陆地棉开花相关基因GhFLP1和GhFPF1功能初步分析

发布时间：2018-06-01 01:27

本文选题：陆地棉 + GhFLP1　；参考：《西北农林科技大学》2016年硕士论文

【摘要】：棉花作为主要的纺织纤维和油料种子作物,是一种重要的经济作物。我国当前土地资源紧张,棉花种植面积锐减,培育短季棉品种,实现麦棉两熟是解决这一问题的重要途径。开花是植物从营养生长进入生殖生长的重要转变过程,作物的早熟性与开花密切相关,所以了解和掌握作物开花规律从而调控这一过程对于提高棉花的早熟性有重要意义。FLP1(flowering-promoting factor 1-like protein 1)基因,是拟南芥中开花促进因子基因FPF1(flowering-promoting factor-1)的同源基因,目前在拟南芥中并没有对FLP1基因进行深入的功能验证。本研究从陆地棉中克隆得到了FLP1的同源基因GhFLP1,对其进行生物信息学分析,表达模式分析以及转拟南芥等试验对其功能进行了深入研究。根据实验室原有研究结果,对转GhFPF1基因棉花进行了功能研究,初步明确了其在棉花中的作用,试验结果如下:1、经克隆测序得到GhFLP1基因长537 bp,无内含子,包含339 bp开放阅读框,编码112个氨基酸。其蛋白质相对分子质量为12.176 kDa,理论等电点为9.13。GhFLP1与其他物种FLP1蛋白序列存在明显保守区域,与雷蒙德氏棉D基因组中的FLP1序列相似性高达99%。2、外源激素处理试验证实GhFLP1能够响应GA和SA的处理,表明GhFLP1可能参与植物开花和逆境胁迫调控。3、在不同品种中表达模式分析发现GhFLP1呈现出比较强烈的组织表达特异性,在花蕾中的表达量远远高于其他组织。结果还表明,该基因在早熟品种中棉所36、中棉所74花蕾中的表达量明显高于晚熟品种中棉所60、鲁棉研28。4、在拟南芥中过表达GhFLP1,转基因拟南芥开花时间提前,莲座叶数目减少,转基因拟南芥中一些开花时间促进基因如AtFT、AtAP1、AtLFY、AtSOC1表达量均有升高,抑制开花基因AtFLC表达量明显下调。5、构建了棉花VIGS载体pCLCrVA-GhFLP1,对棉花进行接种。发病植株的GhFLP1表达量下调,目前植株表型并没有观察到明显变化。6、对转GhFPF1基因棉花进行表型观察,发现转基因株系与野生型相比花期延迟,植株变高。水培结果表明,转基因株系的根系变得更加发达。qRT-PCR结果表明转基因株系中,GhFPF1在根和茎中的表达量远远高于其他组织。
[Abstract]:As the main seed crop of textile fiber and oil, cotton is an important cash crop. At present, the land resources in our country are tight, the planting area of cotton is decreasing sharply, the important way to solve this problem is to cultivate short season cotton varieties and to realize wheat and cotton double cropping. Flowering is an important process of plant transformation from vegetative growth to reproductive growth, and the early maturity of crops is closely related to flowering. Therefore, understanding and mastering the rules of crop flowering to regulate this process is of great significance for improving the precocity of cotton. FLP1flowering-promoting factor 1-like protein 1) gene is the homologous gene of FPF1(flowering-promoting factor-1) in Arabidopsis thaliana. At present, there is no in-depth functional verification of FLP1 gene in Arabidopsis thaliana. In this study, the homologous gene GhFLP1 of FLP1 was cloned from upland cotton, and its function was studied by bioinformatics analysis, expression pattern analysis and transgenic Arabidopsis thaliana test. According to the original results of laboratory studies, the function of transgenic cotton with GhFPF1 gene was studied and its function in cotton was preliminarily determined. The results were as follows: 1. The length of GhFLP1 gene was 537 BP, and the length of GhFLP1 gene was 537 BP, which contained 339bp open reading frame. Encode 112 amino acids. The relative molecular weight of its protein is 12.176 kDa.The theoretical isoelectric point (9.13.GhFLP1) is conserved with other species' FLP1 protein sequence. The similarity of the FLP1 sequence with the D genome of Raymond's cotton is as high as 99.2. The exogenous hormone treatment test proves that GhFLP1 can respond to the treatment of GA and SA. The results showed that GhFLP1 might be involved in plant flowering and stress regulation. The expression patterns of GhFLP1 in different cultivars showed strong tissue expression specificity, and the expression level in flower buds was much higher than that in other tissues. The results also showed that the expression of this gene in the buds of early maturing varieties Zhongmiansuo 36 and Zhongmiaojiao74 was significantly higher than that of late varieties Zhongmiansuo 60 and Lumiyan 28.4.The expression of GhFLP1 in Arabidopsis thaliana was overexpressed. The flowering time of transgenic Arabidopsis thaliana was earlier and the number of rosette leaves was decreased. Some flowering time promoting genes in transgenic Arabidopsis thaliana, such as AtFTFT-AtAP1, AtLFYYN, AtSOC1, were all increased, and the expression of inhibiting flowering gene AtFLC was significantly down-regulated. The cotton VIGS vector pCLCrVA-GhFLP1 was constructed to inoculate cotton. The expression of GhFLP1 in the infected plants was down-regulated and no obvious phenotypic changes were observed at present. The phenotypic changes of transgenic cotton with GhFPF1 gene were observed. It was found that the flowering period of transgenic lines was longer than that of wild-type plants and the plants were higher than those of wild-type plants. The results of hydroponic culture showed that the root system of transgenic lines became more developed. QRT-PCR results showed that the expression of GhFPF1 in roots and stems of transgenic lines was much higher than that in other tissues.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S562

【相似文献】