摘要
采用表达序列标签(EST)和cDNA末端快速扩增技术(RACE)从松江鲈(Trachidermus fasciatus)体内克隆出一个热休克同源蛋白70(即TfHsc70)。其全长2138 bp,理论分子量为71.1 kDa,等电点为5.27,并由一个1947 bp的开放阅读框(ORF)、一个78 bp的5′端未翻译区(UTR)和一个95 bp的3′端带有终止密码子(TAA)的UTR组成。通过与多种鱼类、两栖类和爬行类等动物的热休克同源蛋白70(HSC70)对比发现,其具有88.89%~97.09%的相似性。实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)显示,TfHsc70 mRNA在松江鲈的血液、心脏、肝脏、胃、肠、脾脏、肾脏、大脑、鳃中均有表达,但在皮肤中几乎未检测出。使用脂多糖(LPS)刺激发现,皮肤、血液和肝脏中TfHsc70的表达量在2 h时明显增加,鳃和脾脏中略微上调,而在大脑中呈现先减少再增加的趋势。基于对TfHsc70的蛋白序列以及TfHsc70 mRNA的表达分析,表明TfHsc70是热休克蛋白HSP70家族成员,它可能参与了松江鲈的先天免疫。
Among heat shock proteins,heat shock protein 70(HSP70)is highly conserved in biological evolution,widely found in archaea,plants and animals,and plays an important role in biological resistance to external stimuli,folding and packaging of proteins,presenting antigens and cell proliferation and differentiation.It can be divided into two categories.One type can be induced to express rapidly and in large quantities under different stress conditions,but will be restored to the basic level under normal environment,so they are usually called induced HSP70.The other type has no stress induction process and is expressed constitutively,which is called heat shock homologous protein 70(HSC70).In this study,a heat shock homologous protein(TfHsc70)was cloned from Trachidermus fasciatus by expressing sequence tag(EST)and rapid cDNA end amplification(RACE)technique.It was 2138 bp with a theoretical molecular weight of 71.1 kDa and an isoelectric point of 5.27,and included an open reading frame(ORF)of 1947 bp,a 5′-terminal untranslated region(UTR)of 78 bp,and a 3′-terminal UTR of 95 bp with a stop codon(TAA).Sequence alignment showed that TfHsc70 contained three conserved sequence tags of the HSP70 family(GIDLGTTY,DLGGGTFD and VLVGGSTRIPKIQK)and an ATP-GTP binding site motif(AEAYLGKT).Compared with the heat shock homologous protein 70(HSC70)of many fishes,amphibians and reptiles,it was found that the similarity of HSC70 was 88.89%-97.09%.Quantitative real-time PCR(qRT-PCR)demonstrated that the TfHsc70 mRNA was expressed in blood,heart,liver,stomach,intestine,spleen,kidney,brain,and gill of Trachidermus fasciatus,and the expression level of the TfHsc70 mRNA was the highest in the brain.But it was almost undetectable in the skin.When using lipopolysaccharide(LPS)to stimulate Trachidermus fasciatus,the expression of TfHsc70 significantly increased in skin,blood and liver at 2 h,and was slightly upregulated in gill and spleen,but TfHsc70 showed initial down-regulation followed by a gradual up-regulation in the brain.This was due to the sensitivity of different tissues to LPS.Based on the analysis of the protein sequence of TfHsc70 and the expression of TfHsc70 mRNA,it was shown that TfHsc70 was a member of HSP70 family of heat shock proteins,and it might play an important role in innate immunity of Trachidermus fasciatus.In conclusion,the research explored the sequence characteristics and expression of TfHsc70 mRNA in Trachidermus fasciatus,which laid a foundation for the study of innate immunity in Trachidermus fasciatus and other fish.
作者
贾昌锋
高海霞
祝茜
张雷
JIA Changfeng;GAO Haixia;ZHU Qian;ZHANG Lei(Marine College,Shandong University,Weihai Shandong 264209,China;Weihai Changqing Ocean Sci-tech Co.Ltd.,Rongcheng Shandong 264200,China)
出处
《海洋渔业》
CSCD
北大核心
2021年第4期418-429,共12页
Marine Fisheries
基金
威海市大学共建项目(HXLG20200105)。
关键词
松江鲈
脂多糖
先天免疫
热休克同源蛋白70
Trachidermus fasciatus
LPS
innate immune
heat-shock cognate protein 70
