Please wait a minute...
Journal of Integrative Agriculture  2018, Vol. 17 Issue (10): 2310-2319    DOI: 10.1016/S2095-3119(17)61764-7
Special Issue: 昆虫和植物互作合辑Insect and Plant Interact
Plant Protection Advanced Online Publication | Current Issue | Archive | Adv Search |
Molecular identification and enzymatic properties of laccase2 from the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae)
LIU Zhen-gang, WANG Huan-huan, XUE Chao-bin
Key Laboratory of Pesticide Toxicology and Application Technique, College of Plant Protection, Shandong Agricultural University, Tai’an 271018, P.R.China
Download:  PDF in ScienceDirect  
Export:  BibTeX | EndNote (RIS)      
Abstract  Laccase (EC 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.
Keywords:  laccase        melanization        in vivo expression        enzymatic properties        Plutella xylostella  
Received: 07 June 2017   Accepted:
Fund: 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).
Corresponding Authors:  Correspondence XUE Chao-bin, Tel/Fax: +86-538-8242983, E-mail:   
About author:  LIU Zhen-gang, E-mail:;

Cite this article: 

LIU Zhen-gang, WANG Huan-huan, XUE Chao-bin. 2018. Molecular identification and enzymatic properties of laccase2 from the diamondback moth Plutella xylostella (Lepidoptera: Plutellidae). Journal of Integrative Agriculture, 17(10): 2310-2319.

