The JA Regulation Mechanism On Taxol Biosynthetic In Taxus Chinensis

Taxol,which is an effective and famous anticancer drug in curing breast carcinoma and ovarian cancer, is a kind of rare and complex terpenoid in Taxus sp.. Due to the low content, complex structure and complicated biosynthesis steps, it is hard to fulfil the emmercial requirments of taxol. JA is an efficient inducer of plant secondary metabolites, especially for terpenoids, alkaloids and flavonoids, but the usage is blocked by the side effects in inhibiting cell growth etc. Revealing regulation mechanism of the JA signal could help us to transport specific genes of transcription factors into cells to enhance taxol yield and avoid the harmless of JA molecule. Therefore, our purpose is to clarify the JA signal regulation mechanism of taxol biosynthesis in Taxus chinensis.In this study, the promoters of taxol biosynthetic genes were obtained at first, and then the key element that responds to JA signal(JRE) was verified. The transcription factors were selected according to their affinity with JRE, further functional analysis of them were conducted to clarify the downstream responses of JA signal in regulation taxol biosynthesis. Meanwhile, JAZ and MYC2, both are key factors of JA signal pathway, were analyzed to clarify the upstream of JA responses. Moreover, the functional analysis of MYC2 could link the upstream and downstream responses. Above all, a basic regulation system were concluded to clarify the activities of taxol biosynthesis responding to JA signal. The main results in this study were listed as follows:(1) The targets of JA signal were studied on the taxol biosynthetic genes firstly. Promoters of 6 taxol biosynthetic genes, TASY, T5 H, T10 H, T13 H, TAT and T7 H, were obtained by aligning with simplified Taxus baccata genome data. Promoter analysis showed that E-box, G-box and T/G-box widely presents in these promoters. All of these cis-elements are important elements in responding to JA signal, and they could be regulated by MYC2, suggesting MYC2 is a key transcription factors in regulating taxol biosynthesis by JA. Among the 6 promoters, the presence of T/G-box, series G-box and GCC-box in TASY promoter appear a high similarity with these promoters of PR, STR and PMT2, which are famous JA-responsive genes. Additionally, TASY are common considered as the determining point of the strength of taxol biosynthesis. All these results indicate TASY is the key target of JA regulation network. Thus, effective ERF and MYC2 factors of TASY are the breakpoint to elucidate JA regulation mechanism of taxol biosynthesis.(2) The TcERFs which could bind with GCC-box were selected and functionally analyzed. Deletion analysis showed that T/G-box, CGTCA-motif and GCC-box take part in TASY responding to JA, while GCC-box plays the key role. Thus, 23 ERFs which could specially bind with GCC-box were selected from transcriptome data of Taxus chinensis. Among of them, 13 ERFs were significantly responding to MeJA, 4 was upregulated and 9 downregulated, according to our expression profiling data. Afterwards, TcERF12 and TcERF15 were obtained according to the results of sequence alignment and phylogenetic analysis. TcERF12 and TcERF15 are B1 and B3 subtypes of ERF, which are repressors and activators respectively in plants. Our experiments confirmed that the two JA-responsive ERFs, Tc ERF12 and TcERF15 could repress and activate TASY by binding with GCC-box respectively.(3) TcMYC2 played the key role in upstream of JA signal pathway. Sequence analysis revealed that TcMYC2 was highly similar with AtMYC2, NtMYC2 and CrMYC2, phylogenetic analysis also group them together, indicated that TcMYC2 played a similar role as AtMYC2 did in Taxus chinensis. TcMYC2 could bind with not only T/G-box but also G-box to activate the expression of TASY gene. Moreover, overexpression of TcMYC2 also significantly promote the accumulation of other taxol biosynthetic genes, such as TAT, T5 H, T13 H, BAPT, DBAT and DBTNBT. Additionally, TcERF12/15 are positively regulated by TcMYC2, indicating there are two ways of TcMYC2 regulating taxol biosynthesis. All these results showed that TcMYC2 was an important regulator for biosynthesis of secondary metabolites in JA signal pathway.(4) TcJAZ was the key factor of upstream JA signal pathway. JAZ proteins evolved differently in angiosperm and gymnosperm plants according to our research. Thus, TcJAZ3 was selected to study the JA signal pathway in T. chinensis since it belongs to Group V JAZ proteins which presents in both angiosperm and gymnosperm, indicating a similar function in the JA regulation system. Yeast two-hybrid system certificated that TcJAZ3 could bind with TcMYC2, indicated that T. chinensis have a similar JA signal pathway as well as Arabidopsis do.Afterwards, our results described a JA regulation network consisting of TcERF12/15, TcMYC2 and TcJAZ3 in taxol biosynthesis. Interestingly, a negative regulator, TcERF12 was reported to involve in the taxol biosynthesis, indicating dual roles of JA regulation system in secondary metabolites are important for plant. Our results also lay a foundation to clarify the cross talk of JA, ET, GA, SA signals.