Diversity Research Of Bacillus Thuringiensis Based On The Genomic Characteristics

Bacillus thuringiensis(Bt)belongs to Bacillus cereus sensu lato.It can produce insecticidal proteins with specific and high toxicity against many insects and plays an important role in the control of agricultural,forestry and medicine pests.In addition,in recent years,some other new functions of Bt have been explored,including resistance to nematodes and some mite and tick species,antagonistic effects against some pathogenic fungi and bacteria,plant growth-promoting activities,biosynthesis of metal nanoparticles,and environment bioremediation.And the functional diversity of organisms is determined by genetic diversity.However,the existing typing methods based on genetic diversity can not meet the need for effective typing of strains,such as 16 S r DNA sequence diversity analysis.Therefore,in this study,we analyzed the diversity of Bt at several levels based on the genetic diversity of strains.Firstly,we obtained an efficient ty ping algorithm by screening parameters such as resolution ability and computational efficiency.On this basis,we established a new Bt strain evolutionary typing system.Furthermore,the Bt potential function and the insecticidal activity against Coleoptera pests were analyzed by using the strategy of functional gene annotation,and the characteristics of functional evolution of Bt strains were studied based on the results of strain evolution analysis.In addition,the genome of Coleoptera scarab pests with serious damage and unsatisfactory control effect was studied,which provided a foundation for further analysis of the tolerance mechanism to Bt infection from the genetic point of view.The main results of this study were as follows:(1)Establishment of a new Bt typing system and evolutionary analysis.Firstly,a small number of Bt strain samples with complete genome data were analyzed.And the results showed that the gene mutation of Bt occurred uniformly on chromosomes.Next,we evaluated four typing methods by using five common standard serovar(aizawai,kurstaki,israelensis,thuringiensis,morrisoni,23 strains)Bt strains.The results showed that the analysis method based on conserved core gene base sequence diversity(multi-locus sequence typing MLST and single-copy core genes phylogenetic analysis SCCGPA)might not have sufficient discriminatory power to further differentiate these Bt strains from the same serovar.However,the method based on genome composition characteristics(dispensable genes pattern analysis DGCPA and composition vector typing CVT)showed enough resolution between strains that belong to the same serovar,and the running time of CVT was also faster than the other typing methods.Furthermore,we established the evolutionary typing method of Bt strains based on CVT,and analyzed the evolutionary characteristics and genetic diversity of 190 Bt strains.(2)Evolutionary characteristics analysis of potential functions of Bt variable genes.Variable genes(dispensable genes and specific genes)are the genetic basis of functional diversity of strains.Bt and B.cereus,which belong to B.cereus s.l.,have similar evolutionary and genetic characteristics except for their differences in producing insecticidal genes.We used the strategy of functional gene annotation to analyze the potential functional activity of Bt variable genes,and combined with the results of strain evolution analysis,and studied the potential functional evolution characteristics of Bt.Firstly,the variable genes of 150 Bt strains and 150 B.cereus strains were identified by pan-genome analysis.A total of 38,357 gene clusters were identified,which including 239 core gene clusters,accounting for 0.62% of the total gene clusters,and 38,118 variable gene clusters,accounting for 99.38% of the total gene clusters.Next,we analyzed the diversity of functional gene and insecticidal genes in these variable gene clusters through sequence alignment method.The results were as follows: a,93 different insecticidal genes wer e found in Bt.Most Bt strains(76%,N = 150)carried 1-12 different insecticidal genes.In addition,insecticidal genes were also found in 14 B.cereus strains,which may be caused by contamination or plasmid exchange;b,Enterotoxin,hemolysin,chitinase,collagenase,lanolin sulfobacillin,immunosuppressive factors and other functional genes(clusters)related to nematocidal activit y were also found in Bt and B.cereus strains.Bt also contained insecticidal protein gene with nematocidal activity.And Bt(53.07%,N = 5,851)carried more genes than B.cereus(46.93%,N = 5,851)strains;c,only Bt strain 4BR1 contains a unique class o f cry3 A toxin genes with miticidal activity;d,Bt and B.cereus all contained antifungal-related functional genes(clusters),such as fengycin,surfactin,iturin and fusaricidins genes.Bt(55.41%,N = 1,285)carried more genes than B.cereus(44.59%,N = 1,285);e,Bt and B.cereus all contained antibacterial-related genes,such as bacteriocins and Aii A degrading enzymes genes.And Bt(54.75%,N = 1,989)carried more genes than B.cereus(45.25%,N = 1,989);f,Bt and B.cereus all contained PGPR-related genes,including siderophores,phosphoric acid,polysaccharide,lactic acid,proline,IAA,citric acid,succinic acid,nitrogen metabolic signal transduction protein,2,3-butanediol,ammonium transporter protein,ACC deaminase,oxalic acid,carbamyltransfe rase genes.There are also some auxin export vector genes in Bt,and a specific phytase gene in B.cereus.Overall,Bt(49.80%,N = 16,255)carried less genes than B.cereus(50.20%,N = 16,255);g,Bt and B.cereus