Abstract
BackgroundWe have developed a rice-based oral cholera vaccine named MucoRice-CTB (Cholera Toxin B-subunit) by using an Agrobacterium tumefaciens–mediated co-transformation system. To assess the genome-wide effects of this system on the rice genome, we compared the genomes of three selection marker–free MucoRice-CTB lines with those of two wild-type rice lines (Oryza sativa L. cv. Nipponbare). Mutation profiles of the transgenic and wild-type genomes were examined by next-generation sequencing (NGS).ResultsUsing paired-end short-read sequencing, a total of more than 300 million reads for each line were obtained and mapped onto the rice reference genome. The number and distribution of variants were similar in all five lines: the numbers of line-specific variants ranged from 524 to 842 and corresponding mutation rates ranged from 1.41 × 10−6 per site to 2.28 × 10−6 per site. The frequency of guanine-to-thymine and cytosine-to-adenine transversions was higher in MucoRice-CTB lines than in WT lines. The transition-to-transversion ratio was 1.12 in MucoRice-CTB lines and 1.65 in WT lines. Analysis of variant-sharing profiles showed that the variants common to all five lines were the most abundant, and the numbers of line-specific variant for all lines were similar. The numbers of non-synonymous amino acid substitutions in MucoRice-CTB lines (15 to 21) were slightly higher than those in WT lines (7 or 8), whereas the numbers of frame shifts were similar in all five lines.ConclusionsWe conclude that MucoRice-CTB and WT are almost identical at the genomic level and that genome-wide effects caused by the Agrobacterium-mediated transformation system for marker-free MucoRice-CTB lines were slight. The comparative whole-genome analyses between MucoRice-CTB and WT lines using NGS provides a reliable estimate of genome-wide differences. A similar approach may be applicable to other transgenic rice plants generated by using this Agrobacterium-mediated transformation system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1285-y) contains supplementary material, which is available to authorized users.
Highlights
We have developed a rice-based oral cholera vaccine named MucoRice-cholera toxin B-subunit (CTB) (Cholera Toxin B-subunit) by using an Agrobacterium tumefaciens–mediated co-transformation system
The three MucoRice-CTB lines were selected by the level of CTB protein production and elimination of the marker gene used for the initial transformant selection [11]. We found that these MucoRice-CTB and WT lines are almost identical at the genomic level
In addition to the 12 rice chromosomes, nucleotide sequences of the CTB expression construct, hygromycin resistance gene used as a selection marker, and the binary vector used for Agrobacterium-mediated transformation were added to the reference to examine whether these sequences
Summary
We have developed a rice-based oral cholera vaccine named MucoRice-CTB (Cholera Toxin B-subunit) by using an Agrobacterium tumefaciens–mediated co-transformation system. The advantages of producing PMPs compared to conventional production systems We previously reported MucoRice-CTB, transgenic rice expressing cholera toxin B-subunit (CTB) designed. Kashima et al BMC Genomics (2015) 16:48 as an oral vaccine against cholera [7]. Oral vaccination of mice and macaques with MucoRice-CTB resulted in the induction of antigen-specific serum IgG and mucosal IgA responses with toxin-neutralizing immunity [8]. Because of sequence similarity between cholera toxin (CT) and heat-labile enterotoxin from enterotoxigenic Escherichia coli, MucoRice-CTB successfully induced protective immunity against both Vibrio cholerae–induced and enterotoxigenic E. coli–induced diarrhea [9]. The amount of CTB produced in rice endosperm without RNAi reached only 1/6 of that of MucoRiceCTB with RNAi [10]
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