摘要
采用RACE技术克隆河蚬(Corbicula fluminea)谷胱甘肽-S-转移酶(glutathione S-transferase,GST)基因全长序列,利用DNAMAN 6.0软件序列进行比对,Smart分析保守域和功能位点,SignalIP 4.0预测分析该基因信号肽,MEGA 5.0绘制系统进化树,利用荧光定量PCR和酶活性技术检测Cd^(2+)、Cu^(2+)和Pb^(2+)胁迫后GST在河蚬肝胰腺中的表达特征,为河蚬内脏GST mRNA和酶生物标志物评价指标的建立提供参考。结果表明,GST基因全长952 bp(GenBank登录号:KX211963),编码一个由217个氨基酸组成的多肽,理论等电点PI为5.98,分子量为25.169kD。氨基酸序列中含有GST基因家族特有的GST-N结构域和GST-C结构域,系统进化分析表明,河蚬GST基因与菲律宾帘蛤(Rudiraps philippinarum)同源性最高,为90.32%。重金属胁迫结果显示:GST mRNA表达量和酶活性均高于相同时间点的对照组,并随着胁迫时间的延长呈先升高后降低趋势,每个实验组峰值出现时间略有差异,且峰值结果均显著高于相应的对照组(P<0.05)。Cd^(2+)胁迫后GST mRNA表达和酶活性峰值均出现在24 h,且m RNA在48 h的表达量也显著高于对照组(P<0.05);Cu^(2+)胁迫后GST mRNA表达峰值和酶活性峰值分别出现在12 h和48 h,且在24h胁迫组酶活性也显著高于对照组(P<0.05);Pb^(2+)胁迫后GST mRNA表达和酶活性峰值均出现在48 h,且24 h的mRNA表达量也显著高于对照组(P<0.05)。综上,河蚬GST对重金属刺激敏感,说明河蚬内脏中GST可以作为水体环境重金属污染的指示分子。
To lay a foundation for the establishment of glutathione S-transferase(GST)mRNA and enzyme biomarker evaluation indexes of Corbicula fluminatum viscera,the complete mRNA sequence of GST gene was obtained by RACE,sequence alignment was performed using DNAMAN 6.0 software,conserved domains and functional sites were predicted by Smart program,signal peptide was predicted with signalip 4.0 program.phylogenetic tree was drawn using MEGA 5.0 software,and the mRNA expression changes of GST in hepatopancreas of C.fluminea after Cd^2+,Cu^2+ and Pb^2+ stress were detected by fluorescence quantitative PCR.Results showed that the full-length mRNA sequence of C.fluminea GST gene was 952 bp(GenBank accession number:KX211963),and encoded a protein of 217 amino acids with molecular weight of 25.169 kD and PI of 5.98,containing a GST-N domain and a GST-C domain.The amino acid sequences of GSTs between C.fluminea and Rudirapes philippinarum showed a similarity of more than 90.32%.The results indicated that the mRNA expression and enzymatic activities of GST in groups exposed to heavy metal were higher than those in the control group at each time point and increased first and then decreased with the prolongation of stress time,but the peaking time of each experimental group was slightly different and significantly higher than that in the control group(P<0.05).Under Cd2+ stress,both the mRNA expression level and enzymatic activities of GST reached to peak at 24 h,and the mRNA expression levels at 48 h was that significantly higher than that in the control group(P<0.05);Under Cu^2+ stress,the mRNA expression level and enzymatic activities of GST respectively reached to peak at 12 h and 48 h,and the enzymatic activities at 24 h was significantly higher than that in the control group(P<0.05).Under Pb^2+ stress,both the mRNA expression level and enzymatic activities of GST were peaking at 48 h,and the mRNA expression levels at 24 h was significantly higher than that in the control group(P<0.05).This experiment shows that GST is sensitive to heavy metal stimuli,indicating that GST in viscera of C.fluminea can be used as an indicator molecule to detect heavy metal pollution in water environment.
作者
郑双艳
刘清
张霞丽
谢彦海
ZHENG Shuangyan;LIU Qing;ZHANG Xiali;XIE Yanhai(Science Center of Laboratory Animal,Nanchang University,Nanchang 330006,China;Jiangxi Innovation Fund Management Center for Small and Medium Technological Enterprises,Nanchang 330007,China)
出处
《生态环境学报》
CSCD
北大核心
2019年第2期369-375,共7页
Ecology and Environmental Sciences
基金
国家自然科学基金项目(31460145)
江西省自然科学基金项目(20122BAB204009)
关键词
河蚬
GST
重金属
MRNA表达
酶活性
Corbicula fluminea
GST
heavy metal
mRNA expression
enzymatic activity
