Pseudomonas aeruginosa flagellin for improving disease resistance of plants as well as coding gene and application of pseudomonas aeruginosa flagellin

[0036] The construction of embodiment 1flagellin expression vector

[0037] Using the bioinformatics software Geneious R9 to analyze the gene sequence of flagellin gene flagellin of Pseudomonas aeruginosa, combined with the restriction endonuclease cutting site of pET-28b prokaryotic expression vector, according to the principle of open reading frame code ORF, design The upstream primer with NdeI restriction site and the downstream primer with XholI restriction site.

[0038] Forward primer: 5'-GAC CATATG ATGGCTCTTACTGTTAAT-3' (SEQ ID NO: 3), wherein the underline is the enzyme cutting sequence of NdeI;

[0039] Reverse primer: 5'-TCA CTCGAG TTATCTAAGCAAAGACAACACAGA-3' (SEQ ID NO: 4), where the underline is the restriction sequence of XholI.

[0040] The full-length flagellin gene was amplified by PCR, and the amplification program was as follows: 98°C for 30s; 34 cycles of 98°C for 10s, 55°C for 10s, and 72°C for 30s; 72°C for 2min; and storage at 4°C.

[0041] The PCR ...

[0043] Example 2 Protein expression condition optimization

[0044] The recombinant plasmid pET-28b-flagellin expression vector was transformed into Escherichia coli BL21(DE3) by the heat shock method to obtain a recombinant Escherichia coli containing the recombinant plasmid pET-28b-flagellin, and the recombinant Escherichia coli was named BL21(DE3) / pET-28b-flagellin. The method of extracting the plasmid and transforming the expression host strain is the same as that in Example 1. After the verification is correct, the expression is carried out. The correct expression strain will be verified to be activated overnight, and the strain containing pET-28b empty plasmid will be used as a control. Add 1mL of overnight culture solution to 100mL LB liquid medium containing 100μg / mL kanamycin (1% inoculum size), culture at 37°C, shake at 200r / min for 2-3h until OD 600 It is 0.6-0.8. At this time, set the concentration gradient of the inducer IPTG (add IPTG with final concentration...

[0046] Expression and purification of the protein of embodiment 3

[0047] 1. Inoculate BL21(DE3) / pET-28b-flagellin in LB liquid medium, and culture overnight at 37°C and 200rpm / min with shaking.

[0048] 2. Induce protein expression according to the method and conditions shown in Example 2, that is: transfer the overnight bacterial solution to LB liquid medium containing 100 μg / mL kanamycin at a volume ratio of 1:100, 37°C, 200rpm / min shaking culture to OD 600 The value is 0.6, then add the final concentration of 0.1mmol / LIPTG, shake culture at 30°C and 200rpm / min for 12h, and collect the bacteria.

[0049] 3. Take the bacterial cells obtained in step 2, resuspend them with PBS buffer, and perform ultrasonic crushing (parameters for ultrasonic crushing: total ultrasonic time 30 minutes, every 5 seconds for 5 seconds, power 200W), then centrifuge at 12000rpm for 10 minutes, and collect the supernatants respectively liquid and sediment.

[0050] 4. Perform SDS-PAGE on the s...