Cloning,Identification And Expression Patterns Of AbbHLH Transcription Factor In Agaricus Bisporus

Methyl jasmonate(MeJA)is a very active small molecule signaling substance in plants,which plays an important role in improving the nutritional quality of horticultural products after harvest and inducing the defense of plants.As an important transcription factor in plants and animals,bHLH is widely involved in the growth and development of plants and animals and various stress responses of fruits,vegetables and edible fungi.At present,the mechanism of bHLH transcription factors in JA signaling pathway has been deeply studied in plants and fruits and vegetables,but it has not been reported in edible fungi.In this study,A.bisporus,the model material of edible mushrooms,was used as the research object,and researches that carried out here are shown as followings.(1)The CDS region of bHLH gene in A.bisporus(named AbbHLH)was obtained by molecular cloning.This gene has a total length of 1599 bp and encodes 532 amino acids(aa).In addition,we performed bioinformatics analysis of this gene and its encoding protein.The concrete results were shown as follows:amino acid sequence alignment showed that AbbHLH had a high consistency with the bHLH domain in yeast and plants,containing eight conserved DNA binding sites and the conserved basic helix-loop-helix(bHLH)domain in the 67-119 aa region;The prediction of secondary and tertiary structures shows that AbbHLH is a helix-loop-helix(HLH)-containing structure;Phylogenetic tree analysis showed that AbbHLH transcription factor was closest to bHLH transcription factor in saccharomyces cerevisiae;The prediction of subcellular localization showed that AbbHLH transcription factor was located in the cell nucleus.(2)The constructed recombinant plasmid pET-28a(+)-AbbHLH was transformed into E.coli BL 21(DE3)and expressed by induction.SDS-PAGE results showed that the induced 6×His-AbbHLH fusion protein mainly existed in the form of inclusion body,and the purity of the protein was 80%after purification and renaturation.The polyclonal antibody anti-AbbHLH was obtained by immunizing New Zealand rabbits,and the titer of the antibody was determined as 102400 by indirect ELISA.Western blotting experiment showed that the antibody could specifically identify AbbHLH protein in A.bisporus tissue.Affinity purification of antibody by Protein A and CNBr was used to obtain strong specific antibody.(3)The expression patterns of AbbHLH genes and proteins in A.bisporus were detected.The results of gene expression level showed that the relative expression level of AbbHLH gene in short-term storage was significantly higher than that of 0 h storage,and reached the maximum value at 1 h,which was 25 times compared with AbbHLH gene expressed in 0 h sample.After treatment with MeJA,the expression of AbbHLH gene was significantly inhibited and the inhibition degree was different in different tissue sites(Cap,Gill and Stipe).The inhibition effect by MeJA was strongest in cap.The results of protein expression level showed that there was no significant fluctuation in AbbHLH protein expression during short-term storage,and there was no basically regulated by MeJA.(4)Predictive analysis of promoter binding sites showed that AbbHLH transcription factor could bind to the promoter regions of 12 genes related to the synthesis of ornithine and urea,with the highest degree of binding to the CACGTG motif.In agreement with theoretical predictions,EMSA also verified that AbbHLH transcription factor can directly bind to the CACGTG motif in the promoter of AbASL1、AbASL2、AbUREA、AbSAMS1、AbSAMS2 genes.In summary,the expression of AbbHLH gene was consistently high in short-term storage,and significantly inhibited by MeJA treatment.The AbbHLH transcription factor showed no significant changes in protein levels during post-harvest storage and MeJA treament.EMSA experiments showed that AbbHLH transcription factors can directly bind to the CACGTG motif.The discovery in this study will provide a theory base for solving postharvest quality of A.bisporus and exploring the role of bHLH transcription factor in the regulation of MeJA.