关键词: 褐飞虱, 过氧化物酶, NlPOD1, 表达模式, RNA干扰

Abstract:  【Aim】 To clarify the molecular characteristics, expression patterns and biological function of the peroxidase gene NlPOD1 from the brown planthopper, Nilaparvata lugens. 【Methods】 Based on the transcriptome data of N. lugens, the full-length cDNA sequence of NlPOD1 was cloned by PCR, and its nucleotide and protein sequences were subsequently characterized using bioinformatics tools. The expression patterns of NlPOD1 across different developmental stages (egg, 1 st-5th instar nymphs and newly emerged female and male adults), in different tissues (head, fat body, hemolymph and gut) of the 5th instar nymphs, and in the 5th instar nymphs at different time post injection of different microbes (Escherichia coli, Staphylococcus aureus and Metarhizium anisopliae) at the concentration of 1×10⁷/mL were determined by qRT-PCR. The target gene NlPOD1 in the 5th instar nymphs of N. lugens was further silenced by RNAi, and the survival rates and the median lethal time (LT₅₀) values of the nymphs after target gene silencing and infection with M. anisopliae (1×10⁸ conidia/mL) were determined by bioassay. 【Results】 The full-length cDNA sequence of NlPOD1 (GenBank accession no.: MZ682107) was successfully cloned from N. lugens. Its open reading frame (ORF) is 2 049 bp in length, encoding 682 amino acids with a typical animal heme peroxidase domain (An_ peroxidase domain) and a predicted signal peptide consisting of 29 amino acid residues at the N-terminus. The phylogenetic analysis showed that NlPOD1 is closely related to the PODs of other hemipteran insects, and has the highest homology with the POD of Halyomorpha halys. Developmental expression profiling showed that NlPOD1 was expressed in various developmental stages of N. lugens, with the lowest and highest expression levels in the eggs and the 5th instar nymphs, respectively. Tissue expression profiling revealed that NlPOD1 was expressed in different tissues of the 5th instar nymphs of N. lugens, with significantly higher expression levels in the gut and haemolymph than in the head and fat body. The expression patterns under microbial induction showed that the expression level of NlPOD1 in the 5th instar nymphs of N. lugens was significantly up-regulated within 48 h post injection with E. coli as compared to the control group injected with PBS. However, the expression level of NlPOD1 increased firstly and then stabilized when the 5th instar nymphs of N. lugens were challenged by S. aureus and M. anisopliae. The RNAi results showed that the expression levels of NlPOD1 could be significantly inhibited by microinjection of dsNlPOD1. Inhibition of NlPOD1 expression by RNAi caused no changes in the survival rate of the 5th instar nymphs of N. lugens.  However, the LT50 of the combined treatment with dsNlPOD1 injection and M. anisopliae infection to the 5th instar nymphs of N. lugens was 4.5 d, which was significantly shorter than that of the control group (5.4 d) of combined treatment with dsGFP injection and M. anisopliae infection, indicating that silencing of NlPOD1 could significantly decrease the resistance of N. lugens to the infection with M. anisopliae. 【Conclusion】 NlPOD1 plays important roles in the pathogen defense of N. lugens and can be used as a potential target in the development of N. lugens biocontrol technology mediated by RNAi and entomopathogenic fungi.

Key words: Nilaparvata lugens; peroxidase; NlPOD1, expression pattern, RNA interference