Acta Entomologica Sinica ›› 2020, Vol. 63 ›› Issue (7): 779-787.doi: 10.16380/j.kcxb.2020.07.001

• RESEARCH PAPERS •     Next Articles

Cloning and expression profiling of the serine protease inhibitor gene Nlserpin4 in the brown planthopper, Nilaparvata lugens (Hemiptera: Delphacidae)

WANG Zheng-Liang#, ZHU Hang-Feng#, PAN Hai-Bo, YU Xiao-Ping*   

  1.  (Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine, College of Life Sciences, China Jiliang University, Hangzhou 310018, China)
  • Online:2020-07-20 Published:2020-07-29

Abstract: 【Aim】 To clone and characterize the serine protease inhibitor gene Nlserpin4 from the brown planthopper (BPH), Nilaparvata lugens, and to determine its spatio-temporal expression profiles and its expression pattern induced by entomopathogenic fungi. 【Methods】 Based on the transcriptome and whole genome data of BPH, the full-length cDNA of Nlserpin4 from BPH was cloned by PCR, and its nucleotide and protein sequences were subsequently characterized using bioinformatics tools. The expression patterns of Nlserpin4 across different developmental stages (egg, 1st-5th instar nymphs and newly emerged female and male adults), in different tissues (fat body, gut, hemolymph and carcass) of the 5th instar nymphs, and in the 5th instar nymphs at different time post injection of the entomopathogenic fungus Metarhizium anisopliae were determined by qRT-PCR. 【Results】 The full-length cDNA of Nlserpin4 (GenBank accession no.: MN822802) was successfully cloned from BPH. The open reading frame (ORF) of Nlserpin4 is 1 227 bp in length, encoding 408 amino acids with the predicted molecular weight of 45.91 kD and isoelectric point (pI) of 6.23. The amino acid sequence analysis revealed that Nlserpin4 protein has no putative N-glycosylation site, but contains a predicted signal peptide consisting of 23 amino acid residues at the N-terminal region and a typical RCL region of the serpin family at the C-terminal region with an active cleavage site which can be recognized by target protease. The phylogenetic analysis showed that Nlserpin4 is closely related to the serpins of other hemipteran insects, with the highest homology with Sipha flava serpin4. The qRT-PCR results showed that the expression of Nlsperpin4 had obvious temporospatial characteristics. The expression level of Nlsperpin4 in adults was significantly higher than those in other developmental stages, and the highest expression level was observed in male adult. Nlsperpin4 was expressed in the fat body, gut, haemolymph and carcass, with the highest expression level in the carcass of the 5th instar nymphs. The expression of Nlserpin4 in BPH was significantly down-regulated after infection with M. anisopliae within 48 h post injection, but the expression level of Nlserpin4 was gradually increased with the increase of infection time. 【Conclusion】 The Nlserpin4 of BPH is differentially expressed in different developmental stages, different tissues and different time after infection with the entomopathogenic fungus M. anisopliae. The results of this study provide a theoretical basis for further studying the functions of Nlserpin4 in the growth, development and immune regulation of BPH.

Key words: Nilaparvata lugensMetarhizium anisopliae, serine protease inhibitor, gene cloning, spatio-temporal expression, inducible expression