本文選題：番茄　切入點(diǎn)：抗環(huán)血酸　出處：《山東農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：干旱是限制植物生長的主要環(huán)境因素之一,它誘導(dǎo)活性氧(reactive oxygen species,ROS)的大量積累,影響植物的生長發(fā)育。植物葉綠體是ROS產(chǎn)生的主要部位之一,葉綠體中ROS的有效清除,對于保護(hù)生物膜及光合機(jī)構(gòu)、維持脅迫條件下光合作用的正常運(yùn)行極為重要�？箟难�(ascorbate,As A)是葉綠體ROS清除系統(tǒng)的重要組分之一,它的有效再生是維持植物抗氧化能力的關(guān)鍵。葉綠體單脫氫抗壞血酸還原酶(MDAR)是葉綠體AsA再生的關(guān)鍵酶,它將單脫氫抗壞血酸還原生成AsA,維持葉綠體AsA水平。本研究以野生型(WT)和轉(zhuǎn)反義SlMDAR番茄(Solanum lycopersicum)株系為試材,測定并分析了AsA在AsA-Glu循環(huán)回補(bǔ)途徑中的比例,MDAR酶的活力與干旱脅迫前后葉綠素?zé)晒鈪?shù)、光合速率、活性氧積累量、膜脂過氧化程度等指標(biāo)的關(guān)系,研究SlMDAR的表達(dá)與植物抗干旱脅迫能力的關(guān)系,主要結(jié)果如下:(1)與野生型植株相比,轉(zhuǎn)基因株系的番茄葉片MDAR在信使核糖核酸(Messenger RNA,mRNA)水平上和蛋白水平上均明顯低于野生型。同時,熒光定量PCR實(shí)驗(yàn)表明SlMDAR的表達(dá)明顯受聚乙二醇(polyethylene glycol,PEG)誘導(dǎo)。(2)PEG模擬干旱脅迫處理前后,測定了轉(zhuǎn)基因及野生型番茄植株的MDAR酶活、總AsA、脫氫抗壞血酸(Dehydroascorbate,DHA)含量及AsA/DHA,結(jié)果發(fā)現(xiàn)反義抑制SlMDAR的表達(dá)降低了轉(zhuǎn)基因番茄株系的MDAR酶活性和AsA含量。(3)與WT野生型番茄植株相比,轉(zhuǎn)基因番茄株系在干旱脅迫下相對含水量降低、MDA含量和相對電導(dǎo)率增高,細(xì)胞膜受到的損害更大,反義抑制SlMDAR的表達(dá)降低了轉(zhuǎn)基因番茄植株的干旱脅迫抗性。(4)轉(zhuǎn)基因番茄株系中H_2O_2和O_2·-含量比野生型高,而ROS清除關(guān)鍵酶抗壞血酸過氧化物酶(APX)及超氧化物岐化酶(SOD)的活性差別不大,表明轉(zhuǎn)基因番茄株系具有較低的ROS清除能力,可能與較低的AsA含量相關(guān)。(5)脅迫處理后,相對于野生型,轉(zhuǎn)基因番茄植株光合能力明顯降低,F0明顯升高,PSⅡ的光抑制程度較重,轉(zhuǎn)基因番茄株系的D1蛋白降解速率比野生型高。綜上,反義抑制SlMDAR的表達(dá)降低了MDAR酶活性和AsA的再生能力,從而抑制了ROS的清除,增加了轉(zhuǎn)基因番茄植株光系統(tǒng)受到的氧化損傷,降低了轉(zhuǎn)基因番茄對干旱脅迫的抗性。
[Abstract]:Drought is one of the main environmental factors restricting plant growth. It induces the accumulation of reactive oxygen specieses in reactive oxygen species (Ros) and affects the growth and development of plants. Chloroplast is one of the main parts of ROS production and the effective removal of ROS in chloroplasts. In order to protect the biofilm and photosynthesis, it is very important to maintain the normal operation of photosynthesis under stress. Ascorbate ascorbate as is one of the important components of chloroplast ROS scavenging system. Chloroplast monodehydroascorbate reductase (MDAR) is the key enzyme of chloroplast AsA regeneration. The monodehydroascorbic acid was reduced to AsA to maintain chloroplast AsA level. In this study, wild type WTand antisense SlMDAR tomato Solanum lycopersicum strain were used as experimental materials. The relationship between the activity of AsA and chlorophyll fluorescence parameters, photosynthetic rate, accumulation of active oxygen species and the degree of membrane lipid peroxidation before and after drought stress was determined and analyzed. The relationship between the expression of SlMDAR and the ability of plants to resist drought stress was studied. The main results were as follows: compared with wild-type plants, the MDAR of transgenic tomato leaves was significantly lower than that of wild type at the level of messenger ribonucleic acid (SlMDAR) and protein. Fluorescence quantitative PCR assay showed that the expression of SlMDAR was obviously induced by polyethylene glycol polyethylene glycol (PEG). The MDAR activity of transgenic and wild-type tomato plants was measured before and after simulated drought stress. Total AsA, dehydroascorbate dehydroascorbate (DHA) content and AsA / DHA. The results showed that antisense inhibition of SlMDAR expression decreased MDAR enzyme activity and AsA content of transgenic tomato lines, and compared with WT wild-type tomato plants. Under drought stress, the relative water content of transgenic tomato lines decreased the content of MDA and the relative conductivity increased, and the damage to cell membrane was more serious. Inhibition of SlMDAR expression by antisense decreased drought stress resistance of transgenic tomato plants. The content of H _ 2O _ 2 and O _ 2 路- in transgenic tomato lines was higher than that in wild type. However, the activities of ascorbic acid peroxidase (ROS) and superoxide dismutase (SOD) showed little difference, indicating that transgenic tomato lines had lower ROS scavenging ability, which might be related to lower AsA content. Compared with the wild type, the photosynthetic ability of transgenic tomato plants decreased significantly and the photoinhibition degree of PS-II increased significantly. The degradation rate of D1 protein of transgenic tomato lines was higher than that of wild type. The inhibition of SlMDAR expression by antisense decreased the activity of MDAR enzyme and the regeneration ability of AsA, thus inhibited the removal of ROS, increased the oxidative damage to the light system of transgenic tomato plants, and reduced the resistance of transgenic tomato to drought stress.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q943.2;Q945.78

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