Analysis For Expression Vector Construction In Bacillus Firmus And Bacillus Pumilus

Pribiotics have been widely utilized in industry product and marine culture, ofwhich Bacillus as a significant member creates great economic profit in large-scaleculture of shrimp, which acts as addictive of microbial ecological agent of bio-floc.With the frequent outbreak of diseases in marine animal culture, a technology nameddisease prevention and control of microorganisms is recommended, thus Bacillusbecomes popular for its outstanding advantages. Bacillus firmus and Bacillus pumilusare both wild strains isolated from healthy sea water. To explore a novel access towhite spot syndrome disease (WSS) control, possibilities are to analyze of expressionvector system construction in them. This paper will provide useful approaches forselecting proper hosts harboring an expression vector, and elements to construct astable and highly effective expression-system.This research includes four parts.(1) Cloning and analysis of the polymorphismof the16S-23S rDNA intergenic spacer regions of B. firmus.(2)Electro-transfomation of B. firmus.(3) Cloning of genes p43and hag from B. firmus,and establishing an specific detection method for B. firmus.(4) Potential analysis forapplication of B. pumilus in expressional vector construction.The following are representative results in detail.1. Using16S rDNA sequence analysis and phylogenesis, strains of PC004andPC024were identified B. firmus. Their16S-23S rDNA intergenic spacer regions (ISR)were cloned by PCR. The sequences of11typical bands of ISRs (the length including300bp,400bp,500bp, and600bp, etc.) were obtained from the2strains of B. firmus.Analyses suggested that these sequences contain4types of polymorphic ISRs, namely,ISR0, ISRG, ISRIAand ISRGLV. The types ISR0, ISRG, and ISRIAmay widely exist in B.firmus. Furthermore, the comparative analysis showed the sequences of the same typeof ISRs have higher identity percentage (varying from52.2%to100.0%), while thesequences of different ISR types differentiate greatly. Additionally, the hypotheticalconserved domains different in length flanking the16S and the23S rDNAs may be candidates for targets, according to which the species-specific PCR primer set and/ordetection probes are possibly designed.2. B. firmus was electro-transformed using vector pAD4412, and competent cellswere prepared applying the method reported by Xue et al in1999. However, nopositive recombinat was obtained, indicating a failure.3. Firstly, p43gene of431bp in length was coloned from B. firmus. Secondly,flagellated B. firmus was stained using basic fuchin reported in a simplified Leifsonmethod (Clark,1976). The results indicated2flagella attaching to an end of a cell.Thirdly, eighteen peptides were identified from the protein flagellin of B. firmus usingLC-MS/MS. Flagellin coding gene hag was completely isolated combining FCD-PCDand chromosome walking method. The complete sequence in length of1661bp wassubmitted to NCBI (GenBank Accession No. KC683536). An open reading frameencoding414amino acids with calculated molecular weight of44.2kDa. The analysisof gene hag architecture denoted that the promoter core elements-10region and-35region were specificly recognized by σD, that upstream A+T rich region mightstimulate the initiation of transcription, and that the spacer between the last “G” ofdownstream ribosome binding site (RBS)(GGAGG) and intiation codon ATG was9-nt. Promoter Phag of possible activity was predicted. Besides, a segment ofpeptides encoded by nucleotide sequence was contained in gene hag of B. firmus. Theprotein structural model of flagellin was simulated using I-TASSER. Moreover, aspecies-specific nest-PCR detection method for B. firmus was established on the basisof two primer sets, Bfspec and Bfspec2, which were designed according to thespecific nucleotide sequence contained in hag.4. Bacillus pumilus (201005130501) showed susceptive to ampiciline accordingto the antibiotic susceptibility assay. Utilization about95kinds of carbon sorceassessed by BIOLOG showed it a starch-and arabinose-defective strain. The averagegeneration time was45.51±0.11min. A cryptic plasmid pBP6000, a member of smallrolling replicating plasmid, was isolated from B. firmus, and belonged to pC194family. Its complete sequence was sequenced by BGI (Qingdao) and submitted toNCBI (GenBank Accession No. KC683537). The plasmid was5664bp in lengthconsisted of four orfs, encoding such as replication initiation protein, DNA binding protein and aspartate phosphatase response regulator. PcrA gene (GenBank No.KC683535), encoding DNA helicase which is required for the replication of plasmidpBP6000and the control for the plasmid copy number (PCN), was isolatedThis kindof wild plasmid would provide backbone and element for vector construction toenrich available vector source. Nevertheless, plasmid curing agents such as SDS andacridine orange (AO) failed to knock out the plamid pBP6000.To sum up, in this research the polymorphism of the16S-23S rDNA intergenicspacer regions of B. firmus was elaborated, and the distingishment was analyzed fromgene level between this two strains. In addition, two genes p43and hag were clonedfrom B. firmus, and a species-specific method for detection was established based onnucleotide sequence of hag. With exception no achievement of constructingexpression system in Bacillus, the failure experience would facilitate the breakthroughof this aspect, and provide usefull analysis method. However, reasons for failure ofpartial research need deeper analysis, and approaches to them are to be harder thoughtabout, better concluded and more discussed.