Genetic Manipulation Of Scutellaria Amoena C. H. Wright

Scutellaria amoena C. H.Wright, is a perennial herbaceous plant native to Southwest of China. Recently it has been widely studied with pharmacological activity analysis as well as clinical tests due to its higher contents of baicalin and baicalein than those of S. baicalensis Georgi. The wild populations of Scutellaria amoena C. H.Wright are therefore over-harvested because of its high value. Artificial cultivation has been tried, however, difficulties in seeds' harvest and seedling germination turn to be a great handicap in large scale cultivation. In this dissertation, an efficient system of organogenesis and transformation protocols of Scutellaria amoena C. H.Wright was developed, which provided a powerful means to produce large numbers of uniform plants and hairy roots. Meanwhile, the DHQ-UBGAT cDNA was cloned, which laid the technological foundation for further study on genetic improvement via biotechnique. The results were as follows:(1) The organogenesis and plant regeneration system of Scutellaria amoena C. H.Wright were established on MS medium with different phytohormones, in which non-axillary bud stems fregments were used as the explants. MS medium containing 2mg/L 6-BA and 0.5 mg/L NAA was optimal for callus induction, the frequency of callus induction reached to 100%. Buds were differentiated on the same medium, and grew up on MS medium with 2 mg/L 6-BA,0.2 mg/L NAA and 20 g/L sucrose. Regenerated shoots were rooted and subcultured on half strength of MS medium supplemented with 0.2 mg/L IBA.The genetic stability of regenerated plants was analysed by RAPD markers. The results of RAPD amplification showed that the genetic stability of the regenerated plantlets with repeatedly subculturing was maintained, though slight variations were found. This suggested that in vitro organogenesis was a good rapid propagation way to obtain genetic stable descends of Scutellaria amoena C. H.Wright.(2) The 5% NaOH histochemical method was applied in the study of the localization of flavonoids in the regenerated plantlets of Scutellaria amoena C. H.Wright. The results showed that the flavonoids were mainly distributed in the epidermis and cortex parenchyma of the vegetative organs. The flavonoids were also found to accumulate in the tentacles of the stems and leaves. The content of baicalin in the regenerated plantlets was determined by HPLC. The baicalin contentration was found to be higher in the stems and leaves than that in the roots. Quantitative analysis showed that the content of baicalin was 0.12% in the roots, 0.34% in the stems and 0.55% in the leaves.(3) An efficient protocol of genetic transformation and plant regeneration of Scutellaria amoena C. H.Wright plantlets were established. Hairy roots were induced from the wounded surface of the leaf sections and stem segments on hormone free MS medium after infection by Agrobacterium rhizogenes strain 1.2556. The obtained hairy roots showed the representative characteristics of hairy root. PCR analysis and opine paper electrophoresis confirmed that the T-DNA fragment of Ri plasmid from A. rhizogenes strain had been inserted into the genome of Scutellaria amoena. The contents of baicalin in different hairy roots were evaluated by HPLC analysis. The highest contents of baicalin in hairy roots was 2.59%, which is 7.18 times of that in medicine S. baicalensis Georgi root for the unit time of 3 years.The adventitious buds were obtained directly from hairy root growing on the MS solid medium supplemented with 6-BA 2 mg/L and NAA 0.2 mg/L, and the buds were then formed roots on MS solid medium.(4) The full-length cDNA, encoding the Scutellaria 7-O-glucuronyl transferase (UBGAT), was cloned from leaves of Scutellaria amoena C. H.Wright using one-step RT-PCR method. The DHQ-UBGAT gene, which was 1326 bp length sequence containing 422 coding animo acids, showed extensive homologue(99.0%) with those of Scutellaria amoena (huangqin)species. The sequence was subcloned into pET32a vectors, and expressed in E.coli BL21.