本文選題：玉米　切入點：功能分析　出處：《上海大學》2016年博士論文　論文類型：學位論文

【摘要】：玉米是重要的糧食作物。優(yōu)質(zhì)、高產(chǎn)和耐逆是玉米生產(chǎn)上的主要育種目標,因此相關(guān)功能基因的研究,對玉米生產(chǎn)育種有顯著意義。本研究分別開展了對三個功能基因的研究,包括耐逆相關(guān)基因ZmVPP5,品質(zhì)相關(guān)基因Opaque7,以及產(chǎn)量相關(guān)基因Shrunken4。植物中VPPs蛋白是重要的質(zhì)子泵之一,由多基因編碼,與植物高鹽抗性相關(guān)。Zm VPP5是一個截短形式的VPPs(vacuolar H+-Pyrophosphatase)蛋白,缺乏完成質(zhì)子轉(zhuǎn)運相關(guān)的保守結(jié)構(gòu)域,因此不具有焦磷酸酶活性和質(zhì)子轉(zhuǎn)運活性,但具有完整的VPP1蛋白的二聚化區(qū)段,可能對VPP1具有調(diào)控作用。通過定位分析,發(fā)現(xiàn)該蛋白能夠定位在質(zhì)膜、液泡和細胞核膜上,具有潛在的信號傳導能力。為了研究其生理功能,在酵母和玉米中對ZmVPP5進行了過表達,結(jié)果表明過表達ZmVPP5表現(xiàn)出鹽逆境的超敏感性。對玉米中部分鹽脅迫應答基因進行了轉(zhuǎn)錄水平的研究,發(fā)現(xiàn)過表達Zm VPP5抑制了這些基因的表達。對Zm VPP5進行了酵母雙雜交篩選,得到了互作蛋白ZmBag6,通過LCI(luciferase complementation image)分析和共定位分析表明,兩者可在細胞內(nèi)發(fā)生蛋白互作。Zm VPP5可能作為VPPs蛋白的抑制蛋白,在鹽逆境應答中起作用。該研究擴展了我們對VPPs家族蛋白生物學功能的認識。opaque7是一個經(jīng)典的玉米高品質(zhì)籽粒突變體,具有籽粒柔軟、粉質(zhì)和不透明表型,籽粒賴氨酸含量顯著提高。該基因前期由實驗室通過圖位克隆技術(shù)克隆。序列分析預測Opaque7編碼一個酰基輔酶A合成酶。通過蛋白表達、酶活測定,我們確定該酶的專一性底物為草酸,因此Opaque7是一個草酰輔酶A合成酶,通過煙草融合熒光蛋白細胞定位分析和玉米細胞器分離的方法,證明該蛋白定位在細胞質(zhì)基質(zhì)中。對野生型和突變體關(guān)鍵代謝物進行質(zhì)譜分析,發(fā)現(xiàn)突變體影響了三羧酸循環(huán)(TCA)中草酰乙酸和а-酮戊二酸的含量,從而影響了賴氨酸和谷氨酸/谷氨酰胺等的生物合成,造成醇溶蛋白合成下降,蛋白體減少變小,最終產(chǎn)生opaque表型。shrunken4(sh4)是一個經(jīng)典的粒重下降突變體,籽粒呈皺縮,不透明,頂部凹陷的表型,淀粉和蛋白含量顯著下降。細胞學觀察表明胚乳細胞形態(tài)異常,物質(zhì)合成不足;淀粉粒和蛋白體數(shù)目顯著降低。生化成分測定結(jié)果表明,蛋白、淀粉和油脂三大儲藏物質(zhì)的合成顯著下降,而水解氨基酸,葡萄糖等基礎代謝物也發(fā)生顯著降低。對野生型和突變體進行了RNA-Seq分析,結(jié)果表明sh4突變造成了大量基因表達變化,包括細胞凋亡和自噬相關(guān)基因,物質(zhì)合成代謝相關(guān)基因,以及蛋白合成相關(guān)基因。對野生型和突變體進行活細胞染色觀察發(fā)現(xiàn),sh4存在細胞凋亡提前的現(xiàn)象;透射電鏡觀察到了正在自噬的細胞。通過精細定位,把區(qū)間縮小到了43Kb的范圍,其內(nèi)的候選基因正在進行轉(zhuǎn)基因分析。對上述三個玉米功能基因的研究,推進了我們對玉米抗逆、品質(zhì)和產(chǎn)量性狀相關(guān)機制的認識,為培育高產(chǎn)、優(yōu)質(zhì)和抗逆玉米提供了知識和線索。
[Abstract]:Maize is an important food crop. High quality, high yield and resistance is the main breeding goal of maize production, so the function of genetic studies, is significant for maize production and breeding. This study was carried out to study gene on three functions, including resistance related genes ZmVPP5, Opaque7 and VPPs genes related to quality. Yield genes in plants Shrunken4. protein is one of the important proton pump, by genetic encoding, associated with high salt resistant plant.Zm VPP5 is a truncated VPPs protein (vacuolar H+-Pyrophosphatase), the lack of complete conserved domains related to proton translocation, therefore does not have the pyrophosphatase activity and proton transport activity, dimerization region but with a complete VPP1 protein, may play a role in the regulation of VPP1. Through the positioning analysis revealed that the protein can be localized in the plasma membrane, vacuole and cell membrane, with potential In the signal transduction ability. In order to study its physiological function in yeast and corn were carried out on ZmVPP5 expression, the results showed that over expression of ZmVPP5 showed high sensitivity to salt stress. Salt stress responsive genes in maize were studied in