Cloning and characterization of the nuclear receptor gene <em>AlE75D</em> in<em> Apolygus lucorum</em> (Hemiptera: Miridae)  
昆虫学报
       Home | About Journal | Editorial Board | Subscription | Instruction | Advertisement | Contact Us | Chinese
ACTA ENTOMOLOGICA SINICA  2017, Vol. 60 Issue (9): 984-993    DOI: 10.16380/j.kcxb.2017.09.002
RESEARCH PAPERS Current Issue | Next Issue | Archive | Adv Search Previous Articles  |  Next Articles  
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)
 Download: PDF (2997 KB)   HTML (1 KB)   Export: BibTeX | EndNote (RIS)      Supporting Info
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.  
Service
E-mail this article
Add to my bookshelf
Add to citation manager
E-mail Alert
RSS
Articles by authors
TAN Yong-An
ZHAO Xu-Dong
XIAO Liu-Bin
SUN Yang
ZHAO Jing
BAI Li-Xin
HAO De-Jun
Key wordsApolygus lucorum; AlE75D   gene cloning   expression profile   prokaryotic expression   monoclonal antibody     
Cite this article:   
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.
URL:  
http://www.insect.org.cn/EN/10.16380/j.kcxb.2017.09.002      or     http://www.insect.org.cn/EN/Y2017/V60/I9/984
 
No references of article
[1] YANG Jing, GAO Yue, LIU Zhong-Fang, ZHANG Peng-Jiu, FAN Jian-Bin, NIU Guo-Fei, HAN Zhao-Jun, FAN Ren-Jun. Cloning and expression profiling of chitin synthase 1 gene in the oriental fruit moth, Grapholitha molesta (Lepidoptera: Tortricidae)  [J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(9): 994-1005.
[2] YU Jing, XU Xiao-Xia, GAO Yan-Fu, JIN Feng-Liang. Molecular cloning of the C-type lectin gene PxCTL5 and its mRNA level changes under bacterial stimulation in Plutella xylostella (Lepidoptera: Plutellidae)[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(8): 876-890.
[3] TAN Yao, ZHANG Yu, HUO Zhi-Jia, ZHOU Xiao-Rong, DAN Yan-Min, PANG Bao-Ping. Molecular cloning and expression profiling of the heat shock protein gene GdHsp70 in Galeruca daurica (Coleoptera: Chrysomelidae)[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(8): 865-875.
[4] LI Zhao-Qun, ZHANG Shuai, ZHOU Shu-Fen, LUO Jun-Yu, CUI Jin-Jie. Tissue expression profiling and ligandbinding properties of HarmOBP16 of the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(8): 891-899.
[5] ZHAO Yu, LIU Zhong-Yuan. Cloning, expression and functional verification of the serine protease inhibitor gene ApSerpin-FA72 from Anatolica polita borealis (Coleoptera: Tenebrionidae)[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(8): 857-864.
[6] LI Guo-Ping, FENG Hong-Qiang, HUANG Bo, JIN Yin-Li, TIAN Cai-Hong, HUANG Jian-Rong, QIU Feng. Assessment of toxicities of sixteen insecticides to the plant bug, Apolygus lucorum (Hemiptera: Miridae) by glass-vial and artificial diet bioassays (In English)[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(6): 650-658.
[7] YANG Ji-Gui, PENG Li-Na, LIU Tai-Xing, WU Yun-Fei, DU Guo-Yu, ZHANG Qian, WANG Wei, PAN Min-Hui. Cloning and expression analysis of the Sox family gene BmDichaete in the silkworm, Bombyx mori[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(5): 513-522.
[8] WANG Li-Li, LUAN Bing-Hui, LIU Xue-Qing, WANG Pei-Song, WANG Ying-Zi. Relationship between the contents of nutrients and chlorophylls in leaves of different grape varieties and their resistance to Apolygus lucorum (Hemiptera: Miridae)[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(5): 570-575.
[9] WEI Chang-Ping, JIANG Jian, LI Xiang-Rui, ZHANG Yun-Hui, ZHANG Fang-Mei, CHENG Deng-Fa, GUO Wen-Chao, LIU Huai. Cloning and sequence analysis of heat shock protein gene Ld-HSP60 in Leptinotarsa decemlineata (Coleoptera: Chrysomelidae) and its expression under temperature stress[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(5): 523-532.
[10] WANG Chao-Qun, Zhao-Ying, Cao Ya-Zhong, Wei Hong-Shuang, Li Ke-Bin, Zhang-Shuai, Peng-Yu, Yin-Jiao. Expression and binding characterization of odorant binding protein 1 (AcorOBP1) in Anomala corpulenta (Coleoptera: Scarabaeoidea)[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(4): 363-371.
[11] XU Lu, Zhao Chun-Qing, Xu De-Jin, Xu Guang-Chun, Xu Xiao-Long, Zhang Ya-Nan, Han Zhao-Jun, Gu Zhong-Yan. Molecular cloning and sequence analysis of the estrogen-related receptor (ERR) gene in the small brown planthopper, Laodelphax striatellus (Delphacidae: Hemiptera) and its expression profiles under the stress of sulfoxaflor[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(3): 264-273.
[12] YU Ye-Wei, Xu Yi-Peng, Zhao Chen-Xing, Han Shan-Jie, An-Peng, Yu Xiao-Ping. Gene cloning, polyclonal antibody preparation and expression localization of the endophilins from Nilaparvata lugens (Hemiptera: Delphacidae)[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(3): 274-285.
[13] ZHU Zi-Jie, Wang Gui-Yao, Qiao-Fei, Bai Yue-Liang, CHENG Jia-An, Kong Luen HEONG, Zhu Zeng-Rong. Potential geographical distribution and habitat suitability analysis for two mirid predators Tytthus chinensis and Cyrtorhinus lividipennis (Hemiptera: Miridae) in rice fields based on MaxEnt model[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(3): 335-346.
[14] ZHANG Yuan-Chen, Liu Xiang-Dong. Cloning and expression analysis of lysozyme gene AgLYS in the cotton-melon aphid,Aphis gossypii (Hemiptera: Aphididae)[J]. ACTA ENTOMOLOGICA SINICA, 2017, 60(2): 127-135.
[15] CHEN Li-Peng, DU Yu-Ping, FANG Qi, YAO Hong-Wei, YE Gong-Yin. Molecular cloning, sequence analysis and expression profiling of vitellogenin receptor gene in the green rice leafhopper,  Nephotettix cincticeps  (Hemiptera: Cicadellidae)[J]. ACTA ENTOMOLOGICA SINICA, 2016, 59(9): 925-937.
Copyright © 2011 ACTA ENTOMOLOGICA SINICA
Supported by:Beijing Magtech