Transient expression of optimized and synthesized nattokinase gene in melon (Cucumis melo L.) fruit by agroinfiltration

Abstract

A synthetic gene (sNK) encoding Nattokinase (NK) was constructed by modifying its sequence based on the codon usage in plants. A version (sNKi) incorporating the first intron from the tomato E8 gene was also constructed. The synthetic sNK and sNKi genes were transiently expressed in melon (Cucumis melo L.) fruit. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis and fibrinolytic activity assays showed that the expression level of recombinant NK controlled by E8 promoter was higher than that controlled by 35S promoter, with the maximum fibrinolytic activity of 79.30 U ml−1. The intron could enhance the expression of sNK to an extent of 27.42%. Orthogonal tests determined the optimal agroinfiltration as: an acetosyringone concentration of 0.25 mM, an OD600=0.6 and harvesting 5 days after infiltration.