Clonging And Expression Analysis Of Sporulation-Related Gene (Stpo1) From Pleurotus Ostreatus

Pleurotus ostreatus is the most widely cultivated and consumed edible fungi in China.During the mature of P.ostreatus,the fruiting body will eject a large number of spores.The huge number of spores will not only reduce the quality of fruiting body,but also lead to "mushroom lung".So,breeding of the sporeless P.ostreatus strains has very important significance in solving various problems caused by spores.The nucleic acid sequence of the sporulation-related gene from P.ostreatus,which was named stpo1 herein,was primarily obtained by alignment of the stpp1 gene sequence from Pleurotus pulmonarius with the transcriptome database of P.ostreatus.Then the complete CDS region of stpo1 was cloned by reverse transcription PCR(RT-PCR).To understand the molecular mechanism of stpo1 in the development process of P.ostreatus,bioinformatic analysis and expression pattern analysis of stpo1 were done in this study.To identify the relationship of STPO1 to DNA mismatch repair in vitro,STPO1 was expressed in Escherichia coli BL21(DE3)by using p EASY-E2 expression system.Finally,the stpo1 overexpression vector and RNA interference vector were constructed to clarify the function of stpo1 by Agrobacterium-mediated transformation of P.ostreatus.The results are as follows.1.Using the total RNA of P.ostreatus fruiting body as template,the complete CDS region of stpo1(2,556 bp)was obtained by RT-PCR,whose Gen Bank accession number is KR822419.The full-length genomic DNA(g DNA)of stpo1(3,853 bp)was also cloned by using the P.ostreatus genome as template.The gene structure analysis showed that the stpo1 gene contains 25 introns.The homologous alignment of the coding sequence of stpo1 with that of stpp1,PC9 v1.0 homologous gene and PC15v2.0 homologous gene showed that the stpo1 has 88%,97% and 99% identity with the other three genes,respectively.2.The protein structure prediction showed that STPO1 belongs to the DNA mismatch repair family which involved in DNA mismatch repair.And STPO1 has the typical Mut S domain,ATP binding site and P-loop.The GO-enriched analysis showed that STPO1 has the ability to recognize and bind the mismatched base pair,thus participating in the DNA mismatch repair.Sporulation-related proteins play roles in sporulation by maintaining the meiosis;however,the function prediction of STPO1 protein did not show any correlation of meiosis.3.The expression patterns of P.ostreatus stpo1 gene were analyzed by real-time quantitative PCR.The RT-q PCR results showed that stpo1 gene was highly expressed at the mature fruiting body stage,but unexpressed at other stages.Its expression was significantly upregulated in the gill of fruiting body,but not in stipe or pileus.Those all indicated that stpo1 gene may be related to the sporulation of P.ostreatus.4.The CDS region of stpo1 gene was heterologously expressed in Escherichia coli BL21(DE3),and the STPO1 fusion protein,the molecular weight of which was about 94 k Da,was successfully induced.It further proved that the complete coding sequence of stpo1 was obtained in this study,which laid the foundation for studying the molecular mechanisms of DNA mismatch repair and meiosis in P.ostreatus.5.The P.ostreatus gpd promoter was chosen to replace the CaMV 35 S promoter in the Binary expression vector p CAMBIA1300 to efficiently express hygromycin resistance gene in P.ostreatus.And the stpo1 gene overexpression vector and the stpo1 gene silencing vector were constrcuted by seperately inserting the stpo1 CDS fragment and the hairpin structure downstream of the another gpd promoter.Currently,the transformation of P.ostreatus by the Agrobacterium-mediated transformation is being done.