【作者】 李树芬；

【导师】 杨兴洪；

【作者基本信息】 山东农业大学 ， 植物学， 2010， 硕士

【摘要】 植物在生长发育的过程中,面临着多种逆境胁迫。高温是夏季常见的一种非生物胁迫因子,对作物的生长和产量造成严重的影响。植物在进化的过程中,积累可溶性物质应对各种环境胁迫。甜菜碱是目前研究最多,也是最有效的相溶性物质之一,可以提高植物对低温、高温、盐胁迫等的耐性。本实验以转胆碱氧化酶(codA)基因的番茄和转甜菜碱醛脱氢酶(BADH)基因的番茄为材料,并对野生型番茄施加甜菜碱,研究甜菜碱对提高番茄在种子萌发期、幼苗生长期耐热性的生理机制。主要结果如下:1、番茄种子吸胀时期高温处理后,转codA基因番茄种子相对电导率低于野生型番茄种子,萌发率高于野生型番茄种子;萌发时期高温处理后转codA基因番茄种子萌发率高于野生型番茄种子。转codA基因番茄种子耐热性高于野生型番茄种子。2、外源低浓度的甜菜碱处理提高了野生型番茄种子在高温胁迫下的萌发率,提高了对高温的耐性。3、高温胁迫下,转codA基因番茄种子中热激蛋白基因的表达水平高于野生型番茄种子,HSP70含量高于野生型番茄。4、高温胁迫降低番茄叶片的光合速率。野生型番茄光合速率、表观量子效率、羧化效率的下降幅度明显高于转codA基因番茄。高温胁迫下转codA基因番茄维持较高的光合速率,提高了光合作用对高温的耐性。5、叶绿素荧光分析结果表明:转codA基因番茄提高了PSII对高温胁迫的耐性。这种耐性的提高与PSII反应中心对高温胁迫的耐性提高有关。6、高温胁迫下番茄体内H2O2,O2的积累明显增加,转codA基因番茄叶片中活性氧的积累较少,而且转codA基因番茄叶片中CAT1基因表达水平高于野生型番茄。高温胁迫下转codA基因番茄通过维持较高的抗氧化物酶活性增强活性氧清除能力,减轻活性氧对光合机构的伤害。7、外源施加甜菜碱同样提高了番茄叶片光合作用和PSII反应中心对高温的耐性。8、从盐胁迫的菠菜中克隆甜菜碱醛脱氢酶(BADH)基因,构建真核表达载体,利用农杆菌介导的叶盘法转化番茄,用PCR和western杂交的方法对带卡那霉素抗性的转基因番茄植株进一步检测,获得了转BADH基因的番茄植株。9、转BADH基因提高了番茄叶片光合作用对高温的耐性。10、高温诱导HSP70的积累,转codA基因番茄和转BADH基因番茄叶片中HSP70含量高于野生型番茄;转codA基因番茄类囊体膜上HSP70含量高于野生型番茄;外源施加甜菜碱同样提高了高温下番茄叶片中HSP70的含量。11、高温导致PSII反应中心的损伤,尤其是D1蛋白的降解,造成D1蛋白含量的降低。转codA基因和转BADH基因番茄D1蛋白含量高于野生型番茄,而且外源施加甜菜碱番茄叶片中D1蛋白含量高于未施加甜菜碱的番茄。以上结果表明,转codA基因番茄种子对高温的耐性提高,而且转codA基因和转BADH基因番茄幼苗对高温的耐性也提高,这种耐性的提高与转基因番茄番茄中甜菜碱的积累有关。高温胁迫下,转基因番茄种子和叶片中热激蛋白的积累以及抗氧化酶活性的提高。热激蛋白含量和抗氧化酶活性的提高可能与甜菜碱促进热激蛋白和抗氧化酶基因表达有关。更多还原

