The aim of this article is to successfully express the Bt （Bacillus thuringiensis） toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm （Helicoverpa armigera Hübner） wit...The aim of this article is to successfully express the Bt （Bacillus thuringiensis） toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm （Helicoverpa armigera Hübner） within eukaryotic expression system, which is one of the key links for clarifying the relationship between receptor and Bt resistance. The fragments of aminopeptidase N1 （APN1） gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method, and were separately cloned into pUC 19 vector. After sequencing the gene, the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac. It was cultured in LB medium, which contained Te, Kan, Ge, X-gal, and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis. The results showed that the recombinant baculoviruse was fully capable of expressing APN1. The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationshio of resistance with Bt.展开更多
Trichoplusia ni caterpillars are polyphagous foliage-feeders and rarely likely to encounter aflatoxin B1 （AFB1）, a mycotoxin produced by Aspergillus flavus and A. parasiticus, in their host plants. To determine how ...Trichoplusia ni caterpillars are polyphagous foliage-feeders and rarely likely to encounter aflatoxin B1 （AFB1）, a mycotoxin produced by Aspergillus flavus and A. parasiticus, in their host plants. To determine how T. ni copes with AFB 1, we evaluated the toxicity ofAFB 1 to T. ni caterpillars at different developmental stages and found that AFB 1 tolerance significantly increases with larval development. Diet incorporation of AFB 1 at 1μg/g completely inhibited larval growth and pupation of newly hatched larvae, but 3μg/g AFB 1 did not have apparent toxic effects on larval growth and pupation of caterpillars that first consume this compound 10 days after hatching. Piperonyl butoxide, a general inhibitor of cytochrome P450 monooxygenases （P450s）, reduced the toxicity of AFB 1, suggesting that AFB1 is bioactivated in T. ni and this bioactivation is mediated by P450s. Some plant allelochemicals, including flavonoids such as fiavones, furanocoumarins such as xanthotoxin and imperatorin, and furanochromones such as visnagin, that induce P450s in other lepidopteran larvae ameliorated AFBI toxicity, suggesting that P450s are also involved in AFB 1 detoxification in T. ni.展开更多
基金supported by the grant(30200182)from the National Natural Science Foundation of Chinathe National Basic Research Program of China(2006CB102004)from the Ministry of Science and Technology of China.
文摘The aim of this article is to successfully express the Bt （Bacillus thuringiensis） toxin receptor protein located on the internal membrane of larval midgut of cotton bollworm （Helicoverpa armigera Hübner） within eukaryotic expression system, which is one of the key links for clarifying the relationship between receptor and Bt resistance. The fragments of aminopeptidase N1 （APN1） gene without signal peptide in the susceptible and the resistant H. armigera were cloned separately using PCR method, and were separately cloned into pUC 19 vector. After sequencing the gene, the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into Escherichia coli DH10Bac. It was cultured in LB medium, which contained Te, Kan, Ge, X-gal, and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni and recombinant baculoviruse was obtained. The lysate of cells infected with recombinant baculoviruse was analyzed by SDS-PAGE and blot analysis. The results showed that the recombinant baculoviruse was fully capable of expressing APN1. The APN1 gene successfully expressed in T. ni cell established the base for continuing the research on its function and relationshio of resistance with Bt.
文摘Trichoplusia ni caterpillars are polyphagous foliage-feeders and rarely likely to encounter aflatoxin B1 （AFB1）, a mycotoxin produced by Aspergillus flavus and A. parasiticus, in their host plants. To determine how T. ni copes with AFB 1, we evaluated the toxicity ofAFB 1 to T. ni caterpillars at different developmental stages and found that AFB 1 tolerance significantly increases with larval development. Diet incorporation of AFB 1 at 1μg/g completely inhibited larval growth and pupation of newly hatched larvae, but 3μg/g AFB 1 did not have apparent toxic effects on larval growth and pupation of caterpillars that first consume this compound 10 days after hatching. Piperonyl butoxide, a general inhibitor of cytochrome P450 monooxygenases （P450s）, reduced the toxicity of AFB 1, suggesting that AFB1 is bioactivated in T. ni and this bioactivation is mediated by P450s. Some plant allelochemicals, including flavonoids such as fiavones, furanocoumarins such as xanthotoxin and imperatorin, and furanochromones such as visnagin, that induce P450s in other lepidopteran larvae ameliorated AFBI toxicity, suggesting that P450s are also involved in AFB 1 detoxification in T. ni.
