花鲈OPN5基因克隆及其在光周期处理下的表达特征

Cloning and expression analysis of OPN5 gene in Lateolabrax maculatus under different photoperiodic treatments

【目的】通过克隆花鲈视蛋白5(OPN5)基因(LmOPN5)序列,检测视网膜、血管囊、下丘脑和垂体中LmOPN5基因在不同光周期下的表达水平,探究光周期对LmOPN5基因表达的影响,为深入研究花鲈感知光周期信号提供参考依据。【方法】通过PCR和RACE克隆获得LmOPN5基因全长序列,并进行生物信息学分析;应用实时荧光定量PCR检测LmOPN5基因在各组织中的相对表达量;再比较不同光周期(常光周期组12 L∶12 D,短光周期组8 L∶16D,长光周期组16L∶8D;L:光照时间,D:黑暗时间)条件下,视网膜、血管囊、下丘脑和垂体中LmOPN5基因表达的变化。【结果】LmOPN5基因cDNA全长1732 bp,其中开放阅读框(ORF)长1056 bp,共编码351个氨基酸残基。Lm OPN5蛋白分子量为39.26 kD,理论等电点(pI)为9.17。LmOPN5蛋白二级结构中α-螺旋占40.17%、无规则卷曲占34.76%、延伸链占22.51%、β-转角占2.56%,以α-螺旋和无规则卷曲为主。氨基酸序列同源比对分析结果显示,LmOPN5氨基酸序列与其他硬骨鱼类的OPN5氨基酸序列相似性为82.7%~92.0%,与哺乳动物OPN5氨基酸序列相似性较低。LmOPN5基因在花鲈脑组织分布广泛,尤其在下丘脑、视叶、垂体、端脑和血管囊中的相对表达量较高;同时在视网膜的相对表达量也较高。在视网膜中,LmOPN5基因相对表达量在不同光周期处理下发生显著变化(P<0.05,下同),其中长光照周期促进LmOPN5基因表达,短光照周期抑制LmOPN5基因表达;在血管囊中,长光周期条件下LmOPN5基因相对表达量显著高于常光周期;在下丘脑中,LmOPN5基因相对表达量在3种光周期处理下无显著变化(P>0.05);在垂体中,短光周期条件下LmOPN5基因相对表达量显著高于长光周期和常光周期。【结论】LmOPN5基因序列在硬骨鱼中较保守;不同光周期处理影响视网膜、血管囊和垂体中LmOPN5基因表达,表明花鲈不同组织对光周期的反应不一致,花鲈对光周期的感应具有组织特异性。

【Objective】By cloning the L.maculatus opsin(OPN5)gene(LmOPN5)sequence to investigate the ex-pression level of LmOPN5 gene in retina,saccus vasculosus,hypothalamus,and pituitary of L.maculatus under different photoperiods,the purpose of this study was to explore the influence of photoperiod on the expression level and expression pattern of LmOPN5 gene,which provided reference for further study of L.maculatus sensing photoperiod signal.【Method】Firstly,the full-length sequence of LmOPN5 gene was obtained by PCR and RACE,and bioinformatics analysis was performed.Then,real-time fluorescence quantitative PCR was used to compare the relative expression of LmOPN5 gene in different tissues.Finally,different photoperiods(normal treatment group 12 L∶12 D;short photoperiod 8 L∶16 D;long photoperiod 16 L∶8 D.L∶light time,D:dark time)on LmOPN5 gene expression in retina,saccus vasculosus,hy-pothalamus and pituitary.【Result】The length of LmOPN5 gene cDNA was 1732 bp,and the full length open reading frame(ORF)was 1056 bp,encoding 351 amino acids residues.The molecular weight was 39.26 kD,and the theoretical isoelec-tric point(pI)was 9.17.The results of protein secondary structure analysis showed that LmOPN5 proteins mainly con-tained four stuctures:α-helix(40.17%),irregular coil(34.76%),extended chain(22.51%),andβ-turn(2.56%).There-fore,the proteins were mainly composed of irregular coil andα-helix.Homology comparison analysis of amino acid se-quences showed that the similarity between LmOPN5 amino acid sequence and OPN5 amino acid sequence of other bony fishes ranged from 82.7%to 92.0%,and the similarity with OPN5 of mammals was low.LmOPN5 gene was more widely distributed in brain tissues,with higher relative expression levels in hypothalamus,optic lobe,pituitary,telencephalon and saccus vasculosus.At the same time,it was also highly expressed in the retina of the eye.In the retina,the relative expres-sion of LmOPN5 gene changed significantly under different photoperiod treatments(P<0.05,the same below).Long pho-toperiod promoted the expression of LmOPN5 gene,while short photoperiod inhibited the expression of LmOPN5 gene.In the saccus vasculosus,the expression of LmOPN5 gene in long photoperiod treatment was significantly higher than that in normal photoperiod treatment.In hypothalamus,the expression of LmOPN5 gene did not change significantly under the three photoperiod treatments(P>0.05).In pituitary,the expression of LmOPN5 gene in short photoperiod treatment was significantly higher than that in long photoperiod treatment and normal treatment.【Conclusion】LmOPN5 gene sequence is conserved in bony fish species.Different photoperiod treatments affect the expression of LmOPN5 gene in retina,saccus vasculosus and pituitary.The results indicate that the photoperiod response of different tissues of L.maculatus is not con-sistent,and the sensitivity of L.maculatus to photoperiod is tissue-specific.