Prokaryotic expression of banana MaNPC1 gene and preparation of its polyclonal antibody

【Objective】 Prokaryotic expression of the open reading frame(ORF) of banana fruit non-specific phospholipase C gene(MaNPC1) and preparation of its polyclonal antibody were conducted, to provide theoretical basis for indepth exploration of the mechanism of MaNPC1 in banana fruit resistance to anthracnose.【Method】 The ORF sequence of MaNPC1 gene was cloned, bioinformatics analysis and antigenicity prediction were carried out, and its prokaryotic expression vector was constructed by double enzyme digestion method, and the heat shock method was used to transfer it into Escherichia coli Rosetta 2(DE3) competent cell for induction expression, and the recombinant protein was purified by Ni-NTA resin chromatography column to immunize New Zealand rabbits to prepare polyclonal antibodies. At the same time, Western blotting and real-time fluorescent quantitative PCR were used to detect the MaNPC1 protein expression level and the relative expression level of MaNPC1 gene under the stress of anthracnose infection during banana fruit storage.【Result】 The pGEX-6 p-3-MaNPC1 recombinant plasmid was double-enzyme digested to obtain a specific band of 1650 bp, indicating that the prokaryotic expression vector was successfully constructed and transferred to E. coli Rosetta 2(DE3) competent cell for successful expression. The molecular formula of MaNPC1 protein was C₂₇₄₇H₄₂₄₉N₇₆₉O₇₉₃S₁₀, the molecular weight was 61.06 k D, and the theoretical isoelectric point(pI) was 8.96;the contents of alanine, valine, leucine and serine were high, accounting for 8.7%, 8.2%, 7.8% and 7.7% of the total number of amino acids, respectively;the instability index was 47.14, indicating that it was an unstable protein. The protein antigen segment was rich and the number of hydrophilic amino acids was higher than that of hydrophobic amino acids, which was easy for subsequent antibody preparation. The prepared polyclonal antibody had a higher titer of 1:2048000. Western blotting analysis showed that the MaNPC1 protein expression level of banana fruits showed an upward-decreasing-increasing trend during storage. The MaNPC1 protein expression level of the anthracnose infection group was lower than that of the control group when stored for 15 d, and the other storage time was slightly higher than the control group. Real-time fluorescent quantitative PCR detection showed that the relative expression of MaNPC1 gene showed a gradual upward trend during storage of banana fruits;after storage for 3 to 15 d, the relative expression of MaNPC1 gene in the anthracnose infection group was significantly higher than that in the control group(P<0.05). 【Conclusion】 Anthracnose infection can increase the expression level of MaNPC1 protein, indicating that MaNPC1 protein participates in banana fruit resistance to anthracnose infection.