番茄一个褪黑素合成基因的cDNA克隆与表达分析

Cloning of a melatonin biosynthesis gene and its expression analysis in tomato

为深入研究褪黑素的分子生物学功能,根据番茄基因组数据库的SlSNAT基因序列信息设计引物,以耐盐番茄材LA1401(PI365967)叶片RNA反转录得到的cDNA为模板,利用高保真酶克隆了番茄的褪黑素合成酶基因SlSNAT,基因CDS(coding sequence)序列全长为768 bp,共编码255个氨基酸。利用酶切连接的方法,构建了该基因的超表达载体。实时定量PCR结果表明,SlSNAT基因在叶片中的表达量最高,显著高于其在花、果实、根、种子和萼片中的表达量。在不同非生物逆境处理下的表达结果显示,在干旱处理条件下的表达量最高,其次是在甘露醇的渗透胁迫逆境,在这2种胁迫条件下的表达量均显著高于其在过氧化氢、氯化钠、低温和褪黑素诱导下的表达量。另外,在盐胁迫条件下,SlSNAT基因在根部的表达量受到明显抑制。

To further study the molecular biology function of melatonin,we designed the gene specific primer based on the sequence information from the Tomato Functional Genomics Database, the full-length CDS (coding sequence) of a melatonin biosynthesis gene ( SISNAT) was from salt-tolerant tomato LA1401 (PI365967). The corresponding CDS is 768 bp in length and the deduced protein contains 255 amino acids. The over-expression vector was constructed through enzyme digestion and ligation. Tissue expression profile using Real-time quantitative PCR showed that SISNAT expression level was the highest in leaves, being significantly higher than its expression in flowers, fruits, roots and seeds. Up on differe nt abiotic stresses, SISNAT expression level un der drought treatme nt was the highest followed by mannitol osmotic stress, and its levels under the two stresses were significantly higher than these in the hydrogen peroxide,sodium chloride, cold and melatonin. In addition, under salt stress, SISNAT expression level in roots was remarkablely in hibited. This gene is potential no vel can didate for abiotic tolerance research in tomato.