Abstract
The insect resistant maize YieldGard MON810 was studied to assess the extent to which introduction of a transgene may putatively alter the expression of endogenous genes by comparison of various GM lines vs. their non-transgenic counterparts. To assess the extent to which introduction of a transgene may putatively alter the expression of endogenous genes, GM lines of the insect resistant maize YieldGard MON810 were compared with non-transgenic counterparts. For a more in-depth study, high-throughput deep sequencing together with microarrays were used to compare the transcriptomes of immature embryos of the MON810 variety DKC6575, with a cryIA(b) transgene, and its near-isogenic variety Tietar, grown under controlled environmental conditions. This technique also allows characterisation of the transgenic mRNAs produced. 3′UTR-anchored mRNA-seq produced 1,802,571 sequences from DKC6575 and 1,170,973 from Tietar, which mapped to 14,712 and 14,854 unigenes, respectively. Up to 32 reads from the transgenic embryos matched to the synthetic cry1A(b) sequence, similar to medium-abundant mRNAs. Gene expression analysis, using the R-bioconductor packages EdgeR and DEseq, revealed 140 differentially expressed genes mainly involved in carbohydrate metabolism, protein metabolism and chromatin organisation. Comparison of the expression of 30 selected genes in two additional MON810 and near-isogenic variety pairs showed that most of them were differentially expressed in the three pairs of varieties analysed. Analysis of functional annotation and the precise moment of expression of the differentially expressed genes and physiological data obtained suggest a slight but significant delay in seed and plant maturation of MON810 plants. However, these transcriptomic changes were not associated to undesirable changes in the global phenotype or plant behaviour. Moreover, while most expression changes in MON810 immature embryos were maintained in other transgenic varieties, some gene expression was found to be modulated by the genetic background in which the transgene was introduced through conventional breeding programs.
Highlights
Modified (GM) plants have been cultivated for 17 years, globally covering 160 million ha in 2011
Selected reads were aligned with the B73 maize genome version 5b.60 [17], available at the Maize Genome Sequencing Project web, considering positive those reads showing at least 90% identity with the reference, with a minimum alignment region of 40 nucleotides, which corresponds to the minimum read length given by the 454 software
Using the AgriGO tool [18] for a singular enrichment analysis (SEA) for the GO categories of biological process and molecular function there were no significant GO categories overrepresented comparing a total of 9695 GO categories between DKC6575 and Tietar
Summary
Modified (GM) plants have been cultivated for 17 years, globally covering 160 million ha in 2011. Maize is the second major GM crop, occupying 32% of the global GM area and is the species with the highest number of events approved (65). The insect resistant maize YieldGard MON810 ranks as the second GM event to receive regulatory approval in most countries (23), after the herbicide tolerant soybean GTS-403–2 [1]. MON810 has a transgene cassette carrying an enhanced 35S transcription promoter derived from the cauliflower mosaic virus, the hsp intron and a 59 portion of a Bacillus thuringiensis-derived gene, cryIA(b), encoding an insecticidal protein (h-endotoxin) which controls lepidopteran pest insects such as the European corn borer. The same transgene cassette has been introgressed into several maize varieties. Characterisation of the transgene insertion site has demonstrated a 39 truncation of the cassette resulting in lack of the termination sequence [2] and a complex recombination event associated with transgene integration. As a result of a stop codon at position +7 downstream of this site, the transgenic protein includes two additional amino acids, which is compatible with the reported protein size [4,5]
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