苦瓜果实S-腺苷蛋氨酸脱羧酶基因McSAMDC的克隆、表达及亚细胞定位

Cloning, Expression and Subcellular Localization of S-adenosylmethionine Decarboxylase Gene (McSAMDC) Gene from Fruit in Momordica charantia L.

根据已构建苦瓜果实均一化文库中获得的1个与SAMDC基因相关的EST序列,采用3′RACE技术,克隆获得1个苦瓜SAMDC基因的cDNA全长序列,命名为McSAMDC(GenBank登录号为KC632099)。生物信息分析结果表明,该cDNA全长1 900 bp,5′UTR和3′UTR分别长501、325 bp。该cDNA序列存在3个开放读码框(微型tORF、上游读码框uORF和主读码框mORF),其中mORF长1 077 bp,编码358个氨基酸,预测分子量为39.31 ku,含有酶原剪切位点结构域和蛋白快速降解有关的PEST二个保守结构域。二级结构分析显示,McSAMDC含有无规卷曲(45.53%)、α-螺旋(29.33%)、伸展链(19.83%)、β-转角(5.31%)。序列分析结果表明,McSAMDC与拟南芥(CAA69073.1)、琴叶拟南芥(XP 002882231.1)和雪里红(AAS45435.1)的氨基酸序列相似性分别达到69%、68%和68%。亚细胞定位结果表明,McSAMDC定位在细胞质中。荧光定量结果表明,McSAMDC在果实膨大期表达量最高,并随之迅速下降,并从成熟初期至完全成熟期该基因表达量逐渐增大。

The full length cDNA sequence of S-adenosylmethionine decarboxylase （SAMDC） gene named McSAMDC （GenBank accession No.： KC632099） was cloned by 3＇RACE technique based on the related EST sequence from the normalized Full-Length cDNA library of bitter gourd fruit. The cDNA sequence is 1 900 bp in full length with a 501 bp 5＇-UTR and a 325 bp 3＇-UTR. The cDNA sequence consists of three ORFs （tiny ORF, upstream ORF and main ORF）, and the main ORF was 1 077 bp encoding 358 amino acids with a calculated molecular weight of 39.31 ku. The mORF deduced protein had two conserved function domains （proenzyme cleavage site and rapid degradation of SAMDC protein domain）. In the secondary structure, Random coil, a-helix, Extended strand, and 13-turn was 45.53%, 29.33%, 19.83% and 5.31%, respectively. Amino acid sequence alignment showed the McSAMDC shared high identity with Arabidopsis thationa （CAA69073.1）, Arabidopsis lyrata subsp. （XP 002882231.1）, and Brassica juncea （AAS45435.1） as 69%, 68% and 68%. Subcellular localization analysis showed that the mORF was mainly found in the cytoplasm. Fluorescent quantitative PCR analysis showed the McSAMDC gene was expressed at the highest level at the fruit enlargement stage and rapidly decreased thereafter, and the expression level was continuously increased from the mature green stage until full mature stage.