Molecular cloning and expression of two HSP70 genes in the Wuchang bream (Megalobrama amblycephala Yih)
Molecular cloning and expression of two HSP70 genes in the Wuchang bream (Megalobrama amblycephala Yih)
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DOI：
10.1016/j.fsi.2009.11.018
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发表时间：
2010-03-01
影响因子：
4.7
通讯作者：
Pan, Liangkun
中科院分区：
文献类型：
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作者：
Ming, Jianhua;Xie, Jun;Pan, Liangkun
利用RT-PCR和cDNA末端快速扩增技术（RACE）从五昌鲷（Megalobrama amblycephala Y.）肝脏中分离到两个编码热休克同源蛋白70 （HSC70）和诱导热休克蛋白70 （HSP70）的互补脱氧核糖核酸（cDNA）克隆，分别命名为Ma-HSC70和Ma-HSP70。cdna的长度为2336和2224 bp[不包括poly (A)]，分别包含1950和1932 bp的开放阅读框（orf）。orf编码649和643个氨基酸，预测分子量分别为71.24和70.52 kDa，理论等电点分别为5 25和5 30。基因组DNA结构分析显示，Ma-HSC70基因含有7个内含子，所有内含子均符合GT/AG规则，而Ma-HSP70基因在编码区不含内含子，氨基酸序列分析表明，Ma-HSC70和Ma-HSP70基因均含有HSP70家族的3个特征序列。两个部分重叠的二部核定位信号序列（NLS）和细胞质特征基序（EEVD）同源性分析显示，Ma-HSC70与已知的其他脊椎动物hsp70的同源性超过93.0%，Ma-HSP70与已知的其他脊椎动物hsp70的同源性超过85%，Ma-HSC70与Ma-HSP70的同源性为86.5%。富含B细胞抗原位点，无任何信号肽或跨膜区，两种蛋白还含有许多蛋白激酶C磷酸化位点。n -肉豆蔻酰基化位点、酪蛋白激酶II磷酸化位点和n -糖基化位点，预测它们可能在蛋白质折叠、易位、细胞内定位、信号转导和调控中发挥重要作用。采用荧光实时定量RT-PCR技术研究了热休克（34℃）、拥挤胁迫（100 g L-1）和嗜水气单胞菌攻毒对武昌鲷肝脏两种hsp70 mRNA表达的影响。结果表明，在热休克和拥挤胁迫下，24 h Ma-HSC70 mRNA表达先下降后上升至应激前水平；而Ma-HSP70 mRNA的表达量在热胁迫下呈先升高后降低的趋势，在聚集胁迫下呈持续升高的趋势。细菌攻毒后，Ma-HSC70和Ma-HSP70 mRNA的表达量均呈现先升高后降低的趋势。(C) 2009年Elsevier Ltd .出版
Two complementary deoxyribonucleic acid (cDNA) clones encoding heat shock cognate 70 (HSC70) and inducible heat shock protein 70 (HSP70) were isolated from the liver of Wuchang bream (Megalobrama amblycephala Y.) using RT-PCR and rapid amplification of cDNA ends (RACE) They were named Ma-HSC70 and Ma-HSP70, respectively. The cDNAs were 2336 and 2224 bp in length [not including poly (A)] and contained 1950 and 1932 bp open reading frames (ORFs), respectively. The ORFs encoded proteins of 649 and 643 amino acids with predicted molecular weights of 71.24 and 70.52 kDa, and theoretical isoelectric points of 5 25 and 5 30. respectively Genomic DNA structure analysis revealed that Ma-HSC70 gene contained seven introns with all introns conforming to the GT/AG rule whereas Ma-HSP70 gene did not contain any intron in the coding region Amino acid sequence analysis indicated that both Ma-HSC70 and Ma-HSP70 contained three signature sequences of HSP70 family, two partial overlapping bipartite nuclear localization signal sequences (NLS) and cytoplasmic characteristic motif (EEVD) Homology analysis revealed that Ma-HSC70 shared more than 93.0% identity with the known HSC70s of other vertebrates, while Ma-HSP70 shared more than 85 0% identity with the known HSP70s of other vertebrates, and Ma-HSC70 and Ma-HSP70 shared 86 5% identity Bioinformatics analysis indicated that the proteins encoded by Ma-HSC70 and Ma-HSP70 genes were hydrophilic, rich in B cells antigenic sites, without any signal peptide or transmembrane region The two proteins also contained many protein kinase C phosphorylation sites. N-myristoylation sites, casein kinase II phosphorylation sites, and N-glycosylation sites, predicting that they could play essential roles in protein folding, translocation, intracellular localization, signal transduction and regulation. The predominant secondary structures of the two proteins were a-helix and random coil Fluorescent real-time quantitative RT-PCR was used to Study the effects of heat shock (34 degrees C), crowding stress (100 g L-1) and challenge with bacteria Aeromonas hydrophila on the mRNA expression of the two HSP70s in Wuchang bream liver The results indicated that, during 24 h stress, Ma-HSC70 mRNA expression decreased at first and then rose to the level before stress under heat shock and crowding stress, but Ma-HSP70 mRNA expression increased at first and then decreased under heat stress, and appeared to increase continuously under crowding stress After bacterial challenge, the mRNA levels of both Ma-HSC70 and Ma-HSP70 increased at first and then decreased The cloning and expression analysis of the two HSP70s provide theoretical basis to further study the mechanism of anti-adverseness and expression characteristics under stress conditions of Wuchang bream. (C) 2009 Published by Elsevier Ltd