古代莲超长链脂肪酸延伸相关基因NnCER26的克隆及功能分析

发布时间：2018-04-29 22:01

本文选题：表皮蜡质 + 古代莲　；参考：《中国科学院武汉植物园》2017年硕士论文

【摘要】：表皮蜡质覆盖在陆地植物表面,具有抵御病虫害、防止水分流失等多种功能。因此,蜡质在植物生长发育及适应外界环境方面起着重要作用。而超长链脂肪酸是植物组织中多种脂质的重要前体,包括表皮蜡质、细胞膜上的鞘脂和磷脂、脂肪族木栓质以及种子中的甘油三酯。超长链脂肪酸及其衍生物能够作为植物与环境间的屏障,提供储存于种子中的能量,或作为膜信号分子而发挥作用。目前,关于拟南芥蜡质合成及超长链脂肪酸延伸的研究较多,而对莲蜡质的研究还只局限于成分分析,对其蜡质合成及超长链脂肪酸延伸相关基因的研究尚未见报道。通过序列比对,并参考基因在莲叶中的表达量,我们筛选得到了参于古代莲超长链脂肪酸延伸的候选基因,命名为NnCER26LIKE1和NnCER26LIKE2。克隆到基因后,利用拟南芥遗传转化、酵母异源表达等技术,我们对候选基因的功能进行了分析。实验丰富了蜡质合成与超长链脂肪酸延伸的理论基础,为深入了解莲超长链脂肪酸延伸机制提供了依据。主要结果如下:1、通过扫描电镜观察不同品种莲的表皮蜡质,分析表明它们莲叶正面蜡质结构以颗粒状为主,而背面蜡质结构以棒状为主,同时,不同品种间存在差异。利用薄层层析对其蜡质成分分离,观察结果,不同品种分离得到的条带同样存在差异,其中古代莲543蜡质分离得到的条带较少,蜡质成分相对简单。2、利用序列比对筛选到莲中拟南芥CER26基因的同源基因,即NnCER26LIKE1、NnCER26LIKE2。组织特异性表达分析结果表明,NnCER26LIKE1、NnCER26LIKE2在莲花瓣、花药、浮叶、立叶高表达,符合已报道蜡质超长链脂肪酸延伸相关基因的表达模式。亚细胞定位实验表明,NnCER26LIKE1蛋白定位于内质网,与蜡质相关蛋白的定位情况相一致。3、在拟南芥中过量表达NnCER26LIKE1或NnCER26LIKE2影响了蜡质成分的碳链组成情况,说明两基因都参与了莲超长链脂肪酸延伸,其中NnCER26LIKE1参与了 C30-C32酸延伸,NnCER26LIKE2参与了 C30酸生成。酵母异源表达实验结果分析表明,NnCER26LIKE2与NnCER6共同参与C28酸和C30酸生成。
[Abstract]:Epidermal waxy covering on the surface of terrestrial plants has many functions, such as resisting diseases and insect pests, preventing water loss and so on. Therefore, wax plays an important role in plant growth and development and adaptation to the external environment. Ultra long chain fatty acids are important precursors of many lipids in plant tissues, including epidermal wax, sphingolipid and phospholipid on cell membrane, aliphatic cork and triglyceride in seeds. Ultra-long chain fatty acids and their derivatives can act as barriers between plants and the environment, provide energy stored in seeds, or act as membrane signaling molecules. At present, there are many studies on waxy synthesis and ultra-long chain fatty acid extension in Arabidopsis thaliana, while the study on lotus wax is limited to component analysis, and the research on the genes related to wax synthesis and ultra-long chain fatty acid extension has not been reported. By sequence alignment and the amount of gene expression in lotus leaves, we selected candidate genes named NnCER26LIKE1 and NnCER26LIKE2 for the extension of ultra-long chain fatty acids in ancient lotus. After the gene was cloned, the function of candidate gene was analyzed by means of Arabidopsis genetic transformation and yeast heterologous expression. The experiment enriches the theoretical basis of waxy synthesis and ultra long chain fatty acid extension, and provides a basis for further understanding the extension mechanism of super long chain fatty acids in lotus. The main results were as follows: 1. The epidermal waxes of different varieties of lotus were observed by scanning electron microscope. The results showed that the wax structure in the front surface of lotus leaves was mainly granular, while the wax structure on the back surface was rod-like. At the same time, there were differences among different cultivars. The waxy components were separated by thin-layer chromatography. The results showed that there were also differences in the bands isolated from different varieties, among which there were few bands isolated from ancient lotus 543. The waxy component is relatively simple. 2. The homologous gene of Arabidopsis thaliana CER26 gene, NnCER26LIKE1, NnCER26LIKE2, was screened by sequence alignment. The results of tissue specific expression analysis showed that NnCER26LIKE1NnCER26LIKE2 was highly expressed in lotus petals, anthers, floating leaves and vertical leaves, which was in accordance with the reported expression pattern of waxy ultra-long chain fatty acid extension related genes. Subcellular localization assay showed that NnCER26LIKE1 protein was located in endoplasmic reticulum, which was consistent with the localization of waxy related proteins. Overexpression of NnCER26LIKE1 or NnCER26LIKE2 in Arabidopsis affected the carbon chain composition of waxy components. The results showed that both genes were involved in the extension of ultra-long chain fatty acids of lotus, and NnCER26LIKE1 was involved in the production of C30 acid by C30-C32 acid extension NnCER26LIKE2. The results of yeast heterologous expression analysis showed that NnCER26LIKE2 and NnCER6 were involved in the formation of C28 and C30 acids.
【学位授予单位】：中国科学院武汉植物园
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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