Chemical Control Technique In The Study On Radix Scutellariae And Baicalin Biosynthesis Gene Cloning

Scutellaria baicalensis Georgi is a commonly used Chinese herbal medicines, and its main active compounds is flavonoids, including baicalin, baicalein,wogonoside, wogonin and other factors. The content of active compounds is an important indicator to measure the herbal medicine quality affected by many factors, including developmental stage, environmental and cultivation conditions in the cultivation process. How to optimize these factors to improve the content of medicinal active compounds is one of the hot recently research. This study get around the Chinese herbal harvest research, analyze the impact of different growth stages in the quality, production and accumulation of active compounds, and analyze the correlation of critical gene expression levels and the accumulation of the active compounds, and study the mechanism of phenological phase on the growing development and active compounds in S.baicalensis. Based on the above results,we determine the optimal harvesting period and critical period of the active compound accumulation. So we choose the "daodi" area Chengde of S.baicalensis to conduct tests, to establish a suitable chemical control of production procedures. We analyze the key enzyme genes of the biosynthetic pathway of baicalin resolution, clone and express recombinant protein, and verify their function to build production S.baicalensis active compounds engineered bacteria.In this study, based on the above purpose and content, we get the following conclusions:1.Establishing the optimum growth phenophase and harvest period of S. baicalensisHerbal records the root of S.baicalensis is adopted in February and August, violently dry, but on inconclusive authoritatively. In this study, the pattern and internal quality of S.baicalensis in different growth stages are studied by chemistry, ecology, plant phenology and statistical means of multi-disciplinary, combined with the relationship of climate, soil and other environmental factors and its production and quality.We found that Seedlings and Withered periods may be the key phenological phase affecting the growing development and active compounds in S.baicalensis. As the biomass of the underground sections and the content of active compounds of S.baicalensis are both high, the Withered could be the most suitable harvest period.2. Effects of plant growth regulator on content of active compounds in Scutellaria baicalensisIn this study, based on the effects of plant growth regulator gibberellin and mepiquat chloride(DPC) on the impact of the growth development and content of active compounds of S.baicalensis in cells controlled trials, potted plants controlled trials and field trials research, combined with the key phenological phase of harvesting and accumulating of S.baicalensis,we design procedures of chemical control production, and choose "daodi" area Chengde of S.baicalensis to conduct tests. The results show that plant growth regulator DPC could regulate the growth on the ground and underground effectively, and could enhance the content offlavonoids compounds of S. baicalensis.3Preliminary Study of baicalin biosynthetic gene functionIn this study, we clone the two key enzyme genes from cDN A library, SbF6H and SbUBGAT. Alignments showed that SbF6H comprised an open reading frame of1002bp and there is high amino acid sequence similarity (76%) between Solanum lycopersicum (NP001233840) and SbF6H; SbUBGAT comprised an open reading frame of1431bp, and UBGAT sequences from Perilla frutescens (BAG31952) showed high amino acid sequence similarity(71%) with SbUBGAT. Then connect target gene with the expression vector pET-32a (+) to construct the recombinant plasmid of pET-F6H and pET-UBGAT, transforming the host strain E. coli BL21(DE3).The strain was induced by0.4mmol·L-1IPTG,30℃,for4h and can be expressed about54kD and72kD recombinant fusion protein.