番茄叶绿体单脱氢抗坏血酸还原酶基因对干旱胁迫的响应

发布时间：2018-03-07 16:27

本文选题：番茄　切入点：抗环血酸　出处：《山东农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：干旱是限制植物生长的主要环境因素之一,它诱导活性氧(reactive oxygen species,ROS)的大量积累,影响植物的生长发育。植物叶绿体是ROS产生的主要部位之一,叶绿体中ROS的有效清除,对于保护生物膜及光合机构、维持胁迫条件下光合作用的正常运行极为重要。抗坏血酸(ascorbate,As A)是叶绿体ROS清除系统的重要组分之一,它的有效再生是维持植物抗氧化能力的关键。叶绿体单脱氢抗坏血酸还原酶(MDAR)是叶绿体AsA再生的关键酶,它将单脱氢抗坏血酸还原生成AsA,维持叶绿体AsA水平。本研究以野生型(WT)和转反义SlMDAR番茄(Solanum lycopersicum)株系为试材,测定并分析了AsA在AsA-Glu循环回补途径中的比例,MDAR酶的活力与干旱胁迫前后叶绿素荧光参数、光合速率、活性氧积累量、膜脂过氧化程度等指标的关系,研究SlMDAR的表达与植物抗干旱胁迫能力的关系,主要结果如下:(1)与野生型植株相比,转基因株系的番茄叶片MDAR在信使核糖核酸(Messenger RNA,mRNA)水平上和蛋白水平上均明显低于野生型。同时,荧光定量PCR实验表明SlMDAR的表达明显受聚乙二醇(polyethylene glycol,PEG)诱导。(2)PEG模拟干旱胁迫处理前后,测定了转基因及野生型番茄植株的MDAR酶活、总AsA、脱氢抗坏血酸(Dehydroascorbate,DHA)含量及AsA/DHA,结果发现反义抑制SlMDAR的表达降低了转基因番茄株系的MDAR酶活性和AsA含量。(3)与WT野生型番茄植株相比,转基因番茄株系在干旱胁迫下相对含水量降低、MDA含量和相对电导率增高,细胞膜受到的损害更大,反义抑制SlMDAR的表达降低了转基因番茄植株的干旱胁迫抗性。(4)转基因番茄株系中H_2O_2和O_2·-含量比野生型高,而ROS清除关键酶抗坏血酸过氧化物酶(APX)及超氧化物岐化酶(SOD)的活性差别不大,表明转基因番茄株系具有较低的ROS清除能力,可能与较低的AsA含量相关。(5)胁迫处理后,相对于野生型,转基因番茄植株光合能力明显降低,F0明显升高,PSⅡ的光抑制程度较重,转基因番茄株系的D1蛋白降解速率比野生型高。综上,反义抑制SlMDAR的表达降低了MDAR酶活性和AsA的再生能力,从而抑制了ROS的清除,增加了转基因番茄植株光系统受到的氧化损伤,降低了转基因番茄对干旱胁迫的抗性。
[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.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;Q945.78

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