Phosphate solubilizing gene for promoting organic acid secretion
A phosphorus-dissolving gene and gene technology, applied in the field of phosphorus-dissolving genes that promote the secretion of organic acids
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Embodiment 1
[0032] The cDNA full-length library construction of embodiment 1 Penicillium oxalicum
[0033] The strain Penicillium oxalicum strain I1 used in the experiment was screened from soil samples by the Agricultural Strain Collection Center of the Institute of Agricultural Resources and Agricultural Regional Planning, Chinese Academy of Agricultural Sciences. It was cultivated on an insoluble phosphorus inorganic salt medium for 3 days, and about 0.2 g of fresh mycelium was taken ( Such as figure 1 ), ground with liquid nitrogen, extracted total RNA with RNAiso plus kit, took 1 μg RNA as a template for the synthesis of the first strand of cDNA, and reverse transcribed by SMARTscriptTM under the guidance of 3'SMART CDS Primer IIA and SMARTer IIA oligonucleotide primers The first-strand cDNA was synthesized by reverse transcription at 70°C for 2 min; double-stranded cDNA was synthesized by LD-PCR using 2 μL of the first-strand cDNA product as a template. Use CHROMA SPIN+TE-1000 to p...
Embodiment 2
[0034] Example 2 Screening and Bioinformatics Analysis of Phosphorus-Solubilizing Genes
[0035] Dilute the bacterial culture solution of the library onto the insoluble phosphorus medium containing ampicillin (100 μg / mL), incubate at 37°C for 3-4 days, and observe whether there is a phosphorus-dissolving transparent circle. The results are as follows: image 3 As shown, some strains produced transparent circles on the insoluble phosphorus inorganic salt medium, and the transformants that produced transparent circles were transferred to culture to determine their stability, and the functionally stable colonies were extracted from the plasmids and sequenced to obtain DNA sequences. Blast was used for comparison analysis, and DNAMAN6.0 was used for open reading frame analysis, and the translated proteins were compared and analyzed on the PDB database.
Embodiment 3
[0036] Construction of embodiment 3 expression vector, transformation host, positive clone identification
[0037] Primer design (Forward: 5'-TATTCGGAATTCATGACAGACCTCAAGTCCAT-3'; Reverse: 5'-CAAGTACTCGAGTTAAACGATAGTCTCTAGCTC-3'), using high-fidelity Taq enzyme to amplify the ORF of the above gene, reaction conditions: 94°C for 5min; 94°C for 1min, 60°C for 1min , 72°C 1min cycle 35 times 72°C 10min, 4°C forever. After double digestion with restriction endonucleases EcoRI and XhoI, it was connected to the expression vector pET28a(+), and the expression vector pET28a(+) and restriction site sequence were as follows: Figure 4 , Figure 5As shown, E.coli HST08 was transformed by electric shock method, and the clones that could produce transparent circles were screened on the insoluble phosphorus inorganic salt medium ( Figure 6 ), and the fragment size of the positively transformed clones was selected by PCR identification, and the amplification results were as follows Figur...
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