Screening Of Bacillius Thuringiensis Protein Toxic To Long-horned Beetle And Preliminary Exploration Of Insecticidal Mechanism

Bacillus thuringensis(Bt)is a Gram-positive bacterium with insecticidal specificity for a variety of pests.The study of the insecticidal activity of Bt to long-horned beetles is helpful to develop a new environment friendly approach of control of the pests,and it is an important means to use biological control methods to prevent agriculture and forestry pests.In order to further explore the application of Bacillus thuringiensis in the control of forest pests,in this research,six kinds of Cry8-type crystal protein and two kinds of secreted proteins Vipl and Vip2，were used to test bioactivity against two kinds of larvae——Anoplophora glabripennis(Motsch.)and Semanotus bifasiatus(Matsch.).The Bt active protein was screened from these eight proteins,and the insecticidal mechanism was explored based on the comparative analysis of the midgut slices and the midgut transcriptome data.1.Screening of artificial diets for Semanotus bifaciatus and feed artificial indoor,which can be useful to successfully established the S.bifaciatus experimental population,and the results of this study also provide reference for the development of artificial feed of other insects(Cerambycidae).2.In this study,Cry8Hal protein,which was digested by chymotrypsin,had high activity against the A.glabripennis and the highest lethality rate was 95%.the value of lethal concentration of 50%(LC50)of Cry8Ha1 protein was 90.88 μg/g.Cry8Ga1,Cry8Ea1,Cry8Pa1 and Cry8-like protein digested with chymotrypsin had no obvious lethal effect on the S.bifasiatus,but Cry8Eal showed significant weight inhibition on the species of S.bifasiatus,The weight inhibition rate was 90.15%.3.Observed by larvae midgut transmission electron microscopy threated by Bt protein,compared with the control group,the microvilli structure and function of the midgut larvae fed with Bt protein were severely damaged,and the midgut cells rupture,perforation,the midgut loss of its normal physiological function.4.The transcriptome data of the midgut of A.glabripennis treated with Cry8Hal protein were analyzed,In the differential expression gene analysis,KEGG enrichment of unigenes received 24 metabolic pathways,which on the immune,detoxification of the metabolic pathway was significantly inhibited.The enzymes associated with protein synthesis and membrane repair are induced to be highly expressed,and the metabolism associated with biological processes is inhibited,which is the similar as the previous study of the effect of Cry3Aa-treated larvae on the level of RNA.The study also found that ALP was highly expressed in the intestines of the testes,which was widely reported as a Bt protein receptor.The key proteins expressed in the immune metabolic pathway were screened out by glutathione S-transferase,trypsin,cytochrome P450 and ABC transporter.Analysis of transcriptome data showed that these proteins were down-regulated in Cry8Ha1 protein and inhibited the detoxification and immunoregulatory pathway in the test insects,which may be directly related to the midgut of Bt protein.These genes and proteins will provide ideas and basis for further study of the insecticidal mechanism of Bt protein.