Molecular Mechanisms Of Synergism And Anti-resistance Of PTD To Cry1Ac Toxin, An Insecitcidal Protein Of Bacillus Thuringiensi

The environmentally friendly insecticidal crystal proteins produced by Bacillus thuringiensis are of great scientific interest because of their potency and specificity to a wide range of insect pests. Because of their highly specific toxicity, Bt toxins offer tremendous benefits for insect pest management. The laboratory selection experiments showed that many pests could also adapt to Bt toxins. So far, documented cases of resistance to Bt in open-field populations of pests are only limited to one insect species, the diamondback moth (DBM,Plutella xylostella).Protein transduction domain (PTD) is a kind of peptide fruitfully containing arginine, which has been proved to charge exogenous macromolecules or compounds into living cells. As one of four expression systems,baculovirous-insect expression system has been widely utilized in synthesis of recombinant proteins.This system has many advantages such as high yield regulated by polyhedron promoter, proper post-translation modification, easy purification from cultured cells, and lack of endotoxin contamination.This study built TAT-Cry1Ac fusion insecticidal protein using the capacity with TAT as a vector carrying biological macromolecules through the cell membrane.We researched the synergism and overcome resistance mechanism of PTD on microbial pesticides by the affinities of the insecticidal fusion proteins with DBM BBMV and caderin-like protein on the basis of TAT-Cry1Ac fusion proteins.The results were as follows:1.Leaf-dip method was adopted to measure the toxicity of six fusion insecticidal proteins on the diamondback moth(Plutella xylostella).Bioassay results showed that all the six fusion proteins had all insecticidal activities on both the susceptible and resistant DBM P. xylostella.. Fusion proteins with TAT peptide had higher insecticidal activities than those without TAT. The LC50 values of pET-21b+6His-tat-Cry1Ac(T)and pET-21b+6His -Cry1Ac(P) aganist susceptible DBM were 1.19 mg/L and 6.6 mg/L, respectively. The synergism ratio was 5.54-fold. For the resistant population, the LC50 values of pET-21b-tat-Cry1Ac+6His(T′)and pET-21b-tat-Cry1Ac+6His(P′) were 12.94 mg/L and 27.18 mg/L, respectively, and the synergism ratio was 2.1-fold. These results proved that TAT peptide has significant synergism to Bt insecticidal protein Cry1Ac.2.Cadherin-like protein genes from susceptible and resistant strain of P. xylostella were cloned and sequenced. The full length was 5314 bp nucleotides encoding 1716 amino acids. Multiple alignment showed that amino acid identity of the two strains was 99.42%.Ten amino acids changed in resistant stain compared to the susceptible strain.3. Cadherin-like protein genes were cloned into the Bac-to-Bac baculovirus expression system with transfer vector pFastBacHTb and recombinant plasmid pFastBacHTb/CAD was obtained. Plasmid was transformed into DH10Bac E. coli. Then the recombinant bacmid Bacmid/CAD was obtained. Bacmid/CAD was transfected into cells of the insect Trichoplusia ni (Tn) and recombinant baculovirus was obtained. The recombinant baculovirus was analyzed by SDS-PAGE The results showed that the molecular weiht of expressed production was 200kD which matched with the theoretical value. Western-blot showed that the expressed products combined with CrylAc could combine with multiple cloned antibody of CrylAc, which indicates that CAD was exactly expressed and CAD was the receptor of CrylAc.4.The affinities of the insecticidal fusion proteins with BBMV isolated from the midgut of P.xylotella and the expressed CAD in Trichoplusia ni cells were analysed by ELISA.This has virtue of security and convenience. Results showed that TAT significantly increased the affinity of fusion proteins with BBMV or expressed CAD. The OD450 values of pET-21b+6His-tat-Cry1Ac(T) and pET-21b+6His -Cry1Ac(P)with susceptible BBMV of DBM were 0.207 and 0.165, respectively. The OD450 values of pET-21b+6His-tat-Cry1Ac(T) and pET-21b+6His -Cry1Ac(P)with cadhein-like protein of susceptible DBM were 0.217 and 0.1775, respectively.Further proved that TAT could increase the affinity of insecticidal protein with intestinal epithelial cells, thereby TAT enhanced the insecticidal toxicity and fulfilled the final objective to overcome CrylAc resistance.