1 College of Food and Health, Zhejiang A&F University, Lin'an 311300, China; 2 Zhejiang Provincial Key Laboratory of Characteristic Traditional Chinese Medicine Resources Protection and Innovative Utilization, Lin'an 311300, China; 3 National Innovation Alliance of Lingzhi Sanye Green Industry, Lin'an 311300, China
Abstract：Methyl jasmonate (MeJA) can induce the biosynthesis of Ganoderma terpenes, in order to explore the relationship between Ganoderma terpene biosynthesis and the regulation of basic helix-loop-helix (bHLH) genes and jasmonic acid (JA) signal regulation. In this study, the bHLH gene fragment related to the biosynthesis of Ganoderma terpenoids was cloned from G. lingzhi by homologous alignment. The full length of the gene was further cloned and analyzed its bioinformatics, transcriptional activity, prokaryotic expression, subcellular localization, overexpression and silencing expression. The results showed that a bHLH gene fragment with high homology with the positively regulated tanshinone synthesis transcription factor was obtained from G. lingzhi. qRT-PCR analysis showed that the gene was significantly indigenous in response to MeJA induction. The expression levels of the 3 key genes in the triterpenoid (GA) synthesis pathway of G. lingzhi were significantly increased under MeJA induction (P<0.05), and the expression pattern was consistented with bHLH. A 1 311 bp GlbHLH1 gene (GenBank No. MW981280.1) was cloned, encoding 436 amino acids with a molecular weight of 46.33 kD. Promoter analysis showed that G-box binding to bHLH transcription factor existed in the promoter regions of the 3 key genes in Ganoderma triterpene synthesis pathway, suggested that GlbHLH1 might be involved in the regulation of Ganoderma triterpene metabolism by responding to JA signal. Phylogenetic analysis showed that GlbHLH1 had the closest relationship with GsPIL37177.1 of G.sinense; the transcriptional activation activity in Saccharomyces cerevisiae showed that the gene had transcriptional activation activity. The prokaryotic expression vector of GlbHLH1 gene was successfully constructed, and the fusion protein of expected size (about 75 kD) was expressed in Escherichia coli BL21. Subcellular localization showed that GlbHLH1 was mainly located in the nucleus; the transgenic results showed that the expressions of GlbHLH1 in the overexpression and silencing strains were significantly up- and down-regulated from that in the wild type (P<0.05), the triterpenoid content in the overexpression strains OE-GlbHLH1-1, OE-GlbHLH1-2 and OE-GlbHLH1-3 increased by 25%, 22% and 38%, respectively, and that in the silencing strains Si-GlbHLH1-1 and Si-GlbHLH1-2 was decreased by about 15% and 30%, respectively. In summary, GlbHLH1 is an important transcription factor that controls the synthesis of triterpenoids in G. lingzhi. The above results provide a theoretical basis for further study on the molecular regulation mechanism of bHLH in G. lingzhi.
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