| Phosphorus is one of the necessary nutrients for plant growth and development,and it is a key nutritional factor that limits the primary productivity of land.The content of total phosphorus in soil in China is considerable,but the content of available phosphorus for plant absorption and utilization is on the low sidećIn agriculture,a large amount of phosphate fertilizer is often applied to meet the demand of crops for phosphorus,but this will lead to soil consolidation,environmental pollution,waste of resources and other problems.For this reason,it is necessary to find a way to supply sufficient available phosphorus to crops and has the characteristics of resource saving and environment-friendly.Phosphorus-solubilizing microorganisms(PSM)can convert insoluble phosphorus into available phosphorus in soil,which is a phosphorus supply measure to slow down the decline of soil fertility,improve the utilization efficiency of phosphate fertilizer,solve the waste of resources and do not produce secondary pollution.In this study,two indicators of heat resistance and efficient phosphorus-solubilizing ability were used to screen suitable phosphorus-solubilizing microorganisms from the navel orange rhizosphere to ensure that the strains had certain stress resistance and were suitable for a wider range of agricultural application scenarios.For the screened target phosphorus-solubilizing strains,research on phosphorus understanding characteristics,identification of strain species,optimization of fermentation medium,whole-genome sequencing and annotation,excavation of genes related to stress resistance,and correlation research with phosphorus-solubilizing functional genes The molecular mechanism of the phosphorus-dissolving characteristics of the strain was explored.Below are key research findings:(1)After primary screening and re-screening,two strains QCGJ-01 and QCGJ-04 with heat resistance and good phosphorus-solubilizing ability were isolated from the navel orange rhizosphere soil,and the maximum phosphorus-solubilizing capacity was determined to be 162.07mg/ L and 139.76mg/L,at the same time,it was determined that the amount of phosphorus solubilized by the two strains was significantly negatively correlated with pH.Morphological observation and gene homology analysis were carried out on QCGJ-01 and QCGJ-04,and QCGJ-01 was preliminarily identified as Bacillus velezensis,and QCGJ-04 was identified as Bacillus subtilis.(2)Through single factor experiment and response surface optimization design,the optimal phosphorus solubilizing conditions of phosphate solubilizing bacteria QCGJ-01are: glucose 15.88g/L,ammonium chloride 1.17g/L,inorganic salt 1.13g/L,calcium phosphate It was 6.43g/L,and under this condition,the phosphorus-dissolving capacity of the strain was 260.32mg/L,which was close to the model prediction value of 258.39mg/L;The optimal conditions for phosphorus solubilization by phosphorus solubilizing bacterium QCGJ-04 are: glucose 11.27 g/L,ammonium chloride 1.07 g/L,inorganic salt1.01 g/L,and calcium phosphate 8.02 g/L.Under these conditions,the phosphorus solubilization amount of the strain is 223.51 mg/L,which is similar to the model prediction value of 220.34 mg/L.(3)The whole genome sequencing analysis of Bacillus velezensis QCGJ-01 showed that the genome size of QCGJ-01 was 4016820 bp and the GC percentage was 46.08%.A total of 3976 coding genes were found in the prediction of coding genes,and the functional genes of the strains were abundant.There are 4,51 and 88 related sequences of t RNA,r RNA and s RNA,respectively.There were 434 virulence factor-related genes,95two-component regulatory genes and 1020 transmembrane protein gene sequences,accounting for 25.65% of the total number of coding genes,which were analyzed by.The total number of coding genes annotated by CAZy was 2661,accounting for 66.93% of the total coding genes.The total number of protein coding genes annotated by COG is 3122,accounting for 78.52% of the total coding genes,and the functional genes of the strain are relatively rich.(4)The whole genome of Bacillus subtilis QCGJ-04 was sequenced,and the genome size of QCGJ-04 was 4022320 bp,GC%.A total of 4037 coding genes were found in the prediction of coding genes,and the functional genes of the strains were abundant.There are 3,58 and 87 related sequences of t RNA,r RNA and s RNA,respectively.There were 427 virulence factor-related genes,109 two-component regulatory genes and 851 transmembrane protein gene sequences,accounting for 21.08% of the total number of coding genes,and the total number of coding genes annotated by GO was 2982,accounting for 78.5% of the total number of coding genes.The total number of protein coding genes annotated by COG was 3284,accounting for 81.35% of the total.(5)According to the whole genome data of QCGJ-01 and QCGJ-04,the genome circle maps of two phosphorus-solubilizing strains were constructed.(6)There are many genes related to phosphorus utilization in the genomes of QCGJ-01 and QCGJ-04 strains.The two strains have 23 and 25 genes respectively,among which the most genes related to organic acid production are citric acid(gltA),lactic acid(ilvBH),acetic acid(ackA),pyruvate(pyk,pyc),butyric acid(buk),shikimic acid(aroK)and gluconic acid(gdh).There is a negative correlation between the amount of phosphorus release by combined strains and pH.It is inferred that the secretion of organic acids may be one of the main phosphorus-solubilizing mechanisms of the two phosphorus solubilizing strains.(7)It was found that the two phosphate-solubilizing strains were rich in environmental stress-related genes.Both QCGJ-01 and QCGJ-04 had 11 genes of extensive temperature adaptability,including 7 heat shock protein genes and 4 cold shock protein genes.QCGJ-01 and QCGJ-04 have 17 and 16 salt-tolerant genes respectively,and28 and 29 extensive ph adaptive genes,respectively.The abundance and regulatory pathways of different functional genes are different.They are two phosphorus-solubilizing bacteria with excellent environmental resistance. |
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