华贵栉孔扇贝Akirin 2基因的克隆与表达分析

Cloning and Expression Analysis of Akirin 2 in Scallop Chlamys nobilis

为了解Akirin 在华贵栉孔扇贝(Chlamys nobilis)中的作用,本研究分离了华贵栉孔扇贝Akirin 2 基因(命名为CnAkirin 2),并描述了CnAkirin 2 的特征。使用转录组分析和PCR 方法获得了CnAkirin 2 cDNA序列,通过序列比对分析了Akirin 氨基酸序列在不同物种中的保守性,使用MEGA 7.0 软件的邻接法构建了Akirin 2 的系统进化树,使用实时定量PCR 分析了Akirin 2 在华贵栉孔扇贝6 种不同组织中的表达。结果显示:该基因序列全长1 888 bp,开放阅读框长597 bp,编码氨基酸198 个;CnAkirin 2 的预测分子量为22.11 kD,理论等电点pI 为9.30,具有核定位信号PKRRRCM;不同物种Akirin 氨基酸多序列比对结果显示,推译的CnAkirin 2 氨基酸序列与其他物种Akirin 氨基酸序列在N 端和C 端具有高度相似性;使用邻接法构建的系统进化树显示CnAkirin 2 与牡蛎和其它扇贝等贝类Akirin 聚为一支;实时定量PCR 结果表明CnAkirin 2在不同组织中均有表达,其中在精巢表达量最高,其次为卵巢,在其它组织中的表达水平最低,推测其可能在性腺发育过程中发挥重要作用。本研究为探索CnAkirin 2 在华贵栉孔扇贝中的作用奠定了基础。

In order to investigate the role of Akirin in Chlamys nobilis, Akirin 2 from C. nobilis (named CnAkirin 2) were isolated and characterized in this study. CnAkirin 2 cDNA sequence was determined by transcriptome analyses and PCR method. The conservation of Akirin acid amino sequences from different species were aligned and analyzed. The phylogenetic tree was constructed with Neighbor-Joining method of MEGA 7.0. The expression of CnAkirin 2 in six tissues of C. nobilis were detected with Real time PCR. The results showed that the full-length CnAkirin 2 cDNA was 1 888 bp in length including an open reading frame of 597 bp, which encodes a putative protein of 198 residues with an estimated molecular mass of 22.11 kD, a theoretical isoelectric point of 9.30 and a predicted nuclear localization signal (PKRRRCM). Multiple alignment of the deduced amino acid sequence of CnAkirin 2 with those of other known Akirin protein from various species indicated that CnAkirin 2 shared high similarity with Akirin 2 of other species at N- and C-termini. Phylogenetic tree constructed by the neighbourjoining (NJ) method showed that CnAkirin 2 was clustered with oyster and other scallop Akirin. The result of Realtime quantitative PCR (qRT-PCR) showed that CnAkirin 2 was ubiquitously expressed in all examined tissues of C. nobilis, and that expression level was highest in testis, followed by the ovary, and lowest in other tissues tested,which was suggested that CnAkirin 2 may play an important role during process of gonad development. The research may lay the foundation of elucidating the role of Akirin 2 in C. nobilis. The research may lay the foundation for elucidating the role of CnAkirin 2 in C. nobilis.