Molecular Cloning,Expression Characteristics,and Function Of BpbHLH7 And BpbHLH8 Genes From Birch(Betula Platyphylla SuK.)

Triterpenoids in the bark from brich?Betula Platyphylla Suk.?have broad pharmacological activities,among which botulin and betulic acid have the effect of anti-tumor and anti-AIDS.However,under natural circumstances,the content of triterpenoids in the birch skin is low and the cost of in vitro synthesis is high.Therefore,it is particularly important to use genetic engineering techniques to explore the method of synthesis and production of triterpenoids.Transcription factors can regulate metabolic pathways in their entirety from"multipoint regulation",thereby increasing the content of target metabolites in plants.The bHLH transcription factor is one of the transcription factors involved in the synthesis of terpenoids and in response to MeJA and SA signaling.In this study,the full-length cDNA and promoters of BpbHLH7 and BpbHLH8 from birch were cloned,and we also investigated their expression patterns in diffetenr tissues and under different hormone treatments.Moreover,the function of BpbHLH7 and BpbHLH8 in transgenic yeast and in birch have been identified.The study provided technical support and theoretical basis for metabolic engineering of brich tri terpenoids.The results of this study were as follows:1.Based on the nucleotide sequence from transcriptome database of birch,Full-length of BpbHLH7 and BpbHLH8 from birch were isolated by RT-PCR.A BpbHLH7 cDNA sequence length contained an 168 bp ORF encoding 205 amino acids,and a BpbHLH8 cDNA sequence length contained an 1203 bp ORF encoding 400 amino acids.Both two bHLH-deduced proteins contained a helix-loop-helix domain,E-box and N-box existed in the vicinity,a-helixes,extended strands,and random coils,with hydrophilic and lability.Two amino acid sequences were similar to bHLH family protein?GenBankID:XP₀18817560.1?and?GenBankID:XP₀18813766.1?of.Juglans regia respectively,with a maximum of the amino acid sequence is similarity to bHLH family protein of Juglans regia,up to 67%and 77%.The phylogenetic tree indicated that the encoded amino acid sequences of BpbHLH7 and BpbHLH8 belonged to the same branch as the bHLH transcription factor phylogenetic tree of walnut {Juglans regia),respectively.BpbHLH7 and BpbHLH8 genes obtained in this study may be new members of the bHLH family of birch.2.Tissue-specific and phytohormone treatment expression of BpbHLH7 and BpbHLH8 were investigated by real-time RT-PCR.Results indicated that from mid-June to mid-September,the expression of BpbHLH7 and BpbHLH8 reach to the highest in mid-August and in mid-July.For tissue-specific expression,the expression levels of BpbHLH7 and BpbHLH8 were higher in stems and leaves than roots.The Responses of BpbHLH7 and BpbHLH8 to different abiotic stresses were diffenrent.As for BpbHLH7,the effect of ethylene treatment on 48 h was the most significant than that of other treatments,and the expression levels of BpbHLH7 induced by various stresses rised to the top at different time point.The expression of BpbHLH8 reached the highest level at 12 h after treatment with ABA,JA,SA,GAs and leaf injury treatment,meanwhile,under the treatment of ethylene 48 h,the expression level of BpbHLH8 reach to the highest.3.The promoters of BpbHLH7 and BpbHLH8 were isolated by using genome walking approach,and analysis showed that the promoters are 915 bp and 1285 bp in length and contained multiple eukaryotic cis-acting elements,including TATA-box and CAAT-box,hormone response elements,response elements of defense and stress,such as ABRE-motif,CGTCA-motif.Meanwhile,there was a GARE-motif and a MBS-motif which can bind to MYB transcription factors in BpbHLH8 promoter.In addition,both BpbHLH7 and BpbHLH8 promoters have active functions.4.The yeast expression vectors of BpbHLH7 and BpbHLH8 were constructed using the pYES3 plasmid,and the INVScl-pYES2-5S and INVScl-pYES2-SE obtained previously in the laboratory were transformed into INVScl to obtain recombinant yeast cells.The content and squalene epoxidase activity were examined.The results showed that when compared to other recombinant yeasts,the total triterpene content and squalene epoxidase activity were significantly increased compared to the control when SE and BpbHLH8 were simultaneously transferred,which were 1.15 and 2.76 times higher than those of the control yeast,respectively;at the same time,the squalene content increased most significantly,reaching 11.02 times of the control when SS and BpbHLH8 were transferred.These results indicate that the BpbHLH7 and BpbHLH8 genes are participated in the synthesis of squalene and triterpenoids.5.The over-expression and interference vectors of BpbHLH8 were constructed,named pCAMBIA1303-BpbHLH8 and pRNAi-GG-BpbHLH8.The obtained vector was transferred to Agrobacterium tumefaciens LBA4404,and the pCAMBIA1303-BpbHLH8 and pRNAi-GG-BpbHLH8 were transiently infected by Agrobacterium and achieved transient expression of BpbHLH8 in birch plants.The study found that,in the birch of pCAMBIA1303-BpbHLH8,the BpbHLH8 gene was up-regulated and peaked at 48 h,which was 7.17 times that of the control;the relative expression levels of key enzymes genes?SS,SE,DS,BPW,HMGR,BPX2?of triterpene pathways showed up-regulation of different degrees,among which the expression of SE,was most significantly up-regulated,and its expression peaked at 48 h,11.26 times of the control;the total triterpenoid content analysis results showed that the content of triterpenoid in white birch rose to the highest at 24 h,which was 1.25 times that of the control.In the birch of pRNAi-GG-BpbHLH8,the relative expression levels of BpbHLH8 and key enzymes genes?SS,SE,DS,BPW,HMGR,BPX2?of triterpene pathways were decreased in varying degrees,among the SS gene was the most significant,its expression level was only 34%of the control at 72 hour.