水稻基因启动子Ospz4的克隆与分析

发布时间：2018-05-30 00:45

本文选题：水稻 + 基因启动子　；参考：《湖南农业大学》2016年硕士论文

【摘要】：启动子在基因功能与调控中具有重要的作用,对启动子的研究有助于我们对基因调控复杂性的理解。水稻是世界上重要的粮食作物,也是科学研究中的模式植物之一。为探寻可利用的水稻内源性启动子,在前期研究中,本实验室通过Affymetrix水稻基因表达芯片分析了超级稻两优培九母本培矮64S (Oryza sativa L.)在逆境及正常条件下,不同组织器官在不同的生长发育时期全基因组的表达差异,筛选到一个在正常条件及逆境条件(干旱、低温、高温)下均有较高表达水平的基因OsSG4 (GenBank登录号：AK068991.1)。在此基础上,从水稻日本晴基因组DNA中克隆了该基因上游启动子区域Ospz4 (1 625 bp)及四个不同长度的5'端缺失片段(长度分别为1293 bp、1 006 bp、834bp、492bp)并构建了GUS报告基因植物表达载体。转化农杆菌EHA105后,通过注射本生烟草(Nicotiana benthamiana)进行瞬时表达分析,结果显示Ospz4启动了及四个不同长度的5'端缺失片段均能在烟草叶片中驱动GUS基因的表达,其中最短片段(492 bp)的表达活性最强。为进一步验证该启动子在水稻中的活性,以水稻台北309为受体材料,通过农杆菌浸染法获得各启动子片段相应的阳性转化植株；经GUS组织化学染色验证和实时荧光定量PCR检测,实验结果显示：Ospz4启动子在阳性水稻植株叶片、根、茎、颖花、胚乳及胚芽鞘中均有活性,活性强度为双35S启动子的49%;四个不同长度的5'端缺失片段在阳性水稻植株叶片、根、茎、颖壳及T1代幼苗中均有活性,其中长度为492 bp的片段在阳性水稻植株叶片中的活性强度与Ospz4启动子相当,为双35S启动子的48%；长度1 293 bp、1 006 bp和834 bp片段的活性分别为双35S启动子的的30%、28%和23%,均明显弱于全长片段。对转Ospz4启动子的T1代阳性植株进行逆境处理(干旱、低温、高温)后进行荧光定量PCR检测,结果表明逆境条件下Ospz4启动子均能较稳定的维持较高活性,分别为正常条件下的112%、94%、105%。本实验的初步研究结果表明,Ospz4启动子是一个类似组成型表达的水稻内源性启动子,在水稻研究中具有一定的应用潜力,并为该启动子的进一步改造与应用奠定了基础。
[Abstract]:Promoter plays an important role in gene function and regulation. The study of promoter helps us to understand the complexity of gene regulation. Rice is an important food crop in the world and one of the model plants in scientific research. In order to search for the available rice endogenous promoters, the Affymetrix gene expression microarray was used to analyze the super rice Liangyou peijiu female parent, Pei'ai 64s, Oryza sativa L. Under stress and normal conditions, different tissues and organs expressed different genomes at different stages of growth and development. One of them was selected under normal and stress conditions (drought, low temperature, low temperature). High expression level gene OsSG4 GenBank accession number: AK068991.1. On this basis, the upstream promoter region of Ospz4 1 625 BP and four 5 'terminal deletion fragments (1293 BP 1 006 BP 1 006 bp1 006 BP 834 bp1 992 BP) were cloned from the rice Japanese sunny genomic DNA and the plant expression vector of GUS reporter gene was constructed. After transformation of Agrobacterium tumefaciens EHA105, transient expression analysis was carried out by injecting Nicotiana benthamiana. The results showed that Ospz4 initiated and four 5'terminal deletion fragments of different lengths could drive the expression of GUS gene in tobacco leaves. The expression activity of the shortest fragment 492 BP) was the highest. In order to further verify the activity of the promoter in rice, rice Taipei 309 was used as the recipient material to obtain the corresponding positive transformation plants by Agrobacterium tumefaciens staining, and the results were confirmed by GUS histochemical staining and real-time fluorescence quantitative PCR detection. The results showed that the 1: Ospz4 promoter was active in the leaves, roots, stems, spikelets, endosperm and coleoptile of the positive rice plants, and the activity intensity was 49% of the double 35s promoters, and four 5'terminal missing fragments of different lengths were found in the leaves and roots of the positive rice plants. The activity of stem, glume and T1 generation seedlings was similar to that of Ospz4 promoter, and the length of 492bp fragment was similar to that of Ospz4 promoter in the leaves of positive rice plants. The activity of 1 293 BP 1 006 BP and 834 BP fragment was 30% and 23% of that of the double 35s promoter, respectively, which was significantly weaker than that of the full-length fragment. The T 1 positive plants of transgenic Ospz4 promoter were tested by fluorescence quantitative PCR after stress treatment (drought, low temperature, high temperature). The results showed that the Ospz4 promoter could maintain higher activity under stress conditions, which were 11212994 and 105 under normal conditions respectively. The preliminary results show that Ospz4 promoter is an endogenous promoter similar to the constitutive expression of rice, which has a certain potential for application in rice research, and lays a foundation for the further transformation and application of the promoter.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q943.2

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