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標題: RNAi-SmGI丹參轉殖株之鑑定與分析
Identification and analysis of RNAi-SmGI transgenic Salvia miltiorrhiza
作者: Pei-Yin Yang
關鍵字: Salvia miltiorrhiza;RNAi;GIGANTEA;active compound;丹參;RNAi;GIGANTEA;活性成分
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GI (GIGANTEA)在長日照光週期開花途徑中扮演關鍵性角色。當GI基因突變時,不僅會造成植物(如阿拉伯芥)開花時間延遲,甚至使植物(如蘿蔔)的塊根變大。為了將常用中草藥Salvia miltiorrhiza Bunge的根部丹參進行根部改良,周信堅學長於2011年從丹參選殖出SmGI基因的全長後,以RNAi技術抑制SmGI基因的表現,試圖使丹參的營養生長期得以延長,以期增加丹參產量與活性成分的含量。本研究在觀察到株齡1.5年的丹參植株根部外觀後,發現RNAi-SmGI轉殖株之根部明顯較野生型(wild type, WT)丹參肥大,接著針對已馴化3.5個月之RNAi-SmGI轉殖株進行鑑定與活性成分分析。首先,利用PCR增幅GUS基因及NPT II基因進行分子鑑定,結果得知除了line 2、line 9以外,只有line 13為RNAi-SmGI轉殖株。接著,觀察已馴化3.5個月之丹參轉殖株之根部外觀,發現雖然line 13之根部與野生型丹參沒有顯著差異,但根部乾重比野生型丹參的根部重。由於此結果與已發表的反股AtGI基因之轉殖蘿蔔有雷同之處,故利用qRT-PCR確認RNAi-SmGI轉殖中SmGI基因表現量著實受到RNAi影響而下降。另外,由於丹參是常用中草藥,故必須確認其活性成份是否受到轉基因影響,因此利用HPLC分析3.5個月大野生型丹參及3個RNAi-SmGI轉殖株地上部及根部的活性成分(迷迭香酸、丹參酚酸B、隱丹參酮、丹參酮 I、丹參酮 IIA),結果發現SmGI基因靜默後,根部外觀較野生型良好,雖然轉殖株中迷迭香酸與丹參酚酸B的含量降低,但丹參酮類的活性成分並沒有造成顯著影響。綜合前述,本研究成功鑑定了1個RNAi-SmGI轉殖株,並經由試驗得知抑制SmGI基因可使丹參的根部肥大,因此不僅使其擁有更好以及具商業潛力的外型,而且其有效成分不受影響。

GIGANTEA (GI) plays a key role in photoperiodic flowering pathway. Previous studies showed that GI mutation caused late-flowering in Arabidopsis thaliana and larger root tuber in Raphanus sativus L. var. longipinnatus Bailey. In order to improve the roots size of Salvia miltiorrhiza Bunge, one of the common used traditional Chinese medicine, Chou (2010) isolated SmGI from S. miltiorrhiza and created RNA interference (RNAi)-SmGI mutants. One and half years later, larger roots in RNAi-SmGI mutants were observed. To understand such phenotype was caused by RNAi, different transgenic lines were subcultured, and PCR (GUS and NPT II gene) and qRT-PCR were performed. Although all transgenic lines were with lower SmGI expression, only line 13 was identified as transgenic plant. Moreover, larger roots could not be observed in all 3.5 months-old transgenic plants, but overall, root's dry weight was higher in transgenic ones (similar results were observed in antisense-AtGI transgenic radish).To understand if the active compounds of S. miltiorrhiza Bunge were unchanged in transgenic plants, analysis of five active components (rosmarinic acid, salvianolic acid B, cryptotanshinone, tanshinone I, and tanshinone IIA) in aerial parts and roots were performed. Results showed that all compounds were almost not affected in RNAi transgenic S. miltiorrhiza. Through this study, one RNAi-SmGI transgenic line was identified, which showed larger root after planted for 1.5 years without changing levels of active compounds.
其他識別: U0005-2401201422172500
Rights: 同意授權瀏覽/列印電子全文服務,2017-02-07起公開。
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