鄭石通臺灣大學：植物科學研究所曾雅鈴Tzeng, Ya-LingYa-LingTzeng2007-11-272018-07-062007-11-272018-07-062007http://ntur.lib.ntu.edu.tw//handle/246246/57975Tomato (Solanum lycopersicum) is a temperature-sensitive crop. The harvest of tomato is poor due to the elevated temperature. Studies have pointed out that impairment of tomato pollen during anther development by elevated temperature contributes to decreased fruit set. In this study, we used heat-sensitive (L4783) and heat-tolerant (CL5915) tomatoes cultivated in high temperatures(day35/ night30 and day30/night 25 °C) and optimal temperature (day25/night20 ℃) to investigate the molecular mechanisms of heat tolerance of the tomato pollen grains during anther development. First, we used Real-time PCR to screen candidate genes from anther cDNA library that was constructed from the heat-tolerant tomato by Suppression Subtractive Hybridization (SSH). Thus, Clone 5 and Clone 29 (LwWun1) were selected from the heat-tolerant tomato (CL5915) based on the results of Northern blotting and reverse transcription-polymerase chain reaction (RT-PCR). By means of RACE (Rapid amplification of cDNA end)，I obtained the full-length cDNA of Clone 5 and Clone 29 genes according to the SSH fragment sequence. Through the alignment on the NCBI database, the translation products of Clone 5 and Clone 29 were predicted to be the cupin-superfamily protein and wound-inducible 1 protein (Wun1), respectively. Furthermore, we detect the expression of Clone 5 and LeWun1 by RT-PCR with total RNA extracted from various tomato organs. To study whether these target genes participate in the heat-resistance process of tomato pollen, we generated transgenic tomatoes over-expressing target genes, Clone 5 and LeWun1,in heat-sensitive L4783 tomato and knock-down target genes in heat-tolerant CL5915 tomato by Agrobacteria with pCAMBIA-2301. Additionally, the promoter region of LeWun1 gene was isolated from the tomato by Tail PCR (Thermal asymmetric interlaced PCR), and its function was predicted.中文摘要 1 英文摘要 2 第一章 前言 4 一、 番茄 4 二、 熱逆境 4 三、 番茄與熱逆境 5 四、 使番茄具有耐熱潛力之基因 5 五、 研究目的與方向 7 第二章 材料與方法 9 一、 材料 9 二、 番茄雄蕊RNA的萃取 9 三、 cDNA 之製作 10 四、 Real-time PCR 10 五、 北方墨點轉漬分析(Northern blotting assay) 11 六、 RACE(Rapid amplification cDNA end) 13 七、 聚合酵素連鎖反應(The polymerase chain reaction, PCR) 14 八、 限制酵素切割 15 九、 瓊脂凝膠內DNA片段之回收 15 十、 質粒構築與挑選 16 十一、 蕃茄之轉殖 18 十二、 植物基因組DNA萃取 18 十三、 GUS活性分析 19 十四、 Thermal Asymmetric Interlaced PCR (Tail PCR) 20 十五、 番茄花粉外表型的觀察 22 第三章 結果 24 一、 耐熱番茄中受熱誘導表現的目標基因之確認 24 二、 目標基因序列全長之釣取及比對結果 24 三、 目標基因於野生型番茄各器官表現之情況 25 四、 番茄轉殖載體之構築 26 五、 番茄轉殖株之篩選與鑑定 27 六、 番茄轉殖株花粉性狀之分析 28 七、 釣取LeWun1基因啟動子序列 28 第四章 討論 30 一、 Clone 5與LeWun1基因受熱影響的內生表現情形 30 二、 Clone 5與LeWun1蛋白質序列分析 31 三、 在不耐熱番茄品系4783體內大量表現clone 5 33 四、 LeWun1番茄啟動子之釣取 34 五、 結論 36 第五章 參考文獻 37 第六章 圖表 451707967 bytesapplication/pdfen-US番茄雄蕊tomatostamenotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/57975/1/ntu-96-R94b42019-1.pdf