毛竹赤霉素信号途径相关基因的克隆与功能分析

发布时间：2018-06-25 21:18

本文选题：毛竹 + 赤霉素　；参考：《浙江农林大学》2016年硕士论文

【摘要】：赤霉素是调控植物生长发育的五大激素之一,在如种子萌发、茎的伸长以及果实发育等植物的多种生命活动中发挥重要作用。通过分子遗传学等方法,在水稻、拟南芥等植物中发现赤霉素传导途径的一些重要元件,包括赤霉素的受体蛋白GID1、赤霉素信号的负调控因子DELLA蛋白和DELLA蛋白降解相关的GID2蛋白。目前对赤霉素传导途径的研究表明,在赤霉素不存在或浓度较低的情况下,DELLA蛋白抑制植物的生长,而当活性赤霉素存在时,赤霉素与受体GID1结合后,导致后者构象改变并与DELLA蛋白相互作用,诱导DELLA蛋白泛素化并通过蛋白酶途径降解,激活下游信号转导,进而调控植物生长发育过程。毛竹我国分布最广、经济价值最高的竹种,主要以无性系繁殖方式进行更新生长,毛竹笋有着惊人的生长速度,这种快速生长现象具有重要的学术研究价值。有研究指出在竹笋的快速生长期赤霉素的含量迅速升高,推测赤霉素在笋的快速生长中会发挥关键作用,研究毛竹中的赤霉素合成和信号途径具有重要意义。本研究以毛竹种子幼苗为材料,研究了赤霉素对毛竹种子萌发、幼苗生长影响,并同源克隆获得赤霉素信号转导途径中的三个关键基因,应用生物信息学手段进行基因序列分析。采用实时定量PCR方法研究这些基因在笋的快速生长时期不用部位的表达情况,并利用酵母双杂交系统验证了这些蛋白之间的互作关系,还通过农杆菌介导花序侵染法获得DELLA基因过表达拟南芥,初步探讨了毛竹DELLA基因在植物中的功能,得到了以下结论:1.赤霉素促进了毛竹种子的萌发和毛竹幼苗的生长,赤霉素浓度在100mg.L-1时作用最明显,与对照组相比,种子发芽率提升了19.80%,茎长提升了97.17%;2.通过同源克隆得到了毛竹中的三个基因PhGID1、PhGID2和PhSLR1全长,它们的ORF分别为1065bp、648bp和1866bp,分别编码354、215和621个氨基酸。对氨基酸序列进行生物信息学分析显示,它们和其他植物中的同源蛋白质氨基酸序列一致性较为保守。3.在快速生长期的毛竹笋中,PhGID1、PhGID2和PhSLR1这三个基因在笋的顶部、中部、基部以及根部都有表达,PhSLR1在笋的上、中部表达量较少,在根部较多,而PhGID1和PhGID2的表达情况相似,在笋的上中部表达量高,根部较少,这与PhSLR1基因的表达呈相反趋势。4.原核表达实验表明,当表达菌株为大肠杆菌BL21(DE3),表达载体为pET-32a时,PhSLR1和PhGID2蛋白能够表达且是可溶性的,PhGID1蛋白的表达以包涵体形式存在。5.酵母双杂交结果显示,PhGID2蛋白和PhSLR1有相互作用且作用于PhSLR1蛋白的C端GRAS结构域,PhGID2蛋白和PhGID1蛋白也存在相互作用且不依赖于赤霉素的存在。6.将毛竹基因PhSLR1进行转基因实验,观察表型发现拟南芥转基因植株比野生型要矮小,叶片和果荚也小、少。
[Abstract]:Gibberellin is one of the five hormones regulating plant growth and development, which plays an important role in many life activities of plants such as seed germination, stem elongation and fruit development. Some important elements of gibberellin transduction pathway were found in rice, Arabidopsis thaliana and other plants by means of molecular genetics, including gibberellin receptor protein GID1, the negative regulation factor Della of gibberellin signal and the GID2 protein associated with the degradation of della protein. Studies on the transmission pathway of gibberellin have shown that the Della protein inhibits the growth of plants without or at a low concentration of gibberellin, and when active gibberellin exists, gibberellin binds to the receptor GID1. The latter changes in conformation and interacts with Della protein, which induces the ubiquification and degradation of Della protein through protease pathway, activates downstream signal transduction, and then regulates plant growth and development. Phyllostachys pubescens are the most widely distributed species with the highest economic value in China. They mainly regenerate and grow by clonal reproduction. The bamboo shoots of Phyllostachys pubescens have an amazing growth rate, which has important academic research value. Some studies have pointed out that the content of gibberellin increases rapidly in the rapid growth period of bamboo shoots. It is suggested that gibberellin will play a key role in the rapid growth of bamboo shoots. It is of great significance to study gibberellin synthesis and signal pathway in Phyllostachys pubescens. In this study, the effects of gibberellin on germination and seedling growth of Phyllostachys pubescens were studied, and three key genes of gibberellin signal transduction pathway were obtained by homologous cloning. Gene sequence analysis was carried out by bioinformatics. Real-time quantitative PCR was used to study the expression of these genes during the rapid growth of bamboo shoots, and the interaction between these proteins was verified by yeast two-hybrid system. The overexpression of Della gene in Arabidopsis thaliana was also obtained by Agrobacterium tumefaciens mediated inflorescence infection. The function of Della gene in Phyllostachys pubescens was preliminarily studied, and the following conclusions were obtained: 1. Gibberellin promoted the germination of Phyllostachys pubescens seeds and the growth of Phyllostachys pubescens seedlings. The effect of gibberellin concentration was most obvious at 100 mg 路L ~ (-1). Compared with the control group, the germination rate of the seeds increased 19.80% and the stem length increased 97.17% ~ (2). Three genes, PhGID1, PhGID2 and PhSLR1, were cloned from Phyllostachys pubescens by homologous cloning. Their ORFs were 1065 BP, 648 BP and 1866 BP, respectively, encoding 354215 and 621 amino acids, respectively. Bioinformatics analysis of amino acid sequence showed that the amino acid sequence of homologous protein was conserved with that of other plants. The three genes, PhGID1, PhGID2 and PhSLR1, were expressed in the top, middle, base and root of bamboo shoots at the rapid growth stage, but the expression of PhGID1 and PhGID2 were similar in the middle part of shoot and the root, but the expression of PhGID1 and PhGID2 were similar. The expression of PhSLR1 gene in the upper and middle part of shoot was higher than that in the root, which was opposite to the expression of PhSLR1 gene. E. coli BL21 (DE3) and pET-32a were expressed in E. coli BL21 (DE3). The expression of PhSLR1 and PhGID2 proteins could be expressed in the form of inclusion body. The results of yeast two-hybrid showed that there was interaction between PhGID2 protein and PhSLR1, and the C-terminal GRAs domain of PhSLR1 protein, PhGID2 protein and PhGID1 protein, were also interacted and independent of the existence of gibberellin. 6. The transgenic experiment of Phyllostachys pubescens gene PhSLR1 showed that the transgenic plants of Arabidopsis thaliana were shorter than the wild type and the leaves and pods were smaller and less.
【学位授予单位】：浙江农林大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S795.7

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