Abstract
Safety assessment for genetically modified organisms (GMOs) is required before their release. To date, miRNAs that play important roles in eukaryotic gene regulation have not been considered in the current assessment system. In this study, we identified 6 independent Bt and EPSPS GM rice lines using PCR and immune strip. We analyzed the expression levels of Cry1Ac and EPSPS using quantitative real-time PCR and western blot. Further, miRNAs from the developing seeds of the 6 GM rice lines and the wild-type line were investigated using deep sequencing and bioinformatic approaches. Although these GM lines have different types of integration sites, copy numbers, and levels of gene expression, 21 differentially expressed miRNAs have been found compared to wild type. There is no correlation between transgenic protein expression level and the quantity of differentially expressed miRNAs. This study provides useful data about the miRNA composition of GM plants, and it might be helpful for future risk assessments of miRNA-based GM plants.
Highlights
Differential expression analysis of conserved miRNAs between non-transgenic CK and transgenic lines L1 to L6
We discovered a set of differentially expressed miRNAs from each line compared to the non-transgenic CK, and we compared the tags per million (TPM) values of every sample from all the sets of differential expression miRNAs to make a hierarchical cluster
We found that the rice grain grows in weight quickly during the first 12–18 days after flowering (DAF)[23,29]
Summary
Differential expression analysis of conserved miRNAs between non-transgenic CK and transgenic lines L1 to L6. To identify differentially expressed miRNAs, we compared the expression of conserved miRNAs between non-transgenic CK and the transgenic group lines L1 to L6 (Supplementary Fig. S3). Based on the high-throughput sequencing results, we used a hierarchical clustering algorithm to analyze differentially expressed miRNAs in every sample (Fig. 2). We discovered a set of differentially expressed miRNAs from each line compared to the non-transgenic CK, and we compared the tags per million (TPM) values of every sample from all the sets of differential expression miRNAs to make a hierarchical cluster. The results indicated that 7 miRNAs, i.e. miRNA5337a, miR3979-3p, miR2873c, miR1870-3p, miR169e, miR166e-3p, and miR156l-5p, were significantly up-regulated compared with the non-transgenic CK. 14 miRNAs, i.e.miR5799, miR529b, miR399i, miR2863a, miR2118o, miR2118e, miR1874-5p, miR1874-3p, miR1846d-3p, miR1428f-5p, miR1428e-5p, miR1428d, miR1428c and miR1428b, were significantly down-regulated compared with the non-transgenic CK. There are 547 miRNAs (96% of the total) that showed no difference between non-transgenic CK and the transgenic group
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