Protein HrpNX3 derived from pectobacterium carotovorum as well as genes and application thereof
A pectin bacillus and protein technology, applied in the field of genetic engineering, can solve problems such as unfavorable food and human safety, and achieve the effect of improving the disease resistance of soft rot bacteria, huge economic benefits, and broad application prospects
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Embodiment 1
[0021] Example 1: Pectobacterium carotovorum subsp. carotovorum strain hrpN X3 gene cloning
[0022] Genomic DNA of Pectobacterium carotovorum subsp. carotovorum was extracted.
[0023] According to the conserved sequence of the hrpN gene sequence and the multiple cloning site of the prokaryotic expression vector (to facilitate the construction of the expression vector), design the HrpN structural gene spanning the start codon and the stop codon, and introduce the restriction endonuclease Kpn I A pair of primers with EcoR I restriction site and protective base (the underlined part is the introduced restriction site):
[0024] F1:5'-CG GGTACC ATGCTTAATTCTCTTGGTGGCG-3'
[0025] R1: 5'-CC GAATTC TTAGCTGGAGAGCTTCTTCAACC-3'
[0026] PCR amplification was performed using the total genomic DNA of Pcc X3 as a template. The PCR reaction parameters were: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 45 s, annealing at 50°C for 45 s, extension at 72°C for 1 min, an...
Embodiment 2
[0027] Embodiment 2: recombinant HrpN X3 High protein expression and activity detection
[0028] After verification by sequencing, the p-hrpN X3 hrpN X3 After the gene was cut with Kpn I and EcoR I double enzymes, it was connected with the prokaryotic expression vector pET-30a(+) also cut with Kpn I and EcoR I double enzymes to construct the recombinant expression vector pET30a(+)-hrpN X3 , transformed into the expression strain E.coli BL21(Lyss), to obtain the recombinant strain BL21(Lyss)-hrpN X3 , and identified by restriction enzyme digestion and PCR, using pET-30a (+) empty vector BL21 (Lyss) as a control, adding 1mmol / L IPTG to induce expression for 3 hours, collecting the cells for a small amount of expression test, using 12% SDS -PAGE detection expression ( figure 1 ),Such as figure 1 Arrow indicates, recombinant expression strain BL21(Lyss)-hrpN containing hrpNX3 X3 There is a concentrated target protein band at the relative molecular mass of 35-40kDa, which is ...
Embodiment 3
[0030] Embodiment 3: recombinant HrpN X3 Effects of Protein Treatment on Soft Rot Resistance of Arabidopsis and Dendrobium officinale
[0031] The same batch of seedlings of Arabidopsis thaliana and Dendrobium officinale with basically the same growth potential were selected as the test materials. Spray 30ug / ml HrpN X3 and HrpN Pcc71 , with PBS buffer as the control, after 24 hours, use a needle to gently draw a "ten"-shaped wound on the surface of the leaf to inoculate the soft rot pathogen Pcc BC1 at a concentration of 2×10 8 CFU / ml, the inoculation volume is 5ul, each material is inoculated with 6 seedlings, each seedling has 3 leaves, and repeated 3 times. Place in a constant temperature climate box at 28°C, maintain a relative humidity of 90%, and a photoperiod of 16h (light) / 8h (dark). After HrpN X3 , HrpN Pcc71 The incidence of Arabidopsis thaliana and Dendrobium candidum treated with control PBS are as follows: Figure 4 shown. wxya X3 and HrpN Pcc71 After 24...
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