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马铃薯茄啶-糖基转移酶(solanidine glycosyltransferase,Sgt)家族成员Sgt1、Sgt2和Sgt3参与糖苷生物碱(glycoalkaloids,GAs)合成。已有研究证明,抑制该家族任一成员表达可影响马铃薯块茎中糖苷生物碱合成;然而,对Sgt家族成员的单基因实施调控很难有效降低块茎中总糖苷生物碱的积累。为降低马铃薯块茎中总糖苷生物碱的含量,本研究拟采用RNAi技术,对糖苷生物碱合成代谢途径末端酶基因家族成员Sgt1-3在转录水平进行共调控。为实现这一目的,构建了块茎特异性启动子Patatin驱动的,以Sgt1、Sgt2和Sgt3基因为靶向的RNAi表达载体p CEI-PFR,采用农杆菌介导法转化马铃薯茎段,获得10株可沉默Sgt1、Sgt2和Sgt3基因表达的Patatin-RNAi融合基因的转基因植株。实时定量PCR(RT-q PCR)结果显示,Sgts基因的相对表达量分别降低了大约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)的表达量则增加了约48%~135%。高效液相色谱法(HPLC)证明,尽管转基因株系的绿色组织中糖苷生物碱含量与野生型无显著差异,但块茎中糖苷生物碱分别比野生型降低了46%~59%(庄薯3号)和42%~62%(Favorita)。上述结果提示,复合沉默茄啶-糖基转移酶家族基因可降低马铃薯块茎中糖苷生物碱的积累。此外,该结果可能对研究马铃薯不同组织间糖苷生物碱的分布和积累,以及马铃薯种质资源的创新开发具有一定启示。
Sgt1, Sgt2 and Sgt3, members of the solanidine glycosyltransferase family, are involved in the synthesis of glycoalkaloids (GAs). It has been shown that inhibiting the expression of any member of this family can affect the synthesis of glycoside alkaloids in potato tubers; however, regulation of the single gene of members of the Sgt family can not effectively reduce the accumulation of total glycoside alkaloids in tubers. In order to reduce the content of total glycoside alkaloids in potato tuber, this study intends to use RNAi technology to co-regulate the transcription level of Sgt1-3, an enzyme gene family member at the end of the glycoside alkaloid synthesis pathway. To achieve this goal, tuber-specific promoter Patatin-driven RNAi expression vector pCEI-PFR targeting Sgt1, Sgt2 and Sgt3 genes was constructed and transformed into potato stem segments by Agrobacterium-mediated transformation to obtain 10 strains Transgenic plants that silence the Patatin-RNAi fusion gene for Sgt1, Sgt2 and Sgt3 gene expression. Real-time quantitative PCR (RT-qPCR) showed that the relative expression of Sgts gene was reduced by about 32% -60% (Sgt1), 29% -55% (Sgt2) and 25% -66% (Sgt3) The expression of glyphosate resistance gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) increased by about 48% -135%. High performance liquid chromatography (HPLC) demonstrated that although the content of glycoside alkaloids in transgenic green plants was not significantly different from that in wild type, the content of glycoside alkaloids in tubers was reduced by 46% -59% ) And 42% ~ 62% (Favorita). The above results suggest that the complex silencing of eggplant glycosyltransferase family genes can reduce the accumulation of glycoside alkaloids in potato tubers. In addition, the results may have some implications for the study of the distribution and accumulation of glycoside alkaloids in different tissues of potato as well as for the innovation and development of potato germplasm resources.