收稿日期: 2008-05-26
修回日期: 2008-09-07
网络出版日期: 2010-07-29
基金资助
科技部科研院所社会公益研究专项(2005DIB3J020); 中央级公益性科研院所基本科研业务费专项资金(中国水产科学研究院南
海水产研究所)资助项目(2007ZD08)。
CYP4 gene cloning and expression level analysis of Perna viridis
Received date: 2008-05-26
Revised date: 2008-09-07
Online published: 2010-07-29
Supported by
科技部科研院所社会公益研究专项(2005DIB3J020); 中央级公益性科研院所基本科研业务费专项资金(中国水产科学研究院南
海水产研究所)资助项目(2007ZD08)。
利用简并引物PCR以及cDNA末端快速扩增(rapid amplification of cDNA ends, RACE)技术获得了总长度为1197bp的翡翠贻贝Perna viridis CYP4基因cDNA序列。根据所获得的翡翠贻贝cDNA序列设计定量PCR引物, 利用实时荧光定量PCR(Real-time quantitative PCR)技术测定了CYP4基因在野生翡翠贻贝消化腺、足和性腺中的表达水平。此外, 本研究还应用实时荧光定量PCR技术对经过Aroclor1254暴露处理对翡翠贻贝性腺中CYP4表达水平的影响进行了定量测定。研究结果表明, CYP4基因在野生翡翠贻贝消化腺、足和性腺中均有表达, 且其表达水平具有组织差异和性别差异; Aroclor1254暴露处理对翡翠贻贝性腺CYP4基因的表达水平有明显的诱导作用, 并且这种诱导作用有明显的时间和剂量效应。本研究为CYP4基因作为生物大分子标记物在环境监测领域的应用提供了分子生物学水平的支持。
关键词： CYP4基因; 翡翠贻贝Perna viridis; 荧光定量PCR
周驰,李纯厚,张为民,贾晓平 . 翡翠贻贝CYP4基因克隆及其表达水平分析[J]. 热带海洋学报, 2010 , 29(4) : 82 -88 . DOI: 10.11978/j.issn.1009-5470.2010.04.082
In order to identify and obtain the cDNA sequence of CYP4 gene in Perna viridis, degenerate PCR and RACE (Rapid Amplification of cDNA Ends) were used, and partial CYP4 gene cDNA sequence total of 1197 bp was acquired (GeneBank/NCBI EU429566) from Perna viridis digestive gland. The CYP4 gene expression in digestive gland, foot and gonad of wild Perna viridis and the CYP4 gene expression level in gonad from Aroclor1254 treated Perna viridis were evaluated using real-time quantitative-PCR (RQ-PCR) and Sybr Green I chemistry. The RQ-PCR primers were designed based on the Partial cDNA sequences of Perna viridis CYP4, and the beta-actin target was used as internal reference and included in each RQ-PCR experiment for the normalization of expression data. The results showed that basic CYP4 expression existed in all of the three tissues (digestive gland, foot and gonad) from wild Perna viridis, and the expression of Perna viridis CYP4 had tissue differences and sex differences. The Perna viridis gonad CYP4 gene expression can be induced by Aroclor1254 exposure, and this induction is both exposure-time dependent and exposure-dose dependent. This study has provided useful clues to the inquisition of using CYP4 gene family as biomarker of toxicology effect.
Key words： CYP4 gene; Perna viridis; Quantitative-PCR
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