Identification And Functionalanalysis Of Growth And Development Related Non-coding MilR-1 In Pleurotus Ostreatus

MicroRNAs(miRNAs)are endogenous non-codingRNAs that are about 20-24 nt in length,binding to the UTR region of the target gene mRNA by partial base pairing to degrade the target gene mRNA or inhibit the translation of the target gene protein.In the previous study,we used four different growth and development stages of the Pleurotus ostreatus materias,in turn,are the mycelium(Myc),the primordium(Pri),the young friut body(Yfb)and the mature friut body(Mfb).By softwar spredicting the new milRNAs,the most significant milRNA was named Po-MilR-1.In this study,we learned the function of the Po-MilRNA-1 and poh-1 gene in the development of the fruiting bodies of Pleurotus ostreatus,and the results are as follows:1.The expression of Po-MilR-1 and poh-1 in the mycelium(Myc),the primordium(Pri),young friut body(Yfb),mature friut body(Mfb)stages of Pleurotus ostreatus were analyzed by qRT-PCR.The results of qRT-PCR showed that Po-MilR-1 and poh-1showed a similar spatial expression pattern.Po-MilR-1 and hydrophobe poh-1 didn’t express in mycelium,but showed expression increased in fruiting body with significant differences.When the Po-MilR-1 expression is higher,the poh-1 gene expression is lower.The relative expression level of Po-MilR-1 was about 4.6 times higher than poh-1in primordium,was about 23.5 times in young friut body,and was about 32 times in mature friut body.The results led to a speculation that Po-MilR-1 may inhibit the expression of hydrophobin poh-1 and participate in the development of fruiting bodies.2.There were four Po-MilR-1 precursor loci in Pleurtus ostreatus,according to the mfold analysis,it was found that a precursor sequence could form a stable secondary structure,and its minimum free energy of the hairpin was-49.3 Kcal / mol,which is 272 bp in leagth to be named pre-Po-MilR-1.The DNA sequence of poh-1 gene consist of four introns and three exons was 518 bp and the c DNA sequence was 342 bp in length.The gene encodes 113 amino acids including 8 cysteic acid.By predicting the amino acid domain,we found that this gene is a member of the hydrophobin superfamily.The result of phylogenetic analysis revealed that poh-1 gene from Pleurotus ostreatus had the closest evolutionary relationship with Pholiota nameko.3.The overexpression vector of pGEH-gpd-pre-Po-MilR-1,pGEH-gpd-poh-1 were constructed.Agrobacterium tumefaciens mediated genes to infect the mycelia of Pleurotus ostreatus,and the positive transformants were verified by PCR,qRT-PCR,report gene expression test and Southern blot.Quantitative analysis of qRT-PCR showed that the expression of overexpressing transformants was higher than wild type strains.The maximum expression level in the poh-1 overexpression transformants was about 4.2-fold higher than the wild-type strain.The maximum expression level in the Po-MilR-1 overexpression transformants was about 3.8-fold higher than the wild-type strain.4.The growth rate of Po-MilR-1 overexpressed transformants was slower than that wild type,which indicated that overexpression of Po-MilR-1 affected the growth rate of Pleurotus ostreatus hyphae.In the high temperature stress experiments,poh-1overexpression transformants showed higher thermal stability than wild type.