Scientia Agricultura Sinica ›› 2016, Vol. 49 ›› Issue (21): 4130-4139.doi: 10.3864/j.issn.0578-1752.2016.21.007

• PLANT PROTECTION • Previous Articles     Next Articles

Analysis and Expression of Laccase Gene Stlac2 in Setosphaeria turcica

MA Shuang-xin, LIU Ning, JIA Hui, DAI Dong-qing, XU Miao-miao, CAO Zhi-yan, DONG Jin-gao   

  1. The Key Laboratory of Hebei Province for Molecular Plant-Microbe Interaction, Agricultural University of Hebei, Baoding 071001, Hebei
  • Received:2016-07-11 Online:2016-11-01 Published:2016-11-01

Abstract: 【Objective】The objective of this study is to analyze the bioinformatics and infer the functions of Stlac2 in Setosphaeria turcica, research on the effects of substrates generated in the process of lignin degradation on the relative expression of Stlac2. For further study on the function of Stlac2 in the growth, development, and pathogenicity, the Stlac2 was successfully expressed by prokaryotic expression system. 【Method】The protein sequences of Stlac2 were obtained through NCBI and aligned with the known laccases such as Penicillium marneffei PbrB (PMAA_082060), Aspergillus fumigatus Abr2 (AFUA_2G17530), and Aspergillus nidulans YA (AN6635) by ClustalX for the Cu ion binding sites. The secondary structure and biochemical properties were predicted online by online softwares SOPMA and ProtParam, and the three-dimensional structure was modelled by SWISS-MODEL, and analyzed by SAVES. The effects of lignin degradation substrates on the expression of extracellular laccase were measured by oxidazing ABTS and the effect on the relative expression of specific gene Stlac2 was analyzed by using RT-qPCR. Stlac2 was fused into the pET-30a plasmid and expressed by prokaryotic expression system. The protein Stlac2 expressed by prokaryotic cell was extracted and the laccase activity was detected using ABTS as substrate at 420 nm.【Result】 Stlac2 had typical sites bonding with Cu ion. The proportion of α-helix, extended strand, β-turn and random coil were 18.59%, 25.63%, 11.91% and 43.86%, respectively. The protein molecular weight is 61.64 kD, pI is 5.00, grand average of hydropathicity is -0.37, indicating Stlac2 is a hydrophilic protein. The effect of lignin degradation substrates on the production of extracellular laccase was vanillic acid>vanillin>4-hydroxybenzaidehyde>4-hydroxybenzoic acid>syringaldehyde>CK. The relative expression of Stlac2 was increased about 5-8 folds compared with CK under the substrates 4-hydroxybenzoic acid and 4-hydroxybenzaidehyde conditions. Stlac2 protein was successfully expressed by prokaryotic expression system and a specific protein band of 67 kD was induced. The laccase activity of this specific protein is (40.7±0.3) U·L-1.【Conclusion】The biochemical properties of Stlac2 were analyzed systematically and predicted that Stlac2 is a laccase gene. 4-Hydroxybenzoic acid and 4-hydroxybenzaidehyde could increase the relative expression of Stlac2 significantly, indicating it plays a role in the process of lignin degradation. Stlac2 protein can be expressed by the prokaryotic expression systems of pET-30a and the laccase activity is (40.7±0.3) U·L-1, thus laid a foundation for further study.

Key words:  Setosphaeria turcica, Stlac2, bioformatic analysis, RT-qPCR, prokaryotic expression

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