Cloing and characterization of GIGANTEA gene in Salvia miltiorrhiza and inhibiting its expression by using RNA interference

• Main Authors: Hsing-Chein Chou, 周信堅
• Other Authors: Chin-Fun Chen
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/80992210837994646752

碩士 === 朝陽科技大學 === 生化科技研究所碩士班 === 99 === Salvia miltiorrhiza Bunge, well known as ‘Danshen’, is an ancient Chinese traditional medicine. The roots contain major active compounds such as tanshinone I , tanshinone IIA, cryptotanshinone, and salvianolic acid B. High demand of roots pushed this plant to the brink of extinction. By developing molecular breeding through RNA interference (RNAi) could be possible conserve this species and enhance its metabolite accumulation. Late flowering gene GI (GIGANTEA) in S. miltiorrhiza (SmGI) was cloned by gene homology. SmGI is 3507 bp long and can be deduced to 1169 amino acid. The nucleotide sequences were showing maximum homology with Pisum sativum GI (PsGI) and its deduced amino acids were predicted with three transmembrane sites. The 300 bp fragments from SmGI 3’ end was used for RNAi contruct (pGSA1285). Agrobacterium tumefaciences (GV3101 and EHA105) harboring RNAi construct was used for the gene transformation. In vitro multiple shoot regeneration was done using leaf and petiole on MS basal medium containing 1.5 mg/L BAP and 0.1 mg/L NAA. Regenerated shoots were selected on 20 mg/L kanamycin. Shooting was observed in 80 different lines which were subjected to PCR analysis by using GUS primers and only 13 lines were positive for the GUS, Among 13 transgenic lines, only 6 of them were determined with low SmGI expression.