Construction Of Candida Albican Calmodulin(CMD1) Gene-deleted Yeast And Study On CMD1 Functional Base

To study the effect of fixed-point mutation on the growth cycle and morphogenesis, we constructed the fixed-point mutation expression plasmids, transferred them into CMD 1-defected yeast. It will be helpful for understanding the role of calmodulin in the pathogenesis of Can dida albican. The research work has three parts: 1. Construction and identification of calmodulin gene-deleted yeast. Firstly, plasmid I containing cmdl ::TRPI replacement sequence was transferred into YPH5O1 diploid yeast and the gene replacement colonies were identified by Southernblot. Secondly, plasmid II containing URA3 and CMD1 sequence was transferred into the above TRP1+ yeast and the colonies containing URA3+ plasmid were obtained on Trp/Ura-drop-out culture. Thirdly, the above Trp/Ura+ yeast had meiosis in the sporulation liquid medium, then the TRP1+ haploid strain was obtained on Trp/Ura-drop-out culture after tetrad dissected. Fourthly, plasmid III containing his3TRPl::HIS3trpl replacement sequence was transferred into the above TRP1+ haploid strain and the Trpl::HIS3 gene replacement colony was screened with His/Ura- and Trp/Ura- drop-out culture. Thus we obtained the prospective CMD1 gene-deleted haploid strain. 2. Construction and identification of calmodulin gene fixed-point mutation haploid strain. Firstly single and double fixed-point mutation on the three phenylalanines in the COOH- half of CMD1 were amplified by two step PCR. The PCR products were inserted into shuttle plasmid IV, then transferred into CMDI gene-deleted HIS3+ haploid strain. It showed that CMD1 F89+ F92, Fl4l+ single-mutation and F89+F141+ double-mutation were negative-lethal mutation and CMD1 F89+F92+ and F92+F141+ were lethal mutation. 3. Effects of CMD1 gene fixed- point mutation on yeast. we inoculated cmdl fixed-point mutation haploid strain A,B,Cand E on Trp-drop-out plates, cultured them at 3O℃ 36℃ 37℃ and 37.5℃. then studied on effect of CMDI gene fixed-point mutation on yeast's temperature sensitivity, DNA synthesis cycles, morphogenesis by light scope and electroscope. The results demonstrated that when the 92nd phenylalanine was changed into alanine, the yeast took on temperature-sensitivity; when the 92nd phenylalanine mutation combined with the g9th or 141St phenylalanine mutation, the yeast deceased; the 89th or the 141St phenylalanine single mutation had little effect on form. growth speed DNA synthesis of yeast(P>O.05), but the g9th and the 141st phenylalanine both changed into alanine, the yeast was temperature-sensitive. The temperature-sensitive yeast?form~. growth speed and DNA synthesis of yeast were distinguished markedly from control?s(P