Using Phage-assisted Continuous Evolution To Evolve Bacillus Thuringiensis Toxin-Cry51Aa1

Directed evolution is based on the mechanism of natural evolution,which increases the frequency of gene mutation and produces a large number of mutations.In combination with the actual needs,selective pressure is directed to screen out proteins with expected characteristics.Phage-assisted continuous evolution(PACE)system is able to run continuously,automatically and rapidly,and complete several generations of evolution in a short time without human interference.Hemiptera and Lepidoptera insects are important pests on forestry,including Apolygus lucorμm,as a representative of the Miridae,Trichoplusis ni as the representative of the Noctuidae,different kinds of hosts that include forest trees,fruit trees and so on,the harm seriously affect the growth of trees and the utilization value of lumber.Compared with traditional pest control methods,biological control has the advantages of strong selectivity,safety and protect the ecological balance,Bacillus thuringiensis insecticidal crystal protein is a kind of toxin protein that can effectively kill Lepidoptera and other agricultural and forestry pests,and Bt crops have been planted worldwide,such as Nicotiana tabacum,Gossypium spp and Populus.However,with the increasing resistance of related pests,the resistance ability of Bt toxin protein transgenic crops and the success of pest control strategies are decreasing,which is a disaster for some forest species with precious germplasm resources.This problem can be solved by enhancing the toxicity of Cry toxin.There are two ways to enhance the toxicity of Cry toxin: one is to combine with the toxin protein to use other proteins that inhibit insect growth;the other is to modify the toxin protein.Our laboratory intends to use PACE phylogenetic Cry51Aa1 toxin protein(a Bt toxin protein with specific insecticidal activity against Coleoptera and Hemiptera)to obtain a toxin protein with stronger insecticidal ability and higher specificity,it can effectively control the number of pests,cultivate new varieties of trees with high quality and strong insect resistance,and improve the breeding level of timber,it provides a new and breakthrough technology for tree insect resistance breeding.On the research,this topic first use the immune precipitationmass spectrometry(IP-MS)technology,then establish Apolygus lucorum protein database,,analyzes the Cry51Aa1 toxin proteins in the intestinal receptors,found Apolygus lucorum protein database and there is no common Bt toxin protein receptors,so choose Trichoplusia ni the moth Tn TBR receptor proteins in the body.After testing the PACE system,according to the principle of PACE system evolution Cry51Aa1 toxin proteins.The following results were obtained through experiments and analysis:1.There were no common protein receptors,such as cadherin,aminopeptidase,alkaline phosphatase and so on in the toxin-protein interaction protein Cry51Aa1 searched in the protein database of Plant bug.2.In the phage assisted continuous progression(PACE)system test,the amino acid at position 87 of HA4 protein was successfully transformed from arginine to tyrosine.3.Amino acid mutation sites: I17 T,D32D,V87 V,L115S,G182 D,and V254 L were obtained by using PACE system evolution toxin protein Cry51Aa1 through gene sequence comparison.4.The hydrophilic hydrophobicity of wild-type and evolved toxin proteins was analyzed using Ex PASy software.Compared with the wild-type,the hydrophilicity of the evolved Cry51Aa1 increased,and the mutant amino acid sites I17 V and L115 S affected the hydrophilicity of the proteins.Tmpred method was used to analyze the transmembrane region of wildtype and evolved proteins,and the transmembrane region and direction of wild-type and evolved proteins were different.