發(fā)布時(shí)間：2018-04-19 23:30

  本文選題：蘋(píng)果 + 輪紋病�。� 參考：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文

【摘要】：針對(duì)當(dāng)前蘋(píng)果輪紋病嚴(yán)重影響我國(guó)蘋(píng)果產(chǎn)業(yè)發(fā)展這一問(wèn)題,本文以蘋(píng)果葉片和果實(shí)為試驗(yàn)材料,研究葉片葉綠體和果實(shí)表皮受輪紋病菌脅迫的應(yīng)答反應(yīng)。主要研究結(jié)果如下:1.使用輪紋病菌孢子懸浮液培養(yǎng)蘋(píng)果離體葉片,優(yōu)化了輪紋病菌對(duì)蘋(píng)果葉片的室內(nèi)侵染體系。借助此侵染體系,優(yōu)化了蘋(píng)果品種對(duì)輪紋病抗性的快速鑒定體系,借助此鑒定體系對(duì)常見(jiàn)的蘋(píng)果栽培品種進(jìn)行了輪紋病抗性的鑒定,鑒定結(jié)果與田間調(diào)查結(jié)果一致。經(jīng)過(guò)優(yōu)化的鑒定體系具有操作簡(jiǎn)單、鑒定所需時(shí)間短、重復(fù)性好、待鑒定材料無(wú)損處理等優(yōu)點(diǎn)。2.借助透射電子顯微鏡,可觀察到隨輪紋病菌對(duì)蘋(píng)果葉片侵染時(shí)間的增加,葉片葉綠體先后出現(xiàn)了外被膜解體消失、類(lèi)囊體解體失去原有結(jié)構(gòu)、片層結(jié)構(gòu)疏松解體和消失。這些結(jié)構(gòu)上的變化,使葉綠體的光合代謝、能量代謝等代謝活動(dòng)逐漸減弱和消失。3.經(jīng)過(guò)優(yōu)化的Percoll密度梯度離心法D適于蘋(píng)果葉片葉綠體的分離。借助此方法分離所得蘋(píng)果葉片葉綠體完整度高、純度高、提取率大、提取量大、樣品利用率高,適于后續(xù)研究的開(kāi)展。隨著葉片受輪紋病菌脅迫時(shí)間的增加,葉綠素含量下降,葉綠體中Mg2+-ATPase、Ca2+-ATPase含量下降,葉綠體光合能力下降,葉片質(zhì)膜透性增強(qiáng),葉綠體中防御酶含量上升,葉綠體應(yīng)答脅迫的相關(guān)防御反應(yīng)啟動(dòng)。4.經(jīng)優(yōu)化后的改良酚提法適于蘋(píng)果葉片葉綠體蛋白的提取,優(yōu)化后的2-DE分離體系適于蘋(píng)果葉片葉綠體蛋白的2-DE分離。借助此葉綠體蛋白提取及2-DE分離體系,篩選和鑒定獲得蘋(píng)果葉片應(yīng)答輪紋病菌脅迫前后差異表達(dá)的葉綠體蛋白21個(gè)。其中16個(gè)蛋白參與葉綠體的光合作用、能量代謝及抗氧化反應(yīng),5個(gè)蛋白為蛋白質(zhì)、氨基酸代謝相關(guān)蛋白或功能未知。推測(cè)4個(gè)葉綠體蛋白是蘋(píng)果葉片葉綠體應(yīng)答輪紋病菌脅迫的關(guān)鍵蛋白,值得進(jìn)一步深入研究。5.篩選得到的改良酚提法適于蘋(píng)果果實(shí)表皮總蛋白的提取,優(yōu)化后的2-DE分離體系適于蘋(píng)果果實(shí)表皮總蛋白的2-DE分離。借助此果實(shí)表皮蛋白提取及2-DE分離體系,篩選和鑒定獲得蘋(píng)果果實(shí)表皮應(yīng)答輪紋病菌脅迫前后差異表達(dá)蛋白24個(gè)。其中9個(gè)蛋白分別參與能量代謝及糖代謝,7個(gè)蛋白參與防御反應(yīng)及應(yīng)激反應(yīng),8個(gè)蛋白為蛋白、氨基酸代謝相關(guān)蛋白或未知蛋白。推測(cè)7個(gè)防御與應(yīng)激相關(guān)蛋白是蘋(píng)果果實(shí)表皮應(yīng)答輪紋病菌脅迫的關(guān)鍵蛋白,與蘋(píng)果抗病相關(guān),值得進(jìn)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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S436.611

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