| Bombyx mandarina and Bombyx mori are lepidopterous insects. The comparative study of resistance to pyrethroid in Bombyx mandarina and Bombyx mori will be of great significance to lepidoptera pests prevention and resistive breeding of Bombyx mori. Action drone of pyrethroid is sodium channel protein located in nerve axone, while several genes of the ninth cytochrome P450 family are related to resistance to pyrethroid in insects. In this study, we measured the toxicity of decamethrin on Bombyx mandarina and Bombyx mori, screened Bombyx mandarina with decamethrin resistance, colned sodium channel protein genes in Bombyx mandarina and Bombyx mori, and investigated the transcription of CYP9 family genes induced by cypermethrin in Bombyx mandarina and Bombyx mori. The main results are summarized as follows:1 Research on Pyrethroid resistance and sodium channel protein genes clone in Bombyx mandarina1.1 Comparison of the toxicity of deltamethrin on Bombyx mandarina and Bombyx moriDrop method was used to determinate the toxicity of deltamethrin on Bombyx mandarina and Bombyx mori. The resistance of Wujiang Bombyx mandarina (YWJ), Qidong Bombyx mandarina (YQD) and Soochow University Bombyx mandarina (YSD) was 15.89, 11.67 and 10.11 times of that of Dazao Bombyx mori, respectively, while the resistance of L11 Bombyx mori was 4.37 times of that of Dazao Bombyx mori.1.2 Screening of deltamethrin resistant strains of Bombyx mandarinaDeltamethrin was used to screen resistant strains of Bombyx mandarina. YSD fed with mulberry leaves were screened for three generations under deltamethrin treatment continuously. The results showed that the deltamethrin resistance of S4 was 16.48 times of that of Dazao Bombyx mori, and the LD50 value (0.445 ng/larva) in S4 generation increased by 0.9 times in comparison with the LD50 value (0.237 ng/larva) of S0 generation. YWJ fed with artificial diets for six generations were screened for three generations under deltamethrin treatment. The results suggested that the deltamethrin resistance of S6 was 18.67 times of that of Dazao Bombyx mori, and the LD50 value (0.504 ng/larva) in S6 generation increased by 0.2 times compared with the LD50 value (0.418 ng/larva) of S0 generation.1.3 The cloning and mutation of para sodium channel protein gene structure domainâ…¡in Bombyx mandarina and Bombyx moriAccording to the partial sequence of Bombyx mori sodium channel protein gene (GenBank No. EF521818), the special primer was designed. The cDNA fragments which encoded the sodium channel protein encoding gene structure domainâ…¡of YWJ and Dazao were cloned by RT-PCR. Sequence analysis showed that the length of the two cDNA fragments was about 1135bp, respectively, which encoded 378 amino acids. No point mutation related to kdr resistance and super-kdr was found, while two amino acid point mutations, namely I164L and V196A, were discovered, which might be relevant to deltamethrin resistance of Bombyx mandarina.1.4 Cloning and selective splicing of sodium channel protein genes of Bombyx mandarina and Bombyx moriAccording to the partial sequence of Bombyx mori sodium channel protein encoding gene (GenBank No. BK005824,EF521818), the special primes were designed. The sodium channel protein genes of Bombyx mori Bmpara (GenBank No. EU688970) and Bombyx mandarina Bmmpara were cloned, respectively, by RT-PCR. According to the sequences of the cloned sodium channel protein genes in Bombyx mandarina and Bombyx mori genome, three Bombyx mandarina genome sequences were cloned by PCR. Sequence analysis indicated that the length of Bmpara cDNA is 5 555 bp, which encoded 1851 amino acids. There are six kinds of Bmmpara cDNA, the lengths of which were 5 555 bp, 5 594 bp, 5 432 bp, 5 522 bp, 5 561 bp, 5 399 bp, respectively, and encoded 1 851, 1 864, 1 810, 1 840, 1 853, 1 799 amino acid, respectively. The lengthes of three Bombyx mandarina genome sequences were 1 632 bp, 536 bp and 3 220 bp, respectively. The Blast anlysis and comparison analysis showed that there were at least 34 extrons and 33 introns in Bmpara and Bmmpara. Moreover, there were three optional micro-exons in Bmmpara,including a with eleven amino acids (ENDLGRTKKKK), b with thirteen amino acids (GLKAALCGRCVSS) and c with forty-one amino acids (SLINFVAALCGAGGIQ AFKTMRTLRALRPLRAMSRMQGMRV), six kinds of selective splicing, which established different subtypes of sodium channel protein of Bombyx mandarina.2 Inducement of cytochrome P450 and drug resistance research in Bombyx mandarina and Bombyx mori2.1 P450 oxidase activity determination and induced-transcription of CYP9 family genes in Bombyx mandarinaEnzyme linked immunosorbent assay was used to determine the activity of PNOD in the midgut and fat body of YSD under cypermethrin treatment. Result showed that after 24h inducement, the PNOD activity of fat body and midgut increased by 18.7% and 12.2%, respectively. With the primer designed according to CYP9 family gene of Bombyx mori, CYP9A21 (GenBank No. FJ265741), CYP9A22 (GenBank No. EF535806) and CYP9G3 segments of Bombyx mandarina were cloned, respectively. The expression level of CYP9 family genes of Bombyx mandarina induced by cypermethrin were determined by semi-quantitive RT-PCR. Results indicated that after 24h inducement, the mRNA expression amount of CYP9A21 in midgut is 2.1 times of the control, and the mRNA expression amount of CYP9A20,CYP9A21,CYP9G3 in fat body were 1.9, 3.5, and 1.4 times of the control, respectively. The over expression of these special cypermethrin P450 genes might enhance the oxidation and detoxifcation capacity of Bombyx mandarina to cypermethrin.2.2 Induced-transcription of CYP9 family gene of Bombyx mori and clone and sequence analysis of CYP9A22Semi-quantitive RT-PCR was used to analysis expression level of CYP9 family genes in Bombyx mori induced by cypermethrin. Results indicated that after 24h inducement, the mRNA expression amount of CYP9A20 in midgut, CYP9A22 and CYP9G3 in fat body, were 1.3, 3.1, 1.2 times of the control, respectively. Compared with Bombyx mandarina, the mRNA expression amount of CYP9 family genes in Bombyx mori were lower, which partially accounted for the more susceptivity of Bombyx mori to pyrethroid than Bombyx mandarina. CYP9A22 (GenBank No. EF535804) of cytochrome P450 was cloned by RACE. The total length of the cDNA is 1707 bp, and the length of encoding region is 1596bp encoding five hundred and thirty-one amino acids. The deduced molecule weight of the protein is 61KD with pI 7.71, and the amino acid homology between CYP9A22 and CYP9A14 of CYP9 family attained 62.3%. The transcription start site deduced by NNPP analysis sofeware was coincident with the result deduced according to the total length cDNA of CYP9A22. The combinatopm site of transcription factor analyzed by TFSEARCH 1.3 shows that the upriver sequence of transcription start site contains not only the nuclear structure sequence of promoter such as TATA-box and CAAT-box, but also several combination sites of transcription factors such as GATA-1,CdxA,Dfd, et al.
|
PDF Full Text Request