Genome Sequencing Of Paenibacillus Mucilaginosus 3016 And Screening/identification Of Special Molecular Marker At Species Level

Paenibacillus mucilaginosus is consequently widely used as microbial fertilizer in agriculture. P. mucilaginosus is a multi-function bacteria, such as phosphorus-solubilizing, potassium-releasing, nitrogen-fixing and phytohomone-producing etc. So it has been the focus and hot spot in the field of microbial fertilizers.In this paper, 14 strains of P. mucilaginosus were selected and identified using morphological, physiological and biochemical characteristics, 16S rDNA and gyr B gene sequence. A strain, which have efficiently multifunction including phosphorus-solubilizing, potassium-releasing and nitrogen-fixing, was selected and named as P. mucilaginosus 3016. Results showed that the concentration of soluble phosphorus and potassium were increased to 187.8% and 131.4% in the fermentation broth, respectively. The value of nitrogenase activity was 3.51nmol (C2H4)/h·mg (protein). It is able to produce more than 3.74mg/L, 0.03mg/L and 1.01mg/L Citric acid, IAA and GA3 into the medium, respectively. And the malic acid and acetic acid production were increased 89.9% and 277.7%, respectively.Sanger sequencing and 454 high-throughput sequencing were combined to be employed to complete the genome sequence deciphering of 3016. The detailed map of the whole genome was obtained by using the software of SOAP de novo v1.04 and 454 Newbler v2.3. By means of genome sequence assembly, gap filling and low quality sequence verification, the whole genome sequence map was completed. And the genome was annotated by the bioinformatic methods.A pair of primers were selected which can determined the P. mucilaginosus rapidly and specifically. The primer orf06701-F sequence is 5'-ATGGAGGAAACATGGGGTGA-3' and orf06701-R sequence is 5'-TCAGGAATGAAGGCC CCCTT-3'. The rapid identification was successfully established by optimization of PCR, specificity and sensitivity determination. Results showed a single band (333bp) was consistently amplified from all the strains of P. mucilaginosus tested and negative results were obtained from all the reference strains. The PCR detection limit was 400-1000 cells per assay, indicating the sensitivity of the rapid culture-PCR method. To verify the rapid identification, soil was sampled and cultured. Five of 11 soil isolates were rapidly identified as P. mucilaginosus by the method, and the similarity of 333bp sequences were 100%. In this study, a multifunctional efficient strain was screened named as P. mucilaginosus 3016, which would provide theoretical guidance and technical support in production and application of the microbial fertilizer. And the special primer was identified and the rapid identification was successfully established, which would provide technical supports for the production and ecological evaluation of microbial fertilizer based on P. mucilaginosus.