Journal of Integrative Agriculture ›› 2018, Vol. 17 ›› Issue (10): 2310-2319.DOI: 10.1016/S2095-3119(17)61764-7

所属专题: 昆虫和植物互作合辑Insect and Plant Interact

• 论文 • 上一篇    下一篇

  

  • 收稿日期:2017-06-07 出版日期:2018-10-01 发布日期:2018-09-29

Molecular identification and enzymatic properties of laccase2 from the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae)

LIU Zhen-gang, WANG Huan-huan, XUE Chao-bin   

  1. Key Laboratory of Pesticide Toxicology and Application Technique, College of Plant Protection, Shandong Agricultural University, Tai’an 271018, P.R.China
  • Received:2017-06-07 Online:2018-10-01 Published:2018-09-29
  • Contact: Correspondence XUE Chao-bin, Tel/Fax: +86-538-8242983, E-mail: cbxue@sdau.edu.cn
  • About author:LIU Zhen-gang, E-mail: zhengang2782@163.com;
  • Supported by:
    This work was supported financially by the National Natural Science Foundation of China (31672046), the National Key Research and Development Program of China (2016YFD0200500), and the Funds of Shandong “Double Tops” Program, China (SYL2017YSTD06).

Abstract: Laccase (EC 1.10.3.2) is known to oxidize various aromatic and nonaromatic compounds via a radical-catalyzed reaction, which generally includes two types of laccase, Lac1 and Lac2. Lac1 oxidizes toxic compounds in the diet, and Lac2 is known to play an important role in melanizing the insect exoskeleton. In this study, we cloned and sequenced the cDNA of the diamondback moth, Plutella xylostella Lac2 (PxLac2), from the third instar larvae using polymerase chain reaction (PCR) and rapid amplification of cDNA ends techniques. The results showed that the full-length PxLac2 cDNA was 1 944 bp long and had an open reading frame of 1 794 bp. PxLac2 encoded a protein with 597 amino acids and had a molecular weight of 66.09 kDa. Moreover, we determined the expression levels of PxLac2 in different stages by quantitative PCR (qPCR). The results indicated that PxLac2 was expressed differently in different stages. We observed the highest expression level in pupae and the lowest expression level in fourth instar larvae. We also investigated the enzymatic properties of laccase, which had optimal activity at pH 3.0 and at 35°C. Under these optimal conditions, laccase had a Michaelis constant (Km) of 0.97 mmol L−1, maximal reaction speed (Vm) of 56.82 U mL−1, and activation energy (Ea) of 17.36 kJ mol−1 to oxidize 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid ammonium salt). Type II copper enhanced laccase activity below 0.8 mmol L−1 and reduced enzyme activity above 0.8 mmol L−1 with an IC50 concentration of 1.26 mmol L−1. This study provides insights into the biological function of laccase.

Key words: laccase ,  melanization ,  in vivo expression ,  enzymatic properties ,  Plutella xylostella