日本囊对虾Na-K-2Cl共同转运蛋白基因的克隆与组织表达分析  

Molecular Cloning and Expression Analysis of Na-K-2Cl Cotransporter from Marsupenaeus japonicus

作　　者：刘洪涛[1] 王军[1] 毛勇[1] 乔莹[1] 钟声平[1] 苏永全[1] 

机构地区：[1]厦门大学海洋与地球学院,福建厦门361102

出　　处：《厦门大学学报（自然科学版）》2016年第1期46-54,共9页Journal of Xiamen University：Natural Science

基　　金：国家高技术研究发展计划(863计划)(2012AA10A409);国家星火计划(2015GA720002);国家虾产业技术体系岗位专家项目(CARS-47);福建省科技厅区域重大项目(2013N3004);厦门市南方海洋中心项目(14CZY033HJ07)

摘　　要：利用反转录PCR(RT-PCR)和cDNA末端快速扩增(RACE)等技术在鳃组织中获得日本囊对虾(Marsupenaeus japonicus)Na-K-2Cl共同转运蛋白(NKCC)cDNA全序列,包含14bp的5′非编码区(5′-UTR)、201bp的3′-UTR和3 183bp的开放阅读框(ORF).该ORF共编码1 060个氨基酸,预测蛋白分子质量为117.051ku,理论等电点为6.57,命名为Mj-NKCC.预测Mj-NKCC二级结构由10个跨膜区组成,跨膜区的氨基酸序列和布局均相对保守.同源对比结果显示,Mj-NKCC的氨基酸序列与夏威夷海蚀洞虾(Halocaridina rubra)的Na-K-2Cl共同转运蛋白相似度最高,为77%.系统进化分析表明Mj-NKCC与夏威夷海蚀洞虾、蓝蟹(Callinectes sapidus)等甲壳动物的NKCC聚为一支.实时荧光定量PCR(qRT-PCR)分析表明,Mj-NKCC基因在肝胰腺、鳃、胃、肠、心、眼柄、肌肉和血细胞中均有表达,鳃组织中表达量最高;在盐度骤变时,该基因表达变化显著,表明其很可能参与了对虾的渗透压调节,在对虾的渗透平衡中发挥重要作用.Na-K-2Cl cotransporter gene （Mj-NKCC） from Marsupenaeus japonicus was obtained using reverse transcription PCR （RT-PCR） and rapid amplication of cDNA ends （RACE） in this study. Results show that the full-length cDNA sequence of Mj- NKCC consists of a 14-bp 51 untranslated regions （SCUTR）,a 201-bp 3CUTR and a 3 183-bp open reading frame （ORF）.The deduced amino acids sequence is composed of 1 060 amino acids,with molecular weight of 117. 051 ku and isoelectric point of 6.57.The predicted second structure of Mj-NKCC contains 10 transmembrane domains, which are highly conserved in sequences and localization sites compared to other species.Multiple alignments of the amino acid sequences show that Mj-NKCC has the highest ho mology with Callinectes sapidus （up to 77 ;/0）, and the phylogenetic analysis indicated that it was first clustered together with C. sapidus and Carcinus maenas. Additionally, the quantitative real-time PCR （qRT-PCR） results displayed that Mj-NKCC was expressed in hepatopancreas, gills, stomach, muscle, heart, eyestalk, intestine and hemocytes, with the highest expression level in gills. As the salinity changed sharply, significant changes occurred in the expression of Mj-NKCC,indicating that Mj-NKCC putatively participates in osmotic balance of shrimps and plays an important role in osmoregulation of shrimps.

关 键 词：日本囊对虾 Na-K-2Cl共同转运蛋白(NKCC) 基因克隆 组织表达 盐度骤变 

分 类 号：Q785[生物学—分子生物学] S917.4[农业科学—水产科学]