| Domesticated silkworm (Bombyx mori) and wild silkworm (Bombyx mandarina) originated from a same ancestor. Domesticated silkworm has been tamed for 5700 years. Although the silk production of Bombyx mori has been significantly increased after long-term artificial selection, its adaptability to environment presents the tendency to degradation. Since Bombyx mandarina inhabits in mulberry garden outdoors, its resistance against the external environment especially to the pesticides shows an increasing tendency year by year as a result of natural selection and insecticide pressure. On one side, due to the susceptibility of Bombyx mori to insecticides, many of them were intoxicated and even some areas have given up the rearing of Bombyx mori in autumn. On the other side, because of the increasing insecticide resistance of Bombyx mandarina, it has decreased the production of mulberry leaves significantly. In conclusion, these factors have caused a series of problems that restrict the normal development of silk industry. Bombyx mori (Dazao) and Bombyx mandarina (Suzhou strain) were used as in this paper to study the molecular mechanisms of the organophosphate resistance differences in Bombyx mori and Bombyx mandarina. The susceptibility of Bombyx mori and Bombyx mandarina to organophosphate was investigated, as well as the enzymatic activities of acetylcholinesterase (AChE), the gene structures of two kinds of ace genes, the inductive expression patterns by organophosphate, and the susceptibility of the heterologous expression products of ace2 gene to eserine.Newly molted Bombyx mori and Bombyx mandarina of all instars were used to investigate the phoxim insecticide resistance differences between them by leaf dipping method. The results showed that phoxim resistance differences between them at early stages are comparatively smaller; but as the instars increased, the resistance differences change significantly with the LC50 of Bombyx mandarina 4.43-fold higher than that of Bombyx mori at the stage of 4th instar, and 4.02-fold higher at the stage of 5th instar.The 2nd instar newly molted silkworms and the brain, midgut, fat body, and silk gland of the third day 5th instar Bombyx mori and Bombyx mandarina were collected to assay the differences of enzymatic activities of AChE and I50 inhibited by eserine between them. The results indicated that the enzymatic activities of the newly molted 2nd instar Bombyx mandarina is 1.65-fold compared with that of Bombyx mori; the ratios of enzymatic activities of the brain, midgut, fat body, and silk gland of third day 5th instar Bombyx mandarina and Bombyx mori are 1.90, 2.23, 2.76, and 2.78-fold respectively; the AChE-I50 values of Bombyx mori and Bombyx mandarina tested by eserine are 5.02×10-7M and 5.23×10-7M respectively.We have constructed a cDNA library of brain tissue from Bombyx mandarina. The library qualification evaluation showed the titer of primary cDNA library was 3.5×105 pfu/mL, average inserted size was 1.2 kb. The ORF of chemosensory proteins gene (CSP3)(GenBank Accession No. EU439267) was found through searching cDNA library and was sequenced. Results showed that the CSP3 is 384 bp and encodes a protein of 127 amino acids with predicted molecular weight of 14.6 kD. The amino acids sequence analysis of CSP3 between CSP3 and 17 insect CSP indicated that the CSP3 has 96.85% identities with Bombyx mori CSP3. The detection of the gene has important meaning to research sensitivity to chemistry agrochemical of Bombyx mori and Bombyx mandarina.Acetylcholinesterase genes (Bm-ace1, Bm-ace2) were cloned from Bombyx mori by RT-PCR. Sequence analysis showed that Bm-ace1 contains a 2 025 bp ORF, which encodes a protein of 683 amino acids with predicted molecular weight of 76.955 kD and pI of 6.36; Bm-ace2 contains a 1 917 bp ORF, which encodes a protein of 638 amino acids with predicted molecular weight of 71.675 kD and pI of 5.49, respectively. Both of the two acetylcholinesterase genes contain the characteristic domains. A clustering analysis showed that Bm-ace1(ABY50088)shared highest similarity with Bmm-ace1(ABM66370) from Chinese Bombyx mandarina, which was 99.71%, Bm-ace2 (ABY50089) shared highest similarity with Bm-ace2 (NP001037366) from Bombyx mori, which was 99.84%. Bm-ace1 and Bm-ace2 are located on 15th, 9th chromosome, respectively, according to the information of Bombyx mori genome. This research will help understand the resistance mechanism of Bombyx mori to organophosphorous insecticides.RT-PCR and RACE (rapid-amplification of cDNA ends) were used to study the molecular mechanism of the resistance differences of organophosphorus insecticides between Bombyx mori and Bombyx mandarina. Two full-lenth AChE genes of Bombyx mandarina were cloned and