Genome Analysis And Insecticidal Gene Analysis Of Bt Strain KN11

Bacillus thuringiensis(Bt)is a Gram-positive bacteria which forms spores and crystals during its growth phase.The crystal protein owns insecticidal effection.Due to good insecticidal effection,safety,efficient,Bt has gradually become the most widely used biological pesticides.Bt preparations and transferred Bt gene crops produced by Bacillus thuringiensis have good control effection on pests such as Lepidoptera and Coleoptera,which have achieved great success in pest control.At present,the commercial strain KN11 is well known to havet good insecticidal activity against Hyphantria cunea.The PCR has been shown that the strain KN11 has four Insecticidal genes,including crv1Aa,crv1Ac,crylCa and cry 1 Da.In order to clearify strain KN11's features,explore its potential insecticidal ability.this study,strain KN11 genomic analysis by genome high throughput sequencing technology,based on regular form observation and SDS-PAGE analysis,including gene annotation and prediction,total genome comparison,more thorough identification of insecticidal genes,The insecticidal protein was counted based on the genenoic analysis,The full results are as follows:The Bt strain KN11 vegetative growth cells were rod-shaped,a large number of crystal,double cone.The SDS-PAGE analysis showed that the molecular weight of KN11 insecticidal proteins were about 130 kD and 60 kD.The main protein bands of SDS-PAGE were digested by trypsin and analyzed by LC/MS/MS.The results showed that the expressed proteins were mainly Cry2Ab,CrylAc and Cry1Ca.The strain KN11 was analyzed at genomic level.The KN11 genome was sequenced using double ended 100bp.Sequencing quality evaluation showed that the proportion of high-quality sequences was 86.63%.The further statistics of high quality base distribution of,the results show that with the increase in the number of sequencing cycles,to obtain high quality data base ratio gradually decreased,readl and read2 have the same trend,so in order to improve the reliability of genome splicing,nucleotide sequencing of read should not use too long splice.The high quality reads of genome splicing by genome splicing software IDBA-UD.The results showed that 544 contig length is more than 200bp,the genome size is about 6.42M,the maximum contig 230.28 Kb,N50 genome length is 52.41 Kb,the content of GC is 34.76%,and the same to typical Bt strain.The splicing result is submitted to genebank,the login number is NHPK00000000,the BioProject ID is PRJNA387206,and the BioSample ID is SAMN07141968.Using the NCBI Prokaryotic Genome Annotation Pipeline,the genomic data for gene prediction and annotation,the results showed that the strain encoding 6852 genes,including 6782 CDs,pseudogene 346,tRNAs gene 59,one CRISPR Array.GO enrichment of gene annotation function showed that these genes included 3 parts:cell process,cell component and molecular function.The KN11 strain was compared with the Bt genome which has been sequenced,and the similarity tree(http://tlife.fudan.edu.cn/cvtree/)between KN11 and the sequenced strain was constructed by cvTree.The results show large difference between the strain genome and sequenced Bt strains,Bt subsp.kurstaki strain HD1,Lepidoptera pests with the widely used is not in one Cluster,while other kurstaki strains were clustered in the one Cluster,including HD1,HD73 and YBT1520 strains,The KN11 strain has great difference with widely used the subsp.kurstaki strain.All the protein of Bt insecticidal gene by laboratory database BLAST comparison results showed that the strain encoding 8 Insecticidal Genes,including crylAa1,crylAc1,cry1Ca1,cry1Da1,cry1Ia1,cry2Ab1,cry9Ea1 and vip3Aal.This study used KN11 was produced by Kono corporation in WuhanPolytron Technologies Inc.The field trials was carried out against Pieris rapae,the survey results show that the same control effect between strain KN11 and 5%cascade 2000 times flufenoxuron;using 4000IU/ml strain KN11 suspension against Hyphantria cunea,the effect is very ideal,3 days after 100 times the effect is reached to 96.7%,200 times 92.6%,400 times as high as 73.3%.