Functional Analysis Of Transcription Factor Fst3 Of Pleurotus Ostreatus Involved In Regulation Of Fruiting Body Formation

Pleurotus ostreatus is the most widely cultivated and consumed edible fungus in China.The formation and development of fruiting body have a crucial influence on the economic value of P.ostreatus.However,study on the molecular mechanism of the fruiting body development of P.ostreatus is scarce and fragmentary,especially lacking knowledge about the key functional genes involved in fruiting body development.A transcription factor coding gene(fst3)of P.ostreatus New 831 was obtained by gene differential expression analysis in our preliminary study,which was significantly up-regulated at the stage of primordium.By protein sequence alignment,we found that the Fst3 protein from P.ostreatus has 62% identity with that from Schizophyllum commune,which indicated that the P.ostreatus fst3 gene and the S.commune fst3 gene might share similar function,participating in the formation of fruiting body.In order to clarify the mechanism of the P.ostreatus fst3 gene in the fruiting body development,the fst3 gene was overexpressed and antisense silenced,then the molecular biology and the cultivation biology differences among the wild type strains,the fst3 gene overexpressed strain(fst3+ strain)and the fst3 gene antisense silenced strain(fst3-strain)were analyzed.The main conclusions of this paper are as follows:1.By comparing the Fst3 protein of Pleurotus ostreatus,it was found that it had 62%1.By comparing the Fst3 protein of Pleurotus ostreatus,it was found that it had 62%sequence identity and 71% sequence similarity with Fst3 protein of Schizophyllum commune,respectively,which indicated that the Fst3 protein has a similar function to the fst3 gene of Schizophyllum.2.Using pPo-GPD,a paclitaxel vector constructed in our laboratory,the overexpression vector p Po-GPD-fst3+ and the antisense silencing vector p Po-GPD-fst3-were constructed.3.The p Po-GPD-fst3+ vector and p Po-GPD-fst3-vector were transformed into the mycelium or protoplasts of Pleurotus ostreatus by Agrobacterium tumefaciens-mediated genetic transformation.Several transformants were obtained by screening hygromycin resistance,PCR screening and validation of hyg,Pgpd-hyg,Pgpd-fst3+,Pgpd-fst3-were carried out for some of the proposed transformants further.Finally,16 fst3+ strains and 11 fst3-strains were obtained.4.The m RNA expression of fst3 in fst3+ and fst3-strains was detected by RT-qPCR.The results showed that the fst3 gene was up-regulated by 1.26 ~ 9.59 times in fst3+strains,and decreased by 0.01 ~ 0.30 times in fst3-strains compared with the wild type strains.The results of this part of the experiment fully proved that we successfully obtained fst3 overexpression and antisense silencing strains.5.The fst3+ and fst3-strains were cultivated by plate culture test and bagging test.The results showed that the number of primordia and type of fst3+ strains fruiting bodys were consistent with the wild type strains(fewer primordia and larger fruiting bodys).The number of primordia formed by fst3-strains were more than that of wild type strains,but fruiting bodys were smaller.The results showed that the number of primordia formed by fst3+ strains were similar to that of wild type strains,fewer primordia and primitive differentiation.The number of primordia formed by fst3-strains were significantly higher than that of wild type strains,and the primordia were homogenized,fruiting bodys were smaller.