| Taxol is a valuable secondary metabolite which was mainly isolated from Taxus species.It is in great demand in the market because of its special effects to cancer.However,the biosynthesis of taxol is complex and it also occurs at very low concentrations in Taxus.Meanwhile,it is unclear for its metabolic regulatory mechanism,there is still no effective measures to greatly promote the biosynthesis of taxol.Therefore,it is of great significance for the massive acquisition of taxol to elucidate the regulation mechanism of its biosynthesis.Previously,miR5298 b was screened out to significantly promote the biosynthesis of taxol.In this study,the mechanism of miR5298 b regulating taxol biosynthesis was explored.The main results are as follows:(1)Mi R5298 b could promote the accumulation of taxol,and TcNPR3 and TcWRKY33 were proved to be the direct target gene and key protien of miR5298 b,respectively.It was found that the content of taxol was increased or reduced to 120% and86% respectively when miR5298 b was overexpressed and interfered by Mimicry methods in Taxus chinensis.These results showed that miR5298 b was a positive regulator of taxol biosynthesis,and miR5298 b could activate the expression of DBAT,TASY,T5 H,T10H,T13 H and TAT,thereby increasing the content of taxol.Through the analysis of miR5298 b regulatory network by omics data and bioinformatics analysis,it was found that TcNPR3 and TcWRKY33 constituted the key node of this network,and miR5298 b could downregulated or upregulated their expression respectively.Degradome Sequencing and co-expression assays demonstrated that miR5298 b could splice between the 10 th to 11 th bases of the complementary sequence of TcNPR3,thereby downregulating its expression.However,degradation fragment of TcWRKY33 was not detected of the in the degradation group of miR5298 b.These results suggested that TcNPR3 and TcWRKY33 were the key target regulator and important key regulator in the taxol regulatory network dominated by miR5298 b,respectively.(2)The direct target regulator TcNPR3 could form protein complex with TcTGA6,and transduct SA signals and participate in the regulation of taxol biosynthesis.TcNPR3 was proved to negatively regulate the expression of DBAT,TASY,T5 H and DBTNBT,leading to the reduction of accumulation of taxol by verification of overexpression and RNAi results.TcNPR3 was closely related to At NPR3/4,and shared the same NPR1＿like＿C domain,ANK domain and the EAR motif,suggesting that TcNPR3 also played a role in SA signaling pathway and inhibition to downstream response in Taxus chinensis.Yeast two-hybrid and Bi FC assays showed that TcNPR3 could interact with TcTGA6 independent of SA,and it had a similar interaction mechanism with the reported NPR-TGA complexes.Further,it was proved that TcTGA6 could inhibit the accumulation of taxol by downregulating the expression of several taxol biosynthesis genes via binding to the TGACG-motif in the promoters of those genes such as TASY.Interestingly,further studies showed that the TcNPR3-TcTGA6 complex could play a stronger inhibitory function,which indicated a new mechanism of SA signal transduction in Taxus chinensis.Meanwhile,overexpression of miR5298 b indeed increased the activity of reporter gene by weakening the inhibitory effect of this complex.After SA treatment,taxol biosynthesis genes such as DBAT,TASY,T5 H,T10H,TBT,DBTNBT and TAT were also up-regulated,which was similar to the effect of miR5298 b overexpression on enzyme genes.These results suggested that miR5298 b could reduce the strong inhibitory effects of TcNPR3-TcTGA6 protein complex by degrading TcNPR3,thus mediating SA signal transduction pathway,and then up-regulating the expression of taxol biosynthesis genes.(3)The key protein TcWRKY33 was not only regulated by miR5298 b,but also response to SA signals,and then promoted the accumulation of taxol.Degradation sequence and sequence analysis results showed that TcWRKY33 could not be directly targeted by miR5298 b,but the result of q RT-PCR showed that the expression level of TcWRKY33 was positively regulated by miR5298 b and significantly responded positively to SA signal,suggesting that it was most likely regulated by miR5298 b mediated SA signal.Functional studies have shown that TcWRKY33 activated the expression of DBAT,TASY,T2 H,T5H,T13 H,DBTNBT and TAT by binding to W-boxes in their promoters,thus promoting the accumulation of taxol.Therefore,TcWRKY33 was considered as a SA-responsive factor,activated by miR5298 b and then promoted the biosynthesis of taxol.In conclusion,this work elucidated two pathways by which miR5298 b mediated SA signaling to regulate taxol biosynthesis.Mi R5298 b mediated the SA signaling pathway by degrading the m RNA of the target gene TcNPR3.The decreased expression of TcNPR3 weakened the inhibitory effect of TcNPR3-TcTGA6 protein complex on the downstream genes,so as to increase the expression of taxol biosynthesis genes and promote the accumulation of taxol.In the meanwhile,miR5298 b mediated SA signaling pathway and then indirectly activated the expression of TcWRKY33,which was the downstream response factor of SA signals,thus further up-regulating the expression of taxol biosynthesis genes and promoting the accumulation of taxol.This study not only enrich the transcriptional regulation mode of taxol,but also lay a certain foundation for the establishment of high yield transgenic plants of taxol in the future. |
