Construction of a cDNA library of Pythium carolinianum, a mosquito-killingfungus

A mosquito-killing fungus was isolated in Guiyang City, China, in August 19 94. It was preliminarily identified as Pythium carolinianum based on morpho logical studies. Observations on its extra cellular enzymes found that unde r the induction of chitin, it can produce high activity of substilisin-like proteases (Prl) and trypsin-like proteases (Pr2). These are considered to be related with the process of by the fungus invading into mosquito body. I n order to search for and make use of these genes, a cDNA library has been constructed. The methods used were mainly: 1) isolating total RNA of the fu ngus after 30 h induction of chitin; 2) using the total RNA as a template t o synthesise cDNA using the SMART method; 3) inserting the double stranded cDNA into the vector, Trip IE chi2, and 4) transforming E. coli strain XL I -Blue with the recombinant molecules to make an amplified library. The resu lts of quality tests on the preliminary and amplified libraries showed that this cDNA library was successfully constructed. It can provide a direct so urce for screening genes of Prl and Pr2 and other proteases in the future.