Study Of Bacterial Quorum Behavior Related Genes In Bacillus Amyloliquefaciens PEBA20

Bacteria grope behavior is a regulatory mechanisms which is increasingly widely attentioned. Many bacteria have this ability, the main characterized is quorum and biofiom. In this research, we isolation and characterization of an AHL lactonase gene and the construction of sinR gene mutation strain which is relacted in biofilm formation, the main research results are as follows:1. An AHL lactonase gene (aiiA) was amplified from the genomic DNA of Bacillus amyloliquefaciens, with the intact open reading frame of 753 base pair. The gene shares a high identity to its homologues present in different Bacillus species. This gene was first obtained from B. amyloliquefaciens.2. aiiA gene express vector was constructed and the gene was overproduced in Escherichia coli BL21 (DE3). The product expressed resulted in attenuation and suspension of the infection of Pectobacterium carotovorum subsp. carotovorum on carrot. This study verified the existence of the aiiA gene in B. amyloliquefaciens and provided a prospect of the strain as biocontrol agents with quorum quenching property on bacterial disease control.3. The yqxM-sipW-tasA operon was cloned and the sequenced results show that yqxM operon have 2078 base pair, the intact open reading frame of tasA gene is 786 base pair,and the open reading frame of yqxM is 672 base pair. The BLAST research of the three genes in yqxM operon show that tasA gene is widespread, the identity is 65%-98%. sipW gene and yqxM gene only found in B. amyloliquefaciens and B. subtilis and B. licheniformis.4. The pEASY-T1 empty vector was construct and amplification the kanamycin resistance gene (kna) with the plasmid of pEASY-T1 as template. Connect kna gene with pMD18-T and transferred into E. coli DH5α, Finally, we succeed in obtaining the recombinationg strain contain pMD18-T-kna and the kna resistance gene was successfully expressed.5. An integration plasmid pMD18-T-kna-sinR for the sinR gene disruption in B. amyloliquefaciens PEBA20 was contructed. The plasmid contained upstream region of the sinR gene 650bp and the sinR gene downstream 600bp homologous fragments with insertion of the expression unit of the kna resistance gene between them. Upstream and downstream DNA fragments were synthesized by PCR fom B. amyloliquefaciens PEBA20 DNA.6. The constructed gene targeting vector was linearized with HindⅢ, and electroporated into competent cells of PEBA20. Positive transformaers were screened from the 50μg mL-1 kna resistance plates. 16 transformers were analyzed by PCR to identify sinR gene deletion. Among them the transformers number 6 is detected target band, this research show that the sinR gene disruption vector is successfully recombinationed. Biofilm phenotype analysis showed that, sinR mutant formed a more complex biofilm structure than wild strain.