玉米淀粉合成相关调控基因ZmGRAS17的功能分析

发布时间：2018-06-29 10:40

本文选题：玉米 + GRAS蛋白家族　；参考：《安徽农业大学》2016年硕士论文

【摘要】：玉米是全世界重要的粮食作物之一,同时也是高产量的谷类作物,不仅可以作为人类和动物的粮食,而且还可以为工业生产提供重要的原材料,在人类的生产生活中占据越来越重要的位置。淀粉占有玉米籽粒的大部分比重,提高淀粉含量对于提高作物产量至关重要。由于分子结构的差异可以将淀粉分为直链淀粉和支链淀粉,他们因本身结构以及在淀粉中所占的比重不同从而直接或间接地影响了淀粉的理化特性以及进一步加工性能,最终影响到淀粉的最适用途。因此,研究如何提高和改善玉米籽粒中的淀粉含量以及直链淀粉和支链淀粉的比重,有助于对淀粉代谢途径的进一步研究,对进一步完善淀粉代谢途径,改善淀粉含量和品质的研究有重要的理论和实践意义。本实验利用转录组测序数据并结合生物信息学分析筛选出与淀粉合成相关的基因ZmGRAS17,并通过过表达技术对该基因在玉米淀粉代谢途径中的功能及作用机制进行了初步探索。获得了以下主要结果:1.根据转录组测序结果和生物信息学分析,筛选了一个淀粉合成相关调控基因ZmGRAS17,并从玉米自交系B73中克隆出该基因。该基因编码区全长1527bp,总共编码508个氨基酸,其编码蛋白的相对分子质量为56.08 kDa,等电点为5.91,氨基酸分析显示其属于GRAS家族中的DELLA分支蛋白。2.不同组织荧光定量PCR分析显示,ZmGRAS17在选取的根、茎、叶、雄穗、花丝、果穗、胚和胚乳组织中均有表达,胚乳中的表达量明显高于其他组织,尤其在授粉后20 d的胚乳中表达量最高。3.转录自激活检测实验表明,与对照组相比,转化过pGBKT7-ZmGRAS17载体的酵母细胞在SD/Trp-His-Ade/X-α-GAL三缺板上无法正常生长,只能在SD/Trp-一缺培养平板上生长,没有显示出α-半乳糖苷酶活性,这些结果表明ZmGRAS17在酵母中无自身激活能力。4.亚细胞定位实验表明,ZmGRAS17与GFP载体的融合表达蛋白定位于细胞膜中,是一个膜定位蛋白。5.过量表达ZmGRAS17的转基因植株与野生型和空载体相比,除籽粒长度和厚度变小外,其他生长表型上没有明显差异。通过对籽粒淀粉含量的分析发现,转基因种子籽粒中的总淀粉明显低于野生型和空载体,与野生型相比下降了3.16%,而转基因植株中直链淀粉含量较野生型下降了16.86%;扫面电镜实验发现,ZmGRAS17转基因种子中部和背部的淀粉颗粒较野生型和空载体没有发生变化,但腹部的淀粉颗粒变化非常明显。野生型和空载体种子腹部的淀粉体呈致密均匀分布,无游离淀粉颗粒,而转基因种子腹部的淀粉体排列疏松,其间游离着很多球形单粒淀粉粒;对籽粒可溶性糖含量分析发现,与野生型和空载体相比,转基因植株籽粒的可溶性糖含量略有上升;对籽粒农艺性状的统计研究发现,转基因植株相对于野生型和空载体植株,籽粒长度几乎没有变化,而籽粒宽度和厚度明显下降,较野生型分别降低了3.74%和6.19%,千粒重随之也明显下降,较野生型降低了10.63%。这些结果初步说明ZmGRAS17基因的过表达影响了淀粉颗粒结构和大小。
[Abstract]:Corn is one of the most important grain crops in the world. It is also a grain crop with high yield. It can not only serve as food for human and animal, but also provide important raw materials for industrial production. It occupies a more and more important position in human production and life. Starch occupies most of the proportion of corn grain and improves starch content. Because of the difference in molecular structure, the starch can be divided into amylose and amylopectin, which directly or indirectly affect the physicochemical properties and further processing properties of starch, which will affect the most suitable way of starch. The research on how to improve and improve the starch content in corn grain and the proportion of amylose and amylopectin can help to further study the pathway of starch metabolism. It has important theoretical and practical significance to further improve the pathway of starch metabolism and improve the content and quality of starch. The gene ZmGRAS17 related to starch synthesis was screened by bioinformatics analysis, and the function and mechanism of the gene in maize starch metabolism pathway were preliminarily explored through overexpression technology. The following main results were obtained: 1. a starch synthesis phase was screened according to the sequencing results of the transcriptional group and the bioinformatics analysis. The gene, ZmGRAS17, was cloned from the maize inbred line B73, which encodes a total length of 1527bp and encodes a total of 508 amino acids. The relative molecular mass of the encoded protein is 56.08 kDa and the isoelectric point is 5.91. The amino acid analysis shows that it belongs to the DELLA branching protein.2. in the GRAS family with the fluorescence quantitative PCR analysis of different tissues. ZmGRAS17 was expressed in the selected roots, stems, leaves, spikes, flowers, spikes, spikes, embryos and endosperm tissues. The expression of the endosperm in the endosperm was significantly higher than that of other tissues, especially in the 20 d endosperm after pollination, the highest.3. transcriptional activation test showed that the yeast cells that transformed the pGBKT7-ZmGRAS17 vector were in SD/Trp-Hi compared with the control group. S-Ade/X- alpha -GAL three can not grow normally on the deficient plate and can only grow on a SD/Trp- deficient culture plate and does not show alpha galactosidase activity. These results show that ZmGRAS17 in yeast has no self activation capacity.4. subcellular localization experiment, which indicates that the fusion protein expression protein of ZmGRAS17 and GFP carrier is located in the cell membrane, and is a membrane binding. Compared with the wild type and the empty vector, the transgenic plants with the protein.5. overexpressed ZmGRAS17 had no obvious difference in the other growth phenotypes except the grain length and thickness. The total starch in the grain of the transgenic seeds was significantly lower than that of the wild type and the empty carrier, and the total starch content of the transgenic seeds decreased by 3.16 compared with the wild type. The amylose content of the transgenic plants decreased by 16.86% than that of the wild type, and the scanning electron microscopy showed that the starch granules in the middle and back of the ZmGRAS17 transgenic seeds were not changed, but the changes in the starch granules in the abdomen were very obvious. The amyloid body in the abdomen of the wild type and the empty body showed a compact and uniform distribution. There was no free starch granules, and the starch bodies in the abdomen of the transgenic seeds were loosely arranged and many spherical single starch granules were free between them. The soluble sugar content of the grain was slightly higher than that of the wild type and the empty carrier. Compared with the wild type and the empty carrier, the grain length almost did not change, but the grain width and thickness decreased obviously, which decreased by 3.74% and 6.19% compared with the wild type, and the 1000 grain weight decreased obviously, and the 10.63%. results showed that the overexpression of ZmGRAS17 gene affected the structure and size of starch granules.
【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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