日本沼虾(Macrobrachium nipponense)组蛋白酶B基因克隆及其在组织和卵巢发育过程中的表达  

GENE CLONE OF CATHEPSIN B IN MACROBRACHIUM NIPPONENSE AND GENE EXPRESSION IN TISSUES AND OVARY IN DIFFERENT DEVELOPMENT STAGES

作　　者：赵卫红[1,2] 陈立侨[2] 王资生[1] 张凤英[2,3] 齐志涛[1] 

机构地区：[1]盐城工学院海洋与生物工程学院,盐城224051 [2]华东师范大学生命科学学院,上海200062 [3]中国水产科学研究院东海水产研究所,上海200090

出　　处：《海洋与湖沼》2015年第4期862-869,共8页Oceanologia Et Limnologia Sinica

基　　金：国家自然科学基金资助项目;31101887号;江苏省自然科学基金资助项目;BK2011419号;BK2012675号

摘　　要：组蛋白酶B广泛存在于生物体内,与免疫、消化和繁殖等生理功能息息相关。为了研究组蛋白酶B在甲壳动物体内的作用尤其在卵巢发育过程中的作用,本研究采用3’RACE和5’RACE技术,首次克隆获得日本沼虾(Macrobrachium nipponense)组蛋白酶B(简称Mn CB)基因c DNA全长,并采用实时荧光定量(q PCR)测定了Mn CB在日本沼虾不同组织中和卵巢发育过程中m RNA的表达量。序列结果分析表明:Mn CB序列含有12 bp的5’-UTR,996 bp的ORF和702 bp的3’-UTR。ORF共编码331个氨基酸的多肽,此多肽由16个氨基酸的信号肽、63个氨基酸的前导肽和252个氨基酸的成熟肽组成,其理论p I为6.36,分子量为36.5KDa。q PCR的结果表明:Mn CB在测定的所有组织中均有表达,在心脏中表达量最高,肌肉、肝胰腺和胸神经节中表达量中等,肠、鳃和血细胞中的表达量较低。Mn CB的表达量在卵巢的发育过程中逐步升高,卵巢发育至初级卵黄发生期(Ⅲ期)Mn CB的表达量显著增加(P<0.05),次级卵黄发生期(Ⅳ期)表达量继续增加,并增至最大值,Ⅳ期与Ⅲ期表达量差异不显著(P>0.05),成熟期(V期)表达量显著下降(P<0.05)。上述研究结果表明Mn CB广泛存在于日本沼虾的组织中,并且参与卵黄蛋白原或卵黄蛋白的水解。Cathepsin B is involved in immunity, digestion and reproduction and distributed widely in many organisms. To understand its ovary development in crustacean, we cloned cathepsin B in Macrobrachium nipponense, named Mn CB for the first time, and detected its gene expression. The full sequence of Mn CB was obtained by RACE method based on Mn CB fragment obtained in EST library. Mn CB gene is comprised of 1710 bp, containing a 12 bp 5＇-UTR, a 993 bp ORF and a 702 bp 3＇-UTR. The ORF encodes a polypeptide of 331 amino acids including a 16-amino acid signal peptide, a 63-amino acid propeptide and a 252-amino acid mature peptide. Mn CB m RNA was detected in all the tested tissues including haemocyte, hepatopancreas, muscle gill, intestine, heart, and thoracic ganglia. Real-time quantitative PCR（q PCR） results show that Mn CB was expressed highest in heart, intermediate in muscle, hepatopancreas, and thoracic ganglia, and lowest in gill, intestine, and haemocyte. The level of Mn CB m RNA in ovary increased significantly at the oil globule ovary stage（stageⅢ）（P〈0.05） and reached a maximum value at the yolk granule ovary stage（stage Ⅳ）, then decreased with further ovarian development. moreover, there was no significantly difference in the Mn CB expression in ovary between stages Ⅲ and Ⅳ（P〉0.05）. Therefore, Mn CB plays ubiquitously an important role in hydration of vitellogenin or yolk protein in the ovary of M. nipponense.

关 键 词：日本沼虾 组蛋白酶B RACE 基因表达 

分 类 号：Q789[生物学—分子生物学]