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

作　　者：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] 

机构地区：[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

出　　处：《热带海洋学报》2012年第6期69-75,共7页Journal of Tropical Oceanography

基　　金：国家科技支撑计划项目(2008BAD94B03)

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。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

关 键 词：i-型溶菌酶 克隆 表达 温度 近江牡蛎 

分 类 号：P735[天文地球—海洋生物学]