玉米CCR基因启动子的克隆与功能分析

发布时间：2018-04-17 12:11

本文选题：营养器官 + 启动子　；参考：《安徽农业大学》2016年硕士论文

【摘要】：基因工程中能否使外源基因以一定的表达量在特定的组织部位表达是一个重要问题,而启动子作为能够调控外源基因在特定组织器官表达的分子元件之一,具有重要的研究意义。目前人们普遍使用的是组成型启动子,这类启动子能驱动外源基因在植物中各部位高效的表达,但同时也造成了营养的不必要浪费以及植物细胞的过度负荷。特异性启动子可以通过调控目的基因使其在特定的组织部位表达。因此,对玉米中特异性启动子的研究不仅对改善玉米的品质具有重要意义,而且为基因工程中调控元件的研究提供了参考。本课题在生物信息学的分析结果的基础上,构建了玉米CCR基因启动子以及其筛检片段的表达载体,通过农杆菌介导法获得转基因的水稻植株,并对其表达特性进行了分析,获得如下主要研究结果:1.通过启动子序列分析的相关网站结果显示,该启动子除了基本顺式作用元件TATA-box和CAAT-box外,还含有四种诱导型元件:ARE、Box-W1、CGTCA-motif和MBS;四种光应答元件Box 4、Box I、G-box和Sp1;三种器官表达元件ROOTMOTIFTAPOX1、POLLEN1LELAT52和Skn-1。2.从玉米基因组中分离出一段2145bp的肉桂酰辅酶A还原酶(cinnamoyl-CoA reductase)基因启动子,将其命名为pCCR。通过5’端缺失法对CCR基因启动子的全长进行片段筛检,得到三个不同长度的筛检片段我们分别将其命名为pCCR-1、pCCR-2以及pCCR-3,其片段大小分别为1650 bp、1126 bp以及548 bp。通过双酶切的方法将这四个启动子片段与含有GUS报告基因的双元表达载体pCAMBIA1301连接,构建四个新型表达载体。3.将构建的四个新型表达载体运用农杆菌介导法转入水稻中花11中,共获得42株转基因植株苗,其中CCR有11株,CCR-1有13株,CCR-2有8株,CCR-3有10株。通过PCR及潮霉素检测后,共有27株为阳性植株。4.采用GUS组织化学染色方法,结果显示CCR基因启动子在根、茎、叶、颖壳中表达,花和种子不表达,推测其为营养器官特异性启动子;筛减片段pCCR-1在叶、颖壳、花以及胚中都有表达,其余部位不表达;pCCR-2只在叶中表达,其余组织部位均不表达,即pCCR-2为叶特异性启动子;pCCR-3在茎与颖壳部位表达,其余部位不表达;
[Abstract]:Whether the exogenous gene can be expressed in a certain amount in specific tissues is an important issue in genetic engineering, and promoter is one of the molecular elements that can regulate the expression of exogenous gene in specific tissues and organs.It has important research significance.At present, constitutive promoters are widely used, which can drive the efficient expression of exogenous genes in various parts of plants, but also lead to unnecessary waste of nutrition and excessive loading of plant cells.Specific promoters can be expressed in specific tissues by regulating the target gene.Therefore, the study of specific promoters in maize is of great significance not only to improve the quality of maize, but also to provide a reference for the study of regulatory elements in genetic engineering.Based on the results of bioinformatics analysis, the expression vector of maize CCR gene promoter and its screening fragment was constructed. The transgenic rice plants were obtained by Agrobacterium tumefaciens mediated method, and their expression characteristics were analyzed.The following main results are obtained: 1: 1.In addition to the basic cis-acting elements TATA-box and CAAT-box, the promoter also contains four inductive elements, namely: AREX Box-W _ (1) CGTCA-motif and MBSs; four optical response elements, Box _ 4, Box Ion G-box and Sp1; and three organ expression elements, ROOTMOTIFTAPOX1 POLLEN1LEAT52 and Skn-1.2.A 2145bp cinnamoyl-CoA reductase promoter was isolated from maize genome and named pCCR.The full-length of CCR promoter was screened by 5'end deletion method, and three screening fragments of different length were obtained. We named them pCCR-1pCCR-2 and pCCR-3, respectively, with the size of 1650 BP and 548 BP, respectively.The four promoter fragments were ligated with the dual expression vector pCAMBIA1301 containing GUS reporter gene, and four novel expression vectors. 3 were constructed by double enzyme digestion.The four new expression vectors were transferred into rice Zhonghua 11 by Agrobacterium tumefaciens, and 42 transgenic plants were obtained, of which 11 were CCR-1 and 13 were CCR-2, 8 were CCR-3 and 10 were CCR.After PCR and hygromycin detection, there were 27 positive plants.The results of GUS histochemical staining showed that the promoter of CCR gene was expressed in root, stem, leaf and glume, but was not expressed in flower and seed.There was no expression of pCCR-2 in the other parts of flower and embryo, but no expression of pCCR-2 in other tissues. That is to say, pCCR-2 was expressed in stem and glume, but not in other parts.
【学位授予单位】：安徽农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S513;Q943.2

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