耐熱番茄雄蕊表達基因E8-6和Clone 7之分離與分析

Abstract

本研究以亞洲蔬菜研究發展中心所提供的耐熱番茄5915及不耐熱番茄4783為材料。前人研究中發現於高溫逆境及適溫下其花粉萌發及存活率有所差異。為提高高溫生長下番茄在高溫生長下之花粉萌發率及著果率，以real-time PCR及Northern blot的方式由實驗室所建立雄蕊在高溫表現的Suppression Subtractive Hybridization (SSH) cDNA library中，挑選出耐熱番茄的雄蕊在高溫能持續表現的E8-6以及Clone 7。分別利用CaMV 35S啟動子連接目標基因cDNA全長，於4783不耐熱番茄中過量表現E8-6與Clone 7、及以LeLAT52啟動子連接目標基因RNAi片段，於5915耐熱番茄雄蕊中專一抑制E8-6與Clone 7表現，分析高溫生長下轉殖株之花粉存活率及花粉管萌發率是否因而提升或降低。經花粉存活率與萌發率測試，得知Clone 7會影響花粉發育與萌發時對高溫的耐受性，因此有增加番茄在高溫生殖能力的潛力。 另一方面，E8-6在開花期雄蕊表現量於高溫中有提高的現象。為了進一步瞭解E8-6基因表現在轉錄層次的調控，以及5915與4783間E8-6啟動子序列是否有所差異，使得E8-6在雄蕊中有不同的表現量，進而影響兩品系花粉對於溫度的耐受性，因而分離E8-6啟動子，得到一個2102 bp片段長的啟動子。分析此啟動子序列，發現5915與4783間僅3 bp之差異，且此差異並不影響cis-acting and responsive elements之組成，且兩品系之E8-6啟動子均具有兩個HSE (heat shock element) ，會受高溫所誘導而表現。進一步將E8-6基因啟動子連接GUS報導基因的表現卡匣引入阿拉伯芥中表現，篩選轉殖株以分析E8-6基因組織專一性表現。篩選得到之轉殖株經由GUS染色結果發現，E8-6異位表現於阿拉伯芥之葉片維管束、trichome基部、花萼、柱頭基部、花絲與部分花藥，並且處理乙烯與高溫均可增加葉片中GUS表現量。

Tomatoes incur poor growth and fruit set under hot-dry or hot-wet season in tropics and subtropics, which is a major bottleneck impeding summer production in Taiwan. Recent studies concluded that impairment of pollen and anther development by elevating temperature contributes to decrease fruit set in tomatoes. AVRDC (Asia Vegetable Research and Development Center) has bred serial heat-tolerant tomatoes, yet the molecular mechanism of heat tolerance is still unclear. To analyze these heat-tolerant genes that affect pollen viability, Suppression Subtractive Hybridization (SSH) was used to isolate cDNA fragments from the anther of heat-tolerant tomato CL5915 at high temperature, 35oC. By analyzing temperature effects of these genes from the heat-induced cDNA library using Real-time PCR and Northern blot, Clone 8’ and Clone 7 were selected for further studies. Compared with NCBI nucleotide database, these genes are suggested to be E8-6 and a Lycopersicon esculentum clone 133497F, respectively. E8-6 is a Tomato E8 protein homolog with a related isopenicillin N synthase and related dioxygenases domain, while clone 133497F is presumed to be an Arabidopsis beta-ureidopropionase homolog with a CN hydrolase conserved domain. Several transgenic tomatoes were then created so that overexpression of E8-6 and Clone 7 was built in the heat-sensitive tomato L4783, while knock-down expression was made in heat-tolerant tomato CL5915 in order to characterize the functions of E8-6 and Clone 7. Furthermore, the promoters of E8-6 were isolated from CL5915 and L4783. There is no difference of cis-acting and responsive element between CL5915 and L4783, and both of them have two heat shock elements, indicating that E8-6 may be induced by elevated temperature. Subsequently, the GUS reporter gene driven by E8-6 promoter expressed in Arabidopsis was analyzed. The expression pattern detected by GUS staining indicated that E8-6 promoter expresses in sepal, filament, and stigma of flowers.