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从普通烟草中克隆脯氨酸合成的关键酶基因P5CS,分析其序列特征、进化关系、表达模式,以期为烟草的抗逆研究奠定基础。设计兼并引物获得P5CS基因中间片段,利用RACE技术扩增其全长cDNA;采用生物信息学技术分析该基因的序列结构及其编码蛋白的保守域及基本特性;利用RT-PCR研究其表达模式。从受干旱胁迫诱导的普通烟草品种中烟14中克隆到全长为2584bp的烟草P5CS基因,命名为NtP5CS,GenBank登录号为HM854026,开放读码框为2160 bp,编码719个氨基酸。经Blast同源性比对分析,该序列与番茄tomPRO2基因在核苷酸和氨基酸水平上高度同源。系统进化树分析显示,NtP5CS与番茄tomPRO2基因可能是直系同源基因。RT-PCR分析表明,干旱、低温、ABA、高盐等胁迫条件均可诱导NtP5CS基因的上调表达,且在旺长期和胁迫条件下根和叶中表达量最高。首次从普通烟草中克隆得到P5CS基因,该基因对水分胁迫响应,可能参与普通烟草抗渗透胁迫反应。
The key enzyme gene P5CS of proline synthesis was cloned from Nicotiana tabacum, and its sequence characteristics, evolutionary relationships and expression patterns were analyzed, which laid the foundation for the research of tobacco stress resistance. The middle fragment of P5CS gene was designed by PCR. The full-length cDNA of P5CS gene was amplified by RACE. The sequence structure and the conserved domain of the encoded protein were analyzed by bioinformatics analysis. The expression patterns of the gene were analyzed by RT-PCR. The tobacco P5CS gene with the full length of 2584bp was cloned from tobacco 14 induced by drought stress, named NtP5CS. The GenBank accession number was HM854026, and the open reading frame was 2160 bp, encoding 719 amino acids. According to Blast homology analysis, the sequence was highly homologous to tomato tomPRO2 gene at nucleotide and amino acid level. Phylogenetic tree analysis showed that the NtP5CS and tomato tomPRO2 genes may be orthologous genes. RT-PCR analysis showed that drought, low temperature, ABA, high salt and other stress conditions can induce the up-regulated expression of NtP5CS gene, and the highest expression in roots and leaves under the conditions of vigorous growth and stress. The P5CS gene was cloned from Nicotiana tabacum for the first time. The gene is responsive to water stress and may be involved in the general tobacco resistance to osmotic stress.