Expression Of Glycosyltransferase Gene And Cytochrome P450 Gene In

Objective: Paris polyphylla var. yunnanensis was a kind of local medicine in Yunnan province, which was the main part of the famous Chinese patent medicines such as Yunnan Baiyao, Gongxuening Capsule and so on, and had a high economic value and market prospects. Further more, both cytochrome p450 and glycosyltransferase were significant for the Mevalonate Pathway of the active ingredients of Paris polyphylla var.yunnanensis——synthesis of steroidsaponins. In this paper, glycosyltransferase gene and cytochrome P450 gene in Paris polyphylla var. yunnanensis were cloned,studied and layed the foundation for researching subsequent artificial regulation of Paris polyphylla var. yunnanensis secondary metabolite biosynthetic.Method 1. Cloning: Making analysis and acquisition of the sequence of cytochrome p450 gene and glycosyltransferase gene from the Paris polyphylla var. yunnanensis c DNA library, while primers were designed by the sequence. A template was established from a first strand c DNA by extracting RNA from Paris polyphylla var. yunnanensis and making a reverse transcription. And then the full length of the sequence was obtained by carrying out the PCR from the template. The PCR product purification was connected to a cloning vector, which was transformed into the Trans1- T1 Escherichia coli cells, and the colony PCR test was sequenced.2.Prokaryotic expression: Firstly, the purified PCR product was connected to a expression vector, and was transformed into the Trans1- a large number of T1 Escherichia coli cells. The colony PCR test was sequenced, and then the positive strains of plasmid was extracted. Getting into Trans BL21(DE3) expression of Escherichia coli was induced, and extracting the total protein was extracted and was validated by SDS-PAGE electrophoresis.3. Real-time fluorescent quantitative PCR primers: Making extractions of RNA for the parts of Paris polyphylla var. yunnanensis such as roots, stems and leaves, and reversing transcription to form their first c DNA chain. Actin1 n gene was choosed as internal gene for the real-time fluorescent quantitative PCR.4. Build the construction of eukaryotic expression vector: Making the pPIC9 K carrier,which was accessed into the top 10 E. coli for a lot of preparation, then plasmid extraction. The preparation of yeast GS115 cells, a carrier p PIC9 K was builded and the experimental result was identificated by PCR.Result: Through a series of experiments, the whole length of the glycosyltransferase gene of the plant Paris polyphylla var. yunnanensis was acquired as 870 bp, and coded 289 amino acids. And the protein molecular weight of the gene was 31532.6D, the theoretical potential was 5.63,and it was a hydrophobic protein molecule. Moreover, it had high homology with the glycosyltransferase gene of Amborella plants. They were sharing the same transmembrane region. Prokaryotic expression vector of glycosyltransferase gene was established completely and the protein expression was obtained in E.coli. It showed that there were specific proteins in 31.5KD by SDS-PAGE electrophoresis. The results of real time fluorescent quantitative PCR were showed that the expression levels in the root and in the leaves were rather similar, and two times more than it in the stem.Through a series of experiments, the whole length of the cytochrome p450 of the plant Paris polyphyllais was acquired as 1518 bp and coded 505 amino acids. And the protein molecular weight of the gene was 57735.2D, the theoretical potential was 7.68,and it was a hydrophilic protein molecule. Moreover, it had high homology with the cytochrome p450 gene of date palms and oil palm plants. they were sharing the same transmembrane region.Prokaryotic expression vector of cytochrome p450 gene was established completely and the expression was obtained in E.coli. It showed that there were specific proteins in 57.7KD by SDS-PAGE electrophoresis. The results of real time fluorescent quantitative PCR were showed that the expression levels in the root were the highest, more than twice expression in stems and leaves.Conclusion:In this paper, the whole length of both the glycosyltransferase gene and the cytochrome p450 gene of the plant Paris polyphylla var. yunnanensis were cloned completely. While the prokaryotic expression system had been established successfully. And a reasonable method and basis were offered in other researches of the glycosyltransferase gene and cytochrome p450 gene. Meanwhile, the eukaryotic expression system of the cytochrome p450 gene which was built successfully, and it was provided some references for the further studies of expressions of other genes in eukaryotic cells. The result of real time fluorescence quantitative PCR showed that the expression level of the two genes were the highest in medicated part of plant Paris polyphylla var. yunnanensis ’s root, and it offered some research basis for the further studies in the active component composition of the plant Paris polyphylla var.yunnanensis in the future.