大黄鱼Rac2基因的克隆及其抗病免疫作用分析

Cloning and Analysis of Rac2 Gene in Disease-Resistant in the Large Yellow Croaker( Larimichthys crocea)

克隆了大黄鱼ras-related C3 botulinum toxin substrate 2(Rac2)基因(命名为lycRac2),并从本实验室大黄鱼基因组数据库中获得了lycRac2基因的全长序列。lycRac2基因全长1670.427 kb,包含7个外显子、6个内含子,其c DNA序列全长1221 bp,ORF为579 bp,编码192个氨基酸。同源性比较结果显示,从鱼类到哺乳动物,RAC2蛋白的氨基酸序列相当保守,其基因均具有7个外显子和6个内含子,但不同物种之间7个外显子和6个内含子的长度都不一致。实时定量PCR检测结果显示,lycRac2基因在肝脏、脾脏、肠、胃、肾、头肾、肌肉、皮肤、脑、鳃、心脏、血液中均有不同程度表达,其中在头肾中表达量最高,肌肉中表达量最低。大黄鱼受到LPS(脂多糖)、poly I:C、副溶血弧菌刺激后,肝脏、脾脏、头肾中lycRac2基因表达量明显上调,显示lycRac2基因参与了大黄鱼的免疫反应,其可能具有对细菌和病毒的抗病免疫作用。通过原核表达还获得lycRAC2蛋白,为进一步研究其性质和功能奠定了基础。

The ras-related C3 botulinum toxin substrate 2 （Rac2） gene, was cloned from the large yellow croaker （Larimichthys crocea） （named as lycRac2） , and its full length was obtained by comparing with the genome database of L. crocea in our laboratory. The full length of lycRac2 gene was 1670. 427 kb, including 7 exons and 6 introns. And the cDNA of lycRac2 was 1221 bp, containing a 579 bp open reading frame （ORF） which encoded a deduced protein of 192 amino acids. Homology comparison showed that RAC2 is a very conserved protein in both fish and mammal and all the genes have 7 exons and 6 introns with different lengths. Quantitative real time PCR revealed that lycRac2 gene expressed in liver, spleen, intes- tines, stomach, kidney, head-kidney, muscle, skin, brain, gill, heart and blood, and the highest ex- pression was in head-kidney while the lowest expression was in muscle. After stimulated by LPS, polyI：C and Vibro parahaemolyticus , the expression of lycRac2 gene in liver, spleen, head-kidney was up regulated obviously, this indicated that lycRac2 would participate in the immune activity and play a crucia role in de- fence against bacteria and virus. In this study, lycRac2 gene was expressed successfully in Escherichia coli, which would lay the foundation for further study of character and function of the protein.