Cloning and Functional Analysis of pqq Genes Phosphorus Solubilizing From Bacillus Mycoides Gnyt1

The pqq genes which encodes phosphorus solubilizing from Bacillus mycoides Gnyt1 has been cloned and characterized. The role of this action on the function of pqq genes were detected. The phosphorus soluble pqq genes can secrete some specic organic activity acids to work but don’t destroy the living environment of plants such as soil. In contrast, this plants containing the pqq genes grew well, even better than other plants with lower phosphorus solubilization, in the phosphorus free medium even though its pqq genes activity and turned out phosphorus ring, while pqq dependent expression was induced. Under of each coding of can directly participate directly improving the phosphate capacity have strong


Abstract [Background]
The pqq genes which encodes phosphorus solubilizing from Bacillus mycoides Gnyt1 has been cloned and characterized. The role of this action on the function of pqq genes were detected. The phosphorus soluble pqq genes can secrete some speci c organic activity acids to work but don't destroy the living environment of plants such as soil. In contrast, this plants containing the pqq genes grew well, even better than other plants with lower phosphorus solubilization, in the phosphorus free medium even though its pqq genes activity and turned out phosphorus ring, while pqq dependent expression was induced. [Results] This reaserch that ve pqq genes were cloned from Bacillus mycoides Gnyt1, phylogenetic analysis revealed that pBI-pqqA, pBI-pqqB, pBI-pqqC and pBI-pqqE similarly cluster with Phosphorus-soluble protein. Under the action of ve promoters, each coding region of pqqA, pqqB, pqqC and pqqE from Bacillus mycoides Gnyt1 can directly participate in the phosphate solubilization of the strain, directly improving the phosphate solubilizing capacity of the plant, which have strong nitrogenase activity.
Interestingly, changes in the phosphate solubilizing gene result in complete loss of the phosphate solubilization of the strain. However, the ability to dissolve the phosphorus ring and to dissolve the phosphorus was also exhibited in the medium having ve phosphorus-dissolving genes. [Conclusions] The results indicated that the pqq genes were successfully cloned and a new speci c phosphatedissolving strain was formed. In this research, pqq genes were rst cloned from Bacillus mycoides Gnyt1.

Background
Phosphorus soluble gene was discovered in 1979 as a new type of phosphate-soluble cofactor for several prokaryotic dehydrogenases [1][2][3] . The phenomenon of phosphorus de ciency in soil is widespread and should be given great attention. Pqq genes have been founded and characterized from bactetia, cloned and sequenced of the pqq gene from Acetobacter aceti [4][5] . Several researches indicated that pqq is present in a variety of plants and animals. For example, Kumazawa T et al. [6] and Mitchell et al. were found pqq in human and rat tissues. Kumazawa et al. [7] reseach said that all kinds of food and vegetables contain pqq. The recent research showed that PQQ-lack of mouse liver has low mitochondrial content and elevated plasma glucose concentration [8] . In addition, PQQ is used as a scavenger for reactive oxygen species (ROS) [9][10] and prevents damage to cells of DNA fragments [11] .They are gram-negative of the bacteria famlily, which has a strong phosphorus dissolving ability [12] . This enzyme can only the rst in our Bacillus mycoides Gnyt1, and they have been found in Acinetobacter sp. or Pseudomonas putida [13][14] .
Research progress showed the pqq gene most of the performance was pqq-adhB, which cloned A. polyoxogenes [5] . Kosano et al. [15] reported that PQQ inhibited a-MSH-stimulated melanin-producing B16 melanoma. This inhibition of melanin production is caused by PQQ, which inhibits the expression of tyrosinase mRNA. However, other roles of PQQ, such as the action of tyrosinase-related proteins and the action of Mitf transcription in the pigment system, are not well known [16] . Whereas K. pneumonia is synthesized by gluconate dihydrogen dehydrogenase and coenzyme PQQ. Closely related intestinal bacteria and Salmonella typhimurium can only synthesize decoenzyme, while PQQ cannot [17] . However, the addition of PQQ to E. coli and S. typhimurium cells produces active holoenzyme [18] . Kumazawa et al. [19] report said that all kinds of food and vegetables contained PQQ. Naito et al. [20] reported that PQQ can stimulate DNA synthesis. PQQ is synthesized by a variety of microorganisms [21] from the two amino acids glutamate and tyrosine encoded in the precursor peptide, and is shown as an effective growth factor for plants [22] , bacteria [23] or higher organisms [24] . Although PQQ is an effective growth factor in both in vitro and in vivo models, the detailed mechanism of action remains unclear.
Bacillus mycoides Gnyt1 is a Gram-positive normal isolate from the rhizosphere of the alpine steppe plant of the alpine steppe, which is isolated and named in our laboratory, and has a high ability to dissolve phosphorus [25] . At present, there is no known about the mechanism of phosphorus solubilization and the phosphorus-dissolving gene of this kind of bacteria. In this study, we aimed to clarify the functions of Bacillus mycoides Gnyt1 of pqq genes family, the four pqq genes were cloned and their activities and properties were characterized. We further validated the cloning of the phosphate-dissolving gene and its effect on the ability of the fraction to dissolve phosphorus.

