The VB₁₂ Fermentation And PhbC Gene Research Of Sinorhizobium

Sinorhizobium is a kind of aerobic, heterotrophic, gram-negative bacteria which can be frequently found in soil and work together with Leguminous Plants to fix nitrogen. Now researches mainly focused on the relationship between S. and Leguminous Plants, the rhizobia inoculant, the efficiency of nitrogen fixation and the environmental restoration both at home and abroad.In this paper, we confirmed that the bacteria in our laboratory is one of Sinorhizobium through the 16SrDNA sequence analysis. It is a new-found bacteria and named S. sp. kd23. Both VB12 and poly-hydroxybutyrate (PHB) were found during the fermentation. It seems that VB12 and PHB have inner relationship during their synthesis for the accumulation of PHB earlier and disintegration later. So studying the inner link of the VB12 and PHB analysis have important theory value. So we speculated that the production of VB12 would be increase effectively if the 3-hydroxylbutyryl CoA was accumulated via the knockout of phbC gene.The growth of S. sp. kd23 and VB12 fermentation were studied using conventional process optimizing method. The results showed that the optimal carbon sources for the fermentation was maltose and the best nitrogen sources were corn steep liquor and betaine. It was helpful to the yield of VB12 by keeping sugar at 20 g/L. Cobalt ions played a decisive role in the fermentation process.120 mg/L of VB12 was achieved for 192 h fermentation.The gene phbC which play a critical role in the synthesis of PHB was studied using molecular biology method and gene manipulation techniques. We obtained a new phbC gene of 1859 bp through the conventional and uneven PCR. The identity is 84% with Sinorhizobium meliloti Rm41.The pPHBCAT plasmid vector with chloramphenicol resistance gene and phbC gene homologous arms was constructed via molecular biology techniques. It provided materials for the knockout of phbC gene.There is no report about the bacteria S. sp. kd23. The genetic engineering system was bulit for studying the relationship between PHB and VB12. The 10μg/L chloramphenicol could be used for the screening by antibiotic sensitivity test. The PCR product containing phbC homlogous gene and chloramohenicol resistance gene was used for electroporation. 13 transformants per μg DNA could be obtained at 25 μF,200 Ω 2.2 kV with 2 mm cavette. The PCR results showed that we got the phbC gene silenced mutants.