苹果轮纹病的寄主应答反应研究

发布时间：2018-04-19 23:30

本文选题：苹果 + 轮纹病　；参考：《中国农业科学院》2016年博士论文

【摘要】：针对当前苹果轮纹病严重影响我国苹果产业发展这一问题,本文以苹果叶片和果实为试验材料,研究叶片叶绿体和果实表皮受轮纹病菌胁迫的应答反应。主要研究结果如下:1.使用轮纹病菌孢子悬浮液培养苹果离体叶片,优化了轮纹病菌对苹果叶片的室内侵染体系。借助此侵染体系,优化了苹果品种对轮纹病抗性的快速鉴定体系,借助此鉴定体系对常见的苹果栽培品种进行了轮纹病抗性的鉴定,鉴定结果与田间调查结果一致。经过优化的鉴定体系具有操作简单、鉴定所需时间短、重复性好、待鉴定材料无损处理等优点。2.借助透射电子显微镜,可观察到随轮纹病菌对苹果叶片侵染时间的增加,叶片叶绿体先后出现了外被膜解体消失、类囊体解体失去原有结构、片层结构疏松解体和消失。这些结构上的变化,使叶绿体的光合代谢、能量代谢等代谢活动逐渐减弱和消失。3.经过优化的Percoll密度梯度离心法D适于苹果叶片叶绿体的分离。借助此方法分离所得苹果叶片叶绿体完整度高、纯度高、提取率大、提取量大、样品利用率高,适于后续研究的开展。随着叶片受轮纹病菌胁迫时间的增加,叶绿素含量下降,叶绿体中Mg2+-ATPase、Ca2+-ATPase含量下降,叶绿体光合能力下降,叶片质膜透性增强,叶绿体中防御酶含量上升,叶绿体应答胁迫的相关防御反应启动。4.经优化后的改良酚提法适于苹果叶片叶绿体蛋白的提取,优化后的2-DE分离体系适于苹果叶片叶绿体蛋白的2-DE分离。借助此叶绿体蛋白提取及2-DE分离体系,筛选和鉴定获得苹果叶片应答轮纹病菌胁迫前后差异表达的叶绿体蛋白21个。其中16个蛋白参与叶绿体的光合作用、能量代谢及抗氧化反应,5个蛋白为蛋白质、氨基酸代谢相关蛋白或功能未知。推测4个叶绿体蛋白是苹果叶片叶绿体应答轮纹病菌胁迫的关键蛋白,值得进一步深入研究。5.筛选得到的改良酚提法适于苹果果实表皮总蛋白的提取,优化后的2-DE分离体系适于苹果果实表皮总蛋白的2-DE分离。借助此果实表皮蛋白提取及2-DE分离体系,筛选和鉴定获得苹果果实表皮应答轮纹病菌胁迫前后差异表达蛋白24个。其中9个蛋白分别参与能量代谢及糖代谢,7个蛋白参与防御反应及应激反应,8个蛋白为蛋白、氨基酸代谢相关蛋白或未知蛋白。推测7个防御与应激相关蛋白是苹果果实表皮应答轮纹病菌胁迫的关键蛋白,与苹果抗病相关,值得进一步深入研究。
[Abstract]:In view of the problem that apple rotifer disease seriously affects the development of apple industry in China, the response of leaf chloroplast and fruit epidermis to stress by Rhizoctonia tumefaciens was studied using apple leaves and fruits as experimental materials. The main results are as follows: 1. In order to optimize the indoor infection system, the isolated leaves of apple were cultured with spore suspension of Rhizoctonia tumefaciens. With the help of this infection system, the rapid identification system of apple variety resistance to rosette disease was optimized, and the common apple cultivar was identified with this identification system. The result of identification was consistent with the field investigation. The optimized identification system has the advantages of simple operation, short identification time, good repeatability and nondestructive treatment of the materials to be identified. With the help of transmission electron microscope, it was observed that with the increase of infection time, the chloroplast disintegrated and disappeared, thylakoid disintegrated lost its original structure and lamellar structure loosely disintegrated and disappeared. These structural changes make the photosynthetic metabolism, energy metabolism and other metabolic activities of chloroplast gradually weaken and disappear. 3. The optimized Percoll density gradient centrifugation method D was suitable for the separation of chloroplasts from apple leaves. With the help of this method, the chloroplast of apple leaves has high integrity, high purity, large extraction rate, large amount of extraction and high utilization ratio of samples, which is suitable for further research. With the increase of leaf stress time, the content of chlorophyll decreased, the content of Ca 2 -ATPase in chloroplast decreased, the photosynthetic ability of chloroplast decreased, the permeability of plasma membrane increased, and the content of defense enzyme in chloroplast increased. The defense response of chloroplast in response to stress. 4. 4. The improved phenol extraction was suitable for the extraction of chloroplast protein from apple leaves, and the optimized 2-DE isolation system was suitable for 2-DE isolation of chloroplast proteins from apple leaves. With the help of the chloroplast protein extraction and 2-DE isolation system, 21 chloroplast proteins which were differentially expressed in apple leaves before and after the stress were screened and identified. Among them, 16 proteins were involved in photosynthesis, energy metabolism and antioxidant reaction of chloroplasts, 5 proteins were proteins, amino acid metabolism-related proteins or functions were unknown. It was suggested that four chloroplast proteins were the key proteins in the chloroplast response of apple leaves to the stress of Trichoderma, which was worthy of further study. The improved phenol extraction was suitable for the extraction of apple fruit epidermal total protein, and the optimized 2-DE isolation system was suitable for the 2-DE isolation of apple fruit epidermal total protein. With the help of the extraction of epidermal protein and 2-DE isolation system, 24 differentially expressed proteins were obtained before and after the apple fruit epidermis response to rotavirus stress. Among them, 9 proteins were involved in energy metabolism and glucose metabolism, 7 proteins were involved in defense response and stress response, 8 proteins were proteins, amino acid metabolism-related proteins or unknown proteins. It is inferred that seven proteins related to defense and stress are the key proteins of apple fruit epidermis in response to the stress of Rhizoctonia spp., which are related to the disease resistance of apple, and are worthy of further study.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S436.611

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