三角帆蚌 KSPI cDNA 的分子特征及表达分析

摘要

Kazal-type serines proteinase inhibitor ( KSPI) is very important in the immune reaction of biological body , but the study of KSPI in Hyriopsis cumingii has been rarely reported .In this study , the full length of cDNA sequence of Kazal-type serines proteinase inhibitor(KSPI) gene (accession number:KT901291) of H.cumingii was cloned.It was 1029 bp in length, containing 5′and 3′-UTRs parts of 61 bp and 206 bp, and an ORF of 762 bp encoding 253 amino acids, and the molecular weight was 27397.5 u.The amino acid sequence contains five Kazal domain structures , and the result of homology analysis showed that it has a few of similarity with other species, suggesting that this gene belongs to the typical KSPI gene.Real-time quantitative PCR showed that KSPI gene expressed in a wide range of tissues including the adductor , foot, liver, blood, mantle, gill and gonad, and the highest expression was in mantle , on the contrary, the lowest in liver.After being infected with Aeromonas hydrophila, the expression of KSPI gene had significant increas-ing in 7 tissues compared with the control group .The results suggested that KSPI gene is very important in the immune reaction of Hyri-opsis cumingii.%Kazal型丝氨酸蛋白酶抑制因子（ KSPI）在生物体的免疫应答过程中发挥着重要作用，但在三角帆蚌中尚未进行其相关研究与报道，为研究KSPI对三角帆蚌免疫过程的影响，采用RACE法克隆了三角帆蚌KSPI cDNA全序列（登录号：KT901291），获得了1029 bp的全长，其中包含5′、3′端非翻译区分别为61 bp、206 bp，开放阅读框762 bp，共编码氨基酸253个，分子质量为27397．5u，氨基酸序列包含5个Kazal结构域，与一些已知物种的KSPI编码的氨基酸序列进行同源性分析后发现，与各种物种间具有相似的结构域，属于典型的KSPI。实时荧光定量PCR分析结果表明KSPI在外套膜、血液、肝、腮、闭壳肌、性腺、斧足7个组织中均存在一定的表达量，表达量最高的是外套膜，表达量最低的是肝脏。利用嗜水气单胞菌诱导后检测发现，各个组织呈现出先升高再降低的趋势，在12或24 h达到最大值，之后开始逐渐下降并趋于稳定，推测出KSPI对三角帆蚌的免疫反应发挥重要作用。