关键词: 褐飞虱, 解毒酶, 细胞色素P450单加氧酶, 基因克隆, 表达谱, 水稻品种, 抗虫性

Abstract: To elucidate the function of P450 genes in Nilaparvata lugens, a novel P450 gene CYP4CE1 was cloned from the 4th instar nymphs of brown planthopper N. lugens by reverse transcription-polymerase chain reaction (RT-PCR), rapid amplification of cDNA ends (RACE) and long-distance polymerase chain reaction (LD-PCR). The full-length cDNA (2 160 bp) of CYP4CE1 has an open reading frame (ORF) of 1 626 nucleotides encoding a protein of 541 amino acids. BLAST similarity searches in GenBank databases with command blastx revealed the deduced protein is most similar to CYP4C39 (GenBank accession no.: JC8026) of the common shore crab (Carcinus maenas) with amino acid identity of 43%, with the second highest-level identity (42%) to CYP4C1 (AAA27819) of the tropical cockroach （Blaberus discoidalis） and CYP4C3 （NP_524598） of Drosophila melanogaster, respectively. Multiple alignment of amino acid sequences revealed that CYP4CE1 is a typical microsomal P450 sharing conserved structural and functional domains with other insect CYP4 members, such as helix K (E--R--P), helix I (AG--T), heme-binding domain (PF--G---C-G--F) and CYP4 specific region (EVDTFMFEGHDTT). Temporal expression analysis indicated CYP4CE1 was induced up to 2.1-fold and kept at a constant level in nymphs exposed to moderately resistant rice Minghui 63 (MH63) seedlings during the time course from 12, 24, 48 to 72 h, compared to the nymphs fed on the susceptible rice Taichung Native 1 (TN1) seedlings. Further spatial gene expression analysis demonstrated that CYP4CE1 was expressed at a high level in fat body and at a low level in integument and gut tissue in nymphs fed on TN1 seedlings. However, after exposure of the nymphs to MH63 seedlings for 24 h, CYP4CE1 was slightly upregulated in integument and fat body （about 1.2-fold, respectively）, but dramatically activated up to 12-fold in gut tissue. Whole mount in situ hybridizaion revealed that CYP4CE1 was expressed at a basal level in gut tissue and Malpighian tubules in nymphs fed on TN1 seedlings. After exposure to MH63 seedlings, CYP4CE1 transcripts were significantly accumulated in whole gut tissue including Malpighian tubules. The results suggest a potential role for CYP4CE1 in determining patterns of N. lugens-rice relationships through toxic allelochemical detoxification.

Key words: Nilaparvata lugens, detoxification enzyme, cytochrome P450 monooxygenase, gene cloning, expression profile, rice variety, insect resistance