Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (10): 1956-1965.doi: 10.3864/j.issn.0578-1752.2014.10.009

• PLANT PROTECTION • Previous Articles     Next Articles

Molecular Characteristics and Functional Analysis of Three Cytochrome P450 genes in Locusta migratoria

 REN  Xiao-Yu, YANG  Mei-Ling, GAO  Cui-E, ZHANG  Jian-Zhen, MA  EnBo   

  1. Research Institute of Applied Biology, Shanxi University, Taiyuan 030006
  • Received:2013-11-04 Online:2014-05-20 Published:2014-01-06

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Abstract: 【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 ef?ciency 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 signi?cantly 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 signi?cantly 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, 0.759 μg•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 signi?cant effects on the susceptibility of the locusts to malathion, chlorpyrifos and deltamethrin.

Key words: cytochrome P450 , Locusta migratoria , gene expression , RNA interference , insecticide

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