花斑裸鲤GeHSP70基因的克隆及其对重金属Cu^(2+)胁迫的应答  

Cloning of GeHSP70 gene of Gymnocypris eckloni and its response to Cu^(2+) stress

作　　者：栗瑞红 卫福磊 张霞 王玉晴 朱世海 王国杰[2] 王振吉[2] 李长忠 LI Ruihong;WEI Fulei;ZHANG Xia;WANG Yuqing;ZHU Shihai;WANG Guojie;WANG Zhenji;LI Changzhong(College of Eco-Environmental Engineering,Qinghai University,Xining,Qinghai 810016,China;Qinghai Fisheries Environment Monitoring Station,Xining Qinghai 810012,China)

机构地区：[1]青海大学生态环境工程学院,青海西宁810016 [2]青海省渔业环境监测站,青海西宁810012

出　　处：《西北农林科技大学学报（自然科学版）》2019年第1期1-10,17,共11页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31460700)

摘　　要：【目的】克隆花斑裸鲤GeHSP70基因,并进行生物信息学分析;研究正常和Cu^(2+)胁迫条件下GeHSP70的表达模式。【方法】采用分子生物学技术克隆花斑裸鲤GeHSP70基因的全长cDNA,分析其生物信息学特征。采用Real-time PCR技术分析正常和0.01mg/L Cu^(2+)胁迫(胁迫0,12,24,48h)条件下GeHSP70mRNA在花斑裸鲤肝脏、肾脏、脑和鳃4个组织中的表达情况,并采用ELISA试剂盒检测肝脏、鳃组织中GeHSP70蛋白的表达水平。【结果】花斑裸鲤GeHSP70序列共1 387bp,由123bp的5′-UTR、592bp的3′-UTR和672bp的编码区构成,编码223个氨基酸;GeHSP70蛋白是疏水性蛋白,分子质量为11.15ku,等电点为5.01,包含2个HSP70家族的特征序列:IDLGTTYS(11-18位氨基酸)和IFDLGGGTFDVSIL(199-212位氨基酸)。GeHSP70基因核苷酸序列与鲫鱼的同源性最高(93%),在系统进化树中与同科或属中鱼种聚合成一个分支,显示出高度保守性。Real-time PCR结果表明,正常条件下花斑裸鲤GeHSP70在鳃组织中的表达量最高,其次是肝脏,在脑和鳃组织中表达量较低;随着0.01mg/L Cu^(2+)胁迫时间的延长,花斑裸鲤肝脏、肾脏和鳃组织的GeHSP70mRNA表达量先升后降,在24h表达量最高,而脑组织的表达量持续升高;GeHSP70在花斑裸鲤肝脏和鳃组织的蛋白水平表达量总体均表现为先升后降,表明Cu^(2+)能够诱导GeHSP70的表达。【结论】成功克隆了花斑裸鲤GeHSP70cDNA全长,在0.01mg/L Cu^(2+)胁迫下花斑裸鲤GeHSP70在mRNA水平和蛋白水平呈规律性应答,表明GeHSP70是潜在的反映水环境质量的生物标志物。【Objective】This study cloned Gymnocypris eckloni GeHSP70 gene and analyzed its bioinformatics and expression pattern under normal condition and Cu^2+stress.【Method】The full-length cDNA of Gymnocypris eckloni GeHSP70 gene was cloned by molecular biology technology,and its bioinformatics characteristics were analyzed.The GeHSP70 mRNA expressions in liver,kidney,brain and gill under normal and 0.01 mg/L Cu^2+stress(0,12,24 and 48 h)were analyzed by Real time PCR technology.The ELISA kit was used to determine the protein level of GeHSP70 in liver and gill.【Result】Gymnocypris eckloni GeHSP70 sequence is 1 387 bp in length with 123 bp 5′-UTR,592 bp 3′-UTR and 672 bp encoding region,encoding 223 amino acids.GeHSP70 is a hydrophobic protein with molecular weight of 11.15 ku and an isoelectric point of 5.01.It includes 2 HSP70 family feature sequences of IDLGTTYS(11-18 amino acid)and IFDLGGGTFDVSIL(199-212 amino acid).The nucleotide sequence of GeHSP70 gene has the highest homology with Carassius auratus(93%).Real-time PCR showed that the expression of Gymnocypris eckloni GeHSP70 gill tissue under normal conditions is the highest,followed by liver,brain and gill tissues.With the extension of 0.01 mg/L Cu^2+exposure,GeHSP70 mRNA expressions of Gymnocypris eckloni in liver,kidney and gill tissues firstly increased then decreased with the peak at 24 h,while the expression in brain tissue increased all the time.The protein levels in Gymnocypris eckloni liver and gill tissues first increased and then decreased,indicating that Cu^2+induced the expression of GeHSP70.【Conclusion】This study successfully cloned the full-length Gymnocypris eckloni cDNA of GeHSP70,and GeHSP70 showed regular response at mRNA and protein levels to 0.01 mg/L Cu^2+exposure,indicating that GeHSP70 is a potential biomarker to reflect the quality of water environment.

关 键 词：花斑裸鲤 热应激蛋白70 REAL-TIME PCR 重金属胁迫 

分 类 号：Q785[生物学—分子生物学]