缢蛏ScHsc70 cDNA的分子特性和表达分析

Molecular characteristics and expression analysis of ScHsc70 cDNA in agamaki clam(Sinonovacula constricta)

从缢蛏(Sinonovacula constricta Lamarck)cDNA文库中筛选出一条热休克蛋白70(Heat shock protein 70,HSP70)同源序列,采用EST扩增和5′RACE(rapid amplification of cDNA ends,RACE)扩增方法获得全长cDNA序列,共2 335 bp,包括76 bp的5′非翻译区和309 bp的3′非翻译区,以及1 950 bp的开放阅读框。阅读框共编码649个氨基酸,推算的分子量约为70.89 kD,理论等电点为5.28。氨基酸序列分析结果表明,该基因的氨基酸序列含有HSP70家族的3个签名序列,2个糖基化位点,1个ATP-GTP结合位点,且C末端具有GGXP四肽重复序列以及细胞质特异性调控基序EEVD。该基因是组成型HSC70(Heat shock cognate 70)亚家族基因成员,被命名为ScHsc70。基于氨基酸序列的双壳贝类聚类分析表明,缢蛏与南极帽贝(Laternula elliptica)、文蛤(Meretrix meretrix)的亲缘关系最近。荧光定量表达分析显示,ScHsc70 mRNA在缢蛏水管、鳃、斧足、外套膜、肝和性腺等组织中以不同水平存在,其中在外套膜中的表达量最低,而在水管和肝中的表达量最高。经副溶血弧菌(Vibrio parahaemolyticus)和副鳗弧菌(V.anguillarum)诱导后,ScHsc70 mRNA在缢蛏肝中的水平呈现先升高后下降的变化趋势,且分别在感染后的第20小时和第30小时达到最高。结果提示,缢蛏ScHsc70基因可能参与了对细菌感染的防御反应。

Heat shock proteins consist of several families of highly conserved proteins that play an essential role in a number of cellular processes.Among the 70 kD family of heat shock proteins,heat shock cognate protein 70 kD（Hsc70） and inducible heat shock protein 70 kD（Hsp70） have been extensively studied in vertebrates and invertebrates.Several cDNAs encoding HSP70 have been described in molluscs,including the oyster（Crassostrea gigas）,bay scallop（Argopecten irradians）,Zhikong scallop（Chlamys farreri）,mussel（Mytilus galloprovincialis）,and Asiatic hard clam（Meretrix meretrix）.Recent studies examining HSP70 expression in different species of mollusc have recognized the physiological and ecological importance of heat-shock gene expression in response to changing environments.We isolated an EST sequence with high homology with heat shock protein 70 gene from the cDNA library of Sinonovacula constricta.Then,the complete express sequence of this gene was obtained using PCR and 5′RACE.The cDNA of this gene was 2 335 bp,and consisted of a 76 bp 5′ untranslated region（UTR）,a 1 950 bp open reading frame（ORF） and a 309 bp 3′ UTR.The translated protein consisted of 649 amino acids（70.89 kD）and its calculated isoelectric point was 5.28.Sequence analysis of the protein revealed that this gene contained three signature sequences of the heat shock protein 70 family（HSP70 family）,two glycosylation sites and one ATP-GTP binding site.Four terapeptides of GGXP and a cytoplasm characteristic motif of EEVD were detected in the carboxyl terminal region of the deduced amino acid sequence.This HSP70 is a member of the HSC70（constitutive genes） subfamily in the HSP70 family,and is designated as ScHsc70.Phylogenetic analysis suggested that the protein was most similar to those of S.constricta,Laternula elliptica,and M.meretrix.Quantitative reverse transcriptase（qRT-PCR） analyses revealed that ScHsc70 mRNA was expressed constitutively in all the tissues tested.Expression was lowest in the mantle and highest in the water pipe and liver.Following challenge with Vibrio parahaemolyticus and V.anguillarum,ScHsc70 mRNA expression first increased in the liver of S.constricta then decreased.Expression peaked after 20 and 30 h post-challenge with V.parahaemolyticus and V.anguillarum,respectively.There was a significant increase in ScHsc70 mRNA in the liver of clams challenged with V.parahaemolyticus compared to the control group at 4,12,and 30 h post-challenge.Similarly,there was a significant increase in ScHsc70 mRNA levels in the liver of clams challenged with V.anguillarum 4,8,12,20,30,and 48 h post-challenge.Our results suggest that ScHsc70 may play a role in the immune response of S.constricta to bacterial challenge.