Download Title: Characterization and Comparison of E8

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.