| Flammulina velutipe is a large scale basidiomycete of typical edible fungi,the main edible part is the stipe.Regulatory factors and regulation mechanism of stipe elongation is unclear.Identification of transcriptional factors regulating the elongation of stipe and analysis of their functions are important for suggesting the molecular mechanism of stipe elongation.In this study,the model of the stipe elongation of Flammulina velutipes was first measured,and the transcription factors,JMHY1,WC1 and WC2,which could regulate the elongation of the stipe during the rapid elongation period,were screened at the time.RNA interference and overexpression are used to verify its function,and RNA-Seq sequencing is used to analyze the possible downstream metabolic pathways of the three transcription factors.The molecular mechanism of the transcription factor JMHYl and WC1/2 to regulate the elongation of the stipe is preliminarily revealed.The main results are as follows:1.The elongation at different parts of the stipe was measured at different times.The results showed that the fastest stipe elongation was observed on the 6th to the 8th day after the formation of the primordium,and the highest elongation was observed on the upper part of the stipe;qRT-PCR screening was performed in stipe elongation transcription factors that have been reported to obtain three transcription factors that may regulate stipe elongation of F.velutipes:JMHY1,WC1,and WC2.2.Cloning and bioinformatics analysis of the candidate transcription factors showed:JMHY1 which was 4154bp in length,was located between 105036-109189 on scaffold19.It contained 8 introns and 9 exons and encoded 936 amino acids.The predicted molecular weight is 102.884 KDa,the isoelectric point is 8.75,and the protein secondary structure is mainly α-helix;besides,it contains the JmjN domain.WC1 was located between 85287-87272 on scaffold429,and it was 1995bp in length.There were two introns and three exons,which could encode 627 amino acids.The predicted molecular weight was 68.056KDa,and the isoelectric point was 6.25,protein two.The structure is mainly α-helix,and contains PAS domain.WC2 is located in 49755-50873 on scaffold663 and has a full length of 1071 bp.It contains 3 introns and 4 exons and encodes 303 amino acids.The predicted molecular weight is 33.499 KDa,the isoelectric point is 6.90,and its protein secondary structure is mainlyβ-sheet;it contains GTAT zinc finger domain.3.The analysis of the interference and overexpression functions of the candidate transcription factors showed:Through agrobacterium tumefaciens-mediated transformation assay,8 positive transformants were screened.The overexpression transformants were:JMHY1-OE26,JMHY1-OE30,WC1-OE4,and WC2-OE4;interference transformants were:JMHY1-Ri4,JMHY1-Ri21,WC1-Ri2,and WC2-Ri1.The PCR verified that the eight transformants all had a target fragment inserted into the genome.The mycelial growth rate of JMHY1 RNAi transformants was slow,the primordia apppear 2 days later than the wild-type strain,the stipe elongation rate was obviously slower.There was no significant difference of mycelial growth rate between JMHY1 overexpression transformants and wild-type strain.The phenotypes of transformants of WC1 and WC2 were similar.Compared to wild strains,the primordia were less and stipe elongation slowly after interference.Oppositely,the primordia were more and the fruitbody was neat and thick after overexpression.4.RNA-seq was performed on the transformed strains to analyze the metabolic activities that may be regulated by downstream genes.The downstream regulatory factors of JMHY1 was enriched into the MAPK signaling pathway,and it was speculated that it may regulate stipe elongation through regulating the expression of multiple genes in this signaling pathway.The downstream regulatory genes of WC1 and WC2 may be involved in the regulation of the synthesis of secondary metabolites in cells,thus regulating the stipe elongation. |
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