Analysis Of Polyamine Metabolism And Differentially Expressed Genes In Tomato (Solanum Lycopersicum) Under Heat Stress

The optimal temperature for tomato growth is 20℃to 25℃, but the ambient temperature often reaches 35℃, even up to 40℃. High temperature is one of the most important factors limiting quality and yield in tomato production. Studies on the mechanism of thermotolerance in tomato are of great significance for breeding heat-tolerant varieties and improving cultivation technology. The purpose of the present research emphasised on illustrating the relationships between polyamines and thermotolerance, and to abtain the thermotolerance-relevant genes offering base for developing high efficient, reliable evaluation ways to thermotolerance of tomato and for the molecular biochemistry research. The main results of this investigation are as follows:1. It is important to isolate and characterize unknown DNA sequences flanking known regions, especially regulatory region. Among all the polymerase chain reaction (PCR)-based methods, ligation-mediated PCR (LM-PCR) is one of the most suitable methods for genomic walking. Here We describe a simple and efficient improved LM-PCR, which includes the following step: 1) design the adaptor with several endonucleases; 2)digestion and ligation of the adaptor and DNA ; 3) desired DNA products can be generated from the libraries by Nest-PCR with gene specific primers and adaptor primers. Using this method, upstream regulation sequence of SAMDC genes in tomato had been cloned successfully.2. The SAMDC cDNA from jimsonwead (GenBank accession number: Y07768) was used as a querying probe to blast the GenBank EST database (http://www.ncbi.nlm.nih.gov/BLAST). Based on the assembled homologous cDNA sequences and identification by RT-PCR and RACE, the full-lenghth cDNA(1847bp) of a new member of SAMDC genes in tomato was cloned, with 5'-UTR(523bp)and 3'-UTR(241bp).Corresponding DNA sequence was 3648bp, containing three introns in 5'-UTR, and all the cleave sites obey the GT-AG rule. We designated the gene as SLSAMDC1 (GenBank accession number EF550528). SLSAMDC1 consistes of three ORFs (tiny uORF, small uORF and main ORF), and the main ORF encoding 360 amino acids with a calculated molecular weight of 39kD. A comparison of deduced amino acid sequence revealed that SLSAMDC1 is highly homologous to other plant SAMDC genes, but low to human,E.coli and yeast's. Southern analysis indicated that SLSAMDC1 is encoded by a single copy gene. RT-PCR analysis shows that more intensive expression of SLSAMDC1 in fruit than in any other organs. The upstream regulatory sequence was cloned by genome walking .Further analysis showed the upstream regulatory sequence contains several Cis-acting elements (eg.W-box, TATA-box, CAAT-box).3. Effect of heat stress on polyamine synthesis and net photosynthetic rate in heat-tolerant tomato line Saladette and heat-sensitive cultivar zhongshu 6 was investigated. After heat stress (40/30℃, 24h), the net photosynthetic rate decreased in two cultivars, but the decreased extent in heat-tolerant cultivar was lower than that in heat-sensitive cultivar. Expression of ADC genes and SAMDC genes was repressed by heat stress. Pretreatment of Spd to tomato leaves effectively prevented heat stress-induced decrease in net photosynthetic rate, especially for zhongshu 6. The difference of PAs synthesis between the two cultivars lied in the content of Spd.4. The cDNA-AFLP approach was used to identify genes differently expressed in tomato leaves under heat stress. 768 pairs of primers were used for selective amplification. 187 TDFs were selected for their differential expression under heat stress. Among the 187TDFs, 47TDFs were cloned and sequenced, and expression patterns of 2 TDFs were confirmed by RT-PCR approach. Compared with the publicly available databases, 35 TDFs presented some significant similarity with known plant sequences, which functions were involved in gene regulation, transcription factor, signal transduction, stress defense, etc. At least 18 TDFs were involved in the defense to heat stress in tomato. 12 TDFs with low identity and no match to known genes may represent novel genes.5. A full-lenghth cDNA(SLHSP70) in tomato was cloned by RACE methods. SLHSP70 encodes a protein with 692 amino acids, and shares high homologous to other plant HSP70 located in chloroplast. The sequence comparison and Phylogenetic tree analysis indicate that SLHSP70 is located in Plastid stroma. SLHSP90 was cloned by 3'-RACE with a partial open reading frame(436aa). SLHSP90 is high homologous to Ipomoea nil HSP83(98%).