Media and growth conditions
Bacillus mycoides Gnyt1 was cultured in medium, which contains: 5g yeast powder, 10g Peptone, 10g NaCl, PH 7.0. Optimal growth occurs at 28 • C in aerobic conditions.

Cloning of pqq genes
The complete sequence of the pqq genes were selected from the baseline genome-wide database [26] in our laboratory, all of which have accession numbers in NCBI. Primers are designed from conserved regions. All primers for this study were listed in Table 1. Primer pairs pqqF and pqqR were performed to obtain partially conserved regions. The ampli ed PCR product was puri ed and cloned into pMD-T19 vector (Takara, Japan). The expression vector pBI-121 was obtained from the wheat laboratory of Gansu Agricultural University, and after transformation in bacterial DH5α competent cells, the plasmid was isolated from the positive clone and inserted. DNA was obtained by PCR, and the remaining four clones were cloned by conventional PCR.

Expression of pqq genes in E. Coli
The forward primer pqq with the built-in QuickCutTM SmaI cleavage site and the pqq clone ORF with the QuickCutTM XbaI site and ve pqq genes ( Table 2) were used. The PCR product was digested with SmaI and XbaI and ligated into the PBI-121 expression vector. Positive transformants were identi ed by PCR.
The culture was cultured in Luria-Bertani (LB) medium with a positive concentration of 50 μg/mL. When the OD600 reached 0.5-0.7 at 37 ° C, the culture (5 mL) was transferred to an alternative 500 mL LB medium. In the 2000 mL ask, a 50 μg/mL pseudonym was added to the medium. When the OD600 reached about 0.6, the antibiotic thio-β-galactoside (IPTG) was added to the medium at a nal concentration of 0.1 mmol / L. The bacteria were then centrifuged at 5,000 g for 30 minutes at 16 ° C, washed with PBS, and then cultured with binding buffer (20 mmol / L imidazole, 500 mmol / L NaCl, 20).
Hours to induce expression of the DfSQS1 protein. Methylene / L Na3PO4, pH 7.4) resuspended. Then, a certain amount of lysozyme (0.1 mg / mL) and RDNAse (20 μg / mL) were added to the cell suspension. The cell suspension was stirred and placed at 4 ° C for 30 minutes. The cell suspension was disrupted by sonication 20 and the supernatant was harvested by centrifugation at 4200 rpm for 20 minutes at 4 ° C and replaced by a 750 kDa hollow ber column. After washing with binding buffer (20 mmol / L imidazole, 500 mmol / L NaCl, 20 mmol / L Na3PO4, pH 7.4), protein and exchange buffer (20 mmol / L Na3PO4, 250 mmol / L imidazole, 500 mmol) ) combined / L NaCl, pH 7.4). All collected samples were analyzed by SDS-PAGE. The ve pqq genes clusters were isolated from Bacillus mycoides Gnyt1. Five phosphorus soluble genes were cloned by conventional PCR using individual two genes primers pqqF and pqqR, as the gure shows that the pqq genes related to phosphorus soluble gene after cloning and screening. Moreover, BLAST analysis and sequence comparison revealed that the size of pqqE is 1539bp, which is the longest gene fragment, pqqA is 924bp, pqqB is 897bp, and pqqC is 645bp, which is presumed to be the target fragment. The sequencing datas obtained in this study have been deposited in the GenBank database under the following accession numbers by CP020743. The total of ve genes sequence involving the putative open reading frame(ORF) was obtained, the ORF was ampli ed by using primer pair.

