沉默马铃薯Sgt1-3基因减少块茎糖苷生物碱积累
发布时间：2018-07-14 10:33
【摘要】：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。
[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.
【作者单位】： 甘肃省作物遗传改良与种质创新重点实验室/甘肃省干旱生境作物学重点实验室/甘肃农业大学农学院;甘肃农业大学资源与环境学院;
【基金】：国家自然科学基金(No.31260343) 甘肃省生物技术专项(No.GNSW-2015-11)资助~~
【分类号】：S532
[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.
【作者单位】： 甘肃省作物遗传改良与种质创新重点实验室/甘肃省干旱生境作物学重点实验室/甘肃农业大学农学院;甘肃农业大学资源与环境学院;
【基金】：国家自然科学基金(No.31260343) 甘肃省生物技术专项(No.GNSW-2015-11)资助~~
【分类号】：S532
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