Isolation And Founctional Preliminary Analysis Of JAZ3 And JAZ4 Gene In Ophiorrhiza Pumila

Ophiorrhiza pumila,belonging to Rubiaceae Ophiorrhiza,is a perennial herb,which contains camptothecin(CPT)and its derivatives,which are major ingredients used in anti-cancer drug.The camptothecin-derived drugs are now widely used in the clinical treatment of cancer,however,the drug source shortage has become a huge bottleneck in the development and application of CPT compounds.Currently,with the development of plant secondary metabolic engineering,which aims to solve the shortage of drug source and improve the content of medicinal ingredients,has proved to be a good method.In this study,through transcriptome sequencing of plantlet roots and hariy roots,we screened out the genes that may be involved in CPT biosynthesis and then conduct a preliminary study of thier functions.According to analysis of the O.pumila transcriptome annotation data,we isolated6 Op JAZs,with which we do further analysis by comparing their RPKM number reported in two transcriptome databases,finally we found 4 of them showed a significant difference.In the following experiment we studied the correlation between the gene expression level of Op JAZs and the CPT content,and the correlation coefficient r is calculated by the Person test.And the result showed that OpJAZ3 and OpJAZ4 were able to participate in the regulation of CPT.The over-expression vector of OpJAZ3 and OpJAZ4 were constructed at the same time,through genetic transformation we successfully attained several positive transgenic hairy root lines.Analysis of those them showed the content of CPT did not change significantly among those over-expressing OpJAZ3 lines,while in the media there existed CPT efflux.As for over-expression OpJAZ3,it has a significant effect on the loganin and tryptamine,which were intermediates in CPT synthesis pathway.By over-expressing OpJAZ3 gene,the content of loganin along with tryptamin were increased and decreased respectively.The q RT-PCR analysis of key enzymes in CPT synthesis pathway showed that enzyme genes expression were up or down regulated by the gene transtrfer.This suggests that OpJAZ3 has an effect on multiple transcription factors,and that transcription factors maybe have different effects on CPT synthesis pathways.Over-expression of OpJAZ4 has a certain effect on the synthesis of CPT,which can improve the CPT efflux.Moreover,in the OpJAZ4over-expression lines,the content of loganin was significantly up-regulated but reduced as for tryptamine.Meanwhile,the expression of key enzymes in CPTpathway was detected.It was found that some of the enzymes in the loganin synthesis pathway were significantly down-regulated,which maybe due to the feedback inhibition caused by loganin accumulation.And the tryptamine synthesis key enzymes were significantly declined.At present,it has been widely reported that JAZs are able to interact with the MYC2 gene.Our q RT-PCR result showed that Op MYC2-LIKE1 gene was significantly down-regulated in transgenic hairy roots.The yeast double-hybrid experiments showed that OpJAZ3 and OpJAZ4 could interact with Op MYC2-LIKE1,indicating that the Op MYC2-LIKE1 was the downstream target gene of OpJAZ3 and OpJAZ4.