Sun Yuying, Zhang Jiquan, Xiang Jianhai
College of Life Sciences, Hebei University, Baoding, Hebei 071002, China; College of Marine Life and Fisheries, Huaihai Institute of Technology, 59 Cangwu Road, Lianyungang 222005, China.
College of Life Sciences, Hebei University, Baoding, Hebei 071002, China; Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Gene. 2018 Mar 30;648:12-20. doi: 10.1016/j.gene.2018.01.046. Epub 2018 Jan 12.
Chitin degradation is catalyzed by a two-component chitinolytic enzyme system, chitinase and β-N-acetylglucosaminidase (NAGase). In this paper, the full-length cDNA sequence encoding NAGase (EcNAG) was obtained from Exopalaemon carinicauda. The deduced amino acid sequence of EcNAG open reading frame (ORF) contained one Glycohydro_20b2 domain and one Glyco_hydro_20 domain. Based on the cDNA sequence, the genomic structure of EcNAG was characterized and it was composed of six exons and five introns. EcNAG mRNA majorly expressed in the hepatopancreas and epidermis. During the molting stages, EcNAG mRNA expression was well-regulated and its expression reached the highest level at the molting stage E. In addition, EcNAG was recombinant expressed in Pichia pastoris and the partial enzymatic characterization of recombinant EcNAG was confirmed. After being challenged with Vibrio parahaemolyticus and Aeromonas hydrophila, the expression of EcNAG was up-regulated significantly at 6 h and reached the peak at 12 h. And then, the expression began to down-regulated and came to the normal level at 72 h. It is helpful to research the relationship between the molt-related hormones and chitinlytic enzymes.
几丁质降解由双组分几丁质分解酶系统催化，即几丁质酶和β-N-乙酰氨基葡萄糖苷酶（NAGase）。本文从脊尾白虾中获得了编码NAGase（EcNAG）的全长cDNA序列。EcNAG开放阅读框（ORF）推导的氨基酸序列包含一个糖基水解酶_20b2结构域和一个糖基水解酶_20结构域。基于该cDNA序列，对EcNAG的基因组结构进行了表征，其由6个外显子和5个内含子组成。EcNAG mRNA主要在肝胰腺和表皮中表达。在蜕皮阶段，EcNAG mRNA的表达受到良好调控，在蜕皮阶段E表达达到最高水平。此外，EcNAG在毕赤酵母中进行了重组表达，并对重组EcNAG的部分酶学特性进行了确证。在用副溶血性弧菌和嗜水气单胞菌攻击后，EcNAG的表达在6小时时显著上调，并在12小时时达到峰值。然后，表达开始下调，并在72小时时恢复到正常水平。这有助于研究与蜕皮相关的激素和几丁质分解酶之间的关系。
