Cry1Ab13 Insecticidal Gene PCR Mutagenesis And Functional Verification

Ostrinia nubilalis and Leguminivora glycinivorella Matsumura have serious effects o n crop growth and development,which is one of the important factors contributing to th e reduction of crop yield.It is one of the effective ways to solve the pest problem by using the genetic engineering technology to transfer the insect-resistant gene into the pla nt body and cultivate the insect-resistant traits.Bacillus thuringiensis(Bacillus thuringiensis,Bt)in the formation of spores at the s ame time can produce Lepidoptera,Coleoptera and other pests are toxic crystal protein,these insecticidal activity is high,specific strong,is widely used in pest comprehensive control,But with the expansion of the area gradually expanded,under the pressure of i ts continuous selection,the pests of these active protein resistance,resulting in foreign p lants into the gene resistance to insect resistance weakened.Therefore,in this study,mut ations were introduced into the base sequence of Cry1Ab13 gene by random-error PCR.The mutant library was constructed and the mutant gene was constructed and expressed in vitro.The heterologous expression protein was expressed in vitro.And to identify the insecticidal activity of insecticidal activity,and to improve the insect resistance and enri ch the gene resources by using Cry Bt gene.The main results of this study are as follows:1.Error-prone PCR mutagenesis and construction of mutant libraries.The Cry1Ab13 gene was mutagenized by the error-prone PCR technique,and three mutants were obtai ned by screening.The mutant was ligated with pMD18 T Vector and transformed into E.coli DH5? and coated on LB solid plate to form a mutant library.The nucleotide sequ ence of the mutant gene was 97.79%,97.93% and 98.32%,respectively,and the amino acid sequence of the Cry1Ab13 gene was 96.97%,97.12% and 97.68,respectively.2.Sensitization analysis.The results showed that the three mutant genes were less th an 35% homologous to the known allergen sequences3.Three prokaryotic expression vectors were constructed and induced in E.coli.Th ree heterologous recombinant expression vectors were pET28a-Cry1Ab13-1,pET28a-Cry1Ab13-2,pET28a-Cry1Ab13-3,respectively,by using seamless cloning technique.E.coli BL21(pET28a-Cry1Ab13-1),E.coli BL21(pET28a-Cry1Ab13-2),E.coli BL21(pET28a-B)were transformed into E.coli BL21,and the constructed recombinant expression vect or was transformed into E.coli BL21.Cry1Ab13-3).SDS-PAGE analysis showed that th e E.coli BL21(pET28a-Cry1Ab13-1)and the non-mutated gene control E.coli BL21(p ET28a-Cry1Ab13)were expressed at about 79.5 kDa,respectively,after SDS-PAGE anal ysis using IPTG.Specific bands,consistent with the expected size,indicating that the str ain was correctly expressed.4.The bioassay of the insecticidal activity of the larvae was 18.18%,33.31% and 87.88%,respectively,which were significantly higher than those of the control group(P <0.05).Non-mutated gene-treated group.The results showed that the mortality of lar vae in experimental group was 12.22%,23.89% and 81.11% at 24 h,48h and 72 h respec tively.And the growth and development of surviving larvae were seriously inhibited.Aft er 72 hours,the weight of live insects was only 0.18 g,7% before treatment,and 0.25%of the live animals in the empty control group was 98% before treatment.