沉默马铃薯Sgt1-3基因减少块茎糖苷生物碱积累  

Silencing Sgt 1-3 Genes Decreases Accumulation of Glycoalkaloids in the Potato Tuber

作　　者：安然 张晶晶[2] 郭海霞 石文慧 乔岩 王志伟[1] 石菁[1] 张金文[1] AN Ran ZHANG Jmg-Jlng GUO Hai-Xia SHI Wen-Hui QIAO Yan WANG Zhi-Wei SHI Jing ZHANG Jin-Wen(Gansu Key Laboratory of Crop Improvement ＆ Germplasm Enhancement/ Gansu Provincial Key Laboratory of Aridland Crop Science/College of Agronomy, Gansu Agricultural University, Lanzhou 730070, China College of Resources and Environment, Gansu Agricultural University, Lanzhou 730070, China)

机构地区：[1]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,兰州730070 [2]甘肃农业大学资源与环境学院,兰州730070

出　　处：《中国生物化学与分子生物学报》2017年第8期826-834,共9页Chinese Journal of Biochemistry and Molecular Biology

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~~

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。The solanidine glycosyltransferase （Sgt） family, comprising Sgtl, Sgt2 and Sgt3, participates in the biosynthesis of glycoalkaloids （GAs） in potatoes. It has been shown that inhibition of any one of the Sgt family members influences GA biosynthesis. However, blocking the expression of a single gene of the family does not effectively eliminate the accumulation of total GAs in the tuber. In this study, we employed a strategy to inhibit the expression of Sgtl, Sgt2 and Sgt3 genes simultaneously by RNA interference （RNAi） to reduce the accumulation of GAs in the potato tuber. First, we constructed the RNAi vector pCEI-PFR, in which the expression of siRNAs targeting Sgtl, Sgt2 and Sgt3 genes were driven by the tuber-specific Patatin promoter, and transformed the vector into potato internodal explants by Agrobacterium-mediated transformation. As a result, we obtained ten transgenic plants that contain the Patatin-RNAi fusion gene. Real-time Quantitative PCR （RT-qPCR） showed that the relative expression levels of the three Sgt transcripts decreased by approximately 32% -60% （Sgtl）, 29% ~ 55% （Sgt2） and 25% -66% （Sgt3） , respectively, while the relative expression levels of the 5-enolpyruvylshikimate- 3-phosphate synthase （EPSPS） gene, one of the glyphosate tolerance genes, increased by about 48%- 135%. High performance liquid chromatography （HPLC） revealed that the GA content of green tissues did not show a significant difference between transgenic lines and the wild type. Importantly, the GA content in the tuber of transgenic lines decreased by 46% - 59% （ Zhuangshu No. 3） and 42% - 62% （Favorita） , respectively. We propose that silencing multiple genes of the Sgt gene family reduces the accumulation of GAs in potato tubers. Hence we provide a basis to deeply understand the distribution and accumulation of GAs in different potato tissues, and may germplasm resources. promote innovation and development for potato

关 键 词：马铃薯 糖苷生物碱 代谢调控 RNA干扰 基因沉默 

分 类 号：S532[农业科学—作物学]