卵形鲳鲹组织蛋白酶L基因的克隆及其表达分析

目的]明确卵形鲳鲹组织蛋白酶L基因(TroCatL)的遗传进化规律及其组织表达水平,为研究CatL生物学功能及其对病原的抗病机理提供理论依据.方法]应用RT-PCR和RACE技术克隆TroCatL基因全长cDNA,采用生物信息学方法对其序列特征进行分析;并以实时荧光定量PCR(qPCR)检测TroCatL基因在健康组织中的表达情况及其表达与溶藻弧菌感染的关联性.结果]TroCatL基因全长cDNA为1492 bp(GenBank登录号MH036350),包括开放阅读框(ORF)1011 bp、5'端非翻译区(5'-UTR)120 bp和3'端非翻译区(3'-UTR)361 bp. TroCatL基因编码336个氨基酸残基,其理论等电点(pI)5.7,预测分子质量37.93 kD,且存在CatL特有的保守结构域(ERFNIN、GNFD和GCXGG基序)及由139Cys、279His和303Asn组成的半胱氨酸蛋白酶保守活性位点.TroCatL氨基酸序列与其他鱼类CatL氨基酸序列的同源性高达83.9%~95.2%,尤其与鲈形目鰤鱼的亲缘关系最近. TroCatL基因mRNA在健康卵形鲳鲹组织中均有表达,以在肝脏中的表达最高,在脑组织中的表达最低.经溶藻弧菌感染后,TroCatL基因mRNA在肝脏、脾脏和血液中的表达水平均上调,肝脏和脾脏中的TroCatL基因mRNA在攻毒后24 h达峰值,血液中的TroCatL基因mRNA在感染后12 h达最高值.结论]TroCatL蛋白结构域及其催化活性位点在遗传进化过程中较保守,通过参与机体的免疫应答反应,在卵形鲳鲹抵御细菌或病毒侵染的过程中扮演重要角色.

Cloning and expression analysis of cathepsin L gene in Trachinotus ovatus

Objective]The aim of the study was to definite genetic evolution rule and tissue expression level of ca-thepsin L gene in Trachinotus ovatus(TroCatL),provide theoretical basis for researching biological function and disease resistant mechanism of CatL.Method]The full length cDNA sequence of TroCatL were cloned by using RT-PCR and RACE technology,and the sequence features were analyzed by using bioinformatics methods. Real-time fluorescence quantitative PCR(qPCR)was used to detect the expression of TroCatL gene in healthy tissues and its correlation with Vibrio alginolyticus infection.Result]The full length of TroCatL cDNA was 1492 bp(GenBank accession number:MH036350),including a 1011 bp open reading frame(ORF),a 120 bp 5'untranslated region(5'-UTR)and a 361 bp 3' untranslated region(3'-UTR). The TroCatL gene encoding 336 amino acid residues with theoretical isoelectric point(pI) of 5.7 and predicted molecular weight of 37.93 kD. It contained conservative domain structure(ERFNIN,GNFD and GCXGG motifs),and a cysteine protease active site formed by 139Cys,279His and 303Asn. The amino acid sequences of Tro- CatL shared 83.9%-95.2% homology with CatL amino acids of other fish species,especially sharing the closest relative re-lationship with Seriola dumerili. TroCatL mRNA were expressed in all detected tissues from healthy T. ovatus while the highest level was in liver and the lowest was in brain. TroCatL mRNA were significantly up-regulated in liver,spleen and blood after being infected by V. alginolyticus . TroCatL mRNA in liver and spleen reached the peak value 24 h post infec-tion,while that in blood reached the peak value 12 h post infection.Conclusion]TroCatL protein domains and catalytic active sites are evolutionarily conserved in T. ovatus,and play an important role in against of bacteria or virus infection by participating in host immune response.