| Bacillus thuringiensis (Bt) produces proteinaceous crystlline icclusion bodies upon sporulation, which kill many key insect pests. However, Bt is harmless to the nontarget organisms including humans and enviornment. Therefore, Bt has been used widely in the worldwide. The wide application of Bt formulations and Bt transgenic plants brought out the threat of Bt resistance in insect of field populations. It was reported that some insects have been confirmed to show resitance to Bt in field, but the diamondback moth (DBM, Plutella xylostella) is still the only insect having resistance to Bt formulations in the open field up to now.The major objectives of this present study were to understand the resistance mechanism of P. xylostella, which will provide a theoretical basis in the resistance management in the open field. In this paper, the resistance selection of Cry1Ac toxin in the P. xylostella, biological properties, fitness cost analysis, molecular cloning and expression of CAD (cadherin-like protein) were carried out. The results were as follows.1. Based on the continuous selection on DBM by Cry1Ac in the laboratory for 2 years, Cry1Ac resistant (Cry1AcR) population of DBM was obtained. The resistance level of Cry1AcR population was﹥1,500-fold compared with SS population. Also we tested the effect of the 4℃treatment to pupa and the fecundity from the hybrid the Cry1AcR with SS. The fecundity of the DBM under 4℃treatment for 7d was significantly lower. Compared to the SS and SS♀×Cry1AcR♂, the fecundity of Cry1AcR and SS♂×Cry1AcR♀were significantly lower too.2. The biological characteristics of SS and Cry1AcR populations were observed and compared by constructing the life table. The results showed that the Cry1AcR population has lower the intrinsic rates of increase, gross reproductive rates, net reproductive rates, mean generation time and the relative fitness than the SS population, indicating that the resistant population had distinguished development and reproductive disadvantages. Morecover, the Cry1AcR population has lower number of eggs laid, hatching rate, pupation rate and sex ratio (♀:♂), shorter egg period and lighter pupa weight compared with the SS population.3. The cadherin-like protein genes from P. xylostella of both susceptible and resistant populations were cloned and sequenced. The total length cDNA was 5148bp nucleotides encoding 1716 amino acids. Multiple alignments showed that nucleotide similarity and amino acid identity of the two strains were 98.3% and 98.8%, respectively. 87 nucleotides and 21 amino acids changed in resistant population Cry1AcR compared to the susceptible population SS.4. The Cadherin-like protein was expressed with the Bac-to-Bac system. The Cadherin-like protein protein genes from susceptible and resistant populations of P. xylostella and pFastBacHTb were to form recombinant baculovirus. Then the recombinant bacmid was transfected into cells of Sf9, and the recombinant protein was extracted from the cells. We analyzed the CAD protein through SDS-PAGE electrophoresis. The results showed that the molecular weight of expressed production was about 180D which matched with the theoretical value and the negative control did not express the protein, which indicated that CAD was exactly expressed in Sf9 cells. |
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