Screening And Cloning Analysis Of BHLH Transcription Factors Regulating Crocin Biosynthesis

Saffron is derived from the dried stigma of Crocus sativus L.,a perennial herb of the family Iridaceae.The main medicinal components of saffron are terpenoids such as crocin,crocetin,picrocrocin,safranal.The content of these substances is an important indicator for evaluating the quality of saffron.b HLH transcription factors are one of the largest transcription factor families in plants,which have been confirmed by various studies to regulate the secondary metabolism of plants by responding to jasmonic acid(JA)signaling.Analyzing the biosynthesis and metabolic regulation mechanism of saffron active components on the transcriptional level has become an important mean to reveal the regularity of saffron quality formation and increase the content of active components in the stigma of C.sativus.In this study,the transcriptome sequencing data of C.sativus was used for bioinformatics and cloning analysis of Csb HLH transcription factors,and their expression patterns and functions were studied.The main conclusions were as follows:1.Using methyl jasmonate(Me JA)to treat the stigma of C.sativus,the content of crocin was significantly increased by high performance liquid chromatography(HPLC)compared with the control group.After sequence annotation and deduplication,a total of 57 b HLH family transcription factors were screened from the C.sativus transcriptome sequencing data.The number of amino acids ranged from 180 to 699,and the physicochemical properties were analyzed as unstable hydrophilic proteins.All the 57 Csb HLH genes responded to JA signals to varying degrees.Conserved motif analysis found that Motif 2was a characteristic conserved motif of the Csb HLH transcription family.Only two Csb HLH proteins did not contain Motif 2,but contain Motif 6,which was required for b HLH family functions.Evolutionary analysis showed that the transcriptional proteins of Csb HLH could be matched with the Arabidopsis thaliana Atb HLH transcription factor family,indicating that the b HLH family proteins are highly conserved in plants and have good similarity in sequence and structural formula.The function of the C.sativus Csb HLH transcription family can be predicted according to the evolutionary distance,which provides a reference for exploring the molecular mechanism of Csb HLH regulating crocin.2.Based on bioinformatics analysis and expression analysis,four Csb HLH genes were screened to explore their regulation and mechanism of crocin.Using the Unigene obtained from the C.sativus transcriptome database as a template,four target gene amplification primers were designed respectively,and the coding sequences of Cs MYC2,Csb HLH30,Csb HLH49 and Csb HLH115 genes were successfully cloned from C.sativus,with lengths of 1 932 bp,756 bp,1 140 bp and 675 bp,respectively.Structural analysis found that the four Csb HLH transcription factors all contained secondary structures such as α-helix,extended chain,β-turn and random coil required to maintain their tertiary structure stability.The spatiotemporal expression characteristics of the four Csb HLH genes were analyzed by quantitative realtime PCR.Inducible expression analysis showed that four Csb HLH genes significantly responded to JA signal,among which Cs MYC2 and Csb HLH49 reached the highest expression levels at 3 h and 0.5 h after Me JA treatment,respectively.The Csb HLH30 gene could respond to the JA signal very significantly,and showed a trend of continuous high expression after Me JA treatment;the expression level of Csb HLH115 showed a very significant increase after 1 h of Me JA treatment,and reached the highest expression level at 3 h.Correlation coefficient analysis showed that the expression level of Csb HLH was highly correlated with the content of crocin.Tissue expression analysis showed that Cs MYC2 and Csb HLH30 had the highest expression in stigma and the lowest expression in petal;Csb HLH49 had the highest expression in leaf and lower expression in petal,stamen and stigma;Csb HLH115 had the highest expression in stamen and the lowest expression in leaf.Csb HLH genes have tissue expression specificity.The results of subcellular localization showed that the four Csb HLH transcripts were all expressed in the nucleus.3.Yeast transcriptional activation activity experiments showed that Cs MYC2,Csb HLH49,Csb HLH115 transcription factors had transcriptional autoactivation activity,while Csb HLH30 transcription factor didn’t have transcriptional autoactivation activity.Through yeast one-hybrid experiments,it was found that yeast containing Csb HLH transcription factors could grow on SD/-Leu/Ab A(700 ng/m L)/X-α-gal medium and turn blue,indicating that Csb HLH proteins could recognize and bound to G-box element.The dual-luciferase reporter assay showed that Cs MYC2,Csb HLH30,Csb HLH49 and Csb HLH115 were positive regulators,which could significantly activate the G-box element to initiate the expression of LUC gene,and they were all positive regulators.Verifying the downstream transcriptional regulatory elements that interact with Csb HLH can provide ideas for further research on the molecular mechanism of Csb HLH regulating the biosynthesis of secondary metabolites.4.In order to explore the biological functions of the cloned Cs MYC2,Csb HLH30,Csb HLH49 and Csb HLH115 genes,the p CAMBIA1304-Csb HLH overexpression vectors were constructed,and transformed into Nicotiana tabacum by Agrobacterium-mediated methods.After hygromycin(HYG)resistance screening and molecular identification,transgenic N.tabacum plants were successfully obtained.Using quantitative real-time PCR to analyze the expression of the upstream synthetic pathway enzymes of crocin in transgenic tobacco,it was found that the expression of crocin-related synthase genes changed to different degrees in Csb HLH overexpressing lines,indicating that Csb HLH transcription factor may regulate the accumulation of active components of C.sativus by regulating the genes of crocin synthesis pathway enzymes.This study provided a reference for elucidating the regulatory mechanism of transcription factors in the metabolism of terpenoids in C.sativus.