凡纳滨对虾细胞色素P450(CYP370C2)基因的克隆与表达分析

Molecular Cloning and Expression Analysis of Cytochrome P450( CYP370C2) Gene in Litopenaeus vannamei

细胞色素P450(CYP)酶系在机体的毒素、污染物和药物等代谢过程中发挥着重要作用,为深入了解对虾CYP基因的结构和功能,采用c DNA末端快速克隆技术得到凡纳滨对虾Litopenaeus vannamei CYP家族基因CYP370C2的全长c DNA序列,共1 745 bp,包含1个1 464 bp的开放阅读框,编码487个氨基酸。CYP370C2基因序列中具有CYP蛋白特有的血红素结合区、Ⅰ螺旋保守区、C螺旋保守区和K螺旋保守区。同源性比对结果显示,凡纳滨对虾CYP370C2基因序列与三疣梭子蟹Portunus trituberculatus的CYP基因相似度最高,为64%。CYP370C2基因在所有检测的组织中均有表达,在肝胰腺中的表达量最高,鳃和肠道次之。在48 h的氨氮胁迫试验中,胁迫24 h后,肝胰腺和鳃中的CYP370C2基因相对表达量与对照组相比均显著上调,分别在48 h和24 h达到峰值。脂多糖(LPS)注射后,CYP370C2基因在肝胰腺中的相对表达量在3 h开始显著上升,在12 h达到最大值; CYP370C2基因在鳃中的表达在LPS注射后的3 h和6 h被显著抑制,12 h恢复至对照组水平,随后在24 h和48 h显著上调。这些实验结果显示,CYP370C2基因参与了氨氮胁迫响应和LPS刺激响应,表明CYP370C2基因可能在凡纳滨对虾抗环境胁迫和抗病原体的免疫防御过程中均发挥重要作用。

Cytochrome P450 (CYP) enzyme system plays vital roles in the metabolic processes of toxin, contaminant and medicine. In order to analyze the structure and functions of shrimp CYP, the full-length cDNA sequence of a CYP gene (named CYP370C2 ) was cloned from the Pacific white shrimp (Litopenaeus vannamei) by using rapid-amplification of eD- NA ends technique. The cDNA of CYP370C2 was 1 745 bp in length, containing a 1 464 bp open reading frame encoding 487 amino acids. The sequence of CYP370C2 shared common characteristics with the CYP superfamily, such as the heme- hinging domain, I helix, C helix and K helix. The result of sequence alignment showed that the amino acid sequence of CYP370C2 shared 64% identity with that of the blue crab (Portunus trituberculatus).. Phylogenetic analysis showed that the CYP370C2 gene of L. vannamei was closely related to those of Malacostraca and Crustacea, indicating that CYP gene is rela- tively conserved in aquatic arthropod. The expression of CYP370C2 gene was detected in all the tissues ofL. vannamei test- ed in this study, and the highest expression level was found in hepatopancrea, followed by gill and intestine. In the 48-h ammonia-N exposure experiment, the expression levels of CYP370C2 in hepatopanerea and gill were significantly upregulat- ed after 24 h, and the peak value appeared after 48 h and 24 h, respectively. After lipopolysaccharide (LPS) injection, CYP370C2 transcription in hepatopancrea obviously increased after 3 h, and the highest level occurred after 12 h. By con- trast, the expression of CYP370C2 in gill was inhibited after 3 h and 6 h, and then recovered after 12 h and upregulated af- ter 24 h and 48 h. These results demonstrate that CYP370C2 participate in the host response against ammonia-N stress and LPS stimulation, and thus CYP370C2 may play vital roles in L. vannamei defense system against environmental stress and pathogen infection.