Inducement And Culture Of Transgenic Hair Roots And Functional Analysis Of IFS Gene In Licorice

Glycyrrhiza uralensis Fisch is a plant of leguminous Glycyrrhiza shrubby herb, whose dried root and rhizome are used as medicine. It has been a widely used traditional drugs for a long history in China. It contains flavonoids which has an effect on anti-ulcer, anticancer, anti-HIV, anti-oxidant. In this paper, the genetic transformation and cultural system of Licorice hair roots integrated with IFS over-expressed vector was found after studying on Agrobacterium rhizogenes, explants types, infection method, inducing ways and culture medium. Founction of IFS gene in the flavonoids metabolism was cleared by product and gene expression analysis. The main results are as follows:1、Agrobacterium strains, explant types and infection methods had great influence on the induction of hairy roots. Using four kinds of Agrobacterium rhizogenes--A4, 1025, K599 and Aqua1--containing the target gene to infect explants, only Aqua1 can induce the transgenic hairy root formation, and the highest conversion rate was 46%. Taking into account the number of hairy roots single explants formed and conversion rates, hairy root formation was efficiently(rate 34%)induced by using cotyledonary nodes as explants with infection method.2、Light contributed to the formation of hairy root induction. Under the condition of light, vitrification rate and browning rate of the hairy roots decreased significantly, only 8.3% and 4.2%, than dark conditions and hairy root were stronger in the light conditions.3、Hairy root was different in different culture medium. After studying the effect of culture medium of MS, B5, 1/2MS, SH and MSB5 on the growth of hairy root, it showed that hairy roots in B5, 1/2MS medium containing low ammonium grew well, white, and had more branches and the survival rate were 66.7%, 53.3%, respectively. While hairy roots in MS, MSB5 and SH medium was easy to browning or die. In the later stage of hairy roots culture process, activated carbon can effectively inhibit hairy root browning and callus.4、The result of HPLC analysis showed that the IFS over-expressors did enhance 1.7, 1.9 times of two kinds of isoflavones respectively that compared with the control. Real time PCR results showed that in the five transgenic roots IFS expression was significantly improved, which proved that the IFS gene was highly expressed.In this study, the reasons for the accumulation of liquiritin and glycyrrhizin as the substrate of the isoflavone synthase in licorice were revealed. It laid the foundation for solving the lack of resources, and further optimizing the licorice quality. In this paper, the genetic transformation and cultural system of Licorice hair roots was found,which providetechnical support for the future studying on licorice complex metabolic mechanism and related genes expression and regulation by hairy root system.