| Microbial resources are one of the country's strategic resources.It may be the largest natural resource on the planet that has not been effectively developed and contains huge industrial value yet.The research and utilization of microbial resources centered on modern biotechnology has become the strategic focus of global biological resource competition.As the main branch of microbial resources,plant rhizosphere growth-promoting bacteria occupies a very important position in the utilization of microbial resources in our country,and their full and reasonable utilization is also one of the hot issues that countries around the world pay attention to.In recent years,most researches on PGPR(Plant Growth Promoting Rhizobacteria)at home and abroad are mainly focused on biological performance,and there is little research on molecular mechanism.Therefore,in-depth study of the growth promotion mechanism,metabolic regulation and control promotion effect of PGPR strains To reveal the growth-promoting mechanism of PGPR from the perspective of biomolecular technology,to dig deeper into the internal growth-promoting genes,and to promote the research of molecular biological methods of PGPR.The targeted and more effective use of PGPR strains is a new issue to be solved.In this study,a fine plant rhizosphere strain Bacillus mycoides Gnyt1 with outstanding growth-promoting properties was the research object.On the basis of the whole genome sequencing of the research group,molecular biology techniques were used to study the strain Bacillus mycoides Gnyt1 and strains of the same genus(Six reference strains)compare genomics housekeeping genes and differential genes,predict that there may be functional genes with similar functions in the genome,study the genes related to the nitrogen-fixing and phosphorus-dissolving characteristics of strain Bacillus mycoides Gnyt1,and determine the relative expression levels and main metabolites of the genes.The utilization and in-depth research of plant rhizosphere growth-promoting bacteria provide theoretical basis and scientific and technological support,and the main results are as follows:(1)The comparative genomics analysis of strain Bacillus mycoides Gnyt1 and the reference strain of the same genus showed that the total length of the genome sequence of Gnyt1 strain was 5,597,907 bp,the GC content was 35.57%,and the ORF length of the open reading frame was 792.17 bp.In terms of evolutionary relationship,strain Gnyt1 There is 95% homology in 16 Sr DNA homology comparison with Bacillus strains AH621(CM000719.1)and AH603(CM000737.1).The collinearity between strain Gnyt1 and the reference strain is low,indicating that strain Bacillus mycoides Gnyt1 Compared with the re-arrangement of the genomes of the same genera,the larger differences have the potential to continue research.Different strains have different gene composition and have gene specificity.Among them,the functional genes related to nitrogen fixation and phosphorus solubilization are related to the regulation of gene proteases,pigments,and different secretion systems.(2)The strain Bacillus mycoides Gnyt1 is based on Mauve's strain gene family analysis.There are a total of 3322 identical genes.There are 515 specific genes in strain Bacillus mycoides Gnyt1.Phylogenetic tree analysis found that Bacillus mycoides AH621 strain and Bacillus mycoides Gnyt1 strain have gene function and composition The evolution of expression is relatively recent,and it can be predicted that functional genes with the same expression traits may have the same regulatory mechanism and function of housekeeping genes.(3)Design the upstream and downstream primers according to the sequence of the nitrogen-fixing gene,and clone the five nitrogen-fixing genes(nif D,nif R,nif S,nif U and nif Ux)in the strain Bacillus mycoides Gnyt1,respectively,double enzyme digestion and ligation expression vector p BI121,named as p BI-nif D,p BI-nif R,p BI-nif S,p BI-nif U and p BI-nif Ux,bioinformatics and phylogenetic tree analysis showed that the cloned nitrogen fixation genes were close to rhizobia.The determination of the nitrogenase activity of the strains verified that the cloned nitrogen-fixing genes can be successfully expressed in E.coli,and the five recombinant nitrogen-fixing genes all have nitrogen-fixing activity.(4)Design the upstream and downstream primers according to the sequence of the obtained phosphorus-dissolving gene,and clone the four phosphorus-dissolving genes(pqq A,pqq B,pqq C and pqq E)from the strain Bacillus mycoides Gnyt1,respectively,double enzyme digestion and ligation expression vector p BI121,named p BI-pqq A,p BIpqq B,p BI-pqq C and p BI-pqq E,bioinformatics and phylogenetic tree analysis showed that the cloned phosphate genes were close to the cluster of phosphate family protein groups.The determination of the organic acid content of the strain verified that the cloned phosphorus-dissolving genes can be successfully expressed in E.coli,and the four recombinant nitrogen-fixing genes all have phosphorus-dissolving ability.(5)The whole genome information of strain Bacillus mycoides Gnyt1 and the research results of functional genes related to siderophores show that the genes GYT1 and GYT2 related to siderophore secretion of the research strain Bacillus mycoides Gnyt1 mainly exist in the Porphyrin metabolism pathway.It is mainly related to the transport and metabolism of iron in cells,and the products of genes are related to the chelation and transport of iron,which is of further research significance.(6)The trends in the relative expression of nitrogen-fixing genes of strain Bacillus mycoides Gnyt1 were inconsistent at different culture times.The gene expression of nitrogen-fixing gene recombinant strains p BI-nif D,p BI-nif R,p BI-nif U and p BI-nif Ux at the culture time 4h was relatively The other culture time showed an obvious trend of highest expression,which was significantly higher than that of the control(P<0.05),which were2.4 times,4.3 times,4.9 times,and 4.5 times of the control,respectively.The nitrogenfixing gene recombinant strain p BI121-nif S had a gene at 12 h culture time.Compared with other culture times,the expression level of saccharomyces cerevisiae showed an obvious trend of the highest expression,which was significantly higher than that of the control(P<0.05),and was significantly positively correlated with the control,which was 2.4 times that of the control.(7)The relative expression levels of the phosphate-dissolving genes of strain Bacillus mycoides Gnyt1 at different culture times gradually weakened,and the phosphatedissolving gene recombinant strains p BI-pqq A,p BI-pqq B,p BI-pqq C and p BI-pqq E were cultured for 4 hours.Compared with other culture times,it showed an obvious trend of highest expression,which was significantly higher than that of the control(P<0.05),and was the highest level of gene expression,which was 4.25 times,1.78 times,1.7 times,and4.2 times that of the control,respectively.The strains p BI-pqq A and p BI-pqq E have the highest relative expression levels of phosphate solubilizing genes,and the strain p BI-pqq C has the lowest relative expression levels of phosphate solubilizing genes.(8)The targeted metabolite analysis of the nitrogen-fixing recombinant strain and phosphorus-dissolving recombinant strain of strain Bacillus mycoides Gnyt1 showed that the metabolites secreted by the recombinant nitrogen-fixing strain p BI-nif U are mainly citric acid,isocitrate,malic acid,pantothenic acid,and amber Acid and lactic acid,the metabolites secreted by the recombinant phosphate solubilizing strain p BI-pqq A are mainly pantothenic acid,succinic acid,malic acid,citric acid and lactic acid. |
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