Prokaryotic Expression And Functional Analysis Of Strictosidine Synthase-like Candidate Genes CaSTRL From Camptotheca Acuminata

The Camptotheca acuminata Decne is one of the main plant sources of the anticancer drug camptothecin.Because of the exigent market needs for camptothecin,the finite natural plant resources of C.acuminata,and the low content of camptothecin in C.acuminata,it is particularly important to find a sustainable alternative for camptothecin-producing plants.Unveiling the camptothecin biosynthesis pathway can solve this problem.Strictosidine synthase is the core enzyme involved in the secondary metabolic pathway of indole alkaloids,and its expression level affects the metabolic flow of camptothecin synthesis pathway.Therefore,in order to ascertain the expression pattern of CaSTR gene annotated in C.acuminata and the structure and function of the protein encoded by the CaSTR,this study mined and screened 14 CaSTRL candidate genes from the genomic database of C.acuminata.Their protein physicochemical properties,structural features,phylogenetic relationship and gene expression patterns in different periods and different parts of C.acuminata seedlings were analyzed by bioinformatics and RT-q PCR to further screen candidate genes.The CaSTRL candidate genes were heterologously expressed using the E.coli system,and the functional characterization of the recombinant proteins were performing.The results are as follows:(1)In this study,14 STRL candidate genes were mined and screened from the genomic database of C.acuminata and named CaSTR1-14.Bioinformatics analysis found that the full length of the ORFs of the candidate genes for CaSTR1～14 were 993,990,1176,984,432,831,990,768,822,1086,1113,1128,1053,1104 bp,which respectively encodes 330,329,391,327,143,276,329,255,273,361,370,375,350,367 amino acids.Sequence structure analysis showed that nine CaSTR proteins,CaSTR1,CaSTR2,CaSTR3,CaSTR4,CaSTR7,CaSTR11,CaSTR12,CaSTR13,and CaSTR14,contained a complete six-leaf β-propeller fold structure and five typical XXX#§ conserved domains.Phylogenetic analysis showed that most of the CaSTR sequences and the SSL sequences from Vitis vinifera and Gossypium arboretum were clustered together and closely related.The results of relative gene expression analysis demonstrated that the expression of 9CaSTRL genes in distrinct tissues of C.acuminata seedlings was significantly different,with period and tissue specificity.Except for CaSTR11 and CaSTR14,all genes reached the highest expression levels in leaves at 40 days.(2)The highly expression recombinant plasmids p ET28a(+)-△CaSTR1,p ET28a(+)-CaSTR2,p ET28a(+)-CaSTR3,p ET28a(+)-CaSTR11,p ET28a(+)-CaSTR13 were constructed by codon optimization and whole gene synthesis technology,and were transformed into prokaryotic expression system Escherichia coli BL21(DE3).After denaturing(6 M urea),isolating,purifying(Ni-NTA affinity chromatography),and dilution gradient dialysis renaturation(50 mmol/L potassium phosphate buffer,0.3 mmol/L oxidized glutathione,3 mmol/L reduced glutathione,10% glycerol,6 mol/L gradually reduced to 3mol/L or 2 mol/L urea,p H = 7.0)of five target proteins expressed in the way of inclusion bodies,soluble proteins were obtained after vigourous trials.(3)The functional analysis of five CaSTRL recombinant proteins using 6 groups of substrates,which was TRY and CSE,CFLE,CFRE,CLE,LFLE or SEC respectively,showed that the recombinant proteins CaSTR2,CaSTR11 and CaSTR13 can catalyze TRY and CLE,and produce a small amounts of strictosamide.They have the capability to catalyze a sequencial condensation reaction: Pictet-Spengler condensation and dehydration condensation at the same time.