棉花与烟粉虱互作的蛋白组学分析和GhMLP423基因功能分析

发布时间：2018-05-02 16:33

本文选题：烟粉虱 + 棉花蛋白组　；参考：《华中农业大学》2016年硕士论文

【摘要】：棉花作为我国最重要的经济作物之一,在生产过程中长期遭到各种虫害的严重威胁,近些年来很多棉花种植区烟粉虱频繁发生,给棉花生产带来严重危害,使得棉花产量和纤维品质大幅度下降。烟粉虱刺吸胁迫后,棉花植株响应机制的研究还没有详细报道,其自身的生理机制的变化还不清楚。主要乳状蛋白(MLPs)是Bet v1蛋白亚家族之一,MLPs与另一个亚家族病理机制相关蛋白10(PR10)非常相似,然而目前关于MLP家族蛋白在植物响应胁迫方面的机制还不清楚,我们希望通过相关的分子生物学技术探索MLP蛋白在棉花响应烟粉虱胁迫过程中的作用。我们通过蛋白组手段对烟粉虱抗性较强的JH棉花品种和抗性较弱的ZS棉花品种进行蛋白组表达差异分析,将表达差异的蛋白质进行质谱鉴定分析,成功鉴定出263个棉花蛋白质,去除重复冗余后有176个可供分析的特异蛋白质。通过功能富集分析和代谢通路分析发现这些蛋白质有相当一部分参与了氧化还原反应,而且部分是与活性氧信号路径相关的酶。为了进一步了解烟粉虱侵染棉花后活性氧水平变化情况,我们对抗性材料JH进行酶活力测定发现超氧化物歧化酶活性比对照显著升高,过氧化氢含量初期升高后逐渐降低,而过氧化物酶活力显著降低,表明活性氧信号路径可能参与了棉花抵御烟粉虱侵染的过程。烟粉虱侵染后棉花的GhMLP423蛋白表达上升。为了探索棉花MLP蛋白在棉花响应烟粉虱胁迫中的作用,我们选取了棉花GhMLP423基因进行后续的生物学验证。通过VIGS技术成功干涉了GhMLP423基因并获得了干涉植株。同时,我们对干涉植株进行了酶活力和过氧化氢含量测定。棉花干涉系GhMLP423的过氧化物酶活力低于对照,超氧化物歧化酶活力高于对照,将超氧根离子转化为过氧化氢,因此初期过氧化氢含量高于对照,随着时间点延长而逐渐降低。另外,我们对干涉植株进行了激素水平测定发现GhMLP423干涉系植株叶片SA含量低于正常植株,烟粉虱侵染后明显高于非侵染植株,同时GhMLP423干涉系植株叶片中过氧化氢含量也低于正常植株,烟粉虱侵染后都有所升高,干涉系也明显低于对照,可能由于烟粉虱侵染棉花时SA积累抑制了过氧化氢酶的活性使得过氧化氢积累。与对照植株相比,GhMLP423干涉系没有积累ABA和JA而且对烟粉虱表现出更感的现象,初步推断GhMLP423可能参与了JA信号路径的传导过程。我们通过对烟粉虱不同抗性的棉花蛋白组进行表达差异分析,筛选对抗烟粉虱的棉花抗性相关的基因,同时通过对烟粉虱侵染后的棉花进行生理指标分析,进一步验证在棉花防御烟粉虱过程中相关蛋白的作用。
[Abstract]:Cotton, as one of the most important cash crops in China, has been seriously threatened by various insect pests for a long time in the production process. In recent years, whitefly occurs frequently in many cotton growing areas, which brings serious harm to cotton production. The cotton yield and fiber quality decreased significantly. The study of cotton plant response mechanism has not been reported in detail, and the change of its physiological mechanism is not clear. MLPs, one of the subfamilies of Bet v1 protein, is very similar to that of another subfamily. However, the mechanism of MLP family proteins in response to plant stress is still unclear. We hope to explore the role of MLP protein in cotton response to whitefly stress through molecular biological techniques. We analyzed the protein expression difference of Bemisia Tabaci resistance JH cotton varieties and ZS cotton varieties with weak resistance by proteomic analysis, and identified 263 cotton proteins by mass spectrometry. There are 176 specific proteins that can be analyzed after repetitive redundancy is removed. Through functional enrichment analysis and metabolic pathway analysis, it was found that some of these proteins were involved in the redox reaction, and some were enzymes related to the signal pathway of reactive oxygen species (Ros). In order to further understand the changes of reactive oxygen species (Ros) level in cotton infected with Bemisia Tabaci, we found that the activity of superoxide dismutase (SOD) of resistant material JH was significantly higher than that of the control, and that the content of hydrogen peroxide decreased gradually after the initial increase of the content of hydrogen peroxide. However, the activity of peroxidase decreased significantly, suggesting that the signal pathway of reactive oxygen species may be involved in the resistance of cotton to whitefly infection. The expression of GhMLP423 protein increased in cotton infected with Bemisia Tabaci. In order to explore the role of cotton MLP protein in cotton response to Bemisia Tabaci stress, we selected cotton GhMLP423 gene for further biological verification. The GhMLP423 gene was successfully interfered with by VIGS technique and the interference plants were obtained. At the same time, we determined the enzyme activity and hydrogen peroxide content of interference plants. The activity of peroxidase and superoxide dismutase (SOD) of cotton interference line GhMLP423 was lower than that of the control, and the superoxide dismutase activity was higher than that of the control, so the content of hydrogen peroxide in the initial stage was higher than that of the control, and gradually decreased with the extension of time. In addition, we determined the hormone level of the interference plants and found that the SA content in the leaves of GhMLP423 interference lines was lower than that in normal plants, and that in Bemisia Tabaci infected plants was significantly higher than that in non-infected plants. At the same time, the content of hydrogen peroxide in the leaves of GhMLP423 interference lines was lower than that in normal plants, and the content of hydrogen peroxide in the leaves of Bemisia Tabaci increased after infection. It was possible that SA accumulation inhibited the activity of catalase and resulted in hydrogen peroxide accumulation in cotton infected by Bemisia Tabaci. Compared with the control plant, GhMLP423 interference line did not accumulate ABA and JA and showed more sensitivity to Bemisia Tabaci. It was preliminarily concluded that GhMLP423 might be involved in the transmission of JA signal pathway. By analyzing the difference of expression of cotton protein groups with different resistance to Bemisia Tabaci, we screened the genes related to cotton resistance to Bemisia Tabaci, and analyzed the physiological indexes of cotton infected by Bemisia Tabaci. To further verify the role of related proteins in cotton defense against whitefly.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.622

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