Molecular cloning and RNA interference analysis of β-N-acetylglucosaminidase in Mamestra brassicae L

• Published in: Journal of Asia-Pacific entomology Vol. 19; no. 3; pp. 721 - 728
• Main Authors: Zhang, Huan, Zhao, Kuijun, Fan, Dong
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 01.09.2016; 한국응용곤충학회
• Subjects: Cloning; Expression; Mamestra brassicae L; RNA interference; β-N-acetylglucosaminidase; 농수해양학; Mamestra brassicae L; RNA interference; Expression; β-N-acetylglucosaminidase; Cloning
• ISSN: 1226-8615; 1876-7990
• DOI: 10.1016/j.aspen.2016.06.012

β-N-acetylglucosaminidase plays an important role in insect chitin degradation. In this study, a novel cDNA sequence encoding β-N-acetylglucosaminidase (MbNAG) was cloned from cabbage moth, Mamestra brassicae L. The cDNA sequence, which was 2445bp in length, contained an open reading frame of 1782bp coding for a polypeptide of 594 amino acid residues. The MbNAG open reading frame was cloned into the pET-21b prokaryotic expression vector, and then over-expressed in Escherichia coli BL21 (DE3) cells. After purification and renaturation, the β-N-acetylglucosaminidase enzymatic activity was detected. Transcript analyses on MbNAG sequence during different developmental stages and in specific tissues were carried out by RT-qPCR. The MbNAG mRNA level was significantly higher of the second day in the prepupal stage than at any other time and occurred mainly in the midguts and salivary glands compared with in other tissues, including fat bodies, integuments, malpighian tubules, foreguts and hindguts during the feeding stage of the 5th instar larvae. The highest expression level of MbNAG mRNA occurred 48h after an in vivo injection of 20-hydroxyecdysone. RNA interference reduced the MbNAG mRNA expression level 60% at 48h and 67% at 72h, respectively, and induced both abnormal molting phenotypes and a high mortality rate in M. brassicae larvae. These results suggest that MbNAG plays an essential role in the molting process of M. brassicae. [Display omitted] •One β-N-acetylglucosaminidase cDNA sequence was cloned from Mamestra brassicae.•The enzymatic activity was detected after expression in E. coli.•Higher mRNA level was in the prepupal stage, the midguts and salivary glands.•The highest mRNA level occurred 48h after injection of 20-hydroxyecdysone.•RNAi reduced mRNA level and induced both abnormalities and a high mortality rate.