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Title: Characterization and Comparison of CLONE 5 and WUN1 Genes
between Heat-Tolerant and Heat-Sensitive tomatoes ( Solanum
lycopersicum )
M. S student: Zeng Yaling
Advisor: Dr. Shih-Tong Jeng
Abstract:
Tomato (Solanum lycopersicum) is a temperature-sensitive crop. The harvest of
tomato is poor due to the elevated temperature. Studies have pointed out that
impairment of tomato pollen during anther development by elevated temperature
contributes to decreased fruit set. In this study, we used heat-sensitive (L4783) and
heat-tolerant (CL5915) tomatoes cultivated in high temperatures(day35/ night30 and
day30/night 25 °C) and optimal temperature (day25/night20 ℃) to investigate the
molecular mechanisms of heat tolerance of the tomato pollen grains during anther
development.
First, we used Real-time PCR to screen candidate genes from anther cDNA
library that was constructed from the heat-tolerant tomato by Suppression Subtractive
Hybridization (SSH). Thus, Clone 5 and Clone 29 (LwWun1) were selected from the
heat-tolerant tomato (CL5915) based on the results of Northern blotting and reverse
transcription-polymerase chain reaction (RT-PCR). By means of RACE (Rapid
amplification of cDNA end)，I obtained the full-length cDNA of Clone 5 and Clone 29
genes according to the SSH fragment sequence. Through the alignment on the NCBI
database, the translation products of Clone 5 and Clone 29 were predicted to be the
cupin-superfamily protein and wound-inducible 1 protein (Wun1), respectively.
Furthermore, we detect the expression of Clone 5 and LeWun1 by RT-PCR with total
RNA extracted from various tomato organs. To study whether these target genes
participate in the heat-resistance process of tomato pollen, we generated transgenic
tomatoes over-expressing target genes, Clone 5 and LeWun1,in heat-sensitive L4783
tomato and knock-down target genes in heat-tolerant CL5915 tomato by Agrobacteria
with pCAMBIA-2301. Additionally, the promoter region of LeWun1 gene was
isolated from the tomato by Tail PCR (Thermal asymmetric interlaced PCR), and its
function was predicted.