耐熱番茄中雄蕊表現基因Clone 5 與 LeWun1 之分離及分析

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.