| Based on the merits including high-throughput,long read,fast running,Roche 454 GS FLX becomes one of most important genome sequencing technologies.Of 60,000 Bacillus thuringiensis(Bt)strains globally preserved.12 have been completed sequenced and 24 have finished fine figures.Therefore,genomic sequence studies of Bt strains are of particularly important.In our previous studies,Bt BRC-XQ6,isolated by our lab,showed bacteriostatic effects on Listeria 100526.In this study,Bt BRC-XQ6 genome sequence was investigated by molecular biology,bioinformatics,and other technologies.The sequencing results showed that the reads were mainly 300-600 bp,indicating a high sequencing coverage and good prediction quality.After splicing,366 contigs,representing 6,541,146 bp,were obtained.There are 287 contigs longer than 1000 bp.The longest contig is 176,946 bp.Based on RNAmmer and tRNAscan-SE software online prediction,6 5s rRNA,1 16s rRNA,1 23s rRNA,and 99 tRNA were detected in the genome.31 different types of tRNA,participating in the transit of 20 kinds of essential amino acids,were existed in the life cycle of Bt BRC-XQ6.Based on blast tool,6991 genes,most of which had more than 90%identity with genes in NR database and much lower homology with those of SwissProt database,were predicted from the genome of Bt BRC-XQ6.Based on http://bcam.hzaubmb.orgBtToxin_scannerindex.php,24 insecticidal genes were predicted.Of them,21 Cry,1 Cyt and 2 Vip were found.3 and 2 of 21 Cry were primary rank and secondary rank.According to DBTBS website(http://dbtbs.hgc.jp/)transcription factors of cry genes were predicted.There are 26 transcription factors(P=1%),including DegU,Fur,Zur,PerR,PurR,CtsR,SigF,SigE,SpoⅢD,ComK,AhrC,CodY,PerR,GlnR,GltC,GltR,Hpr,Xre,TnrA,SigH,SigG,SigB,AbrB,RocR,PucR and ResD.Of them,RNA polymerase a factors,including SigB,SigE,igF,SigG and SigH were of particular important because of their regulation on the synthesis of spores.Based on http://bagel.molgenrug.nl/index.php/bagel3,2 bacteriocins were predictied.They showed highest identity with Colicin-Pyocin and Colicin-DNase.The transcriptional regulator of Colicin_Pyocin was predicted to be CodY while that of Colicin-Dnase was Fur,LevR,MntR,Mta and PucR.A pleiotropic regulatory factor plcR(855 bp)widely present in Bacillus cereus group was predicted in the genome.PlcR can activate the expression of a variety of virulence factors,such as phospholipase C.In order to lay the foundation for future study,a cry gene expression vector and a plcR knockout vector wre constructed.KEGG was used to analysis the predicted 6991 genes of Bt BRC-XQ6.There are 179 biological pathways involved in the survival of the microorganism,such as two-component system.Based on BLAST algorithm(blastx/blastp 2.2.24+),the predicted genes were compared with KEGG database and the KO numbers related to Cry proteins were obtained.A Cry protein showed highest identity with UDP-glucose 4-epimerase,involving in pphA and galE paths.KEGG analysis showed that this Cry protein indirectly associated with fructose and mannose metabolism,and a-D glucose,a-D-6P glucose,uridine diphosphate transformation.Since the genome is not fully sequenced,pathways related to other insecticidal proteins were not detected.In this study,we used Roche 454 sequencing technology to sequence Bt BRC-XQ6.It will help us to understand the insecticidal crystal genes and bacteriocins genes in the genome level,providing a good foundation research for Bt Meanwhile,a cry gene expression vectors and a plcR knockout vector wre constructed,providing the foundation for future researches. |
