Construction Of CDNA Library Of A Fungus Producing Perylenequinonoid Derivatives Photosensitizer

This work uses a fungus AL18 producing Perylenequinonoid derivatives photosensitizer which was picked from the mountainous area at the western part of Yunnan.On the basis of establishing the method of total RNA extraction, the cDNA library is constructed.The main results are as follows:1. In order to get over the problem that RNA is easy to degrade and there are a lot of RNase and glucide in this fungus. On the basis of consulting many methods of total RNA extraction, the method is established by experiment for total RNA extraction using guanidinium isothiocyante and β-mercaptoethanol for denaturation, then using water-satured phenol ,chloroform and isoamylalcohole for extraction, RNA stay in water ,while DNA and protein stay in another medium. Finally, RNA is deposited by isopropylalcohol .The integrity and purification of the total RNA isolated are eligible and meet the requirement to construct cDNA library.2. The cDNA is synthesized from the total RNA with the one point mutant of Moloney murine leukemia virus(MMLV) reverse transcriptase,and the cDNA is further size-fractioned by CHROMA SPIN-400 Column to remove the fraction shorter than 500bp. The dscDNA is cloned into the λTriplEx2 vector and the resulting ligating matarial is λ-phage packaged. The unamplifed cDNA library is titered using E.coli XL-Blue, and it turns out that the titer of the cDNA library is 1.04 ×10⁶ pfu/ml,the titer of the amplified cDNA library isl.32×10¹⁰pfu/ml,and the recombinant efficiency is 82% through white/blue screening.It is available to clone the polyketide biosynthase gene for Polyketide biosynthesis.