本文選題：蒺藜苜蓿 + MtDGD1�。� 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：雙半乳糖甘油二脂(digalactosyl diacylglycerol,DGDG)是廣泛存在于高等植物葉綠體和藍(lán)藻中的一類半乳糖脂,作為類囊體膜的主要組成成分之一,DGDG對于植物光合作用功能的發(fā)揮以及植物的生長發(fā)育具有重大意義。而在豆科植物中,DGDG也在其根瘤共生體膜上存在。DGD1是DGDG的主要合成酶之一。在前期工作中,已克隆到該基因的全長,通過免疫熒光和免疫電鏡發(fā)現(xiàn)Mt DGD1定位于根瘤中含菌細(xì)胞的共生體膜上,構(gòu)建了該基因沉默和超表達(dá)載體。本研究以豆科模式植物蒺藜苜蓿A17的雙半乳糖甘油二脂合成酶基因(Mt DGD1)為對象,在前期已有的工作基礎(chǔ)上,對該基因在豆科植物共生固氮中的功能機(jī)制進(jìn)行進(jìn)一步探究。主要研究內(nèi)容和結(jié)果如下:1.通過生物信息學(xué)網(wǎng)站分析、獲取、克隆Mt DGD1啟動子序列,構(gòu)建了啟動子-GUS融合載體,進(jìn)行了組織表達(dá)定位觀察。通過苜蓿基因表達(dá)譜網(wǎng)站及轉(zhuǎn)錄組分析,利用Real_time PCR檢測了Mt DGD1的時空表達(dá)和組織特異性表達(dá),確證該基因與共生固氮作用密切相關(guān)。2.通過植株長勢、根瘤切片的光學(xué)顯微鏡和透射電子顯微鏡觀察、根瘤固氮酶活測定以及根瘤中DGDG脂質(zhì)豐度的測定等多個指標(biāo),檢測和考察了Mt DGD1沉默及超表達(dá)后轉(zhuǎn)基因植株的共生表型,闡明了該基因在共生固氮中的作用;3.通過Real_time PCR和LC-MS分別檢測了低磷條件下Mt DGD1的轉(zhuǎn)錄水平,DGDG及磷脂酰肌醇(PI)、磷脂酰乙醇胺(PE)、磷脂酰膽堿(PC)等脂質(zhì)豐度的變化,初步明確該基因在根瘤發(fā)育過程中的功能機(jī)制。本研究獲得了以上直接實驗證據(jù),確證了Mt DGD1在豆科植物根瘤菌入侵、根瘤發(fā)育和共生固氮中的功能,也表明了DGDG在根瘤共生固氮過程中的重要性。聚半乳糖醛酸酶基因(Polygalacturonase,PG)普遍存在于高等植物細(xì)胞中,因其能夠降解細(xì)胞壁、促進(jìn)細(xì)胞分裂和果實成熟等功能,而與植物生長發(fā)育過程中有著非常密切的聯(lián)系。本研究以豆科模式植物蒺藜苜蓿一個PG(Medtr6g005630)基因為對象,對該基因進(jìn)行了簡單生物信息學(xué)分析,該基因全長4150 bp,其中編碼區(qū)1278 bp,編碼425個氨基酸;對其編碼的聚半乳糖醛酸酶的保守結(jié)構(gòu)域分析結(jié)果發(fā)現(xiàn):該酶包括PL-6 superfamily和Glyco_hydro_28兩個與細(xì)胞壁主要成分--果膠降解有關(guān)的保守結(jié)構(gòu)域;氨基酸序列比對和系統(tǒng)進(jìn)化分析發(fā)現(xiàn)該酶與鷹嘴豆、赤豆等豆科植物中的同源關(guān)系更近。此外,根據(jù)苜蓿基因表達(dá)譜數(shù)據(jù)庫分析和RT-PCR實驗結(jié)果揭示Mt PG在根瘤中特異性表達(dá),而在其他非共生組織如根、莖、葉中表達(dá)量很少;此外,從英國John Innes Centre獲贈3種Tnt1轉(zhuǎn)座子插入突變體材料(NF0999、NF5561和NF4746),通過對該基因Tnt1轉(zhuǎn)座子插入位點(diǎn)分析設(shè)計引物,利用PCR進(jìn)行突變體篩選、驗證及純合突變體共生表型觀察分析等方面揭示了該基因可能對豆科植物共生固氮中的早期侵染過程有很重要的作用。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q945.13

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相關(guān)博士學(xué)位論文 前1條

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本文編號：1860320