草鱼LXRα基因的克隆及表达研究

Cloning and expression of LXRαgene in grass carp(Ctenopharyngodon idellas)

【目的】克隆草鱼肝X受体α亚型（Liver X Receptor α,LXRα）cDNA序列，分析其在草鱼不同组织中的表达情况及n-3 HUFA对草鱼肝胰脏LXRα基因表达的影响。【方法】以草鱼肝胰脏组织为材料，采用RT-PCR技术对其LXRα基因cDNA进行克隆分析，运用生物信息学的方法分析该基因序列同源性。检测LXRα基因在草鱼10种组织中的表达情况。以含0.52% n-3 HUFA的饲料饲喂草鱼95 d后，用实时定量PCR法检测n-3 HUFA对肝胰脏LXRα表达的影响。【结果】克隆得到了草鱼LXRα cDNA序列（GenBank注册号为：FJ965309），其ORF序列长度为1 230 bp，编码409个氨基酸，预测该蛋白分子式为C2083H3343N581O611S30，分子质量为47 263.7 u，等电点pI为7.91，半衰期为30 h。该蛋白质具有哺乳动物的LXRS特征：包括DNA结合位点（DBD）、p-box，配体结合域 （LBD）、激活功能-2（AF-2）区域、D-box、D区域（D region） 和属于2个锌指结构的8个半胱氨酸；与其他物种LXRα的同源性为70.7%～100%，其中与鲤鱼和斑马鱼的同源性分别达100%和94.6%。LXRα基因在草鱼肝胰脏、肌肉、心脏、鳃、精巢、肾脏、脑、脾脏、肠、腹腔脂肪等10种组织中均有表达，其中在精巢中表达水平最高（P＜0.05），在肌肉中表达水平最低 （P＜0.05）；饲喂n-3 HUFA可显著抑制草鱼肝胰脏LXRα基因的表达水平（P＜0.01）。【结论】克隆获得了草鱼LXRα基因cDNA序列，该基因在多种组织中均可表达，其编码的氨基酸序列与其他物种相似性较高，在动物进化中比较保守；n-3 HUFA可通过影响草鱼肝胰脏LXRα基因的表达，调控草鱼的脂质代谢。

【Objective】This study obtained Liver X Receptor α （LXRα） gene cDNA sequence of grass carp （Ctenopharyngodon idellas）,evaluated its expression in different tissues of LXRα,and analyzed the effect of n-3 HUFA on mRNA expression of LXRα.【Method】The cDNA sequence of LXRα was cloned from hepatopancreas of grass carp.The sequence homology of cloned gene was analyzed by bioinformatic methods.And the mRNA expression levels of LXRα in 10 different tissues were analyzed by semi-quantitative PCR.The effect of n-3 HUFA on mRNA expression of LXRα was evaluated after a 95-day feeding trail of diet with 0.52% n-3 HUFA by qRT-PCR.【Result】The cDNA sequence of LXRα was obtained （GenBank accession number FJ965309） and its ORF sequence was 1 230 bp in length encoding 409 amino acids peptides.The predicted formula was C2083H3343N581O611S30 with molecular weight of 47 263.7 u,theoretical pI of 7.91,and estimated half-life of 30 hours.Grass carp LXRα sequence contained characteristic features of mammalian LXRs,including DNA binding （DBD）,P-box,ligand binding （LBD）,activation function-2 （AF-2） domains,D-box,D （hinge） region and eight cysteines belonging to two zinc fingers.The homology with other species was 70.7%-100%,especially it was 100% with Cyprinus carpio and 94.6% with Danio rerio.LXRα widely expressed in hepatopancreas,muscle,heart,gill,spleen,intestine,brain,kidney,spermary,and intraperitoneal fat （IPF） of grass carp,with the highest expression level in spermary,and the lowest in muscle （P＜0.05）.The mRNA relative expression of LXRα in hepatopancreas was very significantly suppressed by n-3 HUFA（P＜0.01）.【Conclusion】The LXRα gene wildly expressed in various tissues of grass carp and it had high similarity to other species,indicating that LXRα was conservative in the course of animal evolution.n-3 HUFA could adjust the lipid metabolism in grass carp by affecting the expression of LXRα gene.