Culture Of Hairy Roots Of Bupleurum Chinense Induced By Agrobacterium Rhizogenes And Bio-Function Analysis Of UGT

Bupleurum chinense DC. is known as one source of Radix Bupleuri, with saikosaponins as its major bioactive components. Uridine diphosphate (UDP) glycosyltransferase are involved in the modification of the triterpene skeleton, which contribute to the diversity of saikosaponins. The gene BcUGT10was shown to be candidates involved in the biosynthesis of saikosaponins. The present work was follow-up bio-function analysis of BcUGT10which was the most revevant one. The induction of hairy roots and plantlet regeneration of B. chinense system was established. These provide a substantial foundation for follow-up function analysis of BcUGT10. Moreover, prokaryotic expression vectors of BcUGT10were constructed and the target protein was espressed. These will be helpful for follow-up bio-function analysis of BcUGT10. The detailed results are described as follows:1The induction of hairy roots of B. chinense was explored and established after experiments on different conditions:A. rhizogenes A4was used to infect the leaves bases of B. chinense tube seedlings. The explants were co-cultured on Phytagel-solidified media for3days and then, were turned into solid media, similar with the co-culture media except that bacteriostat was added. After10days, rootlets began to appear and after4to5weeks, rootlets can be converted into liquid shaking culture stage. Finally, hairy roots and plantlet regeneration of B. chinense was established.2Many methods were used in this process including three different kinds of culture media, B5, modified-White and WPM, also three different types of exogenous hormone, IB A, IAA and NAA. The elicitor of Aspergillusniger can also be used for fungi. Two to four concentration gradients were set for each group, after cultured for three months, weighing and measuring the variation of production, then determining saikosaponin content by means of HPLC. Finally, the production and saikosaponin content for Bupleurum hairy roots can be highest in the culture medium which has been added0.5mg/L IBA,2.4g and9.05mg respectively. Thus we screened out the optimal condition for saikosaponin.3Based on the induction system of B. chinense DC. hairy roots, the transformation of the gene BcUGT10mediated by Agrobacterium rhizogene is in process. Three pieces of hyg-resistant hairy roots were obtained through screening by hygromycin of30mg/L. After measuring by PCR, none of them were positive, so further study is needed.4Overexpression of BcUGT10was conducted by A. tumefaciens-mzdiated genetic transformation of Arabidopsis thaliana using the floral dip method. Hyg-resistant Arabidopsis seedlings were obtained through screening by hygromycin of30mg/L. From12Arabidopsis seedlings were PCR tested, and all of them were positive. 5The PCR products of the ORF sequences of BcUGT10were digested with corresponding restriction enzymes and then were inserted into expression vector PGEX-4T-2. Finally, the prokaryotic expression vector of BcUGT10was obtained. The present work will be helpful for follow-up bio-function analysis of BcUGT10.