Construction of cloned phospholysis gene and plasmid for expression vector
The plasmid for expression vector PBI-121 has been successfully constructed and stored in a laboratory cryogenic refrigerator, which measurement was far more than 15000bp (Fig.2.A). Fuor plasmids of four pqq genes were successfully extracted using a plasmid kit, and the sizes were all less than 2500 bp ( Fig.2.B). As can be seen from the gure, the agarose gel electrophoresis showed that the band was clear and single, and it was proved that the plasmid of the gene was successfully extracted, and the next experiment can be carried out.

Double digestion of plasmid
The enzymatic digestion system was as follows: 10×QuickCut Buffer 3ul, plasmid DNA 6ul, QuickCutTM SmaI 1ul, QuickCutTM XbaI 1ul, ve genes were successfully extracted, and the expression vector pBI121 plasmid DNA was added to the rapidly cleaved enzymes SmaI and XbaI for double digestion, ddh 2 o is up to 30ul. The enzyme was digested at 37 ° C for 6 hours, electrophoresis was detected, and the target fragment was recovered. It can be cut from the gure and has successfully recovered 6 fragments of plasmid DNA ( Fig.3).
Except T4 DNA Ligas, the reaction was incubated at 65 ° C for 3 minutes, then rapidly transferred to ice for a few seconds, then T4 DNA Ligas was ligated for 16 hours or more and stored at 4 degrees for conversion.

Phylogenetic analysis
The amino acid sequences corresponding to the four cloned gene products of the Gnyt1 variable are compared with similar sequences already in GenBank, and the software MEGU is used to generate the phylogenetic tree using the conversion method. The results show that the pqq genes family is derived from a single Gnyt1 pqqA phosphorus dissolving gene that is closer to bacterial phospholytic protein (WP 003194766.1) and clustered together with other distributions ( White colonies cultured overnight were selected, and individual colonies were picked and cultured in LB liquid medium for about 13 hours. At the same time, blue spots were picked as a negative control (Fig.8), and bacterial cells were detected by PCR, and the target bands were ampli ed and correctly sequenced. It is used to detect the phosphate-dissolving activity of phosphate-dissolving bacteria.

Identi cation of phosphate-dissolving activity
Qualitative experiments have shown (Fig.9) that the strains of recombinant phosphorus-solubilizing genes can produce small phosphorus-solubilizing circles on the culture medium, and the phosphorus dissolving characteristics of PBI-pqqA, PBI-pqqB, PBI-pqqC, and PBI-pqqE were identi ed by liquid chromatography. Secreted organic acid content, standard solutions of different concentrations are puri ed and enriched by C18 column mobile phase, and analyzed under speci c chromatographic conditions. The results showed that the four recombinant phosphorus-solubilizing gene strains can all secrete organic acids, and thus have a role in phosphorus dissolution (Fig. 10). The concentration peak of secreted organic acids can be clearly seen from the gure, successfully verifying that the four clones linked to the previous clones Phosphorus gene, indicating that the phosphorus dissolution gene dissolves phosphorus Among them, the peak of oxalic acid is the highest, and the peak of citric acid is the lowest. In addition (Table 3) , PBI-pqqA can secrete 6 kinds of organic acids, with the strongest phosphorusdissolving effect, followed by PBI-pqqB and PBI-pqqC, and can secrete 4 kinds of organic acids, followed by phosphorus-dissolving effect. PBI-pqqA and PBI-pqqC secreted the highest lactate concentration, while PBI-pqqB and PBI-pqqD secreted the highest malate concentration.

