Cloning Of Squalene Synthase Gene From Ganoderma Lucidun And Primarily Analysis Of The Gene Expression

Ganoderma Lucidun, a mushroom, has long been used in China as a traditional medince for the treatment of various kinds of diseases. triterpenoids is the production of Ganoderma lucidun secondary metabolism, triterpenoids formation is dependent on the condensation of two molecules of FPP into squalene and is thus referred to as the first dedicated step in triterpenoids biosynthesis. Squalene synthetase catalyzes the first committed step for sterol biosynthesis and is thought to play an important role in the regulation of isoprenoid biosynthesis in eukaryotes. In order to understand the complex regulation mechanism of triterpenoids synthesis in Ganoderma ]ucidun, a DNA fragment corresponding to Ganoderma ]ucidun squalene synthase and the antibody against the squalene synthase recombinant protein was raised using a rabbit.Amino acid sequences conserved among yeast and human SQS were used to design two degenerate oligonucleotide primers for PCR amplification of genomic DNA. Sequencing of the amplification product revealed a fragment of 395 bp encoding an amino acid sequence corresponding to SQS. Accordcring to the conserved domain of SQS, 57 bp fragment for expressed recombinant protein was designed. This fragmen was inserted into the expression vector pET?2a(+). The recombinant expression plasmid was transferred into the E. coil strain BL21, then cultured and induced with IPTG, the over梕xpressed recombinant protein was detected and purified using preparation electrophoresis, The antibody against the recombinant protein was raised using a rabbit. With the immunological analysis, the anti梥erum showed specific immunological response to standard enterotoxigenic E. coli stain. With the anti梥erum SQS expression of Ganoderma lucidun was performed by Western boltting analysis.