Mining And Analysis Of Key Enzyme Genes In Camptothecin Biosynthesis Pathway

Camptotheca acuminata is a traditional medicinal plant in China,and its main physiologically active substance is camptothecin.Camptothecin has precious medical value and is mainly used for the treatment of gastric cancer,colon cancer,rectal cancer and bladder cancer.Because of the low accumulation of camptothecin in various plant tissues,which leads to its increased needs and expensive price and thus adequate supply in the medical market could not be met.Therefore,study on the biosynthetic pathway of camptothecin can help to increase the yield of camptothecin.In the biosynthetic pathway of camptothecin,various cytochrome P450 monooxygenases(P450s)catalyze most reactions(including hydroxylation,oxidation,carbon-carbon bond cleavage and dehydrogenation reactions).The catalytic activity of most P450 s depends on their reducing partner,cytochrome P450reductase(CPR).Therefore,CPR is of great significance for the study of the biosynthesis pathway of camptothecin.The main contents in our paper are as follows:1.Two CPR gene sequences in Camptotheca acuminata were screened by co-expression analysis transcriptome data and homologous alignment.Specific primers were designed according to the gene sequence,and the c DNAs of suspension cells and young leaves from Camptotheca acuminata were used as templates.PCR technology was used to clone the open reading frames of two CPRs in Camptotheca acuminata.At the same time,the basic characteristics of CPR genes were analyzed by bioinformatic method.The coding sequences of two CPRs in Camptotheca acuminata are separated by 17 introns into 18 exons.In addition,the structural physicochemical properties of the protein of Camptotheca acuminata were analyzed by online protein analysis software.It is found that the two CPRs in Camptotheca acuminata mainly consist of α-helix,random coil and extended chain.Both CPR proteins of Camptotheca have a transmembrane helix region.Multiple alignment of the amino acid sequence of CPR by DNAMAN 6.0 showed that both CPRs in Camptotheca acuminata contain membrane anchoring domain,FMN binding domain,P450 binding domain,cytochrome c binding domain,FAD binding domain and NADPH binding domain.Based on phylogenetic tree analysis,the CPR of Camptotheca acuminata is divided into two categories.Ca CPR1 belongs to class I and Ca CPR2 belongs to class II.2.The open reading frame of the secologanin synthase gene was constructed into the Bam H I/Xho I site of the galactose-inducible yeast double expression vector p ESC-Leu2 d to obtain the recombinant plasmid p ESC-Leu2d-CYP72.Then,Ca CPR1 and Ca CPR2 were inserted into the Not I/Spe I site of the recombinant plasmid p ESC-Leu2d-CYP72,respectively,to obtain the yeast expression vectors p ESC-Leu2d-Ca CRR1-CYP72 and p ESC-Leu2d-Ca CRR2-CYP72.Finally,the vectors p ESC-Leu2 d,p ESC-Leu2d-Ca CPR1,p ESC-Leu2d-Ca CPR2,p ESC-Leu2d-Ca CRR1-CYP72,p ESC-Leu2d-Ca CRR2-CYP72 and p ESC-Leu2d-CYP72 were separately transferred into yeast strain MKP-0 for induction and expression,The enzymatic reaction was carried out with loganin as substrate.Qualitative analysis of yeast extract by HPLC-MS and quantitative analysis of yeast extract by HPLC showed that both Ca CPR1 and Ca CPR2 could provide electrons for P450 monooxygenase and assist P450 monooxygenase to complete catalytic function in vivo.And the substrate conversion rates of Ca CPR1 and Ca CPR2 were 23.09% and 35.23%,respectively.3.This study examined the specific expression of Ca CPRs in different tissues of wild Camptotheca acuminata.The expression level of Ca CPR1 in leaves and roots is about 2-3times higher than that in stems,and the lowest expression in stem apex.Ca CPR2 has the highest expression in young leaves,followed by roots,low expression in mature leaves and stems,and almost no detectable expression in shoot apex.In general,Ca CPR1 has a similar expression pattern to Ca CPR2.In addition,by detecting SA and Me JA-induced changes in the transcription levels of key enzyme genes and P450 reductase genes in the camptothecin biosynthesis pathway in different tissues of Camptotheca acuminata,it is found that the expression levels of Ca CPR1 is the same as Class I CPR in other plants and little change was shown during the induction process.Ca CPR1 may be a constitutively expressed gene.Ca CPR2 is able to respond to the stimulation of salicylic acid and methyl jasmonate,and the expression level changes significantly.Ca CPR2 may belong to the inducible expression gene.