The Whole Genome Bioinformatics Analysis Of Bacillus Pumilus GBSW19 And Cloning Cytochrome P450 Encoding Genes

The genus Bacillus is comprised of Gram-posivite with an aerobic or facultaively anaerobic metabolism.The Bacillus spp.,produce various metabolism product to promote the growth and enhance the resistance of the plant,which is one of the most plant growth promoting rhizobacteria(PGPR).In this study,we found that Bacillus pumilus GBSW19 partcipate in crop residue incoipation to rotation field under low temperature,We detected the soil organic matter,total nitrogen,available nitrogen,total potassium,available potassium and available phsphorus.We also explored the growth characteristic of Bacillus pumilus GBSW19 under different stress conditions and complete the genome-sequencing and bioformatics of GBSW19.Furthermore,three P450 coding genes were successfully cloned from B.pumilus GBSW19 which can be a foundation for further study.The efficient crop residue-decomposing strain Bacillus pumilus GBSW19 was applied in field during the period on December 2013.The results indicated that in winter wheat field,the degradation rate at 30 d was 39.7%,whereas the degradation rate of control at 30 d was just 24.4%.We can get that the degradation of crop residue treated with GBSW19 significantly excelled other treatments.In addition,the application of GBSW19 changed the acidic soil to neutral pH and significantly improved the total nitrogen,available nitrogen and available phosphorus content in soil compared with other treatments.Furthermore,we want to explore the growth characteristic of GBSW19 under different stress conditions.When we cultivated GBSW19 at pH=4.0 preliminary,GBSW19 can grow better than other strains,the amount can get 109CFU/ml,it also change medium' pH at a large degree,When cultivated GBSW19 at pH=10.0 preliminary,GBSW19 can change medium pH at a large extend after cultivated for about 12h,This showed that GBSW19 had a better growth ability in acid and alkaline medium.No matter cultivated GBSW19 under 4? or 10 ?,it had a high growth rate.In addition,GBSW19 can resistant to peroxide stress.Therefore,Bacillus pumilus GBSW19 has great value in future application.The genome size of Bacillus pumilus GBSW19 is 3643817bp which contain 3835 ORF.The annotation of COG.GO.KEGG function was analysised which can help us to understand biological function,cellular component of GBSW19.B.pumilus GBSW19 have 340 specific genes and 2 unique gene families when compared with reference sequences,GBSW19 also have 9 secondary metabolism gene clusters..There are 14 genes may associated with response to acid and alkaline stress.17 genes are associated with forming stress protein,41 genes are parpicipate in forming spore coat,these genes can enhance the resistance to different stress conditions,B.pumilus GBSW19 also have 21 genes which may improved the resistance of GBSW19 under oxidative stress.Nine cellulose and hemicellulase coding genes were identified from B.pumilus GBSW19.The whole genome of Bacillus pumilus GBSW19 have been sequenced,When analysis the data,we found three cytochrome p450 genes in the genome,Three genes were obtained by PCR,To produce the proteins of three genes,we express them in E.coli.The corresponding fragments which were cloned into pET30a(+)and allowed fusion with His-tag-encoding nucleotides at 5,terminal.Then the recombinant strains were induced by IPTG and got the crude proteins after lysed by sonication.The crude proteins were purified by a single chromatographic step using Ni2+-NTA metal-chelating affinity chromatography.The size of purified proteins were tested by SDS-PAGE and were identical to the calculated vavule.We also detected the degradation function to DON toxin by P450.