根据陆地蟹RXR基因保守区序列设计上下游引物,采用逆转录聚合酶链反应(RT-PCR)以及cDNA末端快速扩增(RACE)技术克隆中华绒螯蟹RXR基因并进行各组织间的表达分析。序列分析表明,中华绒螯蟹RXR cDNA全长1 517 bp,编码433个氨基酸。经BLASTn和BLASTx分析表明,中华绒螯蟹RXR基因的氨基酸序列与陆地蟹RXR基因的氨基酸序列相似性最高,为96%。系统进化树分析显示,中华绒螯蟹RXR的氨基酸序列与陆地蟹RXR聚为一支。荧光定量PCR结果显示,RXR在所有检测的组织中均有表达,且在中华绒螯蟹Y器官中表达量最高,肝胰腺、鳃和肌肉中少量表达,心脏、胃、肠、胸神经节和脑神经节中微量表达。在中华绒螯蟹不同的蜕皮时期,Y器官中RXR表达量在D期最高,与AB期、C期呈显著性差异(P＜0.05);肌肉中RXR在AB期表达量最高,与其他蜕皮时期呈显著性差异(P＜0.05);肝胰腺和鳃中RXR在AB期表达量最高,E期表达量最低,但各蜕皮时期表达量之间差异不显著。

Retinoid X receptor(RXR)is a member of the second subfamily of nuclear receptor superfamily.In the insect and the crustacean,RXR is an important signaling protein with varied roles in regulating aspects of reproductive maturation,molting and embryo development.In this study,we cloned RXR gene from Eriocheir sinensis using reverse transcriptase polymerase chain reaction(RT-PCR)and rapid-amplification of cDNA ends(RACE),and primers were designed according to the conserved sequence of RXR from Gecarcinus lateralis.The full-length cDNA sequence of RXR is 1 517 bp and codes a protein of about 433 amino acids.The amino acid sequence comparison results showed that the RXR gene of Eriocheir sinensis shared 96% identity with Gecarcinus lateralis,using BLASTn and BLASTx software.Phylogenetic tree of RXR gene generated by Neighbor Joining method suggested that RXR is clustered closely with that of Gecarcinus lateralis.The expression of the gene in different tissues and molting stages of E.sinensis was analyzed by real-time fluorescent quantitative PCR.The result showed the RXR mRNA was expressed in all tissues examined and highly in Y-organ,with small amount in hepatopancreas,gill and muscle,trace in heart,stomach,intestine,thoracic ganglion and brain ganglion.RXR mRNA was detected with high volume in Y-organ compared to hepatopancreas,muscles and gill at the same molting stages of the crab.RXR mRNA of Y-organ in stage D was significantly(P<0.05)higher than that of stage AB and stage C.It also had significant difference(P<0.05)between muscles at stage AB and stage E,and there was no significant difference expression in hepatopancreas and gill tissues in different molting stages.

国家“八六三”高技术研究发展计划(2012AA10A409 5);国家农业科技成果转化项目(2012GB2C000147);国家自然科学基金项目(31272677);上海教委知识服务平台项目(ZF1206);上海市科委优秀学术带头人项目(12XD1402700)