牙鲆PIK3R3-like基因的表达分析及重组表达

Identification,Expression Analysis and Inducible Expression of PIK3R3-like in Japanese Flounder(Paralichthys olivaceus)

本文从牙鲆(Paralichthys olivaceus)中鉴定得到了PI3K家族的成员之一——PIK3R3-like,并分析其可能为PIK3R3的亚型。由于其部分外显子在转录或修饰时被删除,导致该基因会产生一种较短的mRNA(PoPIK3R3-like-Ⅱ),与完整的转录组相比缺失21 nt。对PoPIK3R3-like的可变剪接长链(PoPIK3R3-like-Ⅰ)进行序列分析发现该基因包含一个1425 bp的开放性阅读框,共编码474个氨基酸,编码的蛋白共包含三个结构域,分别是一个nSH2结构域、一个iSH2结构域和一个cSH2结构域。同源性分析结果显示PoPIK3R3-like-Ⅰ、PoPIK3R3-like-Ⅱ均与其它硬骨鱼的PIK3R3-like相似度较高,系统进化分析显示PoPIK3R3-like与硬骨鱼聚为一支,与两栖类、鸟类和哺乳类相距较远。作者通过qRT-PCR检测了PoPIK3R3-like在各组织中的表达分布,发现其在牙鲆的各组织中均有表达,但在鳃和脑中表达量较高,使用迟缓爱德华氏菌侵染牙鲆鳃细胞及单核-巨噬细胞后,PoPIK3R3-like表达量分别呈现先上升再下降和显著上升的趋势,提示该基因在牙鲆抵抗迟缓爱德华氏菌感染相关的免疫应答中可能发挥作用。利用原核表达的方法在大肠杆菌中异源表达了PoPIK3R3-like-Ⅰ的重组蛋白,SDS-PAGE结果显示得到的重组蛋白与预测大小一致,蛋白纯化结果显示得到的蛋白纯度较高,可用于下一步PIK3R3-like基因功能的相关研究。

As an intracellular phosphatidylinositol kinase,phosphatidylinositol 3-kinase(PI3Ks)can participate in and coordinate cell behavior under a variety of conditions.One of the members of the PI3Ks from the Japanese flounder,termed as PoPIK3R3-like,was identified and characterized in this study.We found that Japanese flounder PIK3R3-like gene can be transcripted into two different mRNAs,namely PoPIK3R3-like-Ⅰand PoPIK3R3-like-Ⅱ.PoPIK3R3-like-Ⅱhas a 21 nt-deletion compared to that of PoPIK3R3-like-Ⅰ.The sequence analysis showed that PoPIK3R3-like-Ⅰcontained an open reading frame(ORF)of 1425 bp and encodes a total of 474 amino acids.Homology analysis demonstrated that PoPIK3R3-like-Ⅰand PoPIK3R3-like-Ⅱshowed a high degree of identity with PIK3R3-like homologues of other teleost.Phylogenetic analysis showed that PoPIK3R3-like-Ⅰclustered with teleost and far away from amphibians,birds and mammals.We detected the expression patterns of PoPIK3R3-like by qRT-PCR,which showed that PoPIK3R3-like was detected in all examined tissues,and the expression level of PoPIK3R3-like in gill cells and monocytes/macrophages were significantly increased after infected by Edwardsiella tarda,suggesting that PoPIK3R3-like may play a role in the immune response of Japanese flounder to resist E.tarda invasion.The recombinant PoPIK3R3-like-Ⅰwas obtained in Escherichia coli by prokaryotic expression.SDS-PAGE showed that recombinant protein was consistent with the predicted size and had a good purification effect,which could be used for the next related research on the function of PIK3R3-like.