泥蚶基质金属蛋白酶组织抑制因子3基因的克隆及镉免疫表达研究  

A novel tissue inhibitor of metalloproteinase 3 in blood clam Tegillarca granose: molecular cloning and immune-related research of Cd

作　　者：李佩芬[1,2] 林志华[2] 包永波[2] 

机构地区：[1]宁波大学海洋学院,浙江宁波315211 [2]浙江万里学院浙江省水产种质资源高效利用技术研究重点实验室,浙江宁波315100

出　　处：《水产学报》2015年第3期336-344,共9页Journal of Fisheries of China

基　　金：国家"八六三"高技术研究发展计划(2012AA10A410);浙江省自然科学重点基金(LZ12C190001);浙江省重中之重学科开放基金(ZS2013008)

摘　　要：通过已构建的泥蚶转录组文库EST,利用SMART RACE技术扩增得到泥蚶Tg-TIMP-3基因全长c DNA序列,并对其进行生物信息学和组织表达相关分析。结果显示,泥蚶Tg-TIMP-3 c DNA全长为804 bp,其中开放阅读框为570 bp,编码189个氨基酸;氨基酸序列比对发现其氨基酸序列与其他动物的相似性并不高,与长牡蛎的相似性仅为35.8%,但TIMP基因的两个功能域(N端和C端)相对保守。qRT-PCR检测结果显示:Tg-TIMP-3 mRNA在鳃中表达最高,外套膜和肝胰腺次之,表达量最低的是血液。在不同浓度重金属镉(Cd)攻毒后,泥蚶鳃中的Tg-TIMP-3 mRNA表达量先略微下调后显著上调达到最高,其中25μg/L和250μg/L镉攻毒后在6 h时表达量达到最高(P<0.01),500μg/L镉攻毒后在9 h时表达量达到最高,48 h后各浓度组Tg-TIMP-3基因表达受到不同程度抑制。研究表明,Tg-TIMP-3对重金属Cd免疫防御或解毒中可能起重要作用,而泥蚶鳃组织中的Tg-TIMP-3 mRNA表达对不同浓度Cd的抗逆免疫反应的灵敏度和时序上存在一定差异。On the basis of cDNA library of Tegillarca granosa,Tg-TIMP-3 cDNA was cloned by SMART RACE（Rapid Amplification of cDNA Ends）techniques and then the bioinformatics,expression and immune defense analysis of Tg-TIMP-3 were carried out. The full length cDNA of Tg-TIMP-3 gene was 804 bp, containing a complete ORF（ Open Reading Frame）of 570 bp encoding 189 amino acids. The deduced amino acid sequence of Tg-TIMP-3 did not share high similarity with others, only shared 35.8% similarity with TIMP-3 from Crassostrea gigas, but the two functional domains of TIMPs were relatively conservative. The results of tissue-specific expression by real time PCR showed that the mRNA transcript level of Tg-TIMP-3 in gill was higher than those in other tissues including haemocytes, adductor muscle, foot, hepatopancreas and mantle. After being challenged by different concentration of Cd, the level of relative expression in gill of Tg- TIMP-3 mRNA was first slightly reduced and then significantly increased the highest. Those challenged by the concentrations of 25 p.g/L and 250 I^g/L of Cd reached the highest at 6 h（P 〈 0.01 ） ,500 p.g/L reached the highest at 9 h ,but finally inhibited to different degrees after 48 h. The results indicated that Tg-TIMP-3 played a vital role in the immune defense responses to Cd. However,the temporal sequence and sensitivity of mRNA expression were different in different challenge groups.

关 键 词：泥蚶 基质金属蛋白酶组织抑制因子3 镉 MRNA表达 免疫反应 

分 类 号：Q785[生物学—分子生物学] S968.3[农业科学—水产养殖]