Analysis Of Bacillus Thuringiensis Genome And Toxic Protein And Their Information Platform Construction Based On NGS

Bacillus thuringiensis(Bacillus thuringiensis,Bt)is an aerobic,spore-forming,gram-positive bacterium that can produce insect-pathogenic crystal proteins ?-endotoxin(Cry)during spore formation.These Cry proteins are activity to a wide variety of insect pests,such as Lepidoptera,Coleoptera and Diptera.Bacillus thuringiensis is known as the most successful microbial insecticide in agriculture and medicine.In addition,the Bt toxin gene has also been used effectively to enhance the resistance of insect pests to genetically modified crops.Over the past few decades,Bt and its toxins have been the most studied biocides.However,some pests are or have been resistant to the commonly used insecticide based on Bacillus thuringiensis,thereby reducing the efficacy of Bt-like insecticides.Therefore,it is urgent to find new Bt strains and toxin proteins with different modes of activity and specificity,which can perpetuate the insecticidal potential of this bacterium.With the development of next-generation sequencing techniques and the introduction of new omics5 concepts,more and more Bacillus thuringiensis has been sequenced,and mass spectrometry analysis of the proteome provides an unprecedented opportunity to identify new strains and new toxin protein.However,integrating and digging these high-throughput data is a very important challenge for experimental biologists,so it is necessary to develop an accurate and efficient platform for experimental scientists to identify new Bt toxins.And genome comparison analysis can enhance our insight to the genome diversity among Bacillus species and the diversity between different Bt subspecies strains,which is of great significance for deep understanding of the genomic characteristics and gene function of Bacillus thuringiensis.In this study,the whole genome sequencing of the six Bt isolates isolated from different regions was carried out.And the completed genome information of Bacillus thuringiensis,which was published before June 2016,was used as reference genome.Using the improved assembly procedure scaffolds were further assembled to construct the genomic draft of these six strains.Then we predict and annotate the coding genes and proteins in these strains'genome.With the functional analysis of these predicted genes,an average of 56.3%of the total CDS can be annotated.The genomic structure of the six Bacillus thuringiensis isolates was analyzed and constructed the genome draft of these six Bt strains.Analysis of genomic characteristics can provide a theoretical basis for the subsequent identification of toxic proteins.Secondly,this study focused on the functional analysis of the predicted genes of Bacillus thuringiensis strain S2160-1,which may lay a theoretical basis for the subsequent identification of toxic proteins.Toxicological test showed that S2160-1 had high mosquito activity.In order to identify and study the toxin proteins in Bt S2160-1,its predicted proteins were blast to Bt toxin proteins database containing 653 amino acid sequences retrieved from the public resources(e-value=0.00001).As a result,46 candidated toxin genes were identified in S2160-1 genome.In order to screen these candidated toxin genes,the primary structure,secondary structure and tertiary structure of these candidates were analysized respectively.The physical and chemical properties analysis results showed that the length of them ranged in 42?1216 amino acids,protein molecular mass was between 4.86 kDa and 137.28 kDa;and the isoelectric point ranged from 4.3 to 10.06,16 pH value of which was larger than 7.Further,tertiary structure domain and PFAM domain analysis displayed that 13 candidated toxin genes in plasmids genome had a typical structure of endotoxin domain:Endotoxin_N,Endotoxin_M,delta_endotoxin_C.Therefore,13 encoding toxin genes were in Bt strain S2160-1.In order to investigate the evolution relationship between these 13 toxins and other Bt toxins,multiple alignment and phylogenetic analysis were carried out among 90 toxins from 11 Bt species and 13 candidate toxins in S2160-1 with MEGA.In addition,the evolutionary relationship between the predicted eight toxin proteins and the known toxin proteins was also studied.The phylogenetic analysis might provide reference for the classification of toxic proteins.With the comparative study,a relatively complete database can be constructed and used to transform hereditary material,which promoting the identification and development of toxic proteins.This study might provide reference for the identification of new toxin proteins with different modes of action and targets,it also might provide an important theoretical basis for the construction of highly toxic engineering bacteria and the application of proteomics in life sciences.Secondly,Bacillus thrringiensis,related Baccillus athracis and closed Bacillus cereus were compared and contrasted,based on publicly available complete genome sequences.The analysis included 17 Bacillus thuringiensis genomes,11 of which were obtained from the public database,13 strains of Bacillus cereus and 8 Bacillus anthracis genomes.The pan-and core genome of each species was defined.And the pan-and core genome of the Bacillus genus,including 17 Bacillus thuringiensis genomes,Bacillus anthracis reference genomes and the Bacillus cereus reference genome was also defined.The number of gene families necessary and the average number of gene families in the three species have been shown to have little difference which resulted in the relationship between the three species is relatively close.Pairwise BLASTP genome comparison was performed within and between strain species,which showed that the Bacillus thuringiensis strains shared less genes than the other two strains.It was also showed that the variation of the genome and nuclear genome curve of Bacillus thuringiensis strain was stronger than that of the other two strains.The clusters of orthologous group classes of all genes in the pan-and core genome of Bacillus thuringiensis and Bacillus anthracis were compared to compare their pan-and core genomes.This study shows how bioinformatics tools ean be used to make a simple eomparison of a wide range of genomes and to gain insight into unknown phenomena through known information.Finally,this study collected the whole genome data of Bacillus thuringiensis and the genomic information and proteomic data of the isolates isolated from our laboratory.Then the Bacillus thuringiensis omics database BtOmics was developed and constructed to facilitate experimental scientists.BtOmics provided the storage of Bacillus thuringiensis genomic information and other information for the experimental scholars.At the same time,we also developed a toxic proteins database of Bacillus thuringiensis BtoxinDB,which collected all information about known toxic proteins with manual excavation and literature search.BtoxinDB provided an online software to predict potential crystal proteins based on sequence similarity.Two databases about the Bacillus thuringiensis database can not only provide storage and query for laboratory data,but also facilitate the sharing of data and experimental scientists to quickly and easily engage in Bacillus thuringiensis bioinformatics analysis.