岷江百合GPAT基因启动子的克隆与功能元件初步分析

发布时间：2017-12-28 00:31

本文关键词：岷江百合GPAT基因启动子的克隆与功能元件初步分析　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：现今社会,研究环境胁迫对植物生长发育的影响已经成为生物学术界的热门。运用基因技术等手段提高植物的抗逆性已成为方法之一。启动子是调控基因表达的关键。所以克隆和构建环境诱导型启动子是培育植物抗逆品种的根本途径。基于近些年的研究结果,诸如CaMV35S、玉米Ubiquitinpromoter等启动子已经被广泛应用。但是这些启动子的特性决定着它们在植物的整个生长发育时期都会高水平表达,不仅会造成资源浪费,严重时会使植物致死,无法达到高效,可调控(定时、定点、定量)表达。而诱导型启动子可以诱导植物在特定的时期、特定条件下表达,避免了不必要的资源浪费,也降低了给植物造成的有害影响。因此诱导型启动子的探究和应用成为研究者们追逐的新潮。本研究基于前人和课题组研究的结果,选取岷江百合作为试验材料,利用有效的基因克隆技术,克隆岷江百合GPAT基因的启动子,并对该启动子做了初步的表达分析,主要结果如下:1.基于本课题组研究得到的岷江百合GPAT基因序列,在该基因编码区设计基因特异性引物,以岷江百合基因组DNA为模板,运用融合嵌套PCR和接头PCR技术克隆岷江百合GPAT基因启动子,共步移得到1007bp的启动子序列,命名为GPATp。2.对步移得到的1007匕p启动子序列进行序列分析,利用NewPLACE、PlantARE等分析预测网站,结果发现在序列中包含许多的TATA-box、CAAT-box等核心顺式元件,另外还有许多与环境胁迫相关的顺式作用元件和组织特异性元件。3.利用缺失克隆,并以GUS为报告基因对启动子进行表达分析,构建了 5个缺失启动子片段:GPATp1(954bp)、GPATp2(644bp)、GPATp3(386bp)、GPATp4(231 bp)、GPATp5(169 bp),分别将这些启动子片段替换植物表达载体pCAMBIA 3301中的35S启动子,构建了 5个植物重组表达载体,分别命名为GPATp1～5.4.烟草转化:利用农杆菌介导的叶盘转化法转化烟草叶盘,以GUS基因为报告基因进行瞬时表达分析,结果显示:该GPATp5(169 bp)组叶盘没有被染成蓝色,说明该启动子片段不能调控GUS基因表达,启动子没有活性;GPATp4(231 bp)组在其他四组之中的表达最弱,结合启动子预测分析结果推测该片段是最小的具有启动子活性的片段;GPATp3(386bp)组比GPATp4(231 bp)组相对染色较深,但弱于GPATp2(644 bp)组和 GPATp1(954 bp)组;GPATp2(644 bp)组深于 GPATp3(386 bp)组;GPATp1(954 bp)组颜色最深,说明该片段活性最强。在进行缺失重组载体转化烟草的同时,每组转化分别进行常温(25 ℃)和低温处理(4℃),结果发现:GPATp1(954 bp)组和 GPATp2(644 bp)组在 4 ℃处理一段时间之后,相对同组常温叶盘颜色略微加深,但不明显,启动子预测分析显示在该启动子片段中含有冷相关顺式元件,说明该启动子在一定程度上受冷诱导,但具体诱导情况还需进一步研究。
[Abstract]:Nowadays, the study of the influence of environmental stress on the growth and development of plants has become a hot topic in the academic community. It has become one of the methods to improve the resistance of plants by means of gene technology. Promoter is the key to regulate gene expression. Therefore, cloning and construction of environmental inducible promoters is the fundamental way to cultivate plant resistant varieties. Based on recent research results, such as CaMV35S, maize Ubiquitinpromoter and other promoters have been widely used. However, the characteristics of these promoters decide that they will be highly expressed in the whole growth stage of plants, which will not only cause waste of resources, but also make plants lethal, and can not achieve efficient, controllable (timing, fixed-point, quantitative) expression. Inducible promoters can induce plants to express at specific times and under specific conditions, avoiding unnecessary waste of resources and reducing harmful effects on plants. Therefore, the exploration and application of the inducible promoter has become a new trend for researchers. This study is based on previous research results and the research group, select the l.regale as experimental materials, the effective use of gene cloning technology, cloning of l.regale promoter of GPAT gene, and the promoter made a preliminary analysis of the expression, the main results are as follows: 1. based on the GPAT gene sequence of l.regale group get this subject. In the design of the gene encoding gene specific primers to l.regale genomic DNA as template, using nested PCR and PCR fusion joint l.regale cloning GPAT gene promoter, were walking by the 1007bp promoter sequence, named GPATp. The 2. step of the 1007 point P promoter sequence and the sequence analysis, prediction of site analysis by NewPLACE and PlantARE, were found in the sequence contains many TATA-box, CAAT-box and other core cis elements, as well as many other related environmental stress cis acting elements and tissue specific element. 3. with the deletion clones, and with GUS as reporter gene expression analysis of promoter constructs 5 deletion promoter fragments: GPATp1 (954bp), GPATp2 (644bp), GPATp3 (386bp), GPATp4 (231 BP), GPATp5 (169 BP), respectively, the promoter fragment replacement plant expression vector pCAMBIA 3301 in the 35S promoter, constructed 5 plant expression vector, respectively named GPATp1 ~ 5.4.: Tobacco by Agrobacterium mediated leaf disc transformation of tobacco leaf disc, using GUS gene as a reporter gene expression analysis, the results showed that: the GPATp5 group (169 BP) leaf disc not be dyed blue, that expression of the promoter fragment of GUS gene promoter is not regulated, no activity; GPATp4 (231 BP) group in the other four groups in the lowest expression and binding to the promoter prediction analysis results suggest that this fragment is the small promoter activity of GP fragment; The ATp3 (386bp) group is relatively darker than that of GPATp4 (231 BP) group, but weaker than that of GPATp2 (644 BP) group and GPATp1 (954 BP) group. GPATp2 (644 BP) group is deeper than that of GPATp3 (386 GPATp2) group, and the color group is the most deep, indicating that the fragment has the strongest activity. In the absence of the recombinant vector into tobacco at the same time, each transformation respectively at room temperature (25 DEG C) and low temperature (4 DEG C), the results showed that: GPATp1 (954 BP) group and GPATp2 (644 BP) group after 4 treatment for a period of time, relative to the same group of normal leaf disc color slightly deep, but no obvious promoter prediction analysis showed that the promoter fragment contains cold related cis elements, indicating that the promoter induced by cold to a certain extent, but the specific induction needs further study.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S682.29

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