›› 2017, Vol. 60 ›› Issue (3): 274-285.doi: 10.16380/j.kcxb.2017.03.005

• RESEARCH PAPERS • Previous Articles     Next Articles

Gene cloning, polyclonal antibody preparation and expression localization of the endophilins from Nilaparvata lugens (Hemiptera: Delphacidae)

YU Ye-Wei#, XU Yi-Peng#, ZHAO Chen-Xing, HAN Shan-Jie, AN Peng, YU Xiao-Ping*   

  1.  (Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China)
  • Online:2017-03-20 Published:2017-03-20

Abstract: 【Aim】 To explore the function of endophilin in the growth and reproduction of the brown planthopper, Nilaparvata lugens. 【Methods】 The endophilin genes of N. lugens were cloned and analyzed by bioinformatics. Then, recombinant prokaryotic expression vectors were constructed and transferred into Escherichia coli Rosetta to induce the expression of fusion proteins. After purification by Ni affinity chromatography, the fusion proteins were used to immunize the New Zealand rabbits to obtain polyclonal antibodies against endophilins. The obtained antibodies were used to detect the expression and localization of endophilins in N. lugens ovary. 【Results】 Two endophilin genes, endophilin A (Endo A) and endophilin B (Endo B), were cloned from N. lugens, with the GenBank accession numbers of KY126096 and KY126095, respectively. Endo A and Endo B encode 387 and 352 amino acids, respectively. Both encoded proteins contain the typical BAR domain and SH3 domain, but show different structure. The titer of the obtained antibodies was estimated as high as 1∶1 000 000 dilution ratio through ELISA, and the antibodies had good specificity as shown by Western blot. Using immunofluorescence, the antibodies were used to detect the expression of Endo A and Endo B in N. lugens ovary. The results indicated that Endo A and Endo B were universally expressed in N. lugens ovary. In ovary, Endo A and Endo B were widely distributed in the extracellular space, cytomembrane and cytoplasm of follicle cells of N. lugens ovary, and this distribution pattern was similar to that of lipids. Meanwhile, Endo A and Endo B were colocalized with yeast-like symbionts invading into N. lugens ovary. 【Conclusion】 The nucleotide sequences and the biological characteristics of Endo A and Endo B were clarified. With the obtained polyclonal antibodies of Endo A and Endo B, the expression of Endo A and Endo B in N. lugens ovary was profiled, and the results suggest that Endo A and Endo B might be associated with the development and maturity of N. lugens ovary as well as the invasion of the yeast-like endosymbionts to N. lugens ovary. These results lay the foundation for further studies on the biological function of Endo A and Endo B in N. lugens.

Key words: Nilaparvata lugens, endophilin, gene cloning, polyclonal antibody, protein localization, ovary