Transcription Analysis And Prokaryotic Expression Of PNAE Gene In Rauvolfia Yunnanensis Tsiangby

Rauwolfia yunnanensis Tsiang is an important kind of medicinal plant. In our country, it is widely used because of it’s functional diversity of alkaloids. In recent years, with Rauvolfia’s medicinal value be continual developed, the resources is consumed continuously, the frequency of regeneration lag behind consumption far. To improve the Rauwolfia yunnanensis’s resource utilization,studying the metabolic pathway of alkaloids is necessary.Ajmaline plays a significant role in medicinal ingredients of Rauwolfia yunnanensis. PNAE (polyneuridine aldehyde esterase) is the key enzyme in the synthetic route of ajmaline. Studying PNAE gene expression regulation is of great significance to control the expression quantity in order to improve the generation of ajmaline. In this paper, Rauwolfia yunnanensis PNAE gene expression analysis and prokaryotic expression lay a foundation for later in-depth study of PNAE protease’s function.This research’s mainly content as follows:1) Taken stem With bud of Rauwolfia yunnanensis for tissue culture, got the aseptic seedling.2) Used leaf of Rauwolfia yunnanensis adult plantas as raw material to extract RNA, reverse transcription, RT-pcr amplified PNAE gene fragment. After purified PCR products were TA cloning, we obtained 795 bp fragments. Predicted the secondary structure of PNAE protease through bioinformatics analysis. Analysis results showed that the protein with 265 amino acid, αα helix, β angle are major components of the whole structure in the protein, a helix spreaded in the whole protein and the it was a hydrophilic protein.3) Used fluorescence quantitative PCR technology to relative quantitative RNA samples. Analysised Rauwolfia yunnanensis PNAE gene ORF sequence published on GenBank, we choosed PNAE gene conservative area, designed and synthesized a pair of specific primers.18s RNA chosen as reference genes, selected adult Rauwolfia yunnanensis plant’s roots, stems, old leaves, leaves, flowers five different organizations, tissue culture seedling’s root, stem and leaves as sample respectively, studied its expression in different organizations through the relative quantitative analysis. The data analysis results showed:the expression of PNAE gene was no obvious difference in adult plant and had no significant differences in tissue culture seedlings also.4) Constructed prokaryotic expression vector, preliminary explored the prokaryotic expression conditions of PNAE gene. Tried the conditions as follow:kind of the composition of culture medium, age of the bacteria, microbial inoculation quantity, shake bottled liquid volume, rotating speed of oscillator, revulsant IPTG concentration and induction opportunity, induction time, training and induction temperature to getting process for efficient expression of recombinant protein PNAE. PGEX-6p-1 for expression vector, BL21 strain for expression,37 ℃,160 rpm, IPTG concentration of 1 mM, after two hours of inducing expression achieved maximum, through SDS-PAGE analysis, it showed that PNAE/pGEX-6p-1 expressed well, restructuring of protein expression was about 55 KDa.Through this study, it has been clear about the RauwoIfia yunnanensis PNAE gene expression pattern, builded the PANE enzyme prokaryotic expression vector and induction training system, and as RauwoIfia yunnanensis parts alkaloid content in the analysis and determination, raw material selection to provide theoretical basis for extraction of alkaloids.