耐熱番茄與不耐熱番茄中RSG與LeHsc70-1基因之分析及比較

Characterization and Comparison of RSG and LeHsc70-1 Genes between Heat-Tolerant and Heat-Sensitive tomatoes (Solanum lycopersicum)

劉浩雲(HAO-YUN LIU)

指導教授 ： 鄭石通

國立臺灣大學/生命科學院/植物科學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.02936

摘要

在台灣濕熱氣候下，番茄常發生生長不良和著果率下降的問題，造成台灣夏季番茄產量降低，近年研究發現，產量降低的原因來自番茄花藥及花粉的發育受到高溫的影響，因此，我們將目標鎖定在花藥與花粉部分，材料為亞洲蔬菜研究發展中心所提供的兩種品系番茄：耐熱品系番茄和不耐熱品系番茄。在花粉活性分析與花粉管萌發分析中，發現高溫逆境接會影響這兩種花粉外表型性狀，尤其是不耐熱品系番茄。利用Suppression Subtractive Hybridization的方法分離出在耐熱品系番茄中受高溫誘導的基因，利用Real-Time PCR確認其中8個基因的確會受到高溫而誘導表現。經過參考相關文獻之後，挑選REPRESSION OF SHOOT GROWTH（RSG）與LeHsc70-1作為目標基因繼續後續研究。LeHsc70-1的全長mRNA序列可由NCBI database獲得，RSG的全長mRNA序列則以RACE的方式釣取獲得。在目標基因的非轉譯區域設計引子，進行Real-Time PCR，結果發現兩個目標基因皆會受到高溫而誘導表現。在兩種品番茄中個別釣取得到RSG基因的啟動子序列1152 bps，但都不含有heat shock element，LeHsc70-1基因的啟動子序列1229 bps，在相同位置含有一個heat shock element。對兩種品系番茄進行轉殖，在不耐熱番茄中過量表現目標基因，而在耐熱品系番茄中以RNAi機制抑制目標基因表現，觀察目標基因是否對耐熱性狀有影響，不過最後無法獲得轉殖成功的番茄轉殖株。

關鍵字

耐熱番茄 ； 不耐熱番茄 ； 熱逆境 ； 花粉

並列摘要

Tomatoes incur poor growth and fruit-set under hot-dry or hot-wet season in the tropics and sub-tropics, which is a major bottleneck impeding summer production. Recent studies concluded that impairment of pollen and anther development by elevated temperature contributes to decreased fruit set in tomatoes. Therefore, we would focus on anther and pollen in this study. The materials used in this study are the heat-tolerant tomato (CL5915-206D4-2-2-0) and the heat-sensitive tomato (L4783) offered by the Asian Vegetable Research and Development Center. Examinations of pollen viability by fluorescin diacetate (FDA) staining and pollen germinability by in vitro pollen germination and tube growth assays indicated that the heat stress can affect both of these traits, especially in the heat-sensitive tomato. From the suppression subtractive hybridization (SSH), the heat-induced genes from heat-tolerant tomatoes were isolated. Also, eight genes induced by the heat stress were further confirmed using the real-time PCR assays. After referring to other researches, REPRESSION OF SHOOT GROWTH (RSG) and LeHsc70-1 were chosen as the target genes. The complete mRNA sequence of LeHsc70-1 was found from the NCBI database, and that of RSG was obtained by RACE analysis. Real-time PCR with primers designed in the UTRs of these genes also indicated that both of two target genes are heat-inducible. 1152 bp-long promoter sequences of RSG gene from two different strains of tomato contained no heat shock element. 1229 bp-long promoter sequences of LeHsc70-1 gene from two different strains of tomatoes contained one heat shock element at the same position. Transgenic tomatoes were constructed with over-expressing target genes in the heat-sensitive tomato and knocking down target genes by RNAi in the heat-tolerant tomato to further understand if the target genes have any effect on the heat tolerance of tomato, but finally it was unsuccessful to obtain transgenic plants.

並列關鍵字

RSG ； LeHsc70-1

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