Resistance Monitoring Of Bombyx Mandarina From Jiangsu & Zhejiang Provinces And Cloning & Sequence Analysis Of CYP6AU1 Gene CDNA

Bombyx mandarina is the main pest in the JiangSu and ZheJiang silkworm producing area, and its population has directly affected the silkworm yields in those areas. Since large amount of insecticide had been used for combating its infestation, many field populations of this pest have developed resistances in some extent, which make more and more frequent control failures in field. So far, we know that the development of insect resistance has close relationship with cytochrome P450 genes.In this study, Bombyx mandarin larvae were used as experiment material. According to the results of large numbers of toxicological experiments, susceptive toxicity baseline was designed, and then the differences of dosage acting on the susceptible strains by different common insecticides such as Methomyl, Dipterex, and Dichlorvos were ascertained. The resistances of Bombyx mandarina to common insecticide were monitored by those experiments. Comparing the monitoring resistances results with that of the laboratory susceptible strains, we found that Bombyx mandarina multiple of resistance to Methomyl went up to 2.4～5.9, and the frequency of the corresponding resistance individual were 5.5%～9.7%; multiple of resistance to Dipterex went up to 2.3～5.8, and the frequency of the corresponding resistance individual were 6.7%～16.7%; multiple of resistance to Dichlorvos went up to 2.3～5.8, and the frequency of the corresponding resistance individual were 4.3%～22.7%. Based on these monitoring results, the instructions to the exact insecticides using methods and tendency in the fields were given. Sequence CYP6AU1 is one of the insecticide resistance P450 sequences from the genome Bombyx mori. According to the sequence of CYP6AU1 on the NCBI, a pair of primers was designed, and CYP6AU1 were successfully cloned from the midguts of the wild silkowrm, Bombyx mandarina, by RT-PCR. The sequences analysis between the Bombyx mori CYP6AU1 (coded with CYP6AU1-H) and Bombyx mandarina CYP6AU1 (coded with CYP6AU1-W) indicated that they both contain a 1,491 bp open reading frame, which encode 496 amino acid (m.w. 57.3KD) with pI 6.69. The comparison of the nucleotide and amino acid sequences of CYP6AU1-H with that of CYP6AU1-W showed they share the highest identity of 98.8% and 99.6%, with 18bp nucleotides and 2 amino acids differences, respectively. This research provides a theory foundation for next study of Bombyx mandarina insecticide resistance genes, and also helps accumulating useful materials for insecticide resistance Bombyx mori breeding.