木薯气孔调节相关基因的表达分析及其与耐旱性的关系

发布时间：2018-08-11 10:28

【摘要】：木薯(Manihot esculenta Crantz)是一种重要的热带块根类作物,具有高光合效率、抗旱、耐贫瘠等特点,是热带及亚热带干旱贫瘠地区主要的粮食作物。气孔是调节植物与外界进行气体和水分交换的重要结构,是影响植物耐旱性的主要因素之一。为了研究气孔密度及气孔调节与木薯抗旱性的关系。我们在干旱胁迫条件下对92个木薯栽培品种的气孔密度进行了统计分析,随后选择了 6个具有典型气孔密度的栽培品种做进一步的生理生化指标的测定,以期探索木薯光合效率、耐旱性与其气孔密度的相关性。同时,我们发现MeMYB2-RNAi转基因木薯的气孔密度明显比非转基因植株大,其光合速率也明显高于非转基因植株、但耐旱性也明显提高。在多个栽培品种以及MeMYB2-RNAi转基因木薯中的研究结果表明木薯气孔密度与抗旱性没有相关性。我们推测木薯在遭受干旱胁迫时很可能是通过快速关闭气孔的方式来减少自身水分的流失,以此避过干旱期。这种高气孔密度、快速关闭气孔的调控机制既能满足木薯作为热带作物高光效的需求,又能保证其耐旱性。植物在受到干旱胁迫时会产生激素脱落酸(ABA),并以此促使活性氧(ROS)的产生,从而关闭气孔,以降低叶片水分的蒸发,保护植物渡过干旱期。但这一过程中产生的大量ROS会破坏细胞内的氧化还原平衡,对植物体造成伤害。谷胱甘肽/谷氧还蛋白系统是植物体内一类重要的蛋白氧化还原系统,通过调控蛋白的氧化还原状态参与到植物的ROS信号传递途径中。CC类谷氧还蛋白(CC-type Glutaredoxins)是高等植物中所特有的一类谷氧还蛋白,能在植物遭受氧化胁迫时有效保护植物细胞免受氧化性损伤。本研究对木薯中多个CC-type GRX基因进行差异表达分析,发现木薯中有多个CC-type GRX基因在叶片中的表达受干旱胁迫的诱导。本课题组已针对木薯CC-type GRX参与ABA对气孔的调节过程做了相关研究,发现在过表达MeGRX232的转基因拟南芥中,ABA诱导气孔关闭的能力明显削弱,转基因拟南芥对干旱胁迫的耐受性降低。CC类谷氧还蛋白可以与TGA类转录因子互作,从而调控下游一系列基因的表达。为了进一步探究木薯中CC-type GRX的互作蛋白,我们通过酵母双杂实验筛选可以与木薯CC类谷氧还蛋白MeGRX058、MeGRX785互作的TGA因子,结果发现MeGRX058和MeTGA304能够相互作用。为进一步验证其与TGA转录因子的相互作用,我们选择了其中的MeGRX058兄和MeGRX785这两个基因作进一步体外蛋白表达分析,以期为木薯GRX后续功能的研究奠定基础。我们利用不同的标签蛋白分别对MeGRX058和MeGRX785进行标记,并分别在酵母和转基因植物中表达上述的两个重组蛋白,并试图通过标签蛋白来纯化重组蛋白。Western blot的结果表明两个重组蛋白均可在酵母和转基因植物中表达,但其表达产物被大量降解,我们推测MeGRX058和MeGRX785蛋白在体内存在泛素化降解。
[Abstract]:Cassava (Manihot esculenta Crantz) is an important tropical root crop with the characteristics of high light efficiency drought resistance and poor tolerance. It is the main food crop in arid and arid areas of the tropics and subtropics. Stomata are important structures regulating the exchange of gas and water between plants and the outside world, and they are one of the main factors affecting plant drought tolerance. In order to study the relationship between stomatal density, stomatal regulation and cassava drought resistance. The stomatal density of 92 cassava cultivars was statistically analyzed under drought stress, and 6 cultivars with typical stomatal density were selected for further determination of physiological and biochemical indexes. In order to explore the correlation between photosynthetic efficiency, drought tolerance and stomatal density of cassava. At the same time, we found that the stomatal density of MeMYB2-RNAi transgenic cassava was significantly higher than that of non-transgenic plants, and its photosynthetic rate was significantly higher than that of non-transgenic plants. The results in several cultivars and MeMYB2-RNAi transgenic cassava showed that there was no correlation between stomatal density and drought resistance of cassava. We speculate that cassava may reduce its water loss by closing the stomata quickly in order to avoid drought. The regulation mechanism of high stomatal density and rapid stomatal closure can not only meet the needs of cassava as a tropical crop with high light efficiency, but also ensure its drought tolerance. Plant plants under drought stress will produce hormone abscisic acid (ABA), and thus promote the production of reactive oxygen (ROS), thus closing stomata, in order to reduce leaf water evaporation, protect plants through the drought period. However, a large amount of ROS produced in this process can destroy the redox balance in cells and cause damage to plant. Glutathione / glutathione system is an important protein redox system in plants. By regulating the redox state of proteins, the glutendoxin (CC-type Glutaredoxins) is a unique class of glutenin in higher plants, which is involved in the ROS signal transduction pathway of plants. It can effectively protect plant cells from oxidative damage under oxidative stress. In this study, the differential expression of multiple CC-type GRX genes in cassava was analyzed. It was found that the expression of multiple CC-type GRX genes in cassava leaves was induced by drought stress. Our team has done some research on cassava CC-type GRX involved in the regulation of stomata by ABA. It was found that the ability of CC-type GRX to induce stomatal closure was significantly weakened in transgenic Arabidopsis thaliana with overexpression of MeGRX232. The reduced tolerance of transgenic Arabidopsis thaliana to drought stress. CC glutendoxin can interact with TGA transcription factors to regulate the expression of a series of genes downstream. In order to further explore the interaction protein of CC-type GRX in cassava, we screened out the TGA factor which could interact with CC glutenin (MeGRX058) and MeGRX785 in cassava by yeast double cross experiment. The results showed that MeGRX058 and MeTGA304 could interact with each other. In order to further verify its interaction with TGA transcription factors, we selected two genes, brother MeGRX058 and MeGRX785, for further protein expression analysis in vitro, in order to lay a foundation for the further study of GRX function in cassava. We use different label proteins to label MeGRX058 and MeGRX785, and express these two recombinant proteins in yeast and transgenic plants, respectively. The results showed that the two recombinant proteins could be expressed in yeast and transgenic plants, but the expressed products were degraded in large quantities. We speculated that the MeGRX058 and MeGRX785 proteins were degraded by ubiquitin in vivo.
【学位授予单位】：海南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S533

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