Hasse) Vauterin (Xac). In the natural conditions, the bacterial pathogen can infect many species of the family Rutaceae and decrease the growth vigor of citru trees, the productity and quality of citrus fruit. Therefore, the disease can cause significant economic losses by seriously affecting the exportation of citrus fruits from China and the development of citrus industry. Copper sulfate, cupric hydroxide and copper chloride are the most widely used chemical agents in the orchard for the control for the disease. In order to understand the effects of the frequent application of these chemicals on the resistance of Xac to copper, two copper resistant related genes copA and copB were cloned and inserted into the prokaryotic expression vectors, and the effects of copper sulfate pressure on the copper resisitance of Xac were also evaluated in this study. Results are as followings:1. Using the DNA extracted from Xac as templates, two copper resistance related genes copA and copB were amplified and cloned. Sequencing results showed that the obtain genes copA and copB were 1798 bp and 1111 bp, respectively. Then, the prokaryotic expression vectors pET-copA and pET-copB were constructed and transformed into Escherichia coli BL21(DE3). The expressed products in Escherichia coli BL21(DE3) under the induction of IPTG were subjected to SDS-PAGE electrophoresis analysis. Results showed that both copA and copB genes were efficiently expressed and the sizes of recombinant proteins were about 69 kDa and 46 kDa, respectively. Polyclonal antibodies against recombinant proteins of copA and copB were produced by immunizing rabbits with recombinant proteins, respectively. The titer of these two antibodies was 1:6400 in indirect ELISA test and their working concentration was ranging 1:2000-1:5000. The reuslts of the western blot anlysis showed that the prepared antisera can react with the prokaryotic expression proteins, and the non-specific reaction can be decreased by the purification of antibodies.2. The effects of the over expression of both genes copA and copB on the copper resistance of Escherichia coli BL21(DE3) were evaluated. Results showd that the growth of Escherichia coli BL21(DE3) transformed with pET-copA and pET-copB was significantly reduced under the induction of IPTG. However, the growth speeds of transformed Escherichia coli BL21(DE3) in media containing copper sulfate at concentrations 100μg/ml-500μg/ml showed no significant differences. It was suggested that the expression of copA or copB could help the bacterium survial on copper sulfate containing medium.3. Four Xac strains were used for the analysis of the variations of copper resistance after culturing on copper sulfate containing media. The half lethal concentration of copper sulfate to all these strains was 100μg/ml. These four strains were continuously cultured on media containing copper sulfate with gradually elevated concentration for 1 year. Results showed that their resistance to copper sulfate could be greatly improved and the lethal concentration of copper sulfate to these strains were raised from 150μg/ml to 270μg/ml-280μg/ml. The expression of both copA and copB in the original and obtained copper-induced cells of Xac strain no.9 was analyzed by western blot using the raised antibodies in this study. Results showed that the gene copA could efficiently expressed under the 100μg/ml copper sulfate induced and non-induced conditions and the expession of the gene in obtained'copper resistant strains'could be improved by the induction of 100μg/ml copper sulfate. However, the expression of gene copB could not be detected by werstern blot under the copper sulfate induced and non-induced conditions.