Study On Synergistic Effect Of Polycalin Protein Fragment Of Plutella Xylostella On Cry1Ac

Plutella xylostella（Px）is a worldwide pest with strong adaptability,fast reproduction and widespread distribution.It is one of the important pests of cruciferous family.Bt toxin is an insecticidal protein produced by the gram-positive bacterium Bacillus thuringiensis.Genetically modified crops and Bt biological agents used to be very effective methods for controlling Px,effectively suppressing the population of target pests such as Px,reducing dependence on conventional pesticides,and increasing farmers’income.However,with long-term using of Bt,the diamondback moth has become increasingly resistant to Bt toxins in the field,and it is particularly critical to accelerate the research on new resistance management strategies.Using synergistic factors of Bt toxins to improve the insecticidal activity of the toxins and field control stability is one of the fastest and most effective ways to be applied in production,and it is also an effective means to effectively delay the Bt resistance of pests.About Bt toxin synergists,the studies about receptor proteins fragments is the most popular.Polycalin protein is a newly discovered potential receptor protein that can bind to Cry toxins.It has been reported in Bombyx mori Linaeus,Plutella xylostella,Helicoverpa armigera and other insects.In this study,a fragment of Polycalin from Px was obtained by PCR technology.The prokaryotic expression method was used to express this fragment.The binding characteristics of the Px.Polycalin protein fragment with Cry1Ac were detected by Western blot;the biological assay method and the Bac to Bac baculovirus expression system were used to study the synergistic effect of the fragment on the toxicity of Cry1Ac.The main findings are as follows:1.Use PCR technology to obtain the Px.Polycalin gene fragment（Pxpolycalinf）,and sequence alignment of the fragment was executed to full length of Px.Polycalin sequence（Gen Bank:MF138149.1）in the NCBI database after sequencing.The recombinant plasmid of the target fragment Pxpolycalinf and the expression vector p ET-32a（+）was constructed prokaryoticly expressed in E.coli BL21 as include bodies and purificatied.Then the binding characteristics of the Px.Polycalin fragment Pxpolycalinf with Cry1Ac were confirmed by Western blot.The results showed that the prokaryoticly express Polycalin fragment（81.48k Da）can bind to activated Cry1Ac.2.Using bioassay method to study the synergistic effect of Px.Polycalin fragment Pxpolycalinf on Cry1Ac toxicity.There was no significant difference in the mortality between the Pxpolycalinf feeding treatment group and the PBS control group by adding only Polycalin protein fragment Pxpolycalinf to the feed;setting different concentrations of Cry1Ac toxin to be fed separately,and calculating the mortality of Px,the LC₅₀ was 33.75ng/m L.1m L 30 ng/m L Cry1Ac toxin and 1μg Polycalin protein fragment Pxpolycalinf were mixed and fed to Px.Compared with the experimental group based on 1ml 30ng/m L Cry1Ac toxin alone,the mortality of Px larvae increased from 45%to 65%,An increase of 20%.After mixing with 1ug of Px.Polycalin fragment Pxpolycalinf,the LD₅₀ of Cry1Ac was 20.42 ng/m L.The monoclonal antibody of the Px.Polycalin fragment Pxpolycalinf was obtained by immunizing animals,and the monoclonal antibody blocking method was used for further testing.It was found that feeding the monoclonal antibody of the Polycalin protein fragment and Cry1Ac toxin together,the lethality rate of Px larvae increased from85%Reduced to 60%,reduced by 25%.It was confirmed by ELISA that Px.Polycalin fragment Pxpolycalinf can bind to BBMV.3.Using the Bac to Bac baculovirus expression system,the Px.Polycalin fragment Pxpolycalinf recombinant bacmid was constructed and transfected into Spodoptera frugiperda Sf9 cells for eukaryotic expression.The laser confocal test results showed that Pxpolycalinf was successfully expressed on the surface of the Sf9 cell membrane.The cytotoxicity test results showed that compared with the Sf9 cells that did not express the protein,200 n M Cry1Ac had no significant change in the death rate of the Sf9 cells that expressed Pxpolycalinf.The above results further verify that the interaction mechanism between the Px.Polycalin protein and Cry1Ac play had synergistic effects on the control of the which may provide a clue for th finding of novel materials in the resistance management of Px.