棉花S-腺苷甲硫氨酸脱羧酶基因的克隆及低温下的表达分析

摘要

根据棉花S-腺苷甲硫氨酸脱羧酶(S-adenosylmethionine decarboxylase,SAMDC)基因已知EST序列设计引物,采用RACE和RT-PCR技术克隆获得该基因的全长cDNA序列,命名为GhSAMDC (GenBank登录号为JN020148).生物信息学分析表明,GhSAMDC cDNA序列全长1874 bp,涵盖tiny ORF (tORF)、upstream ORF (uORF)和main ORF(mORF)3个植物SAMDC基因特征ORF.其中mORF长1064 bp,编码含355个氨基酸残基的SAMDC酶原,预测分子量为38.25 kD,该酶原含有高度保守的酶原剪切位点结构域(LSESSLF)和与SAMDC蛋白快速降解有关的PEST(TIHVTPEDGFSYAS)结构域.GhSAMDC基因组DNA序列全长2743 bp,包含3个内含子,均位于5′UTR,其中一个位于uORF内.聚类分析表明,GhSAMDC与葡萄中该蛋白的同源关系最近,并且与其他双子叶植物聚为一类.荧光定量PCR分析结果表明,GhSAMDC表达受低温诱导,在冷敏感品种新陆早1号中基因表达水平明显高于在耐冷品种新陆早33中.%S-adenosylmethionine decarboxylase is one of the key enzymes in polyamine biosynthesis. With the increase of poly-amine accumulation in plants, SAMDC can improve crop tolerance to environment stresses. We cloned the S-adenosylmethionine decarboxylase gene from Gossypium hirsutum by RACE and RT-PCR, named GhSAMDC (GenBank accession number: JN020148). The cDNA full length was 1 874 bp, with three open reading frames, i.e. tiny ORF (tORF), upstream ORF (uORF) and main ORF (mORF). The mORF was 1 064 bp encoding 355 amino acids with a molecular mass of 38.25 kD, and its deduced protein had two highly conserved function domains (proenzyme cleavage site and PEST domain). The complete genomic DNA sequence of GhSAMDC was 2 743 bp in length with three introns, all of which were located in 5' UTR, and one lay in the uORF. Phylogenetic analysis showed GhSAMDC was the closest to that in Vitis vinifera, and real-time PCR suggested that GhSAMDC was induced by cold stress. It showed lower gene expression level in cold resistant cultivar Xinluzao 33 than in cold sensitive cultivar Xinluzao 1.