›› 2017, Vol. 60 ›› Issue (11): 1247-1254.doi: 10.16380/j.kcxb.2017.11.002

• RESEARCH PAPERS • Previous Articles     Next Articles

Expression, purification and enzymatic characteristics of phosphodiesterase C (AlPLC) of Apolygus lucorum (Hemiptera: Miridae)

TAN Yong-An1, ZHAO Xu-Dong2,3, HAO De-Jun2,3, XIAO Liu-Bin1,*, BAI Li-Xin1, ZHAO Jing1, SUN Yang1, JIANG Yi-Ping1   

  1.  (1. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2. Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; 3. College of Forestry, Nanjing Forestry University, Nanjing 210037, China)
  • Online:2017-11-20 Published:2017-11-20

Abstract: 【Aim】 Based on our previous work of cloning the phospholipase C gene AlPLC from Apolygus lucorum, this study aims to determine the expression profiles of AlPLC gene in different day-old nymphs of A. lucorum, to obtain the recombinant protein which has the phospholipase enzyme activity and to clarify its enzymatic characteristics. 【Methods】 Using the qRT-PCR technique, we determined the expression pattern of AlPLC in one day-old to 13 day-old nymphs of A. lucorum. The recombinant plasmid containing target gene was specifically expressed after induction by IPTG. The recombinant protein was purified by GST agarose affinity chromatography and molecular sieve chromatography. Then the enzymatic characteristics of this recombinant protein under different temperatures and pH were measured by p-nitrophenylphosphorylcholine (p-NPPC). Finally, the optimum temperature and pH for the enzyme activity of PLC were analyzed. 【Results】 AlPLC was found to be continuously expressed throughout the surveyed nymphal stage of A. lucorum, and highly expressed in 4, 8, 12 and 13 day-old nymphs. The recombinant plasmid pCzn1-AlPLC expressed the target recombinant protein of 79 kD after IPTG induction in Escherichia coli. The 12% SDS-PAGE showed that the recombinant protein was mainly present as inclusion bodies. The purified protein of AlPLC with the phosphodiesterase activity was obtained after denaturation and renaturation. This recombinant protein had higher phospholipase activity by using p-NPPC as substrates, and under the protein concentration of 0.25 mg/mL, the most suitable temperature for its reaction was 57℃ (179.54±3.96 nmol/μg·min) and the optimal pH was 8.5 (374.99±2.84 nmol/μg·min). 【Conclusion】 The expression of AlPLC shows the developmental stage-specificity. Higher enzyme activity of AlPLC can be achieved under higher temperature and alkaline environment. The results provide the basis for analyzing the AlPLC function at the protein level.

Key words: Apolygus lucorum, phospholipase C, prokaryotic expression, protein purification, enzymatic characteristics, enzyme activity