Cloning?Expression Analysis And Prokaryotic Expression Of Lignin Biosynthesis Related BnC3H-1 Gene In Boehmeria Nivea

This study of Boehmeria nivea Gaud high flux transcriptome sequencing biological information data,obtained the Boehmeria nivea coumarate-3-hydroxylase?BnC3H-1?gene cDNA sequence,designed for the sequence specific primers,combines the technique of RT-PCR and RACE cloned full length of BnC3H-1 gene sequence,named BnC3H-1,and submit the GenBank,registration number for KY078743.BnC3H-1 for bioinformatics analysis showed that BnC3H-1 gene full length cDNA sequence is 1940 bp and contains a complete open reading code box 1536 bp,5'UTR 96 bp,3'UTR 308 bp,its coding protein containing 511 amino acids,the relative molecular weight is 57.8 kDa,theory of isoelectric point of 8.61.Using BLAST online analysis,the results showed that the gene encoding protein has the Phx-x-x-Gly-x-Arg-x-Cys-x-Gly conservative structure domain,this and other species of C3H gene encoding protein structure domain,the same are CYP450 98 A family.RT-qPCR?Real-time PCR?analysis showed that in different period of ramie BnC3H-1express,xylem and phloem were expressed in the xylem is relatively high.BnC3H-1 in the same period,abundance expressed different parts in the order is:xylem>phloem;BnC3H-1in different periods,the same expression abundance of the sequence is:mature period>rapid growth period>mature late period.Presumably the genes may be formed in the lignin monomer and play a role in lignin biosynthesis.Using protein expression vector pET-22b successfully constructed prokaryotic expression recombinant plasmid pET-22b-C3H,putting the pET-22b-C3H plasmid into E.coli BL21?DE3?,best induced temperature is 37?,best revulsant tendency is 1mmol·L^-1IPTG,induce the expressed fusion protein,SDS-PAGE electrophoresis showed fusion about58 kDa protein size,size and expectations.