The Establishment Of Genetic Reform Manipulation System In Cryptococcus Neoformans And The Research Of Their Capsule Related Genes

The aim of this paper is to research the capsule genes of Cryptococcus neoformans and some of their physical functions. The result of this work were include: (1)screening the ura5 mutant of Cryptococcus neoformans capsule-deficient strains cap60 and cap 70 by the way of suspending mutation, Southern blot analysis indicated that both mutants had URA5 and 0418 selective markers. (2)constructing several shuttle plasmids. All the plasmids could transformate the ura mutants of cap60 and cap 70. Different plasmids had different transformation efficacy, we choosed pCXJ 18 which had high transformation efficacy as a vector for later work. (3)using biolistic method introducing DNA into Cryptococcus neoformans, and we got a high transformation efficacy for the first time, the transformation efficacy is more than 1-2 X 1 03/ug DNA, the protocol lay a good foundation for screening new genes.(4)isolating CAP6O gene of C. neoformans by PCR method, which is required for capsule mutation, the gene was cut into two domain, fusing both domain with green fluorescent protein(GFP) and full expressing to research their function and position. (5)constructing the DNA library of C. neoformans with pCXJ1 8, which founding a good foundation for screening new genes. (6)transformating capsule-deficient strain cap 70 with new gene librarty, the result showed that some of transfornants were positive when reactedwith Cryptococcal antibodies, prepared the ground for screening a new capsule related gene CAP 70 from DNA library.The research of capsule related gene in Cryptococcus neoformans had made some progress in abroad, but almost nothing had be done in our courtry. Early studies demonstrated that capsule formation is one of the most important virulence factors in C. neoformans, so it抯 necessary to research the capsule gene to improve the diagnose and treatment. We hope to bring a new milestone to the Cryptococcosis?therapy and diagnose through our work, and lay a foundation for the better understanding of pathogenic mechanism of C. neoformans and clearing the biochemical biochemical pathway for synthesis of the ploysaccharide capsule.