Synergetic Characteristic Of Five Receptor Fragments From Plutella Xylostella To Cry1Ac Protein

Insect Cadherin, Aminopeptidase N and Alkaline phosphatase localized in the midgut epithelium are identified as three receptors for all Bacillus thuringiensis(Bt) insectical crystal proteins'receptors.They facilitated oligomerization of toxin monomer and then induced the toxin molecule to change the conformation, mediated oligomer specific binding to every receptors. It was reported that Helicoverpa armigera and Manduca sexta'cadherin toxin-binding regions function as synergists or inhibited for Cry1 toxicity against target insects.This research based on some reports that cadherin fragments, Aminopeptidase N fragment and Alkaline phosphatase fragment in toxin binding region in some insect had a synergistic or inhibited effect on Cryl Ac toxin.Five receptor gene fragments (the total length of PxCAD-1 is 735bp, encoded 245 amino acids; the total length of PxCAD-2 is 627bp, encoded 209 amino acids; the total length of PxCAD-3 is 510bp, encoded 170 amino acids; the total length of PxAPN1 is 192bp, encoded 64 amino acids;the total length of PxALPO is 967bp, encoded 322 amino acids) from Plutella xylostella were cloned. The functional region fragments PxCAD-1,PxCAD-2,PxCAD-3, PxAPNl and PxALPO were over expressed in Escherichia coli by vector pGEX-6P-1 respectively.Bioassays used median lethal concentration of CrylAc(1?g/mL)in the presence of PxCADa,PxAPN1a,PxALP1,PxCADband PxCADcwere performed against Plutella xylostella larvaes.1.87 ?g/mL of the Cryl Ac alone can cause the third stage larvaes of Plutella xylostella's mortality was 46.7 percent, when joined a final concentration for 551.25?g/mL of GST-PxCADb, the mortality was 45 percent; When joined a final concentration for 204.67?g/mL of GST-PxCADc, the mortality was 48.9 percent; When joined a final concentration for 187.33 ?g/mL of GST-PxAPNl, the mortality was 45.6 percent,-while the control group for pGEX-6p-1 protein's final concentration of 300.27?g/mL, the mortality was 45.6 percent in the third stage larvaes of Plutella xylostella, there was no significant difference between treatment groups (GST-PxCADb,GST-PxCADc and GST-PxAPNla) and control groups (Cry1 Ac and pGEX-6p-1), the results showed that higher concentration proteins of PxCADb, PxCADc and PxAPNl did not enhance the insecticidal activity of CrylAc.But, when joined a final concentration for 207.58?g/mL of GST-PxCADa, the mortality was 85.6 percent; When joined a final concentration for 290.25 ?g/mL of GST-PxALP1, the mortality rate was 70.0 percent, When joined a final concentration for 187.33?g/mL of GST-PxAPNl, the mortality was 45.6 percent, while the control group for pGEX-6p-1 protein's final concentration of 300.27?g/mL, the mortality was 45.6 percent in the third stage larvaes of Plutella xylostella, there was great significant difference between treatment groups (GST-PxCADa and GST-PxALPl) and control groups (CrylAc and pGEX-6p-1), the results showed that medium or higher concentration proteins of PxCADa and PxALP1 significantly enhanced the insecticidal activity of CrylAc.The results showed there was no significant enhancement of CrylAc toxicity to P. xylostella larvae when including between PxCAD-2, PxCAD-3 and PxAPN1 in bioassays.But there were highly significant enhancement of CrylAc toxicity to Plutella xylostella larvaes when including both PxCAD-1 and PxALPO. The research can provide theoretical bases for Screening Synergetic fragments of Plutella xylostella and the application of transgenic plants and Plutella xylostella endotoxin binding peptide in high efficient insecticidal Bt engineering bacteria.Also can contribute to reveal the toxic mechanism of Bt insecticidal protein and the resistant mechanism of insect pests to Bt insecticidal protein.