中华蜜蜂铜锌超氧化物歧化酶基因的克隆、序列分析及表达特征

摘要

This experiment was conducted to clone the cDNA and study the expression profile of copper/zinc superoxide dismutase (SOD1) gene of Apis cerana cerana. The SOD1 gene of Apis cerana cerana was cloned by nested PCR. The expression level of SOD1 gene in different tissues (brain, thorax and abdomen of adults) and at different developmental stages (3- and 6-day-old larva instars, 1- and 4-day-old pupas and 1- and 7-day-old adults) of Apis cerana cerana was detected using quantitative real-time PCR (qRT-PCR). The results showed that the full length of cDNA of SOD1 gene of Apis cerana cerana (GenBank accession number is JN700517) was 631 bp, encoding 152 amino acids, and the predicted molecular weight and isoelectric point were 15.65 ku and 6. 21, respectively. The deduced amino acid sequence of SOD1 of Apis cerana cerana showed the highest similarity (99% ) with that of Apis mellifera ligustica, and had 67% ~85% similarity with other insects (such as Drosophila melanogaster, Anopheles gambiae, Spodoptera exigua, Bombyx mori, Bom-bus ignitus and so on). The expression level of SOD gene was significantly different at different developmental stages of Apis cerana cerana, and the peak appeared in 6-day-old larva instar, while the lowest value appeared in 4-day-old pupa. The expression level in the brain and abdomen was significantly higher than that in the thorax (P <0. 05). The cDNA of SOD1 gene of Apis cerana cerana is successfully cloned in this study, and the full length is 631 bp, encoding 152 amino acids. The expression profile at different developmental stages and in different tissues is different.%本试验旨在克隆中华蜜蜂铜锌超氧化物歧化酶(SOD1)基因,探究中华蜜蜂SOD1基因的表达特性.利用巢式PCR技术扩增、克隆及分析中华蜜蜂SOD1基因,采用实时定量PCR检测不同发育时期(3和6日龄幼虫、l和4日龄蛹以及1和7日龄成蜂)、不同部位(成蜂的头、胸和腹)SOD1基因的表达量,探究中华蜜蜂SOD1基因的表达特性.结果显示:克隆获得中华蜜蜂SOD1基因的cDNA全长序列,其cDNA全长631bp(GenBank登录号JN700517),编码152个氨基酸,预测蛋白质分子质量为15.65 ku,等电点为6.21,经氨基酸序列比对,与意大利蜜蜂有99％的相似性,与其他典型的昆虫(如果蝇、冈比亚按蚊、斜纹夜蛾,家蚕、熊蜂)也有67％ ～85％的相似性；中华蜜蜂SOD1基因在不同发育时期和部位中均有表达特异性,在6日龄幼虫表达量达到峰值,而在4日龄蛹中最低；头部与腹部表达量显著高于胸部(P＜0.05).本研究成功克隆获得中华蜜蜂SOD1基因,其cDNA全长631 bp,编码152个氨基酸.该基因在中华蜜蜂整个发育时期均有表达,而且不同发育时期以及不同部位的表达量不同.