新型皱纹盘鲍防御素hd-def的cDNA序列分析、基因组克隆及表达研究  

cDNA analysis,genome cloning and gene expression of a novel defensin hd-def from abalone(Haliotis discus hannai)

作　　者：洪旭光[1,2] 孙修勤[2] 郑明刚[2] 昝金东[2] 曲凌云[2] 张进兴[2] 

机构地区：[1]中国海洋大学生命科学与技术学院,青岛266003 [2]国家海洋局第一海洋研究所,青岛266061

出　　处：《高技术通讯》2008年第3期299-305,共7页Chinese High Technology Letters

基　　金：973计划(G1999012005)资助项目

摘　　要：从构建的皱纹盘鲍肝肾 cDNA 文库中筛选到了鲍防御素基因 EST。通过序列分析发现该基因的全长 cDNA 序列编码66个氨基酸残基,其前体由信号肽、前导肽和成熟肽组成。该前体的成熟肽含42个氨基酸(6个 Cys),推测分子量为4323Da,等电点为8.02。氨基酸序列同源性分析表明,该多肽与昆虫防御素的相似性较高,最高可达70%。因成熟肽二级结构具有典型的昆虫防御素结构特征,该多肽应属于抗菌肽中的昆虫防御素亚家族,是一种新型抗菌肽,将其命名为鲍防御素 hd-def。采用基因组步移法获得了全长4032bp 的基因组序列。分析表明,该基因由3个内含子和4个外显子编码组成;3个内含子大小分别为497bp、2357bp 和528bp,其中两个内含子存在于编码信号肽的序列中。用鳗弧菌和金黄色葡萄球菌刺激皱纹盘鲍,能诱导 hd-def 的表达。实验检测了5种组织,发现 hd-def 基因仅在肝胰腺中表达,具有明显的组织表达特异性;其表达属于诱导型表达,提示该基因可能参与皱纹盘鲍的抗细菌感染。A new abalone defensin gene EST was screened from an abalone （Haliotis discus hannai ） liver cDNA library. The sequence analysis reveals that the full length gene encodes 66 amino acid residues and is composed of signal peptide, prepeptide and mature peptide. The mature form has 42 amino acids（6 Cys）, the predictive molecular weight is 4323Da, and the PI is 8.02. The homology analysis for amino acid sequence showed that the similarity between the peptide and inset defensins is up to 70%. The second structure of the mature abalone defensin has a typical structure of insect defensins, therefore, this defensin belongs to the insect defensin family and is called abalone defensin hd-def. From chromosome walking, a 4032 bp gene was cloned. The gene contains 4 exons and 3 introns. The length of the 3 introns are 497, 2357 and 528bp respectively and two of them exist in the signal peptide coding area. 5 tissues were detected with RT-PCR and the results reveled that the gene was only expressed in liver. Stimulated with Vibrio anguillarum and Staphylococcus aureus, abalone hd-def was detected . Therefore, the expression of hd-def is inducible and is connected with bacteria infection.

关 键 词：皱纹盘鲍 鲍防御素 CDNA 基因组 基因表达 抗菌肽 免疫 

分 类 号：S917.4[农业科学—水产科学] Q78[生物学—分子生物学]