DGDG合成酶基因MtDGD1和聚半乳糖醛酸酶基因MtPG在蒺藜苜蓿共生固氮中的功能研究

发布时间：2018-05-08 06:28

本文选题：蒺藜苜蓿 + MtDGD1　；参考：《华中农业大学》2017年硕士论文

【摘要】：双半乳糖甘油二脂(digalactosyl diacylglycerol,DGDG)是广泛存在于高等植物叶绿体和蓝藻中的一类半乳糖脂,作为类囊体膜的主要组成成分之一,DGDG对于植物光合作用功能的发挥以及植物的生长发育具有重大意义。而在豆科植物中,DGDG也在其根瘤共生体膜上存在。DGD1是DGDG的主要合成酶之一。在前期工作中,已克隆到该基因的全长,通过免疫荧光和免疫电镜发现Mt DGD1定位于根瘤中含菌细胞的共生体膜上,构建了该基因沉默和超表达载体。本研究以豆科模式植物蒺藜苜蓿A17的双半乳糖甘油二脂合成酶基因(Mt DGD1)为对象,在前期已有的工作基础上,对该基因在豆科植物共生固氮中的功能机制进行进一步探究。主要研究内容和结果如下:1.通过生物信息学网站分析、获取、克隆Mt DGD1启动子序列,构建了启动子-GUS融合载体,进行了组织表达定位观察。通过苜蓿基因表达谱网站及转录组分析,利用Real_time PCR检测了Mt DGD1的时空表达和组织特异性表达,确证该基因与共生固氮作用密切相关。2.通过植株长势、根瘤切片的光学显微镜和透射电子显微镜观察、根瘤固氮酶活测定以及根瘤中DGDG脂质丰度的测定等多个指标,检测和考察了Mt DGD1沉默及超表达后转基因植株的共生表型,阐明了该基因在共生固氮中的作用;3.通过Real_time PCR和LC-MS分别检测了低磷条件下Mt DGD1的转录水平,DGDG及磷脂酰肌醇(PI)、磷脂酰乙醇胺(PE)、磷脂酰胆碱(PC)等脂质丰度的变化,初步明确该基因在根瘤发育过程中的功能机制。本研究获得了以上直接实验证据,确证了Mt DGD1在豆科植物根瘤菌入侵、根瘤发育和共生固氮中的功能,也表明了DGDG在根瘤共生固氮过程中的重要性。聚半乳糖醛酸酶基因(Polygalacturonase,PG)普遍存在于高等植物细胞中,因其能够降解细胞壁、促进细胞分裂和果实成熟等功能,而与植物生长发育过程中有着非常密切的联系。本研究以豆科模式植物蒺藜苜蓿一个PG(Medtr6g005630)基因为对象,对该基因进行了简单生物信息学分析,该基因全长4150 bp,其中编码区1278 bp,编码425个氨基酸;对其编码的聚半乳糖醛酸酶的保守结构域分析结果发现:该酶包括PL-6 superfamily和Glyco_hydro_28两个与细胞壁主要成分--果胶降解有关的保守结构域;氨基酸序列比对和系统进化分析发现该酶与鹰嘴豆、赤豆等豆科植物中的同源关系更近。此外,根据苜蓿基因表达谱数据库分析和RT-PCR实验结果揭示Mt PG在根瘤中特异性表达,而在其他非共生组织如根、茎、叶中表达量很少;此外,从英国John Innes Centre获赠3种Tnt1转座子插入突变体材料(NF0999、NF5561和NF4746),通过对该基因Tnt1转座子插入位点分析设计引物,利用PCR进行突变体筛选、验证及纯合突变体共生表型观察分析等方面揭示了该基因可能对豆科植物共生固氮中的早期侵染过程有很重要的作用。
[Abstract]:Digalactosyl diacylglycerol (DGDGGGGG) is a kind of galactose lipid found widely in chloroplasts and cyanobacteria of higher plants. As one of the main components of thylakoid membrane, DGDG plays an important role in the development of plant photosynthesis and plant growth. DGDG is one of the main synthase of DGDG in the rhizobial symbiotic membrane of legumes. The full length of the gene was cloned in the previous work. It was found that Mt DGD1 was located on the membrane of symbiotic cells containing bacteria in rhizobia by immunofluorescence and immunoelectron microscopy, and the gene silencing and overexpression vector was constructed. In this study, the double galactosylglycerol synthase gene (Mt DGD1) of Tribulus terrestris A17 was used as the object of this study. Based on the previous work, the functional mechanism of the gene in the symbiotic nitrogen fixation of legume was further explored. The main contents and results are as follows: 1. Through bioinformatics website analysis, the promoter sequence of Mt DGD1 was obtained and cloned, and the promoter -GUS fusion vector was constructed, and the tissue expression localization was observed. By using the website of alfalfa gene expression profile and transcriptome analysis, the spatiotemporal expression and tissue specific expression of Mt DGD1 were detected by Real_time PCR. It was confirmed that the gene was closely related to symbiotic nitrogen fixation. Through the observation of plant growth, optical microscope and transmission electron microscope of nodule sections, the determination of nitrogenase activity in nodules and the determination of DGDG lipid abundance in nodules, and so on, The symbiotic phenotype of transgenic plants after Mt DGD1 silencing and overexpression was detected and investigated, and the role of the gene in symbiotic nitrogen fixation was elucidated. The changes of lipid abundance of Mt DGD1, such as phosphatidylinositol, phosphatidyl ethanolamine, phosphatidylcholine and phosphatidylcholine, were determined by Real_time PCR and LC-MS, respectively. This study obtained the direct experimental evidence above, confirmed the function of Mt DGD1 in the rhizobia invasion, rhizobia development and symbiotic nitrogen fixation of legumes, and also demonstrated the importance of DGDG in the process of nodule symbiotic nitrogen fixation. Polygalacturonase (PGN) is a common gene in higher plant cells, which is closely related to plant growth and development because it can degrade cell wall, promote cell division and fruit maturation. In this study, we studied a PGTR Medtr6g005630) gene of Tribulus terrestris, a leguminous model plant, and analyzed the gene by bioinformatics. The gene was 4150 BP in length and 1278 BP in coding region, encoding 425 amino acids. The analysis of the conserved domain of its encoded polygalacturonase showed that it included two conserved domains, PL-6 superfamily and Glyco_hydro_28, which were related to the degradation of pectin, the main component of cell wall. Amino acid sequence alignment and phylogenetic analysis showed that the enzyme had closer homology with chickpea, red bean and other legume plants. In addition, based on the database analysis of alfalfa gene expression profile and RT-PCR results, it was revealed that Mt PG was specifically expressed in rhizobia, but not in other non-symbiotic tissues such as roots, stems and leaves. Three kinds of Tnt1 transposon insertion mutants, Nf0999, NF5561 and NF4746, were obtained from British John Innes Centre. Primers were designed by analyzing the Tnt1 transposon insertion site of the gene, and the mutants were screened by PCR. Validation and observation of symbiotic phenotypes of homozygous mutants revealed that the gene may play an important role in the early infection process of symbiotic nitrogen fixation in legumes.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q945.13

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