【Abstract】 As sessile organisms, plants are continuously exposed to biotic and abiotic stress conditions during their whole life cycles. High temperature which affects the growth and the production of crops is a common abiotic stress during summer. Over time, plants have evolved mechanisms to overcome environmental stresses. One mechanism is the accumulation of compatible solutes such as glycinebetaine (GB). GB is one of the organic compatible solutes that can accumulate rapidly in many plants under salinity stress, drought and low temperature, and enhanced the tolerance to adverse conditions. In this study, the codA and BADH transgenic tomato were used to elucidate the effect of GB on tomato under high temperature during germination and seedling growth. Additionally, GB was exogenously applied to further investigate the role of GB in the enhancement of the tolerance to high temperature. The main results are as follows:1. After high temperature treatment during imbibition, the relative elective conductivity was greater in wild-type tomato seeds than in the codA transgenic seeds. The germination rate of codA transgenic seeds was higher than that of the wild-type seeds. During the recovery after long time high temperature treatment, the germination rate of codA transgenic seeds was higher than the wild-type seeds. The codA transgenic tomato seeds showed more thermotolerance than that of the wild type seeds.2. Exogenous application of low concentration of GB on MS medium increased the germination rate of the wild-type seeds after high temperature treatment. Imbibition in low concentration of GB also enhanced the germination rate of the wild-type seeds.3. During high temperature treatment, the transcript of gene for HSP70, HSC70 and MT-sHSP were more in the codA transgenic seeds than in the wild-type seeds. The accumulation of HSP70 was more in the codA transgenic seeds than in the wild-type seeds.4. High temperature stress decreased the CO2 assimilation. After high temperature treatment, CO2 assimilation rate, apparent quantum yield and carboxylation efficiency of wild-type tomato leaves decreased greater than that of the codA transgenic tomato leaves.The codA transgenic tomato plants showed higher thermotolerance of photosynthesis than wild type tomato plants.5. The analysis of chlorophyll fluorescence demonstrated that photosystemII (PSII) in codA transgenic plants showed more high temperature tolerance than in wild type plants, suggesting that the accumulation of GB leads to increased tolerance to high temperature-induced photo inhibition. This increased tolerance was associated with an improvement of oxygen-evolving complex and the reaction center of PSII to heat stress.6. Heat stress increased the accumulation of H2O2, O2 in the wild-type and transgenic tomato, but this accumulation was less in transgenic tomato. High temperature decreased the expression of CAT1 in the wild-type and codA transgenic tomato leaves. However the decrease was more in the wild-type plants than in the codA transgenic plants. GB decreased the accumulation of ROS through maintain relatively higher antioxidant enzyme activities.7. Exogenous application of GB also enhanced the thermotolerance of photosynthesis of wild type tomato plants.8. The gene for betaine aldehylde dehydrogenase (BADH) was successfully cloned from spinach which was supplied with 200 mM NaCl for 4 d. After the construction of eukaryote expressing vector, the A. tumefaciens mediated infection was used to infect the tomato leaf discs. After the PCR and western blot analysis of the kanamycin resistant tomato plants, the BADH transgenic seedlings were obtained.9. BADH transgenic tomato plants also showed higher thermotolerance of photosynthesis than that of wild type plants.10. High temperature induced the accumulation of HSP70 in the wild-type and codA, BADH transgenic tomato leaves. The accumulation was greater in the transgenic plants than in the wild-type plants. Exogenous application of GB in wild type tomato plants also increased the accumulation of HSP70 during high temperature stress.11. High temperature induced the degradation of D1 protein. The content of D1 protein was more in the codA, BADH transgenic tomato than that of the wild-type plants. Meanwile, the wild-type tomato plants supplied with exogenous GB also maintained greater content of D1 protein when exposed to high temperature stress.The results in this study indicated that tomato plants transformed with codA or BADH gene enhane the thermotolerance, and this resulted in the biosynthesis of glycinebetaine in transgenic plants. Under high temperature stress, the transgenic seeds and plants maintained more HSP and higher activities of antioxidant enzymes, which may associated with the improvement of the expression of some genes for heat shock protein and antioxidant enzymes by glycinebetaine during high temperature stress.更多还原