Seed Metabolome Analysis of a Transgenic Rice Line Expressing Cholera Toxin B-subunit

Takumi Ogawa,Yoshikazu Yuki,Mio Mejima,Daisaku Ohta,Koji Kashima,Masaharu Kuroda,Shiho Kurokawa,Atsushi Okazawa,Hiroshi Kiyono

doi:10.1038/s41598-017-04701-w

Abstract

Plant-based human vaccines have been actively developed in recent years, and rice (Oryza sativa L.) is one of the best candidate crops for their production and delivery. By expressing a modified cholera toxin B (CTB) subunit, we previously developed MucoRice-CTB, a rice-based vaccine against cholera, which is caused by infection of the intestine with the bacteria Vibrio cholerae. MucoRice-CTB lines have been extensively characterized by whole-genome sequencing and proteome analyses to evaluate the mutation profiles and proteome status, respectively. Here, we report non-targeted metabolomic profiling of the MucoRice-CTB transgenic rice line 51A (MR-CTB51A), MucoRice-RNAi (MR-RNAi), and their non-transgenic parent line by using gas chromatography–time-of-flight mass spectrometry. The levels of several amino acids, organic acids, carbohydrates, lipids, and secondary metabolites were significantly increased in MR-CTB51A compared with the non-transgenic parent line. These metabolomics results complement essential information obtained by genome sequencing and proteomics approaches, thereby contributing to comprehensive understanding of the properties of MucoRice-CTB as a plant-based vaccine.

Highlights

MucoRice-cholera toxin B (CTB) has been developed as a rice-seed-based vaccine against cholera, which is caused by the infection of the intestine with the bacteria Vibrio cholerae
From total ion current (TIC) chromatograms obtained by Gas chromatography (GC)/time-of-flight (TOF)-mass spectrometry (MS) analyses, 198 and 153 ion peaks were listed as metabolite-candidate peaks in the non-polar fractions and the polar fractions, respectively (Supplementary Table S2)
The Principal component analysis (PCA) factor loading plot (Fig. 1b) revealed that the separation of metabolome clusters was due not to a few specific metabolites but to several metabolites (Fig. 1b; Supplementary Table S3). These results indicate that the metabolomic profiles of transgenic lines were distinct from those of non-transgenic rice

Summary

Introduction

MucoRice-CTB (cholera toxin B subunit) has been developed as a rice-seed-based vaccine against cholera, which is caused by the infection of the intestine with the bacteria Vibrio cholerae. A comparative whole-genome analysis using next-generation sequencing demonstrated that MucoRice-CTB transgenic lines (including MR-CTB51A) and their parental non-transgenic lines Fluorescence difference gel electrophoresis and shotgun proteome analyses of MucoRice-CTB brown rice revealed that CTB expression together with RNAi-mediated suppression of seed storage proteins in the endosperm did not up-regulate known rice allergens[17]. The aim of this study was to investigate whether CTB expression and RNAi-mediated suppression of endogenous seed storage proteins influence rice seed metabolism. We compared the brown rice seed metabolome of MR-CTB51A (grown hydroponically) with those of the parental NPB line (grown either hydroponically [NPB-HP] or in a paddy field [NPB-PF]) and MucoRice-RNAi (MR-RNAi), a transgenic line with RNAi-mediated suppression of endogenous seed storage proteins[17]. The metabolic profiles differed among MR-CTB51A, MR-RNAi, and the parental NPB lines, especially in the contents of hydrophilic primary metabolites and lipids, no metabolites unique to these transgenic lines were identified

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Conclusion