Identification and Safety Assessment of Cucumber Mosaic Virus Coat Protein in Genetically Modified Pepper (Capsicum annuum)

Abstract

The great economic losses caused by Cucumber mosaic virus (CMV) infection of peppers has led to the development of genetically modified (GM) CMV-resistant peppers. We developed virus-resistant pepper plants using Agrobacterium tumefaciens-mediated transformation. The expressed recombinant protein was purified using nickel-nitrilotriacetic acid resin and immunoaffinity chromatography, and purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Immunoblot analysis revealed the purified CMV coat protein (CMV-CP) had a molecular mass of 25 kDa. After in-gel digestion and desalting, the internal peptide fragments of CMV-CP were sequenced by matrix-assisted laser desorption/ ionization-time of flight. Most GM pepper and Escherichia coli BL21 internal peptides had identical peptide sequences and contained 137 of 183 whole peptides in CMV-CP. A quantitative enzyme-linked immunosorbent assay was performed to detect CMV-resistant GM peppers. We also provide basic information about the expressed protein in GM peppers for further safety assessment. The contents of soluble protein and CMV-CP were measured in GM and control peppers cultivated in three different areas of Korea. Statistical significance in terms of cultivation areas, harvest times, generations, and plant tissue origin were determined based on a P value of 0.05. The highest amount of CMV-CP was detected at the seedling stage from plant grown in each region. T3 and T5 showed significantly different levels of CMV-CP from T4 in leaves in the whorl stage. No statistical differences were observed among GM peppers at different stages of maturity in any cultivation area. The results from this study contribute to the safety evaluation of newly designed CMV-resistant GM peppers and provide a standard against which to compare other virus-resistant GM peppers.