摘要
本实验利用RACE技术克隆获得了马氏珠母贝HSP70 (Pm-HSP70)基因,并对其基因和氨基酸序列结构特征进行了生物信息学分析,同时采用荧光定量PCR技术分析了该基因在不同组织的表达模式及不同温度下的时序表达模式。序列分析表明,Pm-HSP70 cDNA序列全长为2 215 bp,其中开放式阅读框1 899 bp,5'UTR 107 bp,3'UTR 209 bp,编码632个氨基酸,理论蛋白分子量为69.44 kD,理论等电点为5.61。该蛋白具有HSP70家族典型的结构域HSP70,以及ATP结合位点、细胞质特征性保守序列和3个HSP70家族标签。多序列比对结果表明Pm-HSP70与菲律宾蛤仔HSP70同源性最高,为80%;系统进化分析发现,Pm-HSP70与菲律宾蛤仔等贝类HSP70聚为一支。组织表达定量分析结果显示,Pm-HSP70在马氏珠母贝多个组织中均有表达,在肝胰腺中表达量最高,其次是性腺;对不同温度下鳃组织中Pm-HSP70的时序表达分析发现,在处理后各时间点高温组(32℃)基因表达水平最高,且均显著高于对照组(22℃)和低温组(17℃)。以上结果表明Pm-HSP70可能参与马氏珠母贝的高温胁迫响应。该研究为进一步探索Pm-HSP70在马氏珠母贝温度适应性中的作用提供了基础资料。
In this study,HSP70(Pm-HSP70)gene of Pinctada fucata martensii was cloned by using RACE techno-logy and the structural characteristics of the gene and amino acid sequence were analyzed by bioinformatics,and the expression patterns of the gene(Pm-HSP70)in different tissues as well as its temporal expression patterns under different temperatures were analyzed by using qRT-PCR technology.Sequence analysis showed that the full length of Pm-HSP70 cDNA sequence was 2 215 bp,containing 1 899 bp of the open reading frame(ORF),107 bp of 5'UTR and 209 bp of 3'UTR,which encoded 632 amino acids.The theoretical protein molecular weight was 69.44 kD and the theoretical isoelectric point was 5.61.The protein had typical structure domain HSP70 of HSP70 family,ATP binding site,cytoplasmic characteristic conservative sequences and three HSP70 family tags.Multiple sequence alignment results showed that Pm-HSP70 had the highest homology(80%)with HSP70 in Ruditapes philippinarum.Phylogenetic analysis showed that Pm-HSP70 was clustered with molluscs HSP70 as a branch,such as Ruditapes philippinarum.The results of the tissue expression quantitative analysis showed that Pm-HSP70 was expressed in many tissues of Pinctada fucata martensii,with the most expressive in hepatopancreas,followed by the gonads.And the analysis results of Pm-HSP70 temporal expression at different temperatures in gill indicated that the gene expression level in the high temperature group(32℃)was the highest at each time point after treatment,which was significantly higher than it in the control group(22℃)and hypothermia group(17℃).The above results suggested that Pm-HSP70 might be involved in the resistance to high-temperature stress of Pinctada fucata martensii.This study would provide basic data for the further exploring the role of Pm-HSP70 in temperature adaptability of Pinctada fucata martensii.
作者
刘雅
王庆恒
郑哲
焦钰
李利二
杜晓东
Liu Ya;Wang Qingheng;Zheng Zhe;Jiao Yu;Li Lier;Du Xiaodong(College of Fisheries,Guangdong Ocean University,Zhanjiang,524088;Guangdong Technology Research Center for Pearl Aquaculture and Pro-cess,Zhanjiang,524088;Yinhui Pearl Culture Farmer Specialty Cooperative in Xuwen County,Zhanjiang,524088)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2019年第3期990-998,共9页
Genomics and Applied Biology
基金
广东省海洋渔业与产业发展专项(Z2014009)
马氏珠母贝育珠新技术与产业化(B201601-Z12)
广东省普通高校重大科研项目培育计划(2014GKXM049)共同资助
