| The cotton aphid, Aphis gossypii(Glover)(Homoptera: Aphididae), is one of the most serious polyphagous pests throughout the wordwide by feeding on many kinds of host plants including cotton. It has developed a rapid, broad and high level of resistance to insecticides resulting in the widespread problem for controlling cotton aphid. Imidacloprid(IMI), the first commercialized neonicotinoids, is currently the preferred insecticide for the cotton aphid and other sucking pest control. But, the pests have developed high levels of resistance to IMI due to the broad and repeated application. It has been suggested that the metabolic mechanisms of IMI resistance in pests is mainly related to the increasing metabolic activities of cytochrome P450 s protein encoded by cytochrome P450 s gene(CYP). However, it is less direct evidences to prove the metabolic detoxification of IMI with this P450 s protein expressed by relative CYP gene in vitro, which also involed in different genes in different pests. The increasing of P450 activities has been known to play an important role in the development of cotton aphid resistance to IMI in our previous study. However, the molecular mechanism of metabolism due to the overexpression of P450 genes resulting in the IMI Resistance in Aphis gossypii remains unknown. In this study, the resistant level of cotton aphid to IMI from the major cotton fields in Shandong Province was determined. Then the molecular mechanisms of metabolism was studied in two field resistant strain(XJ and BZ), one selected resistant strain(RS, resistant level is 72.6-fold), and one susceptible strain(SS). Our findings suggest that the overexpression of P450 gene CYP6-like3-Ag could lead to the development of IMI resistance in cotton aphid.We used cpillary microtiter method to test resistance levels to imidacloprid of the 12 cotton aphid field strains in major cotton areas of Shandong Province, and the results showed that 12 cotton aphid field strains developed different level resistance to imidacloprid. and compared with the sensitive strains, the resistance ratio is between 3.73 and 12.90. Among the areas, Xiajin(XJ) and Binzhou’s(BZ) two strains had the highest resistance ratio, resistance ratios were 10.36 and 12.90. Yucheng(YC) and GAOTANG’s two strains had the lowest resistance ratio, resistance ratios were 3.73 and 4.24. The resistance ratio to imidacloprid was between 5.27 and 9.13 in other 8 cotton aphid field strains.Comparison synergies of the inhibitor PBO( piperomyl butoxide) in the susceptible strain(SS) and three resistant cotton aphid strains(XJ, BZ, RS) to imidacloprid, The results showed that the synergism of PBO is most significant in BZ population, 5.43-fold. Followed by the RS strain and XJ strain, were 3.88-fold and 2.65-fold. The Synergistic effects to SS strain was not obvious, was 1.19-fold. Measured the O-demethylation of p- nitroanisole(PNOD), compared the activity of cytochrome P450 in four cotton aphid strains. The results showed that, the activity of cytochrome P450 in RS strains was the highest, was 4.89-fold more than SS strain; followed by BZ strain, was 3.98-fold more than SS strain; there wasn’t significant difference between XJ strain and SS strain, was only 1.53-fold more than SS strain.The LC-MS / MS technology was used to compare the ability of metabolism to imidacloprid of resistant strain(RS) and sensitive strain(SS). The results showed that with the increasing of processing time, the content of the imidacloprid in two strains were first increased and then decreased, and after treatment of 10 hours, the content of the imidacloprid reached the maximum of 0.28 ± 0.03 ng mg-1cotton aphids in sensitive strain. Meanwhile,after treatment of 1h, the content of the imidacloprid reached the maximum of 0.17 ± 0.02ng/mg cotton aphids in resistant strain. After 24 hours, the content of imidacloprid in sensitive strain was 1.91 times more than resistant strain. Nevertheless, the content of 4/5-hydroxy imidacloprid in two groups has been showing an increasing trend, and the 4/5-hydroxy imidacloprid content of resistant strain is always more than the susceptible strain. After 24 hours, the 4/5-hydroxy imidacloprid content of resistant strain and susceptible strain were2910 ± 80 and 990 ± 110 peak area mg-1cotton aphid, the resistant strain was 2.94-fold more than sensitive strain. The results showed that two cotton aphid strains both can metabolize imidacloprid and the main metabolite is 4/5-hydroxy imidacloprid, and the metabolic capacity to imidacloprid of resistant cotton aphid strain was significantly higher than the susceptible cotton aphid strain.According to the amino acid of conserved regions in CYP4 and CYP6 family genes designed the degenerate primers, attained a target gene fragment of approximately 440 bp and390 bp by used the RT-PCR technology, eventually got 15 P450 gene sequences after cloning and sequencing, contrast and exclude the same gene or the allele of the same gene in the samegene family by BLASTX or BLASTP in GenBank, and eventually got 4 CYP4 family gene and 3 CYP6 family genes, contrasting the mRNA expression levels of these 157 P450 gene in RS resistant strains and SS susceptible strains by Real-time PCR technology, The results show that the m RNA expression of CYP4-like2-Ag, CYP4-like4-Ag, CYP6-like1-Ag and CYP6-like3-Ag in RS strains significantly higher than that in SS strains, the expressions in RS strains are 16.77, 6.29, 4.82 and 12.86-fold of SS strains. Further comparing the mRNA expression of the 4 genes in 2 field resistant strains BZ and XJ, the results show that mRNA expression of CYP4-like2-Ag and CYP6-like3-Ag in BZ and XJ strains are higher, among them, CYP4-like2-Ag are 6.05 and 4.74-fold of SS strain respectively; CYP6-like3-Ag are4.97 and 3.07-fold of SS strains respectively. But the mRNA expression of CYP4-like4-Ag and CYP6-like1-Ag in BZ and XJ strains has little difference with SS strain.Using RACE technology, the P450 gene CYP6-like3-Ag was cloned into cotton aphid,the full-length sequence is1386 bp in RS and SS strains, contain all of the CDS sequence,This includes a 5’ untranslated region(UTR) of 265 bp and a 3’ UTR of 481 bp, coding region of 1266 bp, encodes 456 amino acid residues, start codon in ATG, termination codon in TAA,the theoretical molecular weight is 53.112 kDa. according to the naming rules of the International Nomenclature Committee of P450 named CYP6CY3.The recombinant plasmid CYP6CY3 is constructed in the pFast Bac vector. Cotton aphid CYP6CY3 gene is successfully expressed in vitro with Bac-to-Bac baculovirus expression system. Measured the oxidative metabolism of expression products on pattern substrates(PNOD). The results showed that the protein activity is 8.60 mOD/min/mg proteins on the substrate of PNOD(p-nitroanisole). with LC-MS/MS, vitro metabolic effects of the expression product to imidacloprid is measured. The result showed that, in the NADPH regenerating system, the expression product can successfully metabolise imidacloprid to4/5-hydroxy imidacloprid... |
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