【作者】 胡尚杰；

【导师】 卢长明；

【作者基本信息】 中南民族大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 启动子在基因表达调控中起关键性作用,它在很大程度上决定目的基因表达的时间、空间和强度。当前商业化应用的植物基因工程产品大多使用的是组成型启动子,它们驱动外源基因在植物各种组织和所有发育阶段表达,这无疑增加了植物的代谢负担导致物质和能量的浪费,另外可能引起影响植物生长发育的植物形态改变,并且组成型表达的筛选标记基因不利于转基因安全。因此克隆组织特异表达启动子对于提高转基因效率及增加转基因安全性有着重要的意义。油菜中目前研究最多的是种子表达启动子,已经克隆了多个种子特异启动子,而对油菜营养器官启动子研究较少。本研究从油菜基因组中克隆茎部特异性表达基因启动子pSH4,并与GFP报告基因融合,转化拟南芥后对其进行功能的初步验证,该研究的结果可以为油菜茎的基因特异表达提供有价值的启动子。本研究的主要研究结果包括以下几个方面:1、通过筛选拟南芥组织特异表达基因,筛选获得6个茎特异表达基因。2、用甘蓝型油菜中双9号为材料,在盛花期取其根、茎、叶、花器官等,提取RNA进行启动子的各器官表达相对强度进行分析。结果显示候选基因SH4呈在茎中特异性表达。3、检索白菜基因组测序序列,获得油菜SH4启动子的序列,设计引物克隆了油菜茎特异性启动子pSH4,长度1779bp。生物信息学分析显示其具有多种顺式作用元件,包括参与茎组织特异性表达的元件如CCA1ATLHCB1、DOFCOREZM等,激素诱导相关元件ASF1MOTIFCAMV、LTRECOREATCOR15等,以及物理因素诱导的相关元件GT1CORE、GT1CONSENSUS等。4、为进一步验证SH4的功能,成功构建2个植物表达载体:pBI121-SH4-GFP,携带pSH4启动子驱动GFP基因的表达盒,由农杆菌介导转化拟南芥,获得转基因植株,观察T1代植株的GFP的表达情况,结果表明pSH4启动子具有维管束组织表达特异性,是理想的茎表达启动子。本研究对pSH4启动子的分离和组织特异性及其序列特征的生物信息学、表达分布等方面的分析,为深入研究和利用该启动子提供了试验依据和理论基础。另外,本研究构建一种转基因作物通用阳性质粒分子,为转基因生物筛查检测和安全监管服务。通过分析全球商业化种植的转基因作物分子特征,选取常用的调控元件启动子CaMV 35S、FMV35S、nos,终止子NOS 3’、T-35S、g7 3’、E9 3’,和标记基因bar、NPT II、hpt II、pmi作为筛查靶标序列,通过重组PCR技术将这些元件克隆到双元载体pBI121上,构建成包含11个转基因元件的通用阳性质粒分子pBI121-ELEMENTS。质粒序列信息已提交至GeneBank并获得序列号HM047294。该质粒分子对目前国内外批准的5类主要转基因作物(大豆、油菜、玉米、棉花和水稻)理论筛查覆盖率达到91.5%,对我国批准进口的27种转基因作物的理论筛查覆盖率达100%。以pBI121-ELEMENTS为阳性对照,筛查7个转基因品系,结果显示该分子能够有效用于转基因作物定性筛查。更多还原

【Abstract】 Promoter plays a key role in the regulation of gene expression. It is important to achieve temporal and tissue-specific expression of the foreign gene(s) through promoter control. Some constitutive promoters such as the cauliflower mosaic virus (CaMV) 35S promoter and maize ubiquitin are widely used in the current commercial genetic engineering products, and usually result in high and constitutive expression of the genes in host plants. However, constitutive expression of the foreign gene(s) could be harmful to the growth and development of plants because overexpression can lead to metabolic stress. Therefore, application of tissue-specific promoter may increase the safety of genetically modified crops and has an important significance to expression efficiency of target gene. Several seed-specific promoters have been cloned from rape, but few promoters with vegetative organs specificity have been well characterized. In this study, a promoter was cloned from the upstream of the stem-specific expression gene SH4 in rapeseed, and integrated with the GFP reporter gene. A preliminary functional analysis of the pSH4 was made after transformed into Arabidopsis thaliana. The results would provide a valuable promoter for genetic engineering of rapeseed. The results were obtained as follows:1. By screening tissue-specific gene from Arabidopsis thaliana, 6 stem-specific genes were selected as candidates.2. From the rapeseed material Zhongshuang No.9 (in full bloom), RNA was extracted from 8 organs (root, stem, leaf, flower and so on) and quantitatively analysed for the 6 candidate genes. The candidate gene SH4 was shown to express in a stem-specific way.3. According to the known genome sequence (GenBank: AC189510.1) of B.rapa c.v.pekinensis, a 1779bp pSH4 or the 5’flanking sequence of SH4 was isolated. Bioinformatic analysis shows that pSH4 has a variety of cis-acting elements, including the stem tissue-specific elements CCA1ATLHCB1, DOFCOREZM, the hormones-inducable elements ASF1MOTIFCAMV, LTRECOREATCOR15 , physical induced elements GT1CORE、GT1CONSENSUS.4. To characterize the expression features of pSH4, plant expression vectors pBI121-pSH4-GFP was successfully constructed and used to transform Arabidopsis thaliana through the Agrobacterium-mediated method and with the pBI121-GFP as control. We observed the expression of GFP in T1 progeny, indicating that pSH4 is a vascular tissue-specific promoter. This study involved in the isolation and signal scan analysis of pSH4, verification of its tissue-specificity. All these data will give valuable materials and theoretical guidance to study and applification of pSH4 promoter.Genetically modified organisms (GMO) detection is essential for biosafety management but lack of positive control, which become a challenge and a bottleneck of GMO detection. In order to provide a general positive control for GMO screening, plasmid pBI121-ELEMENTS was constructed and confirmed. The pBI121-ELEMENTS contained 11 target elements including promoters (nos, CaMV 35S, FMV35S), terminators (NOS 3’, T-35S, g7 3’, E9 3’) and selective markers (bar, NPT II, hpt II and pmi).The plasmid sequence was submitted to GeneBank and Accession number HM047294 received. These elements covered 91.5% of the worldwide approved transgenic events of major crops including soybean, oilseed rape, maize, cotton and rice. They also covered 100% of transgenic events that China approved. The positive plasmid was used to detect seven related elements and the results showed its effectiveness for GMO screening.更多还原