Molecular Cloning Of Chalcone Synthase And Other Geges Involved In Flavonoid Biosynthesis & Genetic Transformation Of Erigeron Breviscapus

Erigeron breviscapus (Vant.)Hand.-Mazz., a wild medicinal plant used as the raw material for breviscapin extraction, nowadays has been endangered due to over-harvesting. Artificial cultivation of E. breviscapus has been tried in Yunnan Province of China, however, difficulties in seedling reproduction and germplasm degraded turn to be a great handicap in large scale cultivation. In this study, firstly, a rapid seedling raise program was developed by germinating sterilized seeds on MSo (Murashige and Skoog) medium to develop seedlings, which shorted the period of seedling raise to 40~50d.Secondly, an efficient plant regeneration system was established via adventitious shoot from callus of petiole in vitro culture. Results showed that petioles were superior to leaves as explants to induce callus and basal MS medium supplement with 1.0mg/L BA and 1.0mg/L NAA promoted callus induction while 2.0mg/L BA and 1.0mg/L NAA promoted the compact green callus proliferation. Multiple shoots were obtained from callus mass at high regeneration frequency (90.00±1.53%) in the presence of high level of BA (4.0mg/L) and 0.2mg/L NAA. This protocol provided a useful platform for further study on germplasm improvement of E. breviscapus through biotechnique.Thirdly, based on the abundant study on flavonoid, a hypothetic biosynthetic pathway of scutellarin was given and four enzymes involved in this pathway were selected for studying their role in the biosynthesis of scutellarin. Four gene cDNA related to scutellarin biosynthesis were obtained from different species according to previous reports [chs from Gingko biloba L., fs II from perilla frutescens (L.)Britt., f6h from Glycine max L., and ubgat from Scutellaria baicalensis Georgi. ]. After sequence verified, these cDNA fragments were integrated into the plant binary vector pCAMBIA1304 or pCAMBIA2301 by double enzyme digestion. Then these engineered vectors were separately transferred into Agrobacterium tumefaciens EHA105 and Agrobacterium rhizogenes C58C1. The engineered strains were used to transform the petioles and leaf discs of E. breviscapus respectively.Meanwhile, the full-length cDNA of chs gene, which was 1449 bp length containing 1215 bp coding sequence, was isolated from leaves of E. breviscapus using RACE (rapid amplification of cDNA ends) method. Comparative analysis of chs gene and its deduced amino acid sequence showed extensive homology with those of other Compositae species. Southern blotting results revealed that chs was encoded by a multiple gene family. Semi-quantitative RT-PCR analysis revealed that chs mRNA expression could be detected in all the tested tissues among with the...