named Bmm-ace1 and Bmm-ace2 respectively. The sequence analysis showed that Bmm-ace1 and ace1 (Bm-ace1) encoded 681 amino acids, with the homology of 99.71%, and existed two mutated amino acids(G664S,S307P). Bombyx mandarina ace2(Bmm-ace2) and Bombyx mori ace2(Bm-ace2)encoded 634 amino acid with the homology of 99.37%, and existed four mutated amino acid (M18I,N233S,I310V,G621S). The clustering analysis indicates that the ace2 is relatively conserved in the species. By analyzing the 5'UTR (untranslated region ) of the cDNA of Bmm-ace1,and comparing with the 5'URT of cDNA of Bm-ace1, we found that the 5'URT of Bmm-ace1 was 231 bp in length, which is shorter than that of Bmace1 (242 bp), and there are 2 nucleotide mutations and a loss of 11 consecutive base pairs in 5′UTR of Bmm-ace1. Cloning and sequencing of the genome sequence that contains 5′UTR of Bmm-ace1 indicated that both of the Bmm-ace1 and Bm-ace1 follow the GT-AG rule in their exon-intron boundaries, but there are some differences in their splicing patterns in 5′UTRs. The genome sequence of Bmm-ace1 contains the lost 11 consecutive base pairs (TGATTTGAAGG) for Bm-ace1 at +33 site of Bmm-ace1 cDNA sequence, but exists a loss of five nucleotides (ACAGA) at -4 site compared to TGATTTGAAGG nucleotides. This research provides some basic information for deeply understanding the relationship between Bmm-ace1 gene and insecticide resistance.The expression patterns of ace1 and ace2 genes at different development stages in all the five larval instars, and various tissues such as hemolymph, brain, midgut, fat body, and silk gland of third day 5th instar larvae of Bombyx mori and Bombyx mandarina were investigated by using semi-quantitative RT-PCR method, as well as the expression profiles of the larvae exposed to phoxim of different concentrations, and ace1 and ace2 at different tissues of third day 5th instar larvae exposed to the phoxim at a concentration higher than I50. The results showed that the expression of Bm-ace1 and Bm-ace2 decreases first, then increases from 1st instar to 5th instar stage, with the lowest expression of Bm-ace1 at the 3rd instar, and Bm-ace2 at 2nd instar. The expression of Bmm-ace1and Bmm-ace2 decreased gradually from 1st instar to 5th instar stage, with Bmm-ace1 decreasing greatly than Bmm-ace2, which is different from that of Bombyx mori. Tissue expression analysis showed that ace1 genes were expressed only in the brains and fat bodies of Bombyx mori and Bombyx mandarina, while ace2 genes were expressed in all the tissues tested. ace1 and ace2 were expressed highly in brains and fat bodies. The expression of ace1 genes in the brains of Bombyx mori and Bombyx mandarina was nearly identical, while the expression of ace2 genes was 4.17-fold higher in the brain of Bombyx mandarina than that of Bombyx mori. The results of the inductive expression by phoxim showed that below the value of I50, the expression of aec1 genes increased along with the concentrations of phoxim in both Bombyx mori and Bombyx mandarina, while ace2 increased in Bombyx mori, but mained high expression and decreased in Bombyx mandarina. When phoxim was applied at higher concentrations than I50, the expression of ace1 and ace2 decreased in both Bombyx mori and Bombyx mandarina, with Bmm-ace2 decreasing less than others. The results of the tissue expression induced by the phoxim of higher concentrations than I50 showed that these genes all decreased in fat bodies, with Bmm-ace1, Bm-ace1, Bm-ace2 decreasing 82.67%, 81.11%, 84.50% respectively, and Bmm-ace2 decreasing the least at 30.56%.To compare the bioactivities of the expression products of Bm-ace2 and Bmm-ace2 and the differences in insecticide metabolism, Bm-ace2 and Bmm-ace2 were cloned into the transfer vector pFastBacHTB to obtain recombinant vectors pFast-Bm-ace2 and pFast-Bmm-ace2. Then they were transformed into DH10Bac competent cells, and recombinant bacmids were verified by PCR analysis. BmN cells were transfected by the recombinant bacmid using lipofectin to get recombinant viruses. SDS-PAGE and Western blot analysis showed that Bm-ace2 and Bmm-ace2 were expressed successfully. After purification of these expression products, the enzymatic activities of the two proteins were detected as 1 847.94 mOD/ (min.mg) and 1 892.17 mOD/ (min.mg), respectively, and the I50 of Bm-ace2 and Bmm-ace2 to eserine were 1.73×10-7M and 1.87×10-7M, respectively.The results showed that overexpression and mutation of Bmm-ace2 are the main reasons for the differences of phoxim resistance in Bombyx mori and Bombyx mandarina. This research provide insights into the breeding of resistant strains of Bombyx mori and the development of highly effective insecticides against Lepidopteral pests.
|
PDF Full Text Request