the transcription level of Zm overexpression of VPP5 inhibited the expression of these genes. The Zm VPP5 yeast two hybrid screening, the interaction of protein ZmBag6 by LCI (luciferase complementation image) analysis and co localization analysis showed that both in the cell protein interaction.Zm VPP5 may act as a suppressor protein VPPs protein, play a role in the salt stress response. This study expands our.Opaque7 understanding of the biological function of VPPs protein family is a classic high quality corn grain mutant, grain with soft, silty and opaque phenotype, lysine content significantly increased. The early gene from the laboratory through positional cloning techniques. Sequence analysis of Opaque7 encoding a predicted acyl coenzyme A synthetase. The protein expression, enzyme activity assay, we determined the enzyme substrate specificity for oxalic acid, so Opaque7 is a oxalyl coenzyme A synthetase, tobacco fluorescent protein fusion method through analysis and positioning corn cell separation, the protein located in cytoplasmic matrix. Mass spectrometry analysis of wild-type and mutant key metabolites, found mutants affecting three tricarboxylic acid cycle (TCA) content in oxaloacetate and alpha ketoglutarate, thus affecting the biosynthesis of lysine and glutamate / glutamine, caused by alcohol protein synthesis decreased, decreases protein body, resulting in the phenotype of opaque.Shrunken4 (SH4) is a classical particle mutant decreased, grain is shrinking, not transparent, top sag The phenotype, starch and protein content decreased significantly. Cytological observation indicated that abnormal endosperm cell morphology, material synthesis; starch grains and protein body number decreased significantly. The determination results showed that the biochemical composition, protein, starch and oil significantly decreased synthesis of three storage materials, and hydrolyzed amino acids, glucose and other basic metabolites also occurred significantly reduced. The wild type and mutants were analyzed by RNA-Seq. The results showed that the SH4 mutation resulted in the expression of a large number of genes, including cell apoptosis and autophagy related genes, metabolism related genes in material synthesis, and protein synthesis related genes. The wild type and mutant of live cell staining observed SH4 cell apoptosis exists in advance; transmission electron microscope observed are autophagic cells. Through fine mapping, the interval to narrow the scope of the 43Kb, which is a candidate gene in transgenic Analysis of these three maize functional genes has promoted our understanding of the related mechanisms of stress resistance, quality and yield traits, and provided knowledge and clues for cultivating high yield, high quality and resistant maize.

【學位授予單位】：上海大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S513;Q943.2

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