Identification And Functional Analysis Of Growth And Development Related Genes In Pleurotus Ostreatus Based On DGE Technique

Pleurotus ostreatus or Oyster mushroom is a widely cultivated mushroom in the world. Not only does it taste delicious, but also contains high nutritional value, so that it is very popular to many people. At present, the investigation about P. ostreatus was focused on its cultivation, breeding, gene cloning and expression, secondary metabolites isolation and biotransformation, but the function research of development related genes was seriously lagging behind. In this paper, we investigated the effects of promoter length on the activity of gpd promoter from P.ostreatus. A truncated gpd promoter fragment(795 bp) with the highest activity had been chosen to construct the over-expression vector and RNAi vector which were suitable for gene function research in P. ostreatus. Furthermore, the function of development related gene, methionine sulfoxide reductase(Po Msr A) and manganese superoxide dismutase(Po Mn-SOD), were systematically studied with the vectors mentioned above. The main research contents are as follows: 1. Cloning and identification the development related genes of P. ostreatusThe c DNA 3’ end cloning of 119 Tags from DGE library of mycelium stage in P. ostreatus were performed. As many as 83 terminal sequences of 3’ end were successfully obtained and the detection rate was about 69.7%. The obtained sequence of 3’ end were submitted to NCBI database for blast, 41 sequences had a good match to the annotated genes and the total matching rate is 49.4%. Corresponding Tags of 17 enzyme genes with high expression level were found in these 41 sequences. We believe these enzyme genes could play an important role during P. ostreatus differentiation and development, but its specific function needs to be verification. In additionally, 10 terminal sequences of 3’ end had been chosen to perform the 5′RACE experiment and 4 genes’ terminal sequences of 5’ end had been obtained at last. After splicing, the fulllength c DNA sequence of 4 genes, including Msr A gene、Mn-SOD gene、Asp gene and lectin gene, had been acquired fortunately. 2. High level expression of Asp and Mn-SOD genes in Pichia pastorisWe cloned the Asp and Mn-SOD genes in this study. The DNA sequence of ASP gene consisted of 1525 bp which contain six introns and 1212 bp CDS sequence of which encoding 403 amino acids. Similarly, the Mn-SOD also contained six introns with 1031 bp DNA sequence and 663 bp CDS sequence which encodes 220 amino acids. The expression vectors were constructed and electro-transformed in P. pastoris GS115. After multi-copy transformants screening with G418, SDS-PAGE and Western-blot verification, the Asp and Mn-SOD gene were successfully expressed in P. pastoris. The recombinant aspartic protease and manganese superoxide dismutase showed strong biological activity. Then, we optimized the fermentation conditions and found the maximum enzyme activity of crude manganese superoxide dismutase reaches 156.9 U/mg under the condition of initial p H 6.0, the induction time of 6 days and methanol concentration of 0.7 %(v/v). 3. The effects investigation of promoter length on the activity of gpd promoterIn this experiments, a skeleton plasmid p CAMBIA1300 and a cis-driving element of gpd promoter with different lengths(960bp, 795 bp, 633 bp, 403 bp and 231bp) had been used to construct the expression vector p CAMBIA-gpdx-egfp-hph in which the egfp and hph genes were fused expression. By Southern-blot, Western-blot and Quantitative RT-PCR analysis, a truncated gpd fragment(795 bp) drove egfp gene expression much more efficiently than full-length gpd promoter. Further truncated gpd fragment of 633, 403 and 231 bp reduced the egfp gene expression significantly. However, it had been confirmed that gpd promoter as short as 403 bp can sufficiently drive the egfp-hph gene expression. 4. Construction the intermediate vector p GEHUsing the p CAMBIA1302 plasmid as skeleton, an intermediate vector p GEH had been successfully constructed. In which, a 795 bp gpd promoter as a cis-drive element driven egfp and hph gene fused expression and these two genes can be used as selectable markers. On the other hand, it is convenient to be converted on the right border of Ti plasmid vector p GEH. So the gene function research vector, such as over-expression vector or RNA interference vector, could be obtained easily. 5. Constructed the Po Msr A gene overexpression vector p GEX-EM for gene function analysisPo Msr A gene had been cloned in this experiment. 908 bp DNA sequence contained three introns and 612 bp CDS sequence encoded 203 amino acids. We converted the intermediate vector p GEH and successfully constructed the expression vector p GEHEM for Po Msr A gene that via Agrobacterium tumefaciens mediated transformation of P. ostreatus mycelium. Stable integration of the target gene into the genome of P. ostreatus was confirmed by PCR, fluorescence observation, and Southern blot hybridization. The transgenic strains with Po Msr A overexpression exhibited an enhanced tolerance to high temperature, high osmotic stress, and oxidative stress. The results not only enriches the Po Msr A gene function research, but also provided some information for utilization. 6. Constructed the Po Mn-SOD gene overexpression vector p GEX-ES and RNAi vector p GEH-RI for gene function analysisAnother Po Mn-SOD gene has been cloned. The full-length DNA sequence of the gene is 889 bp containing three introns and 615 bp CDS sequence encoding 204 amino acids. By the converted of intermediate vector p GEH, a Po Mn-SOD gene overexpression vector p GEH-ES and three RNAi vector p GEH-RI had been successfully constructed. After transformed P. ostreatus mycelium via A. tumefaciens mediated, we found the overexpression transgenic strains had been conferred ability to against high temperature, high osmotic stress, and oxidative stress, while the RNAi transgenic strains were grow slowly and some strains even stop growth under the same abiotic stress condition. This results indicated that Po Mn-SOD gene was important during P. ostreatus growth. Additionally, quantitative RT-PCR confirmed the interference vector p GEH-RI for Po Mn-SOD gene had produced co-silencing with other homologues. Furthermore, we found that the interference vector p GEH-RI with the 3 ’end fragment had the best interference effect in different growth stages of P. ostreatus using quantitative RT-PCR.