Characterisation of 3′ transgene insertion site and derived mRNAs in MON810 YieldGard® maize
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The construct inserted in YieldGard® 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® 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® maize.
KeywordsCry-hect recombinant mRNAs 3′ insertion site HECT protein ligase YieldGard® MON810 maize
We thank Dr. G. Monastra for providing seeds of MON810 and isogenic control maize. This work was supported by a grant from MIPAF (Ministero delle Politiche Agricole, Alimentari e Forestali), Project: “OGM in Agricoltura”.
- Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK, Zimmerman J, Barajas P, Cheuk R, Gadrinab C, Heller C, Jeske A, Koesema E, Meyers CC, Parker H, Prednis L, Ansari Y, Choy N, Deen H, Geralt M, Hazari N, Hom E, Karnes M, Mulholland C, Ndubaku R, Schmidt I, Guzman P, Aguilar-Henonin L, Schmid M, Weigel D, Carter DE, Marchand T, Risseeuw E, Brogden D, Zeko A, Crosby WL, Berry CC, Ecker JR (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301(5633):653–657PubMedCrossRefGoogle Scholar
- Bogani P, Simoni A, Bettini P, Mugnai M, Pellegrini MG, Buiatti M (1995) Genome flux in tomato auto- and auxotrophic cell clones cultured in different auxin/cytokinin equilibria. I. DNA multiplicity and methylation levels. Genome 38:902–912Google Scholar
- Dunning Hotopp JC, Clark ME, Oliveira DCS, Foster JM, Fischer P, Muñoz Torres MC, Giebel JD, Kumar N, Ishmael N, Wang S, Ingram J, Nene RV, Shepard J, Tomkins J, Richards S, Spiro DJ, Ghedin E, Slatko BE, Tettelin H, Werren JH (2007) Widespread lateral gene transfer from intracellular bacteria to multicellular eukaryotes. Science 317:1753–1756CrossRefGoogle Scholar
- Kohli A, Griffiths S, Palacios N, Twyman RM, Vain P, Laurie DA, Christou P (1999) Molecular characterisation of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination. Plant J 17(6):591–601PubMedCrossRefGoogle Scholar
- Mehlo L, Mazithulela G, Twyman RM, Boulton MI, Davies JW, Christou P (2000) Structural analysis of transgene rearrangements and effects on expression in transgenic maize plants generated by particle bombardment. Maydica 45:277–287Google Scholar
- Rozen S, Skaletsky HJ (2000) Primer3 on the WWW for general users and for biologist programmers. In: Krawetz S, Misener S (eds) Bioinformatics methods and protocols: methods in molecular biology. Humana Press, Totowa, pp 365–386Google Scholar
- Wilson A, Latham J, Steinbrecher R (2006) Transformation-induced mutations in transgenic plants: analysis and biosafety implications. Biotechnol Genet Eng Rev 23:209–234Google Scholar
- Zhang J, Guo D, Chang Y, You C, Li X, Dai X, Weng Q, Zhang J, Chen G, Li X, Liu H, Han B, Zhang Q, Wu C (2007) Non-random distribution of T-DNA insertions at various levels of the genome hierarchy as revealed by analyzing 13 804 T-DNA flanking sequences from an enhancer-trap mutant library. Plant J 49(5):947–959PubMedCrossRefGoogle Scholar