all contained nitrate reductase genes(clusters),related to NPs biosynthesis.And Bt(50.97%,N=1,336)carried more genes than B.cereus(49.03%,N=1,336);h,Bt strains Sbt003 and T13001 carried cry65 Aa toxin genes that inhibited the activity of cancer cells.In addition,we predicted 167 potential secondary metabolite synthesis gene clusters in these variable genes through anti SMASH 3.0 database,which were divided into 15 categories(number in parentheses represents the corresponding number),namely arylpolyene(3),bacteriocin(6),furan(1),ladderane(3),lanthipeptide(37),NRPS(89),NRPS,T1PKS(2),NRPS-like(11),Other(1),PKS-like(1),sactipeptide(3),siderophore(3),T1PKS(4),T3PKS(1),terpene(2).At the same time,KEGG and COG function annotation found a large number of genes with unknown functions.These genes might carried important information related to the functional diversity of Bt strains.In addition,the functional evolution characteristics analysis of 150 Bt strains showed that most of the functional genes(clusters)were distributed in Clade II(71.93%,N = 14,158),a small part genes were distributed in Clade I(21.83%,N = 14,158),and the remaining functional genes(clusters)were distributed in Clade III(5.02%,N = 14,158)and Clade IV(1.21%,N = 14,158).Clade I,Clade II and Clade III strains carried 77-102,75-109 and 85-100 different functional genes(clusters),respectively.Both two strains of Clade IV carried 86 different functional genes(clusters).These results indicated that Bt had a large number of potential functional genes(clusters)to be discovered.Therefore,further exploring of new functions of Bt and application in new fields will be of great significance to promote the efficient utilization of Bt resources.(3)Evolutionary characteristics of Bt Coleoptera insecticidal activity.In recent years,the damage caused by Coleoptera pests is more and more severe.By inquiring the related literature and patent data,we analyzed the diversity of insecticidal protein genes against Coleoptera pests and studied the evolutionary characteristics of Bt Coleoptera insecticidal activity combined with the results of strain evolution analysis.Currently,the most destructive coleopteran pests are mainly from Scarabaeoidea and Chrysomeloidea superfamily.a,Cry3,Cry7,Cry8,Cry23/37 and Vip1/Vip2 from Bt,Cry18 from B.popilliae(Type A)and Cry 43 from Paenibacillus lenorbus(Type B)have been reported to demonstrate insecticidal activity against Scarabaeidae pests;b,Cry1,Cry 3,Cry34/35,Cry55,Cry6,Cry7,Cry8,Cyt2,Sip,Vip1/Vip2,Vip2 from Bt,IPD072 Aa protein from Pseudomonas chlororaphis,AFIP-1A/1B protein from Alcaligenes faecalis and P IP-47 Aa protein from P.mosselii have been reported to demonstrate insecticidal activity against Chrysomeloidea pests.This also provided a new perspective and ideas for the future control of Coleoptera pests.In addition,the evolutionary characteristics of insecticidal activity of 150 Bt strains showed that 45 insecticidal protein genes were related to the control of Co leoptera.Most of them were distributed in Clade II(75.56%,N = 45),and the remaining 11 genes were distributed in Clade I(24.44%,N = 45).Moreover,these Coleoptera insecticidal protein genes in Clade II were concentrated in CVT II 13(29.41%,N=34),CVT II 3(26.47%,N=34)and CVT II 7-8(17.65%,N=34)strains.(4)Genome studies of scarab insects.Scarabs show the strongest tolerance to Bt insecticidal proteins.In order to explore the tolerance mechanism and improve the control effect of these insects,we studied the genomes of two species of scarab insects,Protaetia brevitarsis and Holotrichia oblita.Firstly,k-mer analysis showed that the estimated genome size of the two insects were 810 Mb and 1.34 Gb,and the heterozygosity was 2.35% and 2.17%,respectively,indicating that the two insects had complex genomes with high heterozygosity.In order to solve the assembly problem caused by high heterozygosity,we further designed a hierarchical assembly stratagem,which separated the heterozygous allele sequences to ensure the assembly quality.Based on this method,we have completed the genome assembly and annotation of the P.brevitarsis,solved the current predicament of lack of genetic resources in the research of this insect,and provided valuable data resources for future research on the genetic characteristics and control of this insect.In conclusion,a new evolutionary typing system for Bt strain s was established based on CVT,and the evolutionary analysis of Bt strains was carried out.Furthermore,the potential function of Bt and the Coleoptera insecticidal activity were analyzed by using the strategy of functional gene annotation,and the characteristics of Bt strain's functional evolution were studied based on the results of strain evolution analysis.The results showed that there was a certain correlation between the genetic characteristics and functional characteristics of Bt strain,which pr ovided a basis for further research and utilization of Bt strain resources.Moreover,the new strain typing system CVT could effectively analyze the diversity of strains,help to further remove the redundant strains in the strain resource bank and screen new strains for gene discovery,thereby improving the efficiency of gene discovery including insecticidal protein genes and potential functional genes.In addition,in order to improve the control effect of Coleoptera scarab pests,genome sequencing and research were carried out on the P.brevitarsis,which provided a foundation for further analysis of the tolerance mechanism to Bt infection from the genetic point of view.