玉米ZmVPP5、Opaque7和Shrunken4基因的功能研究

发布时间：2018-03-21 17:21

本文选题：玉米　切入点：功能分析　出处：《上海大学》2016年博士论文　论文类型：学位论文

【摘要】：玉米是重要的粮食作物。优质、高产和耐逆是玉米生产上的主要育种目标,因此相关功能基因的研究,对玉米生产育种有显著意义。本研究分别开展了对三个功能基因的研究,包括耐逆相关基因ZmVPP5,品质相关基因Opaque7,以及产量相关基因Shrunken4。植物中VPPs蛋白是重要的质子泵之一,由多基因编码,与植物高盐抗性相关。Zm VPP5是一个截短形式的VPPs(vacuolar H+-Pyrophosphatase)蛋白,缺乏完成质子转运相关的保守结构域,因此不具有焦磷酸酶活性和质子转运活性,但具有完整的VPP1蛋白的二聚化区段,可能对VPP1具有调控作用。通过定位分析,发现该蛋白能够定位在质膜、液泡和细胞核膜上,具有潜在的信号传导能力。为了研究其生理功能,在酵母和玉米中对ZmVPP5进行了过表达,结果表明过表达ZmVPP5表现出盐逆境的超敏感性。对玉米中部分盐胁迫应答基因进行了转录水平的研究,发现过表达Zm VPP5抑制了这些基因的表达。对Zm VPP5进行了酵母双杂交筛选,得到了互作蛋白ZmBag6,通过LCI(luciferase complementation image)分析和共定位分析表明,两者可在细胞内发生蛋白互作。Zm VPP5可能作为VPPs蛋白的抑制蛋白,在盐逆境应答中起作用。该研究扩展了我们对VPPs家族蛋白生物学功能的认识。opaque7是一个经典的玉米高品质籽粒突变体,具有籽粒柔软、粉质和不透明表型,籽粒赖氨酸含量显著提高。该基因前期由实验室通过图位克隆技术克隆。序列分析预测Opaque7编码一个酰基辅酶A合成酶。通过蛋白表达、酶活测定,我们确定该酶的专一性底物为草酸,因此Opaque7是一个草酰辅酶A合成酶,通过烟草融合荧光蛋白细胞定位分析和玉米细胞器分离的方法,证明该蛋白定位在细胞质基质中。对野生型和突变体关键代谢物进行质谱分析,发现突变体影响了三羧酸循环(TCA)中草酰乙酸和а-酮戊二酸的含量,从而影响了赖氨酸和谷氨酸/谷氨酰胺等的生物合成,造成醇溶蛋白合成下降,蛋白体减少变小,最终产生opaque表型。shrunken4(sh4)是一个经典的粒重下降突变体,籽粒呈皱缩,不透明,顶部凹陷的表型,淀粉和蛋白含量显著下降。细胞学观察表明胚乳细胞形态异常,物质合成不足;淀粉粒和蛋白体数目显著降低。生化成分测定结果表明,蛋白、淀粉和油脂三大储藏物质的合成显著下降,而水解氨基酸,葡萄糖等基础代谢物也发生显著降低。对野生型和突变体进行了RNA-Seq分析,结果表明sh4突变造成了大量基因表达变化,包括细胞凋亡和自噬相关基因,物质合成代谢相关基因,以及蛋白合成相关基因。对野生型和突变体进行活细胞染色观察发现,sh4存在细胞凋亡提前的现象;透射电镜观察到了正在自噬的细胞。通过精细定位,把区间缩小到了43Kb的范围,其内的候选基因正在进行转基因分析。对上述三个玉米功能基因的研究,推进了我们对玉米抗逆、品质和产量性状相关机制的认识,为培育高产、优质和抗逆玉米提供了知识和线索。
[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.

【学位授予单位】：上海大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S513;Q943.2

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