飞蝗3个细胞色素P450基因的分子特性及功能

摘要

【目的】研究飞蝗（Locusta migratoria）细胞色素P450（CYP450）第四家族（CYP4）中3个基因的功能。【方法】以飞蝗为材料，依据飞蝗EST数据库P450基因预测序列设计引物，利用 RT-PCR技术克隆飞蝗细胞色素P450基因的cDNA全长序列，命名为CYP4C69、CYP4C73和CYP4DH1（GenBank登录号： KF857162、KF857163和KF857164）。采用实时定量 PCR 技术分析CYP4C69、CYP4C73和CYP4DH1在飞蝗各发育阶段以及不同组织中mRNA的表达水平。应用RNA干扰技术沉默CYP4C69、CYP4C73和CYP4DH1，实时定量 PCR 技术检测基因的沉默效率。应用农药点滴技术测定2龄若虫期飞蝗对马拉硫磷、毒死蜱和溴氰菊酯杀虫剂的毒力，通过SPSS软件分析得出3种杀虫剂的LC 30，同时应用该技术研究dsRNA干扰过的飞蝗对马拉硫磷、毒死蜱和溴氰菊酯杀虫剂的敏感性。【结果】克隆获得了飞蝗CYP450基因CYP4C69、CYP4C73和CYP4DH1的cDNA全长序列，其中CYP4C69开放阅读框1518 bp，编码506个氨基酸；CYP4C73开放阅读框1521 bp，编码507个氨基酸；CYP4DH1开放阅读框1512 bp，编码504个氨基酸。对飞蝗不同龄期定量PCR结果分析，发现CYP4C69在若虫末期表达量高于成虫期，CYP4C73和CYP4DH1在飞蝗整个发育阶段均有表达，在若虫初期和末期以及成虫期表达量较高；不同组织的定量PCR结果表明CYP4C69、CYP4C73和CYP4DH1均在胃盲囊高表达，CYP4C69和CYP4DH1在脂肪体中高表达。通过RNA干扰，发现CYP4C69、CYP4C73和CYP4DH1均可以被显著沉默。马拉硫磷、毒死蜱和溴氰菊酯对2龄若虫期飞蝗的毒力测定表明2龄若虫期飞蝗对3种杀虫剂的LC 30剂量，分别为59.438、8.215、0.759μg·mL-1。飞蝗对马拉硫磷、毒死蜱和溴氰菊酯杀虫剂敏感性试验结果表明，双链RNA沉默CYP4C69、CYP4C73和CYP4DH1后，飞蝗点滴马拉硫磷、毒死蜱和溴氰菊酯杀虫剂后的死亡率与注射dsGFP的对照组相比，没有显著提高。【结论】获得飞蝗CYP450基因CYP4C69、CYP4C73和CYP4DH1的cDNA全长序列，这些基因均可被双链RNA干扰而沉默，但CYP4C69、CYP4C73和CYP4DH1沉默后，飞蝗对马拉硫磷、毒死蜱和溴氰菊酯杀虫剂的敏感性与对照组相比没有显著提高。%[Objective]The objective of this study is to elucidate the molecular function of the three cytochrome P450 genes of CYP4 family from the locust (Locusta migratoria). [Method]According to the predicted P450 sequences from ESTs database of locust, the primers were designed, the full-length sequences of three novel genes were amplified and named CYP4C69, CYP4C73 and CYP4DH1 (GenBank accession numbers: KF857162, KF857163, KF857164), respectively. The mRNA expression levels of CYP4C69, CYP4C73 and CYP4DH1 were analyzed in different developmental stages and tissues of locusts by real-time quantitative PCR. The RNAi efficiency of dsCYP4C69, dsCYP4C73 and dsCYP4DH1 was evaluated by real-time quantitative PCR. The toxic effects of malathlon, chlorpyrlfos and deltamethrin on 2nd-instar nymphs were analyzed and the LC30 doses of the three insecticides were calculated by SPSS software. As CYP4C69, CYP4C73 and CYP4DH1 transcripts were significantly repressed in locusts by RNAi, the susceptibility of the dsRNA injected locusts to different insecticides, such as malathion, chlorpyrifos and deltamethrin were assessed.[Result]The full-length cDNA sequences of CYP450 genes including CYP4C69, CYP4C73 and CYP4DH1 were obtained by RT-PCR. The cDNA sequences of CYP4C69, CYP4C73 and CYP4DH1 had open reading frames of 1 518, 1 521 and 1 512 nucleotides which encoded proteins of 506, 507 and 504 amino acid residues, respectively. Moreover, the expression level of CYP4C69 was higher in 5th-instar nymph than in the adult stage. However, CYP4C73 and CYP4DH1 expressed during the whole developmental stages and were relatively higher in 1st-, 4th- and 5th-instar nymphs and adult stage. The results of CYP4 genes expression from different tissues indicated that all of the three genes had a high expression level in gastric caeca, CYP4C69 and CYP4DH1 highly expressed in fat body. It was found that CYP4C69, CYP4C73 and CYP4DH1 transcripts were significantly repressed in the locusts after dsRNA injection. The toxic effects of malathion, chlorpyrifos and deltamethrin on 2nd-instar nymphs indicated that the LC30 doses of the three insecticides were 59.438, 8.215, 0.759μg·mL-1, respectively. The susceptibility of locusts to malathlon, chlorpyrlfos and deltamethrin showed that there was no marked difference between locusts’ injection of dsCYP4C69, dsCYP4C73, dsCYP4DH1 and dsGFP.[Conclusion]The full-length cDNA sequences of CYP4C69, CYP4C73 and CYP4DH1 were obtained. And these genes could be silenced by dsCYP4C69, dsCYP4C73 and dsCYP4DH1. However, treatment with dsRNA of CYP4C73, CYP4C69, CYP4DH1 and dsGFP did not show significant effects on the susceptibility of the locusts to malathion, chlorpyrifos and deltamethrin.