中文摘要
持續性大量表現阿拉伯芥COR基因的轉錄活化子 (transcriptional activator) --- CBF1基因於未受冷馴化的阿拉伯芥植株中，能誘導COR6.6, COR15a, COR47和COR78等基因的表現，並提高整棵植株的耐凍能力(Jaglo-Ottoson et al., 1998)。本實驗的目標在於對蕃茄基因組 (genome) 中是否具有與阿拉伯芥CBF1、COR15a、COR47相類似基因的存在作一探究，並試圖建立一套適用於台灣本地蕃茄的農桿菌轉殖系統，以便將阿拉伯芥的CBF1基因轉殖入蕃茄中。
本實驗選擇由亞洲蔬菜研究及發展中心於台灣本地育種而成，具有轉殖商業價值但對冷敏感的蕃茄CL5915-93D4-1-0-3品系為材料。南方轉漬分析的結果發現，在未經轉殖處理的蕃茄之基因組中具有與阿拉伯芥CBF1、COR15a及COR47基因相類似的DNA片段，而北方轉漬分析的結果則顯示：蕃茄的CBF1相似基因之RNA只在1個月大的蕃茄幼苗時期才有表現。
將含有花椰菜鑲嵌病毒35S 啟動子 (Califlower mosaic virus 35S, CaMV35S promoter)、intron GUS報導基因、及Kanamycin (R)篩選基因的雙偶型載體 (binary vector) pCAMBIA2301藉由LBA4404或EHA105菌系感染蕃茄子葉，可建立農桿菌基因轉殖蕃茄系統，並各獲得2 及1個轉殖再生植株。另外，也構築好含CaMV35S啟動子、阿拉伯芥CBF1基因、intron GUS 報導基因、及kanamycin (R)篩選基因的雙偶型載體pJLM1，藉農桿菌LBA4404菌系感染蕃茄子葉。在控制感染用的農桿菌液濃度、單位子葉面積的農桿菌數、及調整篩選培養基的更新速率之後，可提高轉殖效率，並獲得18株轉殖pJLM1的再生植株。
上述轉殖再生植株經kanamycin抗生素篩選、GUS組織染色分析、及PCR檢定證實kanamycin (R)、intron GUS基因都已轉入蕃茄細胞中。另外，南方轉漬分析檢定結果亦顯示阿拉伯芥CBF1基因的片段確已插入蕃茄的基因組之中。這些轉殖過的本地蕃茄可提供為日後探討基因表現及耐冷性狀分析時的實驗材料。

英文摘要
Overexpression of the low-temperature transcriptional activator CBF1 (CRT/DRE-binding factor 1) can induce the expression of a pool of COR -related genes and enhance freezing tolerance of nonacclimated Arabidopsis plants (Jaglo-Ottosen et al., 1998). The objectives of this study were to investigate whether genes homologus to the Arabidopsis CBF1, COR15a and COR47 genes were present in the tomato genome, to establish an Agrobacterium-mediated transformation system for Taiwan local tomato cultivar, and to transfer a constitutively expressed Arabidopsis CBF1 gene into the particular tomato cultivar.
The tomato cultivar CL5915-93D4-1-0-3 from AVRDC was chosen as the target plant due to its sensitivity to chilling, its commercial value, and its previous absence in transformation experiments. To investigate whether homologes of the Arabidopsis CBF1, COR15a and COR47 genes were present in the tomato genome, Southern and Northern blotting analyses were performed. Our results indicated that these counterpart genes did exist in the tomato genome. However, the CBF1-like gene in tomato showed a restricted developmental expression, only being detected in one-month-old seedlings.
To establish an Agrobacterium-mediated transformation system for Taiwan local tomato cultivar, the binary vector pCAMBIA2301 was selected. This vector contains an intron GUS reporter gene and a kanamycin-resistant marker, and both were driven by CaMV35S promoter. Tomato cotyledons were infected by Agrobacterium strain LBA4404 and EHA105 containing pCAMBIA2301, which after kanamycin selection and GUS histochemical staining, resulted in 2 and 1 regenerated transformants, respectively.
This study also transformed tomato with another binary vector, pJLM1 containing CaMV35S promoter and the Arabidopsis CBF1 gene. By optimizing the transformation conditions including, Agrobacterium concentration, numbers of explants per plate while co-culture, and the frequency of changing selection medium, we have obtained eighteen rooted transgenic plants. PCR amplifications showed that Kanamycin-resistant and intron GUS genes had been inserted to the transgenic tomato genome. Southern blotting analysis also demonstrated the existence of Arabidopsis CBF1 gene in the transgenic tomato genome. These transgenic plants should be useful for future chilling tolerance experiments.

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