丹參(Salvia miltiorrhiza Bunge)為中國傳統常用的中藥材,其藥用部位為根,主要活性成分包含丹參酮I、丹參酮IIA、隱丹參酮以及丹參酚酸B等。由於近年來丹參需求增加,因此如何得到高活性成分之丹參便成為重要的研究課題,本試驗之目的是在建立一個以RNA干擾技術抑制丹參開花基因GIGANTEA(GI)表現之方法,期望未來能夠抑制丹參開花並促使丹參體內營養成分重新調配,以期增加其活性成分含量。本研究根據基因同源性,利用5’及3’RACE選殖丹參開花基因SmGI,得基因全長3507 bp,可轉譯成1169個胺基酸。經序列分析得知 SmGI與碗豆(Pisum sativum)PsGI在親源演化、DNA及蛋白質序列均較為相近,並利用TMAP預測得知SmGI有三個穿膜位置。隨後選擇SmGI接近3’之片段,以正反接的方式構築至RNAi載體pGSA1285上,進行基因靜默。利用丹參葉片及葉柄進進行含有pGSA1285農桿菌(EHA105以及GV3101)之感染,其最佳再生條件皆為MS添加1.5 mg/L BAP及0.1 mg/L NAA之培養基,其中葉柄再生能力較佳,因此選擇感染丹參葉柄進行轉殖,並以20 mg/L kanamycin培養基篩選,共獲得80個芽體與再生植株。在利用GUS引子進行PCR鑑定後,確認共得13株轉殖株,且其中6株的SmGI表現量明顯被抑制。
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.
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