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Chinese Journal of Biological Control ›› 2022, Vol. 38 ›› Issue (5): 1202-1212.DOI: 10.16409/j.cnki.2095-039x.2021.11.008

• RESEARCH REPORTS • Previous Articles     Next Articles

Cloning and Function Analysis of a Clip-domain Serine Protease Gene NlSCP4 in the Brown Planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)

WU Wei, CHENG Yiqing, WANG Zhengliang, YU Xiaoping   

  1. Zhejiang Provincial Key Laboratory of Biometrology and Inspection and Quarantine/College of Life Sciences, China Jiliang University, Hangzhou 310018, China
  • Received:2021-09-04 Published:2022-10-20

Abstract: Clip-domain serine protease (CSP),a protein hydrolase with conserved structure and diverse functions,widely exists in different groups of insects and plays important physiological roles in insect growth,development and immunity.In this study,a CSP-encoding gene NlCSP4(GenBank accession No.OK018340) was successfully cloned and identified in the brown planthopper (BPH),Nilaparvata lugens.The full-length cDNA of NlCSP4 is 1911 bp,encoding 636 amino acids with the predicted molecular weight of 69.29 kD and isoelectric point of 9.02.NlCSP4 protein contains a predicted signal peptide consisting of 25 amino acid residues and a clip domain comprising 44 amino acid residues at the N-terminal region,and a typical Tryp_SPc domain at the C-terminus with three specific conserved catalytic sites,namely His,Asp and Ser.The phylogenetic analysis showed that NlCSP4 clustered together with other hemipteran CSPs but were quite distinct from those of lepidopterans in the neighbor-joining tree.The qRT-PCR results showed that the expression of NlCSP4 had obvious temporospatial characteristics.The transcript level of NlCSP4 in old nymphs (4th and 5thinstars) was significantly higher than those in the eggs and young nymphs (1st,2nd and 3rd instars),and the highest expression level was observed in the female adults.NlCSP4 was expressed in the fat body,gut and ovary,with the highest expression level in the fat body of the female adults.The expression of NlCSP4 in BPH was significantly up-regulated after infection with the Gram-positive bacteria Staphylococcus aureus and the entomopathogenic fungus Metarhizium anisopliae within 72 h post injection.However,no obvious induction of NlCSP4 expression was observed when the BPH was challenged by the Gram-negative bacteria Escherichia coli in a short time (within 36 h post injection).Microinjection of NlCSP4 dsRNA could significantly inhibit the expression level of NlCSP4 in the 5th-instar nymphs of BPH.The results of bioassay experiments showed that the survival rate and the ability to resist M.anisopliae infection in the 5th-instar nymphs of BPH were both significantly decreased by RNAi-mediated knockdown of NlCSP4.Our preliminary results confirm that NlCSP4 plays key roles in host growth,development and immune defense,and can be used as an important potential target for RNAi-mediated control of BPH.

Key words: Nilaparvata lugens, clip-domain serine proteinase, gene cloning, gene expression, RNA interference

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