Cloning Of GPD,RAS Promoter And Cytochrome P450 Gene From Lentinus Edodes

Lentinus edodes has been ranked as the world's twise most important cultivated edible mushroom, representing a valuable source both in economy and in nutrition. So it is very important to develop transgenic technology in Lentinus edodes. We aimed in this study to isolate and identify the effective promoter which is adaptive to Lentinus edodes transgenic research to develop the transgenic technology in Lentinus edodes.We isolated Lentinus edodes RAS GPD promoters fragment from genomic DNA by the method of PCR, which based on the reported promoter sequences, specific primers were designed. DNA sequence analysis and homology comparison indicated that it has a about 92% homology with the promoter sequence which has been submitted to GeneBank. Analysis using Promoter Prediction showed that there existed one core promoter region each strand, and many ds-acting regulatory elements were found by PlantCARE.The result of tfsitescan service analysis indicated that several transcription factors were in this region. It can be concluded that it's most probably a promoter sequence.By fusing this three fragments with Escherichia coli beta-glucuronidase (gus ) reporter gene nopaline terminator and the cloned promoter, we get three expression vectors which named pRAS pGPDl and pGPD2, When protoplasts of Volvariella volvacea were mixed with the plasmid DNA in the presence of polyethylene glycol and CaCl2, Hygromycin B -resistant colonies were obtained. This indicated that transformation was successful. Two of 5 transformants obtainedas Hygromycin B-resistant colonies showed two to twenty times higher specific activity of GUS than the recipient. This transformation system will enable us to breed commercial strains oflentinus edodes at the molecular level.The three constructs were delivered into the tissue (Lentinus edodes protoplast) for transient Expression by electroporation.transient expression studies showed that all the promoter fragments can drive strong reporter gene expression.Using the same way, we cloned the Lentinus edodes gene for cytochrome P450, which has 448bp length.The result from BLASTn analysis indicated that fragment is of high similarity at 99% with Le.cypl gene' 5'upstream for cytochrome P450 1. BLASTx analysis provided more information,, Ammo acid sequence encoded by partial sequence of fragment A126 hasthe similarity of 60% compared with cytochrome P450 2 Le.CYP2 .