Functional Characterization And Evolutionary Analysis Of Ta Xadiene Synthase Gene In Plants

Taxa-4(5),11(12)-diene synthease(TS) is the key rate-limiting enzyme in taxol biosynthesis pathway, and can catalyze geranylgeranyl pyrophosphate(GGPP) to taxa-4(5),11(12)-diene, the basic skeleton of all taxanes. The existence of TS gene in plant is the best evidence to prove a particular plant could produce taxol. In this study, we performed transcriptome sequencing of Pseudotaxus chienii, Amentotaxus argotaenia, Torreya grandis,and predicted the TS homologous gene in those transcriptomes and the transcriptome of Corylus heterophylla Fisch. After then, the function of those TS homologous genes was identified by prokaryotic expression. After function identification, we found that only PchTS could catalyze GGPP to taxadiene. It was found that CsTSL could catalyze GGPP to verticiol in our lab previously. Does it have the evolution of TS gene in different plants? We analyze the three protein TchTS, PchTS and CsTSL using molecular docking. The result showed that there were two different active sites between CsTSL and TchTS, and the two active sites were both in the α-domain of TS. In order to recover the TS function of CsTSL, we try to substitute theα-domain of CsTSL with the α-domain of TchTS. But the α-domain substitution of CsTSL withα-domain of TchTS cannot catalyze GGPP to taxadiene. With the analysis of those candidate gene, it was found that the ancient diterpene synthase TS was not ubiquitous in plants. The main conclusions are listed as follows:1. The transcriptome sequencing of P. chienii was performed, and the TchTS homologous genes of P. chienii was predicted and identified. About 8.60 Gb sequence data was generated.Among them, 78,192 Unigenes were assembled. And 42,462 Unigenes were annotated by at least one database. With KEGG analysis, 63 Unigenes were annotated in terpene skeleton biosynthesis pathway and 19 Unigenes were annotated in diterpene biosynthesis pathway. With taxol biosynthesis pathway genes analysis in P. chienii, three taxadiene synthase-like genes,three β-phenylalanine-CoA ligase-like genes, six hydroxylase-like genes, four phenylalanineamino mutase like genes, and five acyltransferase-like genes were identified. Interestingly, a putative P. chienii taxadiene synthase(PchTS) was able to catalyze GGPP to taxadiene. Finally,we detected the chemical extraction of the bark of P. chienii, and did not found taxol. Maybe it was caused by the miss or silence of the downstream enzyme.2. The transcriptome sequencing of A. argotaenia was performed, and the TchTS homologous genes of A. argotaenia was predicted and identified. About 8.14 Gb sequence data was generated. Among them, 82,884 Unigenes were assembled. And 27,495 Unigenes were annotated by at least one database. With KEGG analysis, 43 Unigenes were annotated in terpene skeleton biosynthesis pathway and 20 Unigenes were annotated in diterpene biosynthesis pathway, as well as five GGPS genes. With TS genes analysis in A. argotaenia, 13 taxadiene synthase-like genes were identified. Then, the full length of the highest homology TSL gene AarTSL1 was obtained by RACE-PCR. Through the enzyme function analysis, it was found that AarTSL1 cannot catalyze GGPP to taxadiene. Finally, we analyzed the chemical extraction of the bark of A. argotaenia, and did not found taxol. The result showed that A.argotaenia may not synthesize taxol.3. The transcriptome sequencing of T. grandis was performed, and the TchTS homologous genes of T. grandis was predicted and identified. About 7.68 Gb sequence data was generated.Among them, 85,984 Unigenes were assembled. And 29,418 Unigenes were annotated by at least one database. With KEGG analysis, 5,893 Unigenes were annotated. With TS genes analysis in T. grandis, 3 taxadiene synthase-like genes were identified. Then, the highest homologous TSL gene TgrTSL1 was cloned. And with the enzyme function analysis, it was found that TgrTSL1 cannot catalyze GGPP to taxadiene. Finally, we checked the chemical extraction of the bark of T. grandis, and did not found taxol. The result showed that T. grandis may not synthesize taxol.4. The research of TSL gene in C. heterophylla was performed. Firstly, we obtained a TS homologous Unigene from the transcriptome of C. heterophylla. And then, the full length of the gene was obtained by RACE-PCR, and was named as CheTSL. Next, with the enzyme function analysis, it was found that CheTSL cannot catalyze GGPP to taxadiene. We foundCheTSL gene is a kaurene synthase kaurene synthase encoding gene. So far, nobody find the TS gene in C. heterophylla, so the presence of taxol in C. heterophylla is still a mystery.5. The research of the active sites of the three protein TchTS, PchTS and CsTSL was performed. Firstly, with the analysis of the three protein TchTS, PchTS and CsTSL using molecular docking with 2-fluorogeranylgeranyl diphosphate(FGP), the different active sites among them were obtained. The result showed that there were no difference between PchTS and TchTS, and two different active sites between CsTSL and TchTS, namely VAL610 of TchTS and PHE637 of CsTSL, and PHE834 of TchTS and ILE860 of CsTSL. The two active sites were both in the α-domain. So we try to substitute the α-domain of CsTSL with theα-domain of TchTS. The α-domain subsititution of CsTSL with the α-domain of TchTS cannot catalyze GGPP to taxadiene. Finally, we detected the chemical extraction of the bark of C.sinensis, and did not found taxol. In conclusion, CsTSL gene may be the mutated TS gene without TS function.