罗氏沼虾组织蛋白酶D基因的克隆及其组织表达分析

Molecular Cloning and Tissue Expression Analysis of Cathepsin D Gene from Macrobrachium rosenbergii

采用cDNA末端快速扩增技术(RACE)从罗氏沼虾肝胰腺中克隆了组织蛋白酶D基因(CATD)全长cDNA,采用实时荧光定量PCR技术对罗氏沼虾CATD在肝胰腺、眼柄、鳃、心脏、肌肉、胃等组织中的表达进行了研究.结果显示,罗氏沼虾CATDcDNA全长1 361bp,开放阅读框为1 158bp,编码385个氨基酸,CATD蛋白预测分子质量为42.1kDa,理论等电点为6.602;基于CATD氨基酸同源序列比对和系统发育分析显示,罗氏沼虾与脊尾白虾、日本囊对虾、斑节对虾和美洲螯龙虾紧密聚为一枝,相似度分别是94%,88%,87%和82%;罗氏沼虾所有组织中均检测到CATD的表达,其在眼柄中的相对表达量最高,其次是心脏.

The full length cDNA of cathepsin D gene（CATD）was cloned from hepatopancreas of Macrobrachium rosenbergii by rapid amplification of cDNA ends（RACE）method.The expression of CATDin hepatopancreas,eyestalk,gill,heart,muscle,stomach of M.rosenbergii was analyzed by real-time fluorescence quantitative PCR method.The results are as follows：the full length cDNA sequence of CATDis 1 361 bp,which contains a 1 158 bp open reading frame（ORF）that encodes 385 amino acid,which has a calculated molecular mass of 42.1kDa and the isoelectric point of 6.602.Phylogenetic analysis based on CATD amino acid sequence showed that M.rosenbergii CATD clustered with Palaemon carinicauda,Marsupenaeus japonicus,Penaeus monodon,and Homarus americanus cathepsin D2,and the similarities were 94%,88%,87%and 82%respectively.Expression of CATD was detected in all tested tissues of M.rosenbergii,with the highest expression level in eyestalk and the second in heart.