Cloning and characterization of the nuclear receptor gene AlE75D in Apolygus lucorum (Hemiptera: Miridae)
TAN Yong-An1,2, ZHAO Xu-Dong1,2, XIAO Liu-Bin3, SUN Yang3, ZHAO Jing3, BAI Li-Xin3, HAO De-Jun1,2,*
(1. Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; 2. College of Forestry, Nanjing Forestry University, Nanjing 210037, China; 3. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
Abstract 【Aim】 To clone the nuclear receptor gene from the cotton mirid bug, Apolygus lucorum, analyze its expression profiles, obtain the recombinant protein and prepare the highly specific monoclonal antibody against E75D protein. 【Methods】 The full-length cDNA of the nuclear receptor gene was cloned using RT-PCR and RACE techniques. The relative expression levels of this gene in female adults of different ages (1-16 day-old) and six tissues (fat body, flight muscles, ovary, Malpighian tubules, midgut and cuticle) in 7 day-old female adults of A. lucorum were determined using qRT-PCR method. The T vector containing the target gene was digested with the restriction enzyme NdeI/XbaI and subcloned, and the recombinant plasmid was specifically expressed after induction by IPTG. The recombinant protein was purified by GST agarose affinity chromatography and molecular sieve chromatography. The monoclonal antibody was obtained by mice immunity, cell fusion and ascites preparation, and its specificity was determined by Western blotting. 【Results】 A nuclear receptor gene was cloned from A. lucorum and named AlE75D (GenBank accession no.: KX912700). Its ORF is 1 911 bp in length, encoding a polypeptide with a predicted molecular mass of 75.68 kD. Amino acid sequence comparison indicated that AlE75D has the classic characteristics of the nuclear hormone receptor, including a highly conserved ligand-independent A/B activation domain (23 a.a.), a hinge region (D domain, 85 a.a.), a ligand-binding domain (LBD) (E domain, 190 a.a.) and F domain (338 a.a.), but has no C domain. AlE75D showed the highest similarity to E75 from the hemipteran insect Lygus hesperus (98% amino acid sequence identity). AlE75D was found to be continuously expressed throughout the whole adult stage of A. lucorum, and its expression level reached the peak in the 7 day-old female adult. The transcription levels of AlE75D were higher in the fat body and ovary; however, only small amounts of mRNAs were detected in flight muscles and Malpighian tubules. The vector containing AlE75D gene was digested and linked to the vector pCzn1. The recombinant plasmid pCzn1-AlE75D expressed the target recombinant protein of 75 kD after induction by IPTG. The recombinant protein was purified by using Ni-NTA agarose and used as the antigen, and one cell line which could stably passage and secrete monoclonal antibody against AlE75D protein was obtained. The monoclonal antibody could specifically bind to the total protein of A. lucorum as well as the recombinant AlE75D. 【Conclusion】 The expression of the nuclear receptor gene AlE75D in A. lucorum showed developmental stage- and tissue-specificity. The obtained monoclonal antibody against AlE75D recombinant protein is highly specific. The results could provide the basis for analyzing the gene’s function at the protein level.
TAN Yong-An,ZHAO Xu-Dong,XIAO Liu-Bin et al. Cloning and characterization of the nuclear receptor gene AlE75D in Apolygus lucorum (Hemiptera: Miridae)
[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(9): 984-993.