Andersen S O. 1978. Characterization of a trypsin-solubilized phenoloxidase from locust cuticle. Insect Biochemistry, 8, 143–148.
Arakane Y, Muthukrishnan S, Beeman R W, Kanost M R, Kramer K J. 2005. Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning. Proceedings of the National Academy of Sciences of the United States of America, 102, 11337–11342.
Baldrian P. 2006. Fungal laccase - occurrence and properties. FEMS Microbiology Reviews, 30, 215–242.
Barrett F M, Andersen S O. 1981. Phenoloxidases in larval cuticle of the blowfly, Calliphora vicina. Insect Biochemistry, 11, 17–23.
Cao Z S, Liu X M, An S H, Jiang J W. 2014. Molecular identification of Laccase2 gene in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). Acta Entomologica Sinica, 57, 150–160. (in Chinese)
Cao Z Y, Zheng T, Xie B F, Lu C P. 2004. Advances of industrial application of laccase. Biotechnology Letters, 15, 414–416.
Chen P, Qu M, Yang J, Yang Q. 2014. Cloning and expression of two laccase genes Oflac1 and Oflac2 from the insect Ostrinia furnacalis. Scientia Agricultura Sinica, 47, 1341–1350. (in Chinese)
Claus H, Faber G, König H. 2002. Redox-mediated decolorization of synthetic dyes by fungal laccase. Applied Microbiology and Biotechnology, 59, 672–678.
Claus H, Filip Z. 1997. The evidence of a laccase-like enzyme activity in a Bacillus sphaericus strain. Microbiological Research, 152, 209–216.
Dittmer N T, Gorman M J, Kanost M R. 2009. Characterization of endogenous and recombinant forms of laccase-2, a multicopper oxidase from the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology, 39, 596–606.
Dittmer N T, Kanost M R. 2010. Insect multicopper oxidases: Diversity, properties, and physiological roles. Insect Biochemistry and Molecular Biology, 40, 179–188.
Dittmer N T, Suderman R J, Jiang H, Zhu Y C, Gorman M J, Kramer K J, Kanost M R. 2004. Characterization of cDNAs encoding putative laccase-like multicopper oxidases and developmental expression in the tobacco hornworm, Manduca sexta, and the malaria mosquito, Anopheles gambiae. Insect Biochemistry and Molecular Biology, 34, 29–41.
Elias-Neto M, Soares M P, Simões Z L, Hartfelder K, Bitondi M M. 2010. Developmental characterization, function and regulation of a Laccase2 encoding gene in the honey bee, Apis mellifera (Hymenoptera, Apinae). Insect Biochemistry and Molecular Biology, 40, 241–251.
Fang J L, Xia D R, Yang R X. 1988. Studies on the semi-synthetic diet and rearing technique of diamondback moth. Acta Phytophylacica Sinica, 15, 167–171. (in Chinese)
Furlong M J, Wright D J, Dosdall L M. 2013. Diamondback moth ecology and management: Problems, progress, and prospects. Annual Review of Entomology, 58, 517–541.
Futahashi R, Banno Y, Fujiwara H. 2010. Caterpillar color patterns are determined by a two-phase melanin gene prepatterning process: new evidence from tan and laccase2. Evolution & Development, 12, 157–167.
Futahashi R, Tanaka K, Matsuura Y, Tanahashi M, Kikuchi Y, Fukatsu T. 2011. Laccase2 is required for cuticular pigmentation in stinkbugs. Insect Biochemistry and Molecular Biology, 41, 191–196.
Garavaglia S, Cambria M T, Miglio M, Ragusa S, Iacobazzi V, Palmieri F, D’Ambrosio C, Scaloni A, Rizzi M. 2004. The structure of Rigidoporus lignosus laccase containing a full complement of copper ions, reveals an asymmetrical arrangement for the T3 copper pair. Journal of Molecular Biology, 342, 1519–1531.
Gorman M J, Dittmer N T, Marshall J L, Kanost M R. 2008. Characterization of the multicopper oxidase gene family in Anopheles gambiae. Insect Biochemistry and Molecular Biology, 38, 817–824.
Gorman M J, Sullivan L I, Nguyen T D, Dai H, Arakane Y, Dittmer N T, Syed L U, Li J, Hua D H, Kanost M R. 2012. Kinetic properties of alternatively spliced isoforms of laccase-2 from Tribolium castaneum and Anopheles gambiae. Insect Biochemistry and Molecular Biology, 42, 193–202.
Hattori M, Konishi H, Tamura Y, Konno K, Sogawa K. 2005. Laccase-type phenoloxidase in salivary glands and watery saliva of the green rice leafhopper, Nephotettix cincticeps. Journal of Insect Physiology, 51, 1359–1365.
He N, Botelho J M, McNall R J, Belozerov V, Dunn W A, Mize T, Orlando R, Willis J H. 2007. Proteomic analysis of cast cuticles from Anopheles gambiae by tandem mass spectrometry. Insect Biochemistry and Molecular Biology, 37, 135–146.
Hu P P, Fu S Y, Li G R. 2001. Methods of assaying laccase activity by Panus Conchatus. Guangzhou Chemistry, 26, 22–27. (in Chinese)
Hüttermann A, Mai C, Kharazipour A. 2001. Modification of lignin for the production of new compounded materials. Applied Microbiology and Biotechnology, 55, 387–394.
Kumar S V, Phale P S, Durani S, Wangikar P P. 2003. Combined sequence and structure analysis of the fungal laccase family. Biotechnology and Bioengineering, 83, 386–394.
Liu C X, Han Z J, Li F L. 1993. Study on the large scale propagation of diamondback moth by using the method of vermiculite and radish seedling. Entomological Knowledge, 6, 341–344. (in Chinese)
Niu B L, Shen W F, Liu Y, Weng H B, He L H, Mu J J, Wu Z L, Jiang P, Tao Y Z, Meng Z Q. 2008. Cloning and RNAi-mediated functional characterization of MaLac2 of the pine sawyer, Monochamus alternatus. Insect Molecular Biology, 17, 303–312.
Palmer A E, Lee S K, Solomon E I. 2001. Decay of the peroxide intermediate in laccase: Reductive cleavage of the O-O bond. Journal of the American Chemical Society, 123, 6591–6599.
Riedel F, Vorkel D, Eaton S. 2011. Megalin-dependent yellow endocytosis restricts melanization in the Drosophila cuticle. Development, 138, 149–158.
Strong P J, Claus H. 2011. Laccase: A review of its past and its future in bioremediation. Critical Reviews in Environmental Science and Technology, 41, 373–434.
Sugumaran M, Giglio L, Kundzicz H, Saul S, Semensi V. 1992. Studies on the enzymes involved in puparial cuticle sclerotization in Drosophila melanogaster. Archives of Insect Biochemistry and Physiology, 19, 271–283.
Thomas B R, Yonekura M, Morgan T D, Czapla T H, Hopkins T L, Kramer K J. 1989. A trypsin-solubilized laccase from pharate pupal integument of the tobacco hornworm, Manduca sexta. Insect Biochemistry, 19, 611–622.
Wu X, Zhan X, Gan M, Zhang D, Zhang M, Zheng X, Wu Y, Li Z Y, He A. 2013. Laccase2 is required for sclerotization and pigmentation of Aedes albopictus eggshell. Parasitology Research, 112, 1929–1934.
Xu J H, Huang Z P, Guan X, Yu Y P, Yang B M, Song Q. 1997. Artificial rearing and low temperature keeping of Plutella xylostella. Journal of Fujian Agricultural University, 26, 187–190. (in Chinese)
Yamazaki H I. 1972. Cuticular phenoloxidase from the silkworm, Bombyx mori: Properties, solubilization, and purification. Insect Biochemistry, 2, 431–444.
Yatsu J, Asano T. 2009. Cuticle laccase of the silkworm, Bombyx mori: Purification, gene identification and presence of its inactive precursor in the cuticle. Insect Biochemistry and Molecular Biology, 39, 254–262.
Ye F Y. 1999. The hypothesis and verification of signal peptide. Foreign Journal of Medical Molecular Biology, 21, 377.
Yoshida H. 1883. Chemistry of lacquer (Urushi). Part I. Journal of the Chemical Society, 43, 472–486.
Zhang P. 2007. Test method for the laccase activity with ABTS as the substrate. Textile Auxiliaries, 24, 43–45.
Zhukhlistova N E, Zhukova Y N, Lyashenko A V, Zaitsev V N, Mikhailov A M. 2008. Three-dimensional organization of three-domain copper oxidases: A review. Crystallography Reports, 53, 92–109.

[1] LIU Ning, ZHANG Qian-qian, JIA Hui, ZHAO Bin, ZHU Zi-ping , CAO Zhi-yan, DONG Jin-gao. Characterization of laccase gene StLAC6 involved in the pathogenicity and peroxisome function in Setosphaeria turcica[J]. >Journal of Integrative Agriculture, 2022, 21(7): 2019-2030.
No Suggested Reading articles found!