近江牡蛎i-型溶菌酶基因分析及温度对基因表达的影响

doi:10.11978/j.issn.1009-5470.2012.06.011

热带海洋学报 ›› 2012, Vol. 31 ›› Issue (6): 69-75.doi: 10.11978/j.issn.1009-5470.2012.06.011cstr: 32234.14.j.issn.1009-5470.2012.06.011

近江牡蛎i-型溶菌酶基因分析及温度对基因表达的影响

许友卿1, 吴卫君1, 丁兆坤1, 潘志忠2, 何为2, 吴铁军2, 陈秀荔2, 李咏梅2,蒋伟明2

1. 广西大学水产科学研究所，广西南宁 530005；2. 广西水产研究所，广西南宁 530021

收稿日期:2013-02-06 修回日期:2013-02-06 出版日期:2012-12-01 发布日期:2013-02-06
通讯作者: 蒋伟明, 副研究员, 主要从事水产动物生物学研究。E-mail: nnjwm@qq.com
作者简介:作者简介：许友卿(1958—), 女, 广东省汕头市人, 博士, 教授, 博士生导师,主要研究环境生物学、鱼类营养、生理生化与分子生物学。
基金资助:
基金项目：国家科技支撑计划项目(2008BAD94B03)

Analysis of i-type lysozyme gene from Crassostrea hongkongensis and the effect of temperature on the gene expression

XU You-qing1, WU Wei-jun1, DING Zhao-kun1, PAN Zhi-zhong2, HE Wei2, WU Tie-jun2, CHEN Xiu-li2, LI Yong-mei2, JIANG Wei-ming2

1．Institute for Fishery Sciences, Guangxi University, Nanning 530004, China;

Received:2013-02-06 Revised:2013-02-06 Online:2012-12-01 Published:2013-02-06

摘要/Abstract

摘要： 溶菌酶是机体先天免疫系统中一个重要的效应分子, 参与机体多种免疫反应。本研究克隆了近江牡蛎Crassostrea hongkongensis i-型溶菌酶的保守cDNA序列, 并探讨其在不同组织的表达、不同温度对其表达的影响。结果表明, 近江牡蛎i-型溶菌酶保守cDNA序列长634 bp, 包含一个长420 bp开放阅读框, 编码139个氨基酸；其蛋白序列具有典型的i-型溶菌酶特征, 包含特定氨基酸残基序列CL(E/L/R/H)C(I/M)C、部分高度保守序列SCG(P/Y)FQI和酶活性中心位点(Glu³⁴、Asp⁴⁵、Ser⁴⁸、Trp⁶¹)；另外, 还发现一个新的氨基酸保守序列HNGGPRGC。i-型溶菌酶基因在近江牡蛎消化腺、肌肉、鳃、外套膜、唇瓣等5种组织的表达量以消化腺最高, 鳃次之, 肌肉最低。i-型溶菌酶基因的表达与温度相关, 水温20—25℃, 其表达量比较稳定；水温6℃或13℃, 表达量都呈下降趋势；在27℃, 表达量在整个实验过程都维持一个相对高的水平；在34℃, 其表达量在第12小时达到最高, 第48 小时后急剧下降；表明温度可影响i-型溶菌酶基因表达, 从而调节其免疫功能和对外环境的应激反应。

关键词: i-型溶菌酶, 克隆, 表达, 温度, 近江牡蛎

Abstract: Lysozyme is one of the important effectors involved in a variety of immune responses in innate immune system of organisms. A conservative i-type lysozyme cDNA sequence was cloned from Crassostrea hongkongensis and its expression in different tissues was studied. The effects of different temperatures on the gene expression in the experiment were also studied. The cloned conservative i-type lysozyme cDNA sequence contained 634 bp with a 420 bp open reading frame and the cDNA encoded 139 amino acids. A new conservative amino acid sequence, HNGGPRGC, and the typical features were found in the protein sequence of the i-type lysozyme from C. hongkongensis, including specific amino acid residue sequence, CL(E/L/R/H)C(I/M)C, a partial highly conservative sequence, SCG(P/Y)FQI, and the enzyme active site (Glu³⁴, Asp⁴⁵, Ser⁴⁸, Trp⁶¹). Real-time quantitative PCR was used to assess the expression pattern of the i-type lysozyme gene in the digestive gland, muscle, gill, mantle, and labial palps of C. hongkongensis. The highest expression level of the i-type lysozyme gene was found in the digestive gland, the second in the gill, and the lowest in the muscle. A temperature stress experiment was conducted to study the expression control of the i-type lysozyme gene. The expression level was stable when the water temperature was at 20?25℃. A tendency to decrease expression was found at 6 or 13℃. The expression was maintained at a relatively high level throughout of 96 h at 27℃. The expression level was found the highest at 12 h and sharply declined after 48 h at 34℃. It is concluded that the expression of the i-type lysozyme gene was affected by temperature, which could be used to control the immune function of C. hongkongensis in response to external environment

Key words: i-type lysozyme, clone, expression, temperature, Crassostrea hongkongensis

中图分类号:

P735

许友卿吴卫君丁兆坤潘志忠, 何为, 吴铁军, 陈秀荔, 李咏梅,蒋伟明. 近江牡蛎i-型溶菌酶基因分析及温度对基因表达的影响[J]. 热带海洋学报, 2012, 31(6): 69-75.

XU You-qing, WU Wei-jun, DING Zhao-kun, PAN Zhi-zhong, HE Wei, WU Tie-jun, CHEN Xiu-li, LI Yong-mei, JIANG Wei-ming. Analysis of i-type lysozyme gene from Crassostrea hongkongensis and the effect of temperature on the gene expression[J]. Journal of Tropical Oceanography, 2012, 31(6): 69-75.

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近江牡蛎i-型溶菌酶基因分析及温度对基因表达的影响

Analysis of i-type lysozyme gene from Crassostrea hongkongensis and the effect of temperature on the gene expression

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