【摘要】：馬鈴薯茄啶-糖基轉(zhuǎn)移酶(solanidine glycosyltransferase,Sgt)家族成員Sgt1、Sgt2和Sgt3參與糖苷生物堿(glycoalkaloids,GAs)合成。已有研究證明,抑制該家族任一成員表達可影響馬鈴薯塊莖中糖苷生物堿合成;然而,對Sgt家族成員的單基因?qū)嵤┱{(diào)控很難有效降低塊莖中總糖苷生物堿的積累。為降低馬鈴薯塊莖中總糖苷生物堿的含量,本研究擬采用RNAi技術(shù),對糖苷生物堿合成代謝途徑末端酶基因家族成員Sgt1-3在轉(zhuǎn)錄水平進行共調(diào)控。為實現(xiàn)這一目的,構(gòu)建了塊莖特異性啟動子Patatin驅(qū)動的,以Sgt1、Sgt2和Sgt3基因為靶向的RNAi表達載體p CEI-PFR,采用農(nóng)桿菌介導法轉(zhuǎn)化馬鈴薯莖段,獲得10株可沉默Sgt1、Sgt2和Sgt3基因表達的Patatin-RNAi融合基因的轉(zhuǎn)基因植株。實時定量PCR(RT-q PCR)結(jié)果顯示,Sgts基因的相對表達量分別降低了大約32%~60%(Sgt1)、29%~55%(Sgt2)和25%~66%(Sgt3),而草甘膦抗性基因——5-烯醇式丙酮酸-3-磷酸莽草酸合酶(5-enolpyruvylshikimate-3-phosphate synthase,EPSPS)的表達量則增加了約48%~135%。高效液相色譜法(HPLC)證明,盡管轉(zhuǎn)基因株系的綠色組織中糖苷生物堿含量與野生型無顯著差異,但塊莖中糖苷生物堿分別比野生型降低了46%~59%(莊薯3號)和42%~62%(Favorita)。上述結(jié)果提示,復(fù)合沉默茄啶-糖基轉(zhuǎn)移酶家族基因可降低馬鈴薯塊莖中糖苷生物堿的積累。此外,該結(jié)果可能對研究馬鈴薯不同組織間糖苷生物堿的分布和積累,以及馬鈴薯種質(zhì)資源的創(chuàng)新開發(fā)具有一定啟示。
[Abstract]:Solanidine glycosyltransferase (SGT) family members Sgt1, Sgt2 and Sgt3 are involved in the synthesis of glycoside alkaloids. It has been shown that inhibiting the expression of any member of the family can affect the synthesis of glycoside alkaloids in potato tubers, however, it is difficult to effectively reduce the accumulation of total glycoside alkaloids in tubers by regulating the single gene of SGT family members. In order to reduce the content of total glycoside alkaloids in potato tubers, RNAi technique was used to co-regulate the transcriptional level of Sgt1-3, a member of terminal enzyme gene family of glycoside alkaloid biosynthesis pathway. In order to achieve this goal, the tuber specific promoter Patatin driven RNAi expression vector pCEI-PFRwas constructed, which was targeted at Sgt1, Sgt2 and Sgt3, and was transformed into potato stem segment by Agrobacterium tumefaciens. Ten transgenic plants with Patatin-RNAi fusion genes that silenced the expression of Sgt1 Sgt2 and Sgt3 genes were obtained. The results of real-time quantitative PCR (RT-Q PCR) showed that the relative expression of Sgts gene decreased by about 32% (Sgt1), about 55% (Sgt2) and 25% (Sgt3), while the expression of glyphosate-resistant gene, 5-enolpyruvylshikimate-3-phosphate synthase3-phosphate shikimate synthase (EPSPS), increased about 48135. High performance liquid chromatography (HPLC) showed that although the content of glycoside alkaloids in green tissue of transgenic lines had no significant difference with wild type, the glycoside alkaloids in tubers were 4659% (Zhuangshu 3) and 42% (Favorita) lower than wild type, respectively. These results suggest that the compound silencing of Solanidine-glycosyltransferase family genes can reduce the accumulation of glycoside alkaloids in potato tubers. In addition, the results may be helpful to the study of the distribution and accumulation of glycoside alkaloids in different tissues of potato and the innovative development of potato germplasm resources.
【作者單位】： 甘肅省作物遺傳改良與種質(zhì)創(chuàng)新重點實驗室/甘肅省干旱生境作物學重點實驗室/甘肅農(nóng)業(yè)大學農(nóng)學院;甘肅農(nóng)業(yè)大學資源與環(huán)境學院;
【基金】：國家自然科學基金(No.31260343) 甘肅省生物技術(shù)專項(No.GNSW-2015-11)資助~~
【分類號】：S532

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