Expression Of Histidine Kinase Gene From Cordyceps Militaris And Isaria Cicadae Miq.,Detection Of Histidine Kinase In Cordyceps Militaris

Histidine kinase is an important part of two-component system. Two-component system is the important mechism that fungus feel the external stimuls, and it makes the fungi respond and adapt to the changes in the environment. Cordyceps cicadae and Cordyceps militaris are traditional medicinal and edible fungus in China. The regulational and expressional differences of histidine kinase of two entomogenous fungi will be found by the researches on the histidine kinase gene of them.By Cordyceps militaris histidine kinase gene which was found, the primers were designed to obtain the IcHK gene sequences. The ORF of IcHK was got by sequencing and alignment. The results of bioinformatics analysis showed that the total length of IcHK-ORF sequence is 2811 bp, and the translation products of IcHK gene showed that there are 4 HAMP functional domains, 1 HisKA functional domain, 1 HATPase domain and 1 REC functional domain in Ic HK sequence.The researches on histidine kinase are focused on its expression and regulation for external signals for a long time and studies on fungicides are increased in recent years. Three kinds of fungicide, Fludioxonil, Iprodione and Captan, were chosed to study their effect on on the expression of Cordyceps militaris histidine kinase gene CmHK and Cordyceps cicadae histidine kinase gene IcHK by Real-time PCR. The results showed the sensitive of the expression of CmHK and IcHK to the three kinds of fungicides are different. They could not result in the significant increases of the relative transcription level of CmHK gene, so the expression of CmHK gene was not sensitive to the three kinds of fungicides. But they could lead to the increases of the relative transcription level of IcHK gene, which suggested the expression of IcHK gene was sensitive to the three kinds of fungicides, especially more sensitive to fludioxonil and captan.Two methods of preparing antibody were carried out according to the putative amino acids sequence of histidine kinase protein CmHK according to the CmHK gene. A method was obtaining fusion expression protein by the prokaryotic expression of partial sequence of CmHK gene. The fusion expression protein was used as antigen for the preparation of antibodies. Based on the ORF sequence and the analysis of functional domain of CmHK gene, it was divided into two parts then the primers were designed respectively. Two partial sequences Cm HK1 and CmHK2 gene obtained by PCR were connected to the expression vector pET-41a(+) respectively, and transformed into BL21(coli Escherichia) receptor cells to construct prokaryotic expression system of CmHK1 and CmHK2 ultimately. IPTG was used to induce the expression of the fusion proteins,and ProteinIsoTM Ni-NTA Resin was used to purify the fusion proteins. Then two antibodies anti-CmHK1 and anti-CmHK2 were obtained by using the purified fusion protein CmHK1 and CmHK2 as antigen to immunize rabbits respevtively.The other method was that the antigenic determinant short peptides of chemical synthesis were coupled to protein carriers for the preparation of antibodies. The three short peptides of chemical synthesis, HK3, HK4 and HK5, were coupled to BSA to prepare three antibodies, anti-HK3, anti-HK4 and anti-HK5.The antibodies obtained by the two methods were used to detect CmHK of C. militaris by Western blotting analysis. Western blotting analysis showed that specific hybrid bands were observed using all antibodies besides anti-HK2. It could be saw two specific hybrid bands, 85 kDa and 45 kDa and it showed the possible existence forms of CmHK in mycelium of C militaris were 85 kD and 45 kD.