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Title: Characterization and Comparison of E8-6 and a Homolog of Arabidopsis beta-UP Gene between Heat-Tolerant and Heat-Sensitive tomatoes (Solanum lycopersicum) M.S. student: Yu-Huei Chen Advisor: Professor Shih-Tong Jeng Abstract: 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 elevated temperature contributes to decreased fruit set in tomatoes (Peet et al., 1998; Sato et al., 2006). Earlier study of our laboratory show that continuous exposure of tomato to high temperature (day / night temperature of 35℃ / 30℃ and 30℃ / 25℃) decreased pollen viability and pollen tube germination rate (Wang and Liu, 2006). AVRDC of the World Vegetable Center has bred serial heat-tolerant tomatoes since 1970’s, 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 with elevated expression level at high temperature from heat-tolerant tomato CL5915 anther (Wang and Liu, 2006). By analyzing temperature effect of these genes in the heat-induced cDNA library using Real-time PCR and Northern blot, Clone 8’ and Clone 7 were isolated from these heat-induced genes for further studies. We get the complete sequence by 5’ RACE and 3’ RACE from cDNA of tomato anther. Blasts with NCBI nucleotide database, these genes are E8-6 and a Lycopersicon esculentum clone 133497F. Blasts with NCBI protein database, E8-6 is an Tomato E8 protein homolog with a related dioxygenases conserved domain, while clone 133497F is presumed an Arabidopsis beta-ureidopropionase homolog with a CN hydrolase conserved domain. To confirm that if E8-6 and Clone 7 play some part in heat tolerance of anther or pollen, we proceed with the transgene of candidate heat-tolerant genes by over-expressing E8-6 and Clone 7 in heat-sensitive tomato L4783 and knock-down E8-6 and Clone 7 in heat-tolerant tomato CL5915 anther to compare the pollen viability and pollen tube germination rate of transgenic plants and wild type plants under high growth temperature. Up to now we get transgenic plants of over-expressing Clone 7 in L4783 (Line 6-11, 6-12) and knock-down Clone 7 in CL5915 (Line 8-1, 8-5, 8-7, 8-8). Pollen germination test show that pollen of Line 8-1 has decreasing germination rate than CL5915. Furthermore, by cloning and element presuming of promoters, I will study the heat-induced and tissue specific property of E8-6. Poster 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 elevated temperature contributes to decreased fruit set in tomatoes. AVRDC of the World Vegetable 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 with elevated expression level at high temperature from heat-tolerant tomato CL5915 anther. By analyzing temperature effect of these genes in the heat-induced cDNA library using Real-time PCR and Northern blot, Clone 8’ and Clone 7 were isolated from these heat-induced genes for further studies. Blasts with NCBI nucleotide database, these genes are E8-6 and a Lycopersicon esculentum clone 133497F. E8-6 is a Tomato E8 protein homolog with a related dioxygenases conserved domain, while clone 133497F is presumed an Arabidopsis beta-ureidopropionase homolog with a CN hydrolase conserved domain. To confirm that if E8-6 and Clone 7 play some part in heat tolerance of anther or pollen, we proceed with the transgene of these candidate heat-tolerant genes by over-expressing or knock-down target genes to compare the pollen viability and pollen tube germination rate of transgenic plants and wild type plants under different growth temperature. Pollen germination test show that pollen of Clone 7 knock-down plant has decreasing germination rate than heat-tolerant tomato CL5915. In order to study the tissue specific and ethylene induced expression of E8-6, we get E8-6 the promoter and proceed with the promoter assay. GUS reporter gene driven by E8-6 promoter is expressed in Arabidopsis and the expression pattern is detected by GUS staining. We find that E8-6 promoter expresses in sepal, filament and stigma of flowers.