三疣梭子蟹(Portunus trituberculatus)CYP4C基因的cDNA克隆及表达分析  

CLONING AND EXPRESSION OF CYP4C GENE IN PORTUNUS TRITUBERCULATUS

作　　者：张晓燕[1,2] 李健[1] 刘萍[1] 陈萍[1] 孙铭[1,3] 杨爱国 

机构地区：[1]中国水产科学研究院黄海水产研究所,青岛266071 [2]上海海洋大学,上海201306 [3]中国海洋大学,青岛266071 [4]青岛汇泉海洋科技开发有限公司,青岛266071

出　　处：《海洋与湖沼》2014年第1期73-79,共7页Oceanologia Et Limnologia Sinica

基　　金：国家863计划课题;2012AA10A409号;中央级公益性科研院所基本科研业务费项目资助;20603022012021号

摘　　要：采用RT-PCR及Smart-TM Race技术,首次获得三疣梭子蟹CYP4C基因cDNA全长序列。该基因全长1888bp,编码一个由514个氨基酸组成的多肽,预测分子量大小为59.54kDa,理论等电点为8.08。氨基酸序列中含有CYP基因家族特有的K螺旋保守序列(ExxR)和血红素结合区(FxxGxxxCxG)。同源性及系统进化分析表明,三疣梭子蟹CYP4C基因与岸蟹、凡纳滨对虾、日本沼虾、中国对虾、沟虾、红螯螯虾的同源性分别为88%、72%、66%、59%、60%、59%。荧光定量RT-PCR结果表明,CYP4C在肝胰腺、肌肉、眼柄、鳃和心脏中均有分布,在肝胰腺中表达量最高。高盐(45)胁迫后,三疣梭子蟹CYP4C的表达量显著高于对照组,随着胁迫时间的延长出现逐渐降低的趋势,在胁迫48h后表达量达到对照组水平;低盐(11)胁迫条件下,CYP4C的表达水平随着胁迫时间的延长呈现逐渐下降的趋势,且从胁迫6h开始显著低于对照组。研究推测,CYP4C基因参与三疣梭子蟹盐度适应调节,是蜕皮机制的调控因子之一。A complete cDNA sequence of CYP4C gene in Portunus trituberculatus was first cloned in RT-PCR and Smart-TM Race technology. The length of the CYP4C gene is 1888bp encoding a protein of 514 amino acids. Bioinformatics analysis deduced that the CYP4C gene protein molecular weight is 59.54kDa with a theoretical pI 8.08. The amino acid sequence has CYP conserved domains of helix K(ExxR) and heme-binding motif(FxxGxxxCxG). Blast analysis revealed that the similarities of CYP4C with C. maenas, L. vannamei, Macrobrachium nipponense, Fenneropenaeus chinensis, O. limosus, C. quadricarinatus were 88%, 72%, 66%, 59%, 60%, 59%, respectively. Real-time fluorescent quantitative PCR was used to assess the mRNA expression of CYP4C in different tissues and its expression level of CYP4C in salinity stress. The results show that CYP4C could be expressed in all tested tissues of P. trituberculatus, including hepatopancreas, muscle, eyes, gills, and heart, among them the highest expression level was observed in hepatopancreas, while the lowest in heart. The expression level of CYP4C was up-regulated distinctly in the hepatopancreas at salinity 45, and then gradually reduced with time to the level of control group after 48 h, while at salinity 11, the CYP4C expression dropped significantly below that of the control group 6 h later. The results imply that CYP4C is involved in the accommodation to salinity change and play an important role in its molting.

关 键 词：三疣梭子蟹(Portunus trituberculatus) CYP4C 基因克隆 表达 盐度胁迫 

分 类 号：Q78[生物学—分子生物学]