基于lECT2的EST序列设计特异引物,采用rACE技术,成功克隆获得赤点石斑鱼lECT2 CdnA全序列,全长601 bP,包括了79 bP的3′uTr区、468 bP的开放阅读框(Orf)以及54 bP的5′uTr区,共编码155个氨基酸.通过与21个物种的氨基酸序列进行同源比对发现,所推导氨基酸序列与大黄鱼的相似性最高,为78%,与其他物种的相似性在40%--73%之间,表明lECT2氨基酸序列具有较高的保守性.基于lECT2基因的氨基酸序列构建的脊椎动物系统进化树与传统物种树基本吻合.同时,通过rT-PCr方法得出,lECT2在健康赤点石斑鱼多种组织中均有表达,肝组织表达量最高,感染哈维氏弧菌(VIbrIO bArVEyI)后lECT2在脾、头肾、鳃等免疫器官中有较高表达量,而在肝脏中表达量上调最为显著,其结果证实了lECT2与赤点石斑鱼免疫应答相关,肝脏可能是赤点石斑鱼lCET2蛋白最主要的表达场所.Based on the sequences from EST library,the full length cDNA of LECT2 was successfully obtained from Epinephelus akaara by RACE technique.The full-length of LECT2 is 601 bp,including 54 bp of 5′UTR,79 bp of 3′UTR,and a 468 bp open reading frame encoding a protein of 155 amino acids.The comparison of LECT2 amino acid sequence in E.akaara with its homologous sequences in other 21 species showed that it shared the highest identity(78%) with LECT2 amino acid sequence in Pseudosciaena crocea,and with the other 20 species,the identity percentages ranged from 40% to 73%.This result indicated that LECT2 is conserved among species.In this study,it was also found that the phylogenetic tree based on LECT2 amino acid sequences is congruent with their evolutionary relationships.Semi-quantitative RT-PCR analyses showed that LECT2 could be detected in various tissues,and the highest expression was found in the liver of E.akaara.Expression of LECT2 in the liver of V.harveyi-challenged fish dramatically increased comparing to that of the healthy fish.Besides the liver,expression in kidney,spleen and gill was slightly higher than those of other examed issues in the V.harveyi-challenged fish.These results revealed that LECT2 might be involved in the process of immunity reactions,and the liver might be the most significant location of LECT2 gene expression.福建省高等学校新世纪优秀人才支持计
