苦瓜果实β-半乳糖苷酶基因的克隆、表达及亚细胞定位

Cloning,Expression,and Subcellular Localization of β-galactosidase Gene from Momordica charantia L. Fruit

根据已构建苦瓜果实均一化文库中获得的1个与β-Gal基因相关的EST序列,采用经3'RACE技术,克隆获得1个苦瓜β-Gal基因的cDNA序列McGAL,全长为2261 bp,开放阅读框2160 bp,编码719个氨基酸。该基因在GenBank基因数据库的登录号为AFD54987.1。应用生物信息学软件对McGAL氨基酸序列分析表明,McGAL含有糖苷水解酶家族35的保守结构域G-G-P-[LIVM]-x-Q-x-E-N-E-[FY],C端不含凝集素结构域。二级结构显示,该酶含有α-螺旋(19.89%)、伸展链(26.98%)、β-转角(6.54%)和无规卷曲(46.59%)。McGAL氨基酸序列与鹰嘴豆、苜蓿、绿豆、大豆、羽扇豆的氨基酸序列的同源性分别达到73%、73%、73%、72%和71%。亚细胞定位结果表明,McGAL定位在线粒体膜上。荧光定量结果表明,McGAL在果实绿熟期表达量最高并随之下降,该基因可能与果实成熟软化初期相关。

Abstract ：The cDNA sequence of^-galactosidase gene named McGAL was cloned by 3＇RACE technique based on the related EST sequence from the normalized full-length cDNA library of bitter gourd fruit. The full length of cDNA sequence was 2261 bp, including a 2160 bp open reading frame （ORF） that encoded 719 amino acids. The se- quence had been deposited in GenBank database with the accession number of AFD54987. 1. Sequence analysis showed that the gene contained the putative active site-containing consensus sequence pattern G-G-P-[ LIVM ]-x-Q- x-E-N-E-[ FY] belonged to glycosyl hydrolase family 35 without lectin domain at their C-termini. In the secondary structure, ct-helix, extended strand, β-turn, and random coil were 19.89% ,26.98% ,6.54% and 46.59% , respec- tively. Amino acid sequence alignment indicated the McGAL had higher identity with Cicer arietinum, Medicago sa- tiva, Vigna radiate, Glycina max, and Lupinus angustifolius of 73% , 73% ,73% , 72% and 71% , respectively. Subcellular localization revealed the McGAL was located in mitochondrial inner membrane. Fluorescent quantitative PCR analysis displayed the McGAL gene expressed the highest levels at the mature green stage and decreased there- after,which meaned the gene may be related to early ripening stage.