3.1 Cloning of five pqq genes from B. mycoides Gnyt1
The five 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 figure 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 five genes sequence involving the putative open reading frame(ORF) was obtained, the ORF was amplified by using primer pair.
3.2 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 figure, 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.
3.3 Double digestion of plasmid
The enzymatic digestion system was as follows: 10×QuickCut Buffer 3ul, plasmid DNA 6ul, QuickCutTM SmaI 1ul, QuickCutTM XbaI 1ul, five genes were successfully extracted, and the expression vector pBI121 plasmid DNA was added to the rapidly cleaved enzymes SmaI and XbaI for double digestion, ddh2o 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 figure and has successfully recovered 6 fragments of plasmid DNA ( Fig.3).
3.4 Linked expression vector
The four pqq genes and the expression vector PBI121 were ligated, and the ligation system was: 10×T4 DNA Ligase Buffer 1ul, pqqA / pqqB /pqqC/ pqqE 6ul, pBI121 DNA 1ul, T4 DNA Ligase 1ul, dd H2O 10ul. 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.
3.5 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 (Figure 5.5), and the phosphorus dissolving gene pqqB is closer to PqqB Thiazoline antibiotic biosynthetic protein (WP 018985147.1) (Figure 5.6), while pqqC is clustered closer to the Bacillus pqqC family protein group (Figure 5.7), and pqqE is closer to the Bacillus and Bacillus cereus dissolved phosphorus family protein cluster (Figure 5.8).
3.6 E. coli transformation
Using competent cells, each product was transformed into E. coli, and E. coli was used as a vector for propagation and growth, and the constructed PBI-pqqA, PBI-pqqB, PBI-pqqC, PBI-pqqE vectors were transformed into Escherichia coli DH5α in the large intestine. A large number of bacteria are amplified and positively transformed by bacterial liquid phase PCR.
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 amplified and correctly sequenced. It is used to detect the phosphate-dissolving activity of phosphate-dissolving bacteria.
3.7 Identification 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 identified by liquid chromatography. Secreted organic acid content, standard solutions of different concentrations are purified and enriched by C18 column mobile phase, and analyzed under specific 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 figure, 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 phosphorus-dissolving 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.
Table 3 Content of organic acids in recombinant strains
Stains
|
Organic acid content (mg/L)
|
oxalic acid
|
tartaric acid
|
Malic acid
|
Citric acid
|
Lactic acid
|
Acetic acid
|
pqqA
|
14.93±0.41
|
14.10±0.90
|
14.81±0.69
|
14.39±0.46
|
18.23±0.94
|
18.18±0.05
|
pqqB
|
3.21±0.15
|
5.12±0.64
|
106.82±2.75
|
13.94±0.76
|
_
|
_
|
pqqC
|
24.24±0.34
|
_
|
_
|
20.17±0.97
|
0.48±0.01
|
0.29±0.01
|
pqqD
|
32.09±0.77
|
_
|
119.58±7.30
|
_
|
0.63±0.14
|
_
|