背角无齿蚌抗菌肽theromacin基因cDNA全序列克隆及表达分析  

Full-length cDNA cloning and expression analysis of theromacin gene in Anodonta woodiana

作　　者：张果苹 汪桂玲[1] 郭诗照[1] 白志毅[1] 李家乐[1,2] 

机构地区：[1]上海海洋大学农业部淡水水产种质资源重点实验室,上海201306 [2]上海海洋大学上海高校水产养殖学E研究院,上海201306

出　　处：《水产学报》2014年第5期662-670,共9页Journal of Fisheries of China

基　　金：国家自然科学基金(31101939);上海市教委科研创新重点项目(13ZZ128);上海高校知识服务平台项目(ZF1206)

摘　　要：采用RACE方法,克隆了背角无齿蚌抗菌肽theromacin基因的cDNA全序列。结果显示,该cDNA序列全长为870 bp,5'端非翻译区104 bp,3'端非翻译区460 bp,开放阅读框长为306 bp,共编码101个氨基酸,相对分子量为11 166.9 u。同源性分析显示,该基因编码的蛋白与三角帆蚌theromacin基因氨基酸序列的相似度最高,为73%;与贻贝中发现的抗菌肽defensins、mytilins、myticins和mytimycins等4种类型相似度较低,属于Macin家族(登录号:KJ598604)。实时荧光定量PCR分析结果显示,该基因在血液、肝脏、外套膜、鳃、斧足和肠等组织中均有表达,在外套膜中的表达量最高,在其他组织中的表达量较低;经嗜水气单胞菌诱导后,各个组织的表达量出现明显上调的趋势且与对照组显著差异,推测theromacin基因可能在背角无齿蚌的免疫反应中起重要作用。Anodonta woodiana belongs to the Unionidae that consists of freshwater bivalve mollusks and it is the main freshwater mussel in China. Theromaicn is an antimicrobial peptide that is an important component of the natural defenses of freshwater mussel. In this study,a theromacin gene from A. woodiana was identified and characterized. The full-length cDNA sequence of theromacin gene of A. woodiana was cloned by rapid amplification of cDNA ends（ RACE）. A 870 bp cDNA sequence contained a 306 bp open reading frame（ ORF） encoding 101 amino acids,a 104 bp 5＇ untranslated region（ 5＇ UTR）,and a 460 bp 3＇ untranslated region（ 3＇ UTR）. The molecular weight of this mature peptide was estimated to be 11 1 66. 9 u. Homology analysis indicated that amino acid sequence showed the highest similarity with theromacin in Hyriopsis cumingii（ 73%）,and lower similarity with defensins,mytilins,myticins and mytimycins in mussels. It belongs to the macin family. Real-time quantitative PCR showed that theromacin gene expressed in a wide range of tissues including the mantle,blood,gill,foot,liver and intestine,with the highest level of transcripts in mantle,followed by foot,while in other tissues it was lowly expressed. After being challenged with Aeromonas hydrophila,the expression of theromacin gene was increased significantly in 6 tissues compared with the control mussel. The results suggested that theromacin gene might play an important role in the immune reaction of A. woodiana.

关 键 词：背角无齿蚌 theromacin基因 分子特征 cDNA末端快速扩增(RACE) 实时荧光定量PCR 

分 类 号：Q785[生物学—分子生物学] S917.4[农业科学—水产科学]