Cloning And Functional Analysis Of P-hydroxybenzoate M-geranyltransferase In Arnebia Euchroma (Royle) Johnst

Shikonin derivatives belonging to naphthoquinone compounds, are not only a group of important secondary metabolites in the comfrey plant, but main active ingredients of a traditional Chinese herb Zicao, which has the effect of anti-inflammatory, antioxidant, anti-cancer, liver protection, inhibition on DNA topoisomerase etc. Shikonin derivatives are composed of p-hydroxybenzoate (PHBA) and an isoprenoid moiety derived from geranyl pyrophosphate (GPP). PHB A is synthesized through the phenylpropanoid (PP) pathway, whereas GPP can be synthesized through the cytosolic mevalonate (MVA) pathway or plastid 2-Cmethyl-D-erythritol-4-phosphate (MEP) pathway. Enzyme p-hydroxybenzoate m-geranyltransferase (PGT) catalyses the coupling of GPP and PHB to yield m-geranyl-p-hydroxybenzoate (GBA), the precursors of shikonin derivatives. Previous research had revealed that the activity of the enzyme correlated closely with shikonin production. Therefore, excavation and clone new PGT genes can lay the foundation for the regulation of the production of shikonin derivatives using cell cultures and construction of engineered strain that can produce shikonin derivatives efficiently. In the preceding work of our project group, transcriptome of Arnebia euchroma was completely sequenced and then built a transcriptome database. And on this basis, this research intend to clone PGT genes and study the functions of them.Objective:(1) Obtaining full-length sequences of PGT from transcriptome database and providing a target gene for revealing the catalytic mechanism and identifying function; (2) Identifying function of candidate PGT genes by enzymatic reaction in vitro and ferment cultivation of yeast strains for protein expression. In this way, laying the foundation for elucidating the enzymatic characteristics of PGT and shikonin biosynthetic pathway; (3) Revealing the important significance of PGT to the biosynthesis of shikonin derivatives by detecting the expressional differences of PGTs between different cell lines, different tissues and organs, different treatments. (4) Sheding light on the absense of the catalytic function of several homologous sequences by constructing chimeric protein and verifying the functions; (5) Transient expression in Arabidopsis mesophyll protoplasts was used to determine the subcellular localization of AePGTs. And then trying to clarify the correlations between the MEP pathway and MVA pathwy in the generation of shikonin derivatives.The research contents are as follows:(1) 17 PGT genes were firstly gained from transcriptome database of Arnebia euchroma, eventually narrowing down the candidate genes to 8. Then bioinformatically analyze the full-length sequence of the 8 candidate genes to lay the groundwork for the cloning and function analysis of PGT genes.(2) 6 candidate genes and 1 known PGT genes were successfully cloned, named AePGTl, AePGT2, AePGT3, AePGT4, AePGTS, AePGT6, AePGT. The full-length of these 7 PGT genes were cloned into vector pESC-His and transformed to yeast strains HMT1. After the corresponding proteins were expressed, enzymatic reactions in vitro were carried out with GPP and PHBA as substrates in order to identify function of candidate genes, while the fermentation of yeast were dectected to verify results. The results showed that only AePGT4, AePGT6, AePGT have biological catalysis to cause GPP react with PHBA to form GBA. Through the above study. PGT genes which can encode proteins with biological activity were cloned, which laid a strong foundation for studying the characteristics of enzyme and shikonin biosynthetic pathway.(3) Detecting the expressional differences of AePGTs between shikonin-proficient and shikonin-deficient cell lines, overground segment and underground segment of plants. The results showed that the overall AePGTs expression levels of shikonin-proficient cell lines and underground segment of plants which contained more shikonin derivatives were significantly higher than that of shikonin-deficient cell lines and overground segment. Besides, after treated with MeJA, the expression level of AePGTs increased markedly, especially in shikonin-proficient cell lines. These results strongly suggest the positive correlations exist between expression levels of AePGTs and shikonin derivatives biosynthesis.(4) Sequence alignment showed the N-terminal of AePGT2, AePGT3, AePGT5 lacked about 50～60 amino acids compared with the catalytically active PGTs. So the missing N-terminal Sequence were merged with the full-length of the 3 inactive AePGTs and got 3 new chimeric genes, AePGT-Cp2, AePGT-Cp3, AePGT-Cp5. Next, heterogenous expression and enzymatic reaction in vitro were performed. The results of enzymatic reaction suggested all 3 chimeric proteins could catalyze the formation of GBA. Therefore, it is reasonable to presume that the missing N-terminal Sequence may contains an important domain related to substrates binding.(5) Transient expression in Arabidopsis mesophyll protoplasts was used to determine the subcellular localization of AePGTs. But unfortunately, the results showed that AePGT4, AePGT6, AePGT didn’t located in plastid memebrans. However, combinding with the location of PP pathway and previous or current subcellular localization analysis in mitochondria and plastids, we deduced that PGT localized in the endoplasmic reticulum.