<p style="text-align: justify;">MIR604 maize was produced by Agrobacterium-mediated transformation of the inbred maize line A188. The T-DNA segment of the vector plasmid pZM26 contained a modified <i>cry3A</i> (<i>mcry3A</i>) gene encoding the mCry3A insecticidal protein and the <i>pmi</i> (<i>manA</i>) gene from <i>Escherichia coli</i> encoding the enzyme phosphomannose isomerase as a selectable marker.<br>
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The maize-optimised modified <i>mcry3A</i> gene encodes a protein of 598 amino acids. The native <i>cry3A</i> gene was modified to incorporate a cathepsin-G serine protease recognition site within the expressed protein. The original N-terminal region of this protein has been removed and the mCry3A protein commences at a methionine residue in position 48 of the native protein. The entire coding region of the <i>mcry3A</i> gene was synthesised using codons that are preferred in maize. The amino acid sequence of the synthetic version of Cry3A is the same as the native protein, except for the modified serine-protease recognition site. Transcription of the <i>mcry3A</i> gene was directed by the MTL promoter derived from the <i>Zea mays</i> metallothionein-like gene, which demonstrates root-preferential expression. Terminator and polyadenylation sequences were derived from the nopaline synthase gene from <i>Agrobacterium tumefaciens</i>.<br>
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The <i>pmi</i> gene represents the <i>E.coli manA</i> gene and encodes the enzyme phosphomannose isomerase (PMI). It was used as a selectable marker gene during the transformation process. Mannose, a hexose sugar, is taken up by plants and converted to mannose-6-phosphate by hexokinase. This product cannot be further utilised in plants as they lack the PMI enzyme. The accumulation of mannose-6-phosphate inhibits phosphoglucose isomerase, causing a block in glycolysis. It also depletes cells of orthophosphate required for the production of ATP. Therefore, while mannose has no direct toxicity on plant cells, it causes growth. This does not occur in plants transformed with the <i>pmi</i> gene as they can utilise mannose as a source of carbon. Expression of the <i>pmi</i> gene was driven by the promoter and first intron from the <i>Z. mays</i> polyubiquitin gene which provides constitutive gene expression in monocots. The nopaline synthase gene from <i>Agrobacterium tumefaciens</i> was again used for termination and polyadenylation sequences. The left border sequence (from octopine Ti plasmid pTi15955) and right border (from nopaline Ti plasmid pTiT37) contained non-coding sequences essential for the transfer of the T-DNA segment.<br>
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