Discussion
In the present study, we found that the same pathway, Bacillus was used as bene cial bacteria in our research because it has high phosphorus dissolving ability [27][28] . PQQ genes stably catalyzes sustained redox cycling as a cofactor and does so more e ciently than most other redox active cofactors [29] . However, like all redox active compounds, PQQ can still promote oxidation under certain conditions and induce oxidative protein modi cation, including oxidation of cysteamine thiol [30][31][32] . It has been demonstrated that terpenoids including PQQ undergo an electron redox cycle in the presence of ascorbate, NAD(P)H and thiol compounds (such as glutathione), thereby forming corresponding semiquinone free radicals [33][34][35][36] . In addition, PQQ oxidizes the redox regulatory site of the N-methyl-Daspartate (NMDA) receptor, thereby protecting NMDA or glutamate-mediated neuronal cell damage [37] . Therefore, the permanent phosphorus solubilization gene may have a certain basic signi cance for supplementing the ability of plants to dissolve phosphorus.
In this report, we successfully identi ed the four pqq genes in Bacillus for use as a phosphorus solubilization. We obtained conventional PCR clone of pqqA, pqqB, pqqC and pqqE. Compared with other known pqq genes, three key genes were found in our strain [38][39] . The phosphorus-dissolving family is an important gene for bacterial phosphorus solubilization. We identi ed the pqq genes of Bacillus mysoides Gnyt1. This is the rst clone and complete sequence description of the phosphate solubilizing gene in Bacillusmysoides Gnyt1. The plasmids of the four genes extracted were all within 15000 bp, and were well aligned with the partial sequence of Bacillus (partial sequence 94% identical). A high level of protection indicates that the Gnyt1 strain has a phosphate soluble gene similar to the reported corresponding phosphate-dissolving family gene [40][41] .
The cloning of the pqq genes of Bacillus provides interesting and important suggestions for the regulation of phosphorus and phosphorus-soluble organic acid metabolites. Bacillus mysoides Gnyt1 has the largest total number of acidic and basic residues for each protein. Explaining the mechanism of phosphorus solubilization is of great signi cance for the biotechnological application of such proteins. In this regard, the Bacillus Gnyt1 pqq genes may be a good model for research. Studies have shown that micro-spatial analysis of gene expression shows that changes in different PQQ genes can signi cantly affect metabolites, cell signaling and immune function-related genes [42] . Strict control of the activity and expression of the phosphate-dissolving gene, transcription or post-translational regulation depends on the speci c environment of the host organism. B. Gnyt1 is at ambient temperature under aerobic and anaerobic conditions and uses hydrogen hydride as the electron donor. The amino-acid sequences of pqqA and pqqB of B. Gnyt1 are more similar to the corresponding proteins in bacterial phospholytic protein and PqqB quinolinone biosynthetic protein (WP 003194766.1, WP 018985147.1).
Although the research was carried out in antibiotics, the results showed important information. The expression of four phosphorus-dissolving genes increased the expression rates of PBI-pqq, PBI-pqq, PBIpqq, and PBI-pqq by increasing the temperature to 37 °C. Finally, we will obtain a single, expressible phosphate soluble gene that is recombined. In one study, the phosphate solubilizing gene was also present in other plants or phosphate solubilizing bacteria. However, other studies have shown that the phosphorus-dissolving family includes many genes, and different ones contain different phosphorusdissolving genes. Among them, pqqA, pqqC and other reports have the same length [43][44][45] , and pqqE are rarely reported in other studies and can be used as a resource for further research.
There are few reports on phosphorus solubilization in Bacillus mysoides, our research will enrich the knowledge of phosphorus solubilization in these bacteria.
In the present study, we found that the same pathway, Bacillus was used as bene cial bacteria in our research because it has high phosphorus dissolving ability [27][28] . PQQ genes stably catalyzes sustained redox cycling as a cofactor and does so more e ciently than most other redox active cofactors [29] . However, like all redox active compounds, PQQ can still promote oxidation under certain conditions and induce oxidative protein modi cation, including oxidation of cysteamine thiol [30][31][32] . It has been demonstrated that terpenoids including PQQ undergo an electron redox cycle in the presence of ascorbate, NAD(P)H and thiol compounds (such as glutathione), thereby forming corresponding semiquinone free radicals [33][34][35][36] . In addition, PQQ oxidizes the redox regulatory site of the N-methyl-Daspartate (NMDA) receptor, thereby protecting NMDA or glutamate-mediated neuronal cell damage [37] . Therefore, the permanent phosphorus solubilization gene may have a certain basic signi cance for supplementing the ability of plants to dissolve phosphorus.
In this report, we successfully identi ed the four pqq genes in Bacillus for use as a phosphorus solubilization. We obtained conventional PCR clone of pqqA, pqqB, pqqC and pqqE. Compared with other known pqq genes, three key genes were found in our strain [38][39] . The phosphorus-dissolving family is an important gene for bacterial phosphorus solubilization. We identi ed the pqq genes of Bacillus mysoides Gnyt1. This is the rst clone and complete sequence description of the phosphate solubilizing gene in Bacillusmysoides Gnyt1. The plasmids of the four genes extracted were all within 15000 bp, and were well aligned with the partial sequence of Bacillus (partial sequence 94% identical). A high level of protection indicates that the Gnyt1 strain has a phosphate soluble gene similar to the reported corresponding phosphate-dissolving family gene [40][41] .
The cloning of the pqq genes of Bacillus provides interesting and important suggestions for the regulation of phosphorus and phosphorus-soluble organic acid metabolites. Bacillus mysoides Gnyt1 has the largest total number of acidic and basic residues for each protein. Explaining the mechanism of phosphorus solubilization is of great signi cance for the biotechnological application of such proteins. In this regard, the Bacillus Gnyt1 pqq genes may be a good model for research. Studies have shown that micro-spatial analysis of gene expression shows that changes in different PQQ genes can signi cantly affect metabolites, cell signaling and immune function-related genes [42] . Strict control of the activity and expression of the phosphate-dissolving gene, transcription or post-translational regulation depends on the speci c environment of the host organism. B. Gnyt1 is at ambient temperature under aerobic and anaerobic conditions and uses hydrogen hydride as the electron donor. The amino-acid sequences of pqqA and pqqB of B. Gnyt1 are more similar to the corresponding proteins in bacterial phospholytic protein and PqqB quinolinone biosynthetic protein (WP 003194766.1, WP 018985147.1).
Although the research was carried out in antibiotics, the results showed important information. The expression of four phosphorus-dissolving genes increased the expression rates of PBI-pqq, PBI-pqq, PBIpqq, and PBI-pqq by increasing the temperature to 37 °C. Finally, we will obtain a single, expressible phosphate soluble gene that is recombined. In one study, the phosphate solubilizing gene was also present in other plants or phosphate solubilizing bacteria. However, other studies have shown that the phosphorus-dissolving family includes many genes, and different ones contain different phosphorusdissolving genes. Among them, pqqA, pqqC and other reports have the same length [43][44][45] , and pqqE are rarely reported in other studies and can be used as a resource for further research.
There are few reports on phosphorus solubilization in Bacillus mysoides, our research will enrich the knowledge of phosphorus solubilization in these bacteria.

