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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。
According to a EST sequence homologous to β-Gal gene obtained from a homozygous library of bitter melon, a McGAL cDNA was obtained by 3’RACE cloning of a β-Gal gene from bitter melon, with a total length of 2261 bp, Open reading frame 2160 bp, encoding 719 amino acids. The gene in GenBank database accession number AFD54987.1. The amino acid sequence analysis of McGAL using bioinformatics software showed that McGAL contains the conserved domain G-G-P- [LIVM] -x-Q-x-E-N-E- [FY] of glycoside hydrolase family 35 and the C-terminus does not contain a lectin domain. The secondary structure shows that the enzyme contains alpha-helix (19.89%), extension chain (26.98%), beta-turn (6.54%) and random coil (46.59%). The amino acid sequences of McGAL were 73%, 73%, 73%, 72% and 71% homologous to the amino acid sequence of chickpea, alfalfa, mung bean, soybean and lupine. Subcellular localization results show that McGAL is located on the mitochondrial membrane. Fluorescence quantitative analysis showed that the expression of McGAL was the highest during fruit ripening and then decreased, which may be related to the early ripening stage of fruit.