【摘要】 为了解淡水贝类是否存在组织蛋白酶L的亚型及其亚型的免疫相关作用,本实验利用已构建的池蝶蚌血细胞全长c DNA文库,筛选获得与之同源的EST序列,结合RACE技术进一步克隆了池蝶蚌一个新的组织蛋白酶L基因的c DNA全长,命名为Hs Cts L1-like基因(Gen Bank登录号为KF015273)。该序列全长为1280 bp,5′-非翻译区(5′UTR)为31 bp,3′-非翻译区(3′UTR)为256 bp,开放阅读框区(ORF)为993 bp,编码330个氨基酸,预测蛋白相对分子量为36.86 ku,理论等电点为6.23。序列分析结果显示,Hs Cts L1-like与其他软体动物相对应序列具有共同结构特征,包含信号肽、前肽抑制域和成熟肽三部分,在其他物种中已鉴定的Cts L签名序列标签(ERF/WNIN、GNFD、GCXGG和QCHN等)在Hs Cts L1-like中均可找到。其氨基酸序列同缢蛏Cts L1(AGL33704.1)同源性最高,达67%;与报道的三角帆蚌Cts L(ADV03094)和池蝶蚌中另一个Cts L(AEX88474)仅均为52.91%;系统进化分析表明,Hs Cts L1-like与缢蛏、长牡蛎和合浦珠母贝的Cts L1聚为一分支,推测Hs Cts L1-like属于Cts L家族中的亚型1。实时荧光定量PCR(q RT-PCR)检测显示,Hs Cts L1-like m RNA在肝脏中表达量最高,其次是卵巢和精巢。注射鳗弧菌后,血细胞和肝脏Hs Cts L1-like m RNA转录水平显著升高,暗示其是一个免疫有关的基因,参与了池蝶蚌的先天免疫应答反应。更多还原

【Abstract】 In order to study the role of another Cathepsin L(Cts L) isoform from Hyriopsis schlegelii in immune responses, we isolated and characterized a novel Cts L gene from the freshwater pearl mussel Hyriopsis schlegelii using c DNA library screening and rapid amplification of c DNA ends(RACE), and it was named Hs Cts L1-like(Gen Bank accession No. KF015273). The Hs Cts L1-like c DNA was 1280 bp long, and consisted of a 5′-untranslated region(UTR) of 31 bp, a 3′-UTR of 256 bp with a polyadenylation signals(AATAAA) at 11 nucleotides upstream of the poly(A) tail, and an open reading frame(ORF) of 993 bp encoding a polypeptide of330 amino acids with 36.86 ku predicted molecular weight. The theoretical isoelectric point was 6.23. Sequence analysis showed that the putative Hs Cts L1-like protein sequence sharing similar structural features with other reported Cts Ls containing a typical signal peptide sequence with 16 amino acids(Met1~Ser16), a propeptide inhibitor domain(Try25~Phe84) and a mature domain(Leu114~Val330). And some signature sequence tags were identified for Cts L family proteins in other species including the oxyanion hole(Gln), the active triad formed by Cys, His and Asn, and the conserved ERF/WNIN, GNFD, GCXGG and OCHN motifs can all be found in the Hs Cts L1-like protein sequence. Moreover, homology analysis revealed that Hs Cts L1-like shared the highest identity(67%) with Cts L1(AGL33704.1) from the razor clam, Sinonovacula constricta, while the similarities of Hs Cts L1-like with the reported Hyriopsis cumingii(ADV03094) and H. schlegelii Cts L(AEX88474) were both52.91%. The phylogenetic tree revealed that the Hs Cts L1-like clustered with the invertebrate Cts L cysteine proteases and was also closely related to S. constricta, Crassostrea gigas and Pinctada fucata Cts L1. Real-time quantitative reverse transcription PCR(q RT-PCR) showed that Hs Cts L1-like is expressed in a wide range of tissues including the liver, gills, foot, heart, ovary, testis, kidney, blood, intestines and mantle, with the highest level of transcripts in liver, followed by ovary, testis, intestines and blood, while there is rare expression in other tissues. After Vibrio anguillarum stimulation, the expression level of Hs Cts L1-like m RNA was significantly upregulated at 2 h and 8 h in liver and blood. These results suggested that Hs Cts L1-like belongs to subtype 1 of the Cts L subfamily, and it might play an important role in the freshwater mussel, H. schlegelii immune response.更多还原