Pseudomonas Sp.wj1 Transcriptome Sequencing And Functional Analysis Of Inositol Monophosphate Gene(IMP)

Phosphorus is one of the essential nutrients needed for life activities,and plays a key role in biological metabolism.Soil phosphorus solubilizing microorganisms make the soil of poorly soluble phosphorus converted into soluble phosphorus which are easily absorbed and used by plants.It can significantly improve the soil phosphorus content,and promote plant growth.At present,the molecular mechanism of phosphorus solubilizing microorganisms is mainly focused on the direct oxidation pathway,and other studies are vague.In this experiment,the transcriptomes of phosphorus solubilizing bacteria wj1 with strong ability to dissolve phosphorus grown in soluble and insoluble phosphate were compared and analysed by using Illumina HiSeqTM 2000 RNA-Seq technology to discovere functional genes,reveal the adaptive regulation process and the mechanism of phosphate solubilization in the presence of insoluble phosphorus.At the same time,the differences of key enzyme genes in the process of phosphorus metabolism were analyzed from the perspective of multiple metabolic pathways,cloning and functional analysis for differentially expressed genes,and the main results were as follows:1.By using Illumina HiSeqTM 2000 platform sequencing,a total of 2,463,507,800 nt clean reads were generated and assembled into 8989 unigenes with an average length of 850 bp and an N50 of 1530 bp.The unigenes were annotated the non-redundant(NR),Swiss-Prot,Gene Ontology(GO),Clusters of Orthologous Groups(COG)and Kyoto Encyclopaedia of Genes and Genomes(KEGG)databases were 5115,2211,4486,2901,and 3755,and 5140 unigenes(57.19%)were annotated in at least one database.The predicted CDS is 12087.SSR is 17.Quantitative PCR technique was used to detect some of the data in the transcriptome,and the results were consistent with the results of transcriptome sequencing.It’s proved that the transcriptome sequencing data is accurate and reliable.2.According to the Unigenes in the KEGG database notes,4486 Unigenes were annotated to the KEGG database,and positioned in 183 metabolic pathways,and there are 20 metabolic pathways related to phosphorus metabolism.According to the analysis of differentially expressed genes,687 genes were identified differentially,421 up-regulated genes(61.28%)involved in 130 metabolic pathways,and most of them concentrated on amino acid biosynthesis,TCA cycle,carbon metabolism,lipid metabolism,glucose metabolism and other metabolic pathways.These differentially expressed genes reflected the metabolic processes and regulatory pathways of wj1 in response to phosphorus stress.3.In the analysis of the wj1 transcriptome,we found that the gene expression was significantly different after the treatment of phosphorus stress.The gene was cloned and named IMP,which was 885 bp.The results were similar to those of transcriptome sequencing by 99.89%.Quantitative analysis showed that it was highly expressed in insoluble phosphorus.The results indicated that IMP gene played a role in the solubilization of insoluble phosphorus.The IMP gene was linked to the expression vector PGEX-4T-1 and transformed into E.coli BL21 to induce the expression of 56 KDa protein,the fusion protein was detected by SDS-PAGE protein electrophoresis.The method of enzyme activity determination of IMP gene was studied,and the activity of IMP was determined according to the method of phytase activity measurement.The activity of IMP in BL21(PGEX-4T-1-IMP)strain increased with the increase of time in 4 h,the change of enzyme activity was significantly different,and the maximum enzyme activity reached 543.48 U·mg-1 in 4 h.4.Quantitative analysis of phospholipase C,protein kinase C in the phosphatidylinositol signaling pathway and PhoB,PhoU,PhoR,PstB,PstS1 and PstS2 genes in the PhoR/PhoB two component system at the same conditions.All of these genes were highly expressed in the presence of insoluble phosphorus as the sole source of phosphorus,which showed that the IMP gene played an important role in the above two pathway.IMP gene and the two pathway of all genes under low phosphorus conditions are activated in a state of high expression,also shows that multiple systems of bacteria participate in the regulation of phosphorus metabolism,PI pathway and PhoR/PhoB two-component system coordinate regulation.