L Solation,Bioinformatics Analysis And Expression Evaluation Of C4H Gene From Asarum Sieboldii Miq.

Cinnamate 4-hydroxylase enzyme(C4H),one of the key enzymes in phenylpropanoid compounds biosynthesis pathway,catalyzs trans-cinnamic acid to transform being 4-coumaric acid.Methyleugenol,major component in Asarum sieboldii Miq.volatile oil,belong to phenylpropanoid compounds.Thus,isolation of C4 H gene and functional verification from Asarum sieboldii Miq.is theoretical basis to clarify molecular mechanisms in biosynthesis pathway,to grasp accumulation dynamic rule,to conduct genetic improvement,to enhance quality of Asarum.The sequences of homologous C4 H gene were searched from NCBI.We found conservative segment through blasting C4 H from diffrent plants,designed degenerate primer to perform reverse transcription Polymerase Chain Reaction(RT-PCR)using cDNAs as templates.An approximate850 bp fragment was obtained eventually.5’ end fragment and 3’ end fragment were successfully acquired through RACE(Rapid Amplification of cDNA Ends).A full length cDNA of AsC4 H was acquired after assembling the conservative fragment,5’ end fragment and 3’ end fragment.Bioinformatics analysis of the AsC4 H encoding proteins were performed using many softwares.Meanwhile,fluorescent quantitative PCR(RQ-PCR)was used to compare the As C4 H expression in the different periods and different parts.C4H gene was isolated,analyzed and detected expressive quantity from Asarum sieboldii Miq.The above is of significance for researching regulatory mechanism of main active ingredient in biosynthesis pathwayfrom Asarum sieboldii Miq.,genetic improvement and secondary metabolism engineering.