Characterisation of 3′ transgene insertion site and derived mRNAs in MON810 YieldGard® maize

The construct inserted in YieldGard (R) MON810 maize, produced by Monsanto, contains the CaMV 35S promoter, the hsp70 intron of maize, the cryI(A)b gene for resistance to lepidopterans and the NOS terminator. In a previous work a truncation event at the 3' end of the cryI(A)b gene leading to the complete loss of the NOS terminator was demonstrated. The 3' maize genome junction region was isolated in the same experiment not showing any homology with known sequences. The aim of the experiments here reported was therefore to isolate and characterize a larger portion of the 3' integration junction from genomic DNA of two commercial MON810 maize lines. Specific primers were designed on the 3' integration junction sequence for the amplification of a 476 bp fragment downstream of the sequence previously detected. In silico analysis identified the whole isolated 3' genomic region as a gene putatively coding for the HECT E3 ubiquitin ligase. RT-PCR performed in this region produced cDNA variants of different length. In silico translation of these transcripts identified 2 and 18 putative additional aminoacids in different variants, all derived from the adjacent host genomic sequences, added to the truncated CRY1A protein. These putative recombinant proteins did not show homology with any known protein domains. Our data gave new insights on the genomic organization of MON810 in the YieldGard (R) maize and confirmed the previous suggestion that the integration in the genome of maize caused a complex recombination event without, apparently, interfering with the activity of the partial CRY1A endotoxin and both the vigor and yield of the YieldGard (R) maize.