Cloning And Expression Analysis Of Proline Synthetase Gene NtP5CS And Ntδ-OAT In Nicotiana Tabacum

Tobacco is mainly planted in mountainous or semi-mountainous areas with water shortage. Drought is an important factor that limits the improvement of tobacco yield and quality in China. The study of genes relating to water stress response and their clone expressions is significant for revealing the drought-defying mechanism at molecular level and improving the drought resistance of tobacco. In this study, the full-length cDNA sequence of the key proline synthetase gene was separated from tobacco(Nicotiana tabacum). The sequence signature, evolutionary relationship and expression patterns induced by various adverse stresses were then analyzed to reveal the response mechanism to stress induction and provide theoretical guidance for improving the stress resistance of tobacco(Nicotiana tabacum)using this gene. The results were as follows.1. The homologous fragment of known genes was designed to obtain the middle segment of P5CS and its full-length cDNA was amplified by RACE. The sequential structure of this gene, the basic and conserved domain characteristics of its encoding proteins were analyzed using bioinformatics. Its expression patterns were studied by RT-PCR. We cloned the P5CS gene with a full length of 2,584 bp from tobacco induced by drought stress, which was termed NtP5CS (GenBank accession HM854026). The ORF was 2160 bp in length, which encoded 719 amino acid residues. GenBank Blast analysis showed that the sequence was highly homologous with the level of nucleotide and amino acid of tomPRO2 gene obtained from tomato. According to the phylogenetic tree, NtP5CS and tomPRO2 might be orthologous genes. RT-PCR showed the upregulated expression of NtP5CS could be induced by drought, low temperature, ABA and high salt and it had the highest expression in root and leaves under vigorous growing stage and water stress condition. It was the first time to gain the P5CS from tobacco, which responded to water stress and might play a role in response to osmotic stress.2. Ornithine aminotransferase is a key enzyme for Synthesis of proline based on Ornithine, so it plays a critical role for plants in adaptation to environmental stresses. A 425-bp middle segment was cloned from tobacco(Nicotiana tabacum, zhongyan 14)using degenerate primer designed from the known conserved regions of Ornithine aminotransferase gene. Special primers derived from the 425-bp segment were used to amplify the 5'and 3'ends of the cDNA using RACE and verified by BLAST. Ornithine aminotransferase gene was obtained for the first time from tobacco. The full-length cDNA was termed Ntδ-OAT (GenBank accession GU144571) which was 1,781 bp in length and encoded 477 amino acids. According to the phylogenetic tree, the evolution of Ntδ-OAT corresponded with traditional biological classification and its functional domain had a high conservative property in the process of biological evolution. Real-time PCR showed that the expression of Ntδ-OAT was induced by multiple stresses, such as drought, high salt, low temperature as well as ABA. It was concluded that Ntδ-OAT might play a role in role in response to osmotic stress...