文档介绍：
日本沼虾血蓝蛋白基因 cDNA 全长克隆及
表达分析
王文锋1,2,夏西超1,王雪参1,吕黎1,杨洪1,宁黔冀1**
5
10
15
(1. 河南师范大学生命科学学院,河南新乡 453007;
2. 新乡医学院生命科学技术系,河南新乡 453003)
摘要:目的克隆日本沼虾血蓝蛋白基因,进行生物信息学及时空表达分析。方法利用 RACE
技术从肝胰腺中克隆血蓝蛋白基因 cDNA 全长序列,用生物软件对其序列进行生物信息学分
析,时空表达分析采用 Real-time PCR 方法。结果日本沼虾血蓝蛋白基因 cDNA 全长 2151
bp,包含 145 bp 的 5′UTR、14 bp 的 3′UTR 和 1991 bp 的开放阅读框(ORF)。ORF 编码
663 个氨基酸,预测分子量为 KDa,理论等电点为 ,含有典型的三个血蓝蛋白结
构域和两个保守的铜离子结合位点。与淡水螯虾、凡纳滨对虾血蓝蛋白相似性分别为 70%和
63%。该基因在肝胰腺中的相对表达量最高,肌肉和血细胞表达较弱,大颚器官、表皮和卵
巢几乎不表达;肝胰腺血蓝蛋白基因的表达量在蜕皮间期最高,蜕皮后期和蜕皮前期较低;
嗜水气单胞菌刺激后肝胰腺中的表达量显著增加。结论与其它甲壳动物相似,日本沼虾血
蓝蛋白基因具有典型的结构域和保守的铜离子结合位点,在蜕皮间期的肝胰腺中呈高表达,
是参与机体免疫防御反应的一种重要分子。
关键词:日本沼虾;血蓝蛋白;基因克隆;RACE;实时荧光定量 PCR;时空表达
中图分类号:Q459
20
Molecular cloning and expression analysis of hemocyanin
gene from Macrobrachium nipponense
Wang Wenfeng1,2, Xia Xichao1, Wang Xueshen1, Lv Li1, Yang Hong1, Ning Qianji1
(1. College of Life Sciences, Henan Normal University, HeNan XinXiang 453007;
25
30
35
40
45
2. Xinxiang medical university, HeNan XinXiang 453003)
Abstract: Objective The aim of this study was to clone the hemocyanin gene from Macrobrachium
nipponense, and to do the bioinformatics and spatial and temporal expression analysis of the gene.
Methods The hemocyanin gene was cloned from hepatopancreas of Macrobrachium nipponense
using the rapid amplification of cDNA ends (RACE) method, and its sequence was analyzed with
biological software, spatial and temporal expressions of the gene were detected by real-time PCR.
Results The full-length cDNA of hemocyanin was 2151bp and contained a 145bp of
5'-untranslated region (UTR), a 14bp of 3'UTR and a 1991bp of open reading frame (ORF) that
encoded a protein of 663 amino acids with a calculated molecular mass of kDa and pI at
. The deduced amino acid sequence containing three hemocyanin domains and two
copper-binding sites exhibited 70% similarity to that of Pacifastacus leniusculus, 63% to that of
Litopenaeus vannamei. The two copper-binding sites and six