›› 2010, Vol. 53 ›› Issue (3): 257-268.doi:

• 研究论文 • 上一篇    下一篇



  • 出版日期:2010-05-07 发布日期:2010-03-20
  • 通讯作者: 杨之帆

Molecular cloning and expression profiling of cytochrome P450 monooxygenase gene CYP4CE1 in Nilaparvata lugens (Homoptera: Delphacidae)

YANG Zhi-Fan, LIU Xiao-Li, ZHANG Yan-Yan   

  • Online:2010-05-07 Published:2010-03-20

摘要: 为了解P450基因在褐飞虱Nilaparvata lugens适应水稻品种过程中的重要作用,利用反转录聚合酶链式反应(RT-PCR),快速扩增cDNA末端(RACE)和长距离聚合酶链式反应(LD-PCR)技术,克隆了褐飞虱4龄若虫的CYP4家族的一个P450单加氧酶基因,被命名为CYP4CE1。该基因的全长cDNA序列(2 160 bp)含有一个1 626 bp的开放阅读框(ORF),编码541个氨基酸残基的蛋白质。通过GenBank数据库中的blastx搜索引擎进行同源性分析,结果表明CYP4CE1编码的蛋白与岸蟹 Carcinus maenas的CYP4C39(GenBank登录号:JC8026)的相似性最高,两者的氨基酸序列同源性达43%;其次与热带蟑螂 Blaberus discoidalis的CYP4C1(AAA27819)及黑腹果蝇Drosophila melanogaster的CYP4C3(NP_524598)的相似性也较高,氨基酸序列同源性分别达42%。氨基酸序列比对表明该蛋白含有CYP4家族成员的所有保守特征序列,如螺旋K(E--R--P),氧结合结构域即螺旋I(AG--T),血红素结合区(PF--G---C-G--F)以及CYP4成员的特有特征序列(EVDTFMFEGHDTT)等。使用Northern杂交检测CYP4CE1随时间的表达变化,结果表明:与饲养于感虫水稻台中1号(Taichung Native 1,TN1)上的若虫相比,在取食中度抗性水稻Minghui 63(MH63)秧苗12,24,48,72 h的各时间段的褐飞虱体内,该基因有2.1倍的过量表达且表达水平保持稳定。进一步通过Northern杂交检测该基因的组织表达特异性,结果显示:该基因在取食TN1秧苗的若虫脂肪体中表达量最高,在肠道组织及体壁中的表达水平较低;褐飞虱取食MH63秧苗24 h后,该基因在体壁及脂肪体中的表达量略有上升(各约1.2倍),而在肠道组织中的表达量则大幅升高(约12倍)。肠道整体原位杂交表明,CYP4CE1在取食TN1秧苗的若虫的肠道组织及马氏管中均有本底水平的表达;若虫取食MH63秧苗后,该基因在上述肠道各区段表达水平明显增强。结果提示,在褐飞虱与水稻互作过程中CYP4CE1的重要功能之一可能是参与水稻有毒次生物质的代谢。

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