【作者】 孙晓琳；

【导师】 赵世杰； 孟庆伟；

【作者基本信息】 山东农业大学 ， 植物学， 2011， 硕士

【摘要】 低温冷害是影响作物正常生长发育的环境因素之一。细胞膜系统是植物遭受低温冷害的首要部位,冷害的根本原因是细胞膜系统受损。植物类囊体膜受到低温伤害后,结构和功能遭到破坏,导致光合功能下降,而类囊体膜不饱和脂肪酸含量的上升则有利于光合功能的维持。番茄甘油-3-磷酸酰基转移酶（LeGPAT）优先选择C18:1-ACP作为底物,使其转移到叶绿体类囊体膜中磷脂酰甘油（PG）的sn-1位上,导致PG含有较高比例的18:1脂肪酸,从而能够决定叶绿体类囊体膜上磷脂酰甘油（PG）顺式不饱和脂肪酸含量。因此,探讨低温胁迫下类囊体膜PG顺式不饱和脂肪酸含量维持光合功能的作用具有重要的理论意义和应用价值。本实验以野生型（WT）,转正义番茄叶绿体LeGPAT基因株系T₂-19（+）、T₂-5（+）和转反义LeGPAT基因株系T₂-16（-）、T₂-2（-）为试材,通过测定类囊体膜脂脂肪酸组成、光合及叶绿素荧光参数、叶绿体活性氧的产生、叶绿体活性氧清除酶活性以及D1蛋白修复等,研究了类囊体膜脂PG脂肪酸不饱和程度与番茄耐冷性的关系。主要结果如下:（1）番茄类囊体膜PG脂肪酸组成的分析。结果表明,和WT相比,转基因植株类囊体膜脂PG脂肪酸含量发生了明显变化。转正义基因番茄植株类囊体膜脂PG的18:2和18:3含量明显增加,脂肪酸不饱和程度升高,而转反义基因番茄植株PG的18:2和18:3含量下降,脂肪酸不饱和度明显下降。（2）长期低温胁迫下,野生型和转基因株系的生长量受到了不同程度的抑制,其中转反义基因植株的抑制程度最高,转正义基因植株的抑制程度最低。对低温下野生型和转基因株系的光合放氧速率和PSII最大光化学效率（Fv/Fm）进行测定后发现,植株生长量受低温抑制的程度与低温下光合及Fv/Fm受抑制的趋势相同。表明低温下植株生长量减少与光合受到抑制有关,而类囊体膜脂PG不饱和度的提高有利于维持光合机构的稳定性,减轻对PSII的光抑制。经过4d的低温胁迫,T₂-5、T₂-19、WT、T₂-2和T₂-16的相对电导率分别增加到46.72、49.11、67.07、73.21和79.96%。这表明在低温胁迫条件下,各个株系的番茄叶片均遭到了明显的伤害,与WT相比,转正义基因株系的膜系统受到的伤害程度较轻,而转反义基因株系番茄的膜系统受到的伤害程度较重。（3）与野生型相比,低温胁迫下转正义基因植株维持较高的叶绿体超氧化物歧化酶（SOD）和抗坏血酸过氧化物酶（APX）活性,产生较少的O₂（?）和H₂O₂;而转反义基因植株叶绿体抗氧化酶SOD及APX的活性较低,O₂（?）和H₂O₂的含量较高。与其它株系相比,转正义基因株系叶绿体SOD和APX维持较高的活性,有利于清除活性氧,能够降低由于活性氧的积累而造成的膜脂过氧化程度,维持细胞膜的完整性。（4）低温胁迫对D1蛋白修复速率的影响。分离类囊体膜,进行SDS-PAGE和Western杂交,结果表明,低温胁迫下野生型和转基因番茄植株D1蛋白总含量均下降。与野生型相比,转正义基因番茄植株能够维持较高的D1蛋白含量,而转反义基因植株D1蛋白含量较低。用蛋白合成抑制剂硫酸链霉素处理后,转正义基因番茄植株D1蛋白含量少于转反义基因植株中D1蛋白含量。上述结果表明,低温胁迫下PG不饱和脂肪酸含量的增加可能提高了D1蛋白的修复速率,降低膜的伤害度,提高抗氧化酶的活性,进而提高番茄植株的耐冷性。更多还原

【Abstract】 Plants are incapable of escaping from unfavorable environment, such as low temperature. The membrane is the primary location that was damaged under chilling stress. Chilling stress can damage the structure and function of thylakoid membrane, causing the decrease of photosynthesis, but the increase of unsaturated fatty acid of thylakoid membrane can enhance the photosynthesis ability of plants. GPAT from chilling-resistant plants prefers oleoyl-ACP （18:1-ACP） to palmitoyl-ACP （16:0-ACP） as a substrate. Thus, the proportion of oleic acid （18:1） changes at the sn-1 position of PG in chilling resistant plants. So, it is significant to study the relationship between the proportion of unsaturated fatty acids and chilling tolerance by using GPAT transgenic tomato plants.In this study, wild type （WT）, sense transgenic lines [T₂-19（+）, T₂-5（+）] and antisense transgenic lines [T₂-16（-）,T₂-2（-）] of tomato were used to determine the fatty acid composition of PG in thylakoid membrane, oxygen evolving activity, the maximal photochemical efficiency of PSII （Fv/Fm）, the production of chloroplastic reactive oxygen species （ROS） and activities of antioxidase in chloroplasts, and the repair of D1 protein under chilling stress. The results were showed as follows:（1） The results showed that the content of fatty acids of PG changed markedly. Compared with WT, a higher content of unsaturated fatty acids in PG was detected in sense transgenic plants. In contrast, the decreased unsaturation of PG in antisense transgenic plants was observed.（2） Under long time chilling stress, the growth of WT and transgenic lines were all decreased. But the fresh weight of the sense transgenic plants was higher than that of WT and antisense plants. Under chilling stress, the O2 evolution rates and Fv/Fm in WT and transgenic plants obviously decreased. Those decreases were more obvious in WT than those in transgenic plants. These results indicated that decrease of the fresh weight was owing to inhibition of the photosynthesis. The relative electronic conductance of leaves increased in both WT and transgenic plants under chilling stress for 4 days. But it increased more quickly in antisense plants than that in WT and sense transgenic plants. These results indicated that higher unsaturation ratio of PG reduced membrane damage under chilling stress.（3） Compared with WT, the sense transgenic plants could maintain higher activities of SOD and APX, and produce lower content of O₂（?） and H₂O₂ under chilling stress. In contrast, activities of SOD and APX were lower and the content of ROS was higher in the antisense transgenic plants. Relative to other lines, the higher activities of SOD and APX can scavenge more ROS, mitigate the extent of lipid peroxidation caused by the accumulation of ROS, and maintain the integrity of the cytomembrane.（4） We analysed the influence of chilling stress on the repair of D1 protein. During chilling stress for 8 h, the total content of D1 protein decreased among five lines. Compared with WT, there was a higher level of D1 protein in sense transgenic plants and lower level of D1 in antisense ones. After the treatment of streptomycin （SM） and low temperature, the content of D1 was lower in sense trangenic plants than in antisense ones.We concluded that the increase of unsaturation in PG can enhance the ability of repair of D1 protein, alleviate the injury of membrane, thus improve the activities of antioxidase in chloroplasts.更多还原