Functional Analysis In Triterpenoid Synthesis Of Five CYP450 Family Genes In Betula Platyphylla Suk.

Triterpenoids of the bark of birch(Betula Platyphylla Suk.),including betulin,betulinic acid and oleanolic acid have broad pharmacological activities,such as anti-tumor,anti-inflammatory,anti-HIV,anti-malarial.However,the natural secondary metabolites of birch is low,and their chemical synthesis is costly,which restricts its application in production practice.Cytochrome P450 monooxygenase(CYP450)is the largest family of enzymes involved in plant metabolism,catalyzing the triterpene skeleton to form functional groups having various functions such as a hydroxyl group,a carbonyl group,and a carboxyl group and the members of this family play keys role in the diversification and functional modifications of triterpenoid.In this study,thefull-length cDNA of BpCYP450 from birch in response to SA and MeJA signals we cloned,the expression characteristics of BpCYP450 and triterpenoid synthesis key genes were also identified.Moreover,we explored the expression characteristics and function in triterpenoid synthesis of five BpCYP450 genes in tobacco and birch.The results of this study were as follows:1.Based on the nucleotide sequence from transcriptome database of Betula platyphylla SuK.,special primers were designed.Full-length of five BpCYP450 from Betula platyphylla SuK.wereisolatedby RT-PCR reaction,named CYP94B89,CYP89S1,CYP97B62,CYP86B54 and CYP86A182.The results of bioinformatics analysis indicated that the open reading frames(ORFs)of the five genes were 1284 bp,1533 bp,1188 bp,1704 bp and 1539 bp,respectively,encoding 427,510,395,567 and 512 amino acids,respectively.They have conserved regions of CYP450 and significant transmembrane structures.The proteins encoded by CYP94B89,CYP97B62,CYP86B54 and CYP86A182 have the highest similarity to the CYP94B3,CYP97B2,CYP86B1 and CYP86A32 of Quercus suber,respectively,which are 85%,89%,81%and 86%,respectively.The protein encoded by CYP89S1 has a similarity to the Capsicum chinense CYP89A2 protein of up to 45%.Combining multiple sequence alignments and phylogenetic tree showed that the five genes obtained in this study were new members of the cytochrome CYP450 family.2.Tissue-specific and MeJA,SA,GA3,ABA,ethylene and mechanical treatment expression of BpCYP450 and triterpenoid synthesis key genes were investigated by using real-time RT-PCR.Results indicated that,the expression levels of CYP89S1 and CYP97B62 and BpSE and BpDS genes were highest in leaves,CYP86B54,CYP86A182 and BpSS,BpW and BpY were higher in roots and stems,and CYP94B89 gene was the highest in stems.The expression of BpCYP450 and triterpenoid synthesis key genes were induced by MeJA,SA,GA3,ABA,ethylene and mechanical treatment at different degrees.MeJA mainly induced the expression of BpY,CYP89S1,BpSS,BpDS,CYP97B62 and Bp W genes,SA mainly induced the expression of CYP86A182,CYP86B54 and CYP89S1 genes,GA3 mainly induced the expression of BpY,BpW and CYP89S1 genes,and ABA mainly induced the expression of CYP86B54,CYP89S1,CYP94B89 and BpY,ethylene and mechanical damage mainly induce CYP89S1 and BpY gene expression.The trend of hormone-induced co-expression of five BpCYP450 and birch sinensis synthase genes were analyzed.Results showed that,the expression of CYP94B89,CYP86B54 and BpDS were highly similar by MeJA,SA,GA3,ethylene and mechanical damage induce.The expression of CYP86A182 similar to BpW in stems by GA3 and ethylene induce.The expression of CYP89S1 in leaves similar to BpSS and BpSE by GA3 and mechanical damage induce.In addition,CYP89S1 in leaves showed similar expression trends with BpSS,BpY and BpW by SA induction.The above results indicate that the four BpCYP450 genes and the triterpene synthase gene have synergistic effects on different hormone-induced responses.3.The promoters of CYP94B89,CYP89S1,CYP86B54 and CYP86A182 were isolated by walking approach,and analysis showed that the promotersare were 778 bp,791 bp,843 bp and 1057 bp in length.The binding site and function of BpCYP450 were predicted by the PlantCare database.It was found that the promoter transcriptional elements TATA-box and CAAT-box were present in the CYP94B89,CYP89S1,CYP86B54 and CYP86A182 promoter sequences and located at multiple sites.The four CYP450 promoters has CGTCA-motif and TGACG-motif could response the MeJA elements.At the same time,the CYP89S1,CYP86B54 and CYP86A182 promoters also have GA response elements.In addition,the ABA response element ABRE in the CYP94B89 promoter sequence,the SA response element TCA-element in the CYP86B54 promoter was also found.The CYP89S1,CYP86B54 and CYP86A182 promoters all contain MBS cis-acting elements capable of binding to the MYB site.Agrobacterium tumefaciens-mediated BpCYP450 gene,promoter and transcription factor BpMYB21 were transiently expressed in N.benthamiana.The results showed that BpMYB21 transcription factor can bind to CYP94B89,CYP86B54 and CYP86A182 promoters,and up-regulate the expression of these three BpCYP450 genes.4.To verify the function of five BpCYP450 for triterpenoid synthesis,CYP94B89,CYP89S1,CYP97B62,CYP86B54,CYP86A182 and triterpenoid sinensis synthase genes BpW and BpY were co-expressed in tobacco by transient infection.The results showed that CYP97B62 and CYP86A182 were able to up-regulate the expression of BpW.The content of betulin in the over-expressed CYP97B62 and CYP86A182 genes was 3.23 mg/g and 3.15 mg/g,respectively,which was 1.50 and 1.46 times that of wild tobacco.The content of betulinic acid was 0.45 mg/g and 0.43 mg/g,respectively,which was 12.36 times and 11.91 times that of wild tobacco.The content of betulin and betulinic acid was the highest among the tobaccos co-expressed by BpW+CYP89S1+CYP97B62+CYP86B54+CYP86A182,which were 3.99 mg/g and 0.49 mg/g,respectively,which were 1.85 times and 13.62 times of wild tobacco.Among the tobaccos co-expressed by BpW+CYP89S1,the content of betulinic acid was higher than that of BpW and CYP89S1,which was 8.99 times that of wild tobacco.The above results indicate that CYP97B62 and CYP86A182 play important roles in the synthesis of betulin and betulinic acid.CYP89S1 has a function in the synthesis of betulinic acid.It is speculated that CYP89S1,CYP97B62 and CYP86A182 may promote the conversion of lupinol to betulin and betulinic acid.In the N.benthamiana co-expressing BpCYP450 and BpY genes,the content of oleanolic acid was significantly lower than that of wild tobacco.By analyzing the metabolites,it is speculated that five BpCYP450 may convert oleanolic acid into other triterpenoids.5.Eight strains of birch plants overexpressing CYP89S1 were successfully obtained,and 5 positive CYP89S1 transgenic plants were identified by PCR.The overexpression of CYP89S1 gene was the best in the CYP89S1-5 strains.The expression characteristics of key enzyme genes BpSSy BpSE,BpW,BpY and BpDS in CYP89S1-5 transgenic birch seedlings were detect by QPCR.The relative expression levels of key enzyme genes BpSS,BpSE,and BpW were up-regulated,and the up-regulation of BpW was the most obvious.The relative expression of BpDS and BpY genes was down-regulated.The above results indicated that the overexpression of CYP89S1 gene had co-expression characteristics with BpW,which was consistent with the transient expression of CYP89S1 and BpW in tobacco to promote the increase of betulinic acid.