| Research objective: Astragalus membranaceus is a commonly used medicinal herb,the demand of which has been increased in recent years with the contradiction of reduced resource of wild stocks.To satisfy the market need and decrease the devastation of environment due to the evacuation of Astragalus,researchers are motivated towards the improvement of Astragalus membranaceus quality.In this study,cytochrome CYP450 genes possibly participate in the biosynthesis of Astragalus saponin were selected based-on Astragalus transcriptomics data and literatures.Seven CYP450 genes were cloned and three of them were separately constructed into plant expression vectors and yeast expression vecotors to study their function in the biosynthesis of Astragalus saponin,which might contribute further to upgrade the quality of Astragalus membranaceus..Methods: The transcriptome data of Astragalus membranaceus was obtained by de novo RNA sequencing.It has been reported that the CYP450 genes of 71 clan are related to the biosynthesis of saponin.Thus,CYP450 genes in 71 clan were selected based on Astragalus transcriptomics data annotated against SWISSPROT,KOG,GO,and KEGG database and referred literatures that stated the expression level of the downstream genes of astragaloside biosynthesis were consistent with that of squalene epoxidase(SE)gene.Therefore,genes consistently expressed to SE were selected that may be involved in the biosynthesis of the triterpenoids of Astragalus membranaceus applying real-time quantitative PCR(RT-q PCR).5’-/3-RACE were applied to get the full length c DNA of those genes.Am CYP82D47,Am CYP71A26,and Am CYP93A3 was analyzed by online tools such as NCBI,SOPMA,and TMHMM Server v.2.0.p CAMBIA1302 plasmid was used as the plant expression vector,and p AUR123 and p RS424 plasmids as the yeast expression vector to construct individual expression vectors for plant and yeast expression,respectively.After that,the recombinant plasmids were transformed into Saccharomyces cerevisiae and the final products were detected by HPLC.Results:(1)A total of thirty-six CYP450 genes belonging to 71 clan,possibly related to the biosynthesis of Astragalus saponins were screened and analyzed.Q-PCR were performed and fourteen genes were consistent up-regulated expressed with SE in SE-overexpression hairy root strains.The expression levels of the fourteen CYP450 genes screened in different tissues of Astragalus membranaceus were examined.Among them,9 genes were relatively high in root expression,and 4 genes were relatively high in leaves.One gene was The expression level in the stem is relatively high.(2)Three genes,Am CYP82D47,Am CYP71A26,and Am CYP93A3 were cloned.(3)Three plant expression vectors and three yeast expression vectors harboring individual candidate CYP450 genes were constructed,namely p CAM-Am CYP82D47,p CAM-Am CYP71A26,p CAM-Am CYP93A3,p RS424-Am CYP82D47,p RS424-Am CYP71A26,and p RS424-Am CYP93A3.The yeast recombinant plasmids were transformed into Saccharomyces cerevisiae.(4)The fermentation products of three genetically engineered strains of Am CYP82D47,Am CYP71A26,Am CYP93A3 were detected by HPLC.Conclusion:Based on the transcriptome data of Astragalus membranaceus,36 P450 genes related to the biosynthesis of Astragalus saponins were screened,and 7 full-length sequences were obtained.Then three of these genes were used to construct plant expression vector and yeast expression vector.The recombinant plasmid of the yeast expression vector was transformed in Saccharomyces cerevisiae.Further research on subsequent experiments will be conducted to explore different substrates or optimizing fermentation conditions to elucidate the function of the three CYP450 genes... |
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