Cloning And Expression Analysis Of Fst3 Gene Involved In Regulation Of Fruiting Body Formation Of Pleurotus Ostreatus

Pleurotus ostreatus is an important source of food. At present, the information about the development process of the fruiting body is limited, especially the lack study of fruiting body development related functional genes, so we can not describe the mechanism of fruiting body development accurately. Some reports suggest that the fst3 gene of Schizophyllum commune can be activated by signals from the fruiting body development, which will prevent the surrounding aerial hyphae’s formation and compete for more nutrients for the fruiting body. However, the studies and reports about the fst3 gene of Pleurotus ostreatus still can not be found.In this study, the fst3 gene of Schizophyllum commune was compared with Pleurotus ostreatus genome in the JGI and the primer sequence was designed according to the homologous sequence. The full-length c DNA sequence of fst3 gene of Pleurotus ostreatus had been cloned by RT-PCR and RACE-PCR techniques. To further understand the molecular mechanism in the development process of Pleurotus ostreatus, bioinformatic analysis and expression profile analysis were done, and the expression vector was constructed and transformed into E.coli BL21(DE3).Overexpression vector and antisense expression vector were constructed, which will be transformed into Pleurotus ostreatus by Agrobacterium tumefaciens-mediated method.The main conclusions of the research are as follows:1. The full-length c DNA of fst3 gene of Pleurotus ostreatus from total RNA extracted from Pleurotus ostreatus New 831 mycelia by RACE technology, and the Gene Bank accession number was KJ716894. Bioinformatics analysis showed that the full-length fst3 c DNA was comprised of 2549 bp, encoding a putative protein of 795 amino acids.NCBI conserved domain analysis showed that the gene belongs to fungi MHR super family of transcription factors, with the typical zinc finger DNA binding domain.2. The fst3 expression levels at different developmental stages were determined by semi-quantitative RT-PCR and shown to be highest at the mature fruiting body stage,which could indicate that the fst3 gene may be related with Pleurotus ostreatus fruiting body development. The fst3 gene was expressed in Escherichia coli BL21 and showed expression activity in prokaryotic cells. The SDS-PAGE analysis showed that relative molecular mass of recombinant protein was approximately 88 k Da and consistent with the predicted molecular weight.3. The fst3 eukaryotic overexpression vector p-gpd1300-overfst3 and antisense expression vector p-gpd1300-antisensefst3 were constructed by enzyme digestion and enzyme linked method, which prepared the experiment materials for the Agrobacterium tumefaciens-mediated transformation of Pleurotus ostreatus New 831.