马铃薯StSUT2基因干扰载体的构建及遗传转化

发布时间：2018-05-29 03:31

本文选题：马铃薯 + RNA干扰　；参考：《兰州理工大学》2017年硕士论文

【摘要】：马铃薯(Solanum tuberosum L.)是世界公认的主要粮食作物之一,它的块茎不仅可以当作粮食作物,而且还可以用于工业生产淀粉等。在植物光合作用产生的碳水化合物,通过韧皮部筛管向储存库器官运输并积累的过程中,起到主要作用的就是蔗糖转运蛋白(Sucrose transporter)。双子叶植物中的蔗糖转运蛋白包括SUT1、SUT2和SUT4三类,主要功能就是对植物光合产物蔗糖的装载、运输和卸载过程的调节作用。在马铃薯中,StSUT1蛋白主要对蔗糖的装载进行调控,StSUT4蛋白主要对蔗糖的卸载过程进行调控,并且还与光周期相互作用来调节马铃薯植株的开花、茎长短和块茎形成,但是StSUT2蛋白的功能仍是未知的。根据国内外多年来对高等植物SUT2的研究分析来看,它的结构与葡萄糖信号感受器类似,因此推测SUT2有可能是蔗糖信号感受器而不是蔗糖转运体。本研究主要通过构建马铃薯StSUT2基因的干扰载体,并对其转化至马铃薯中,为对StSUT2蛋白进行功能分析奠定了基础。实验主要分为两部分:一是干扰载体的构建,用Gateway克隆技术可将StSUT2基因干扰片段快速重组到表达载体中,通过对重组子进行测序,确定为干扰表达载体;二是马铃薯遗传转化,将构建好的干扰载体通过农杆菌介导法转化至马铃薯块茎中,使其再分化产生含有干扰基因的转基因植株。取得的主要成果如下:(1)利用TOPO克隆,将扩增出的干扰片段整合到pENTR中,构建出入门载体命名为pENTR-SUT2。(2)根据Gateway克隆技术进行LR重组,根据入门载体与表达载体的att L和attR位点重组,构建出干扰表达载体pSUT2-RNAi。(3)用冻融法将干扰载体pSUT2-RNAi转至农杆菌GV3101中。(4)将干扰载体转化至夏波蒂(SHB)和陇薯3号(L3)等马铃薯品种的块茎中,通过卡那霉素筛选和PCR扩增检测,初步确定干扰基因已经成功转入马铃薯植株中。用实时荧光定量PCR对转化株进行相对表达量检测表明,干扰基因已经转入马铃薯植株,并对StSUT2基因表达产生抑制作用。
[Abstract]:Solanum tuberosum L.) It is recognized as one of the main food crops in the world, its tuber can be used not only as food crops, but also in industrial production of starch. In the process of carbohydrate transport and accumulation from photosynthesis to storage organ through phloem sieve tube, the sucrose transporter protein Sucrose transporterium plays a major role. Sucrose transporter in dicotyledonous plants consists of sucrose transporter (SUT2) and SUT4. The main function of sucrose transporter in dicotyledonous plants is to regulate the loading transport and unloading process of plant photosynthate sucrose. In potato, StSUT1 protein mainly regulates the loading of sucrose. StSUT4 protein mainly regulates the unloading process of sucrose, and also interacts with photoperiod to regulate the flowering, stem length and tuber formation of potato plants. But the function of the StSUT2 protein is still unknown. According to the research and analysis of SUT2 in higher plants in recent years, its structure is similar to that of glucose signal receptor. Therefore, it is speculated that SUT2 may be a sucrose signal receptor rather than a sucrose transporter. In this study, the interference vector of potato StSUT2 gene was constructed and transformed into potato, which laid a foundation for functional analysis of StSUT2 protein. The experiment is mainly divided into two parts: one is the construction of interference vector, the interference fragment of StSUT2 gene can be quickly recombined into the expression vector by Gateway cloning technology, and the recombinant gene can be identified as interference expression vector by sequencing the recombinant gene, and the second is the genetic transformation of potato. The constructed interference vector was transformed into potato tuber by Agrobacterium tuber and transformed into transgenic plants containing interference gene. The main achievements are as follows: (1) using TOPO cloning, the amplified interference fragments were integrated into pENTR, and the entry vector named pENTR-SUT2.PU 2 was constructed. LR recombination was carried out according to Gateway cloning technology, and recombination according to the att L and attR sites of the entry vector and expression vector. The interference expression vector pSUT2-RNAi.f3 was constructed. The interference vector pSUT2-RNAi was transferred to Agrobacterium tumefaciens GV3101 by freeze-thaw method. The interference vector was transformed into the tubers of potato varieties such as Hepatitis B) and Longshu No.3 (L3), and was screened by kanamycin and detected by PCR amplification. It was preliminarily confirmed that the interfering gene had been successfully transferred into potato plants. The relative expression of the transformed plant was detected by real-time fluorescence quantitative PCR. The results showed that the interfering gene had been transferred into potato plant and the expression of StSUT2 gene was inhibited.
【学位授予单位】：兰州理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S532

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