池蝶蚌组织蛋白酶L1-like基因的克隆及表达特征分析

发布时间：2019-05-24 07:43

【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。
[Abstract]:In order to investigate the immune correlation of cathepsin L subtypes and their subtypes in freshwater shellfish, the constructed full-length c DNA library of blood cells from Hyriopsis cumingii was used to screen the homologous EST sequences. The full length of c DNA of a new cathepsin L gene, named Hs Cts L1-like gene (Gen Bank accession number KF015273, was further cloned by RACE. The full length of the sequence is 1280 bp,5'- untranslated region (5'UTR) 31 bp,3'- untranslated region (3'UTR) 256 bp, open reading frame region (ORF) is 993 bp, encoding 330 amino acids, and the predicted relative molecular weight of the protein is 36.86 ku,. The theoretical isoelectric point is 6.23. The results of sequence analysis showed that, Hs Cts L1-like had common structural characteristics with other molluscs, including signal peptide, propeptide inhibitory domain and mature peptide, and identified Cts L signature sequence tags (ERF/WNIN,) in other species. Both GNFD,GCXGG and QCHN can be found in Hs Cts L1-like. The amino acid sequence of Sinonovacula constricta L 1 (AGL33704.1) had the highest homology with Cts L 1 (AGL33704.1), and only 52.91% with the reported Cts L (ADV03094 and another Cts L (AEX88474). Phylogenetic analysis showed that, Hs Cts L1-like and Cts L _ 1 of Sinonovacula constricta, Oyster longus and Pinctada repellent were one branch, suggesting that Hs Cts L1-like belonged to the subgroup of Cts L family. Real-time fluorescence quantitative PCR (q RT-PCR (real-time fluorescence quantitative RNA) showed that the expression of, Hs Cts L1-like m RNA was the highest in the liver, followed by ovaries and spermatozoa. After injection of Vibrio eel, the transcription level of Hs Cts L1-like m RNA in blood cells and liver increased significantly, suggesting that it is an immune related gene, which is involved in the innate immune response of Hyriopsis cumingii.
【作者单位】：南昌大学生命科学学院;
【基金】：江西省科技支撑项目(20121BBF60036) 江西省自然科学基金(20151BAB204035) 江西省教育厅自然科学青年基金项目(GJJ14219) 江西省科技落地项目(KJLD12001) 国家自然科学基金(31260282;31260642;31160534)~~
【分类号】：Q78;S917.4

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