Conclusion
PqqA, pqqB, pqqC and pqqE genes were the rst clone analysis and E. coli transformation. In this case, four genes cloned from B. Gnyt1 were full-length DNA encoding pqqA, pqqB, pqqC and pqqE, and were named pBI-pqqA, pBI-pqqB, pBI-pqqC and pBI-pqqE. Bioinformatics analysis of pBI-pqqA, pBI-pqqB, pBI-pqqC and pBI-pqqE have been completed. Phylogenetic analysis trees re ect the accumulation of the pqq genes in higher clade, slightly closer to Phosphorus-soluble protein. Four pqq genes were expressed in E. coli, and four recombinant phospholytic genes were obtained, which proved that PBI-pqqA, PBI-pqqB, PBI-pqqC and PBI-pqqE have phosphorus-dissolving properties. This is the rst report on the cloning and identi cation of the phosphate solubilizing gene from Bacillus mycoides Gnyt1. Finally, the B. Gnyt1 phosphate-dissolving gene a promising model for studying the mechanism of phosphorus solubilization by speci c phosphorus-dissolving bacteria, a feature that makes it an attractive candidate for biotechnology applications. Blue and white spots of recombinant genes transformed into E. coli. A: PBI-pqqA blue and white spots. B: Blue and white speckle diagram of PBI-pqqB. C: Blue and white speckle diagram of PBI-pqqC. D: Blue and white speckle diagram of PBI-pqqE.

Figure 10
Qualitative determination of phosphorus-solubilizing gene strains