Bt Xtra™ maize

Insect-resistant and glufosinate ammonium herbicide tolerant maize developed by inserting the cry1Ac gene from Bacillus thuringiensis subsp. kurstaki and the phosphinothricin acetyltransferase (bar) gene from Streptomyces hygroscopicus.

pDPG165, pDPG320 and pDPG699

Corn DBT418 was co-transformed using three vectors pDPG699, pDPG165 and pDPG320.

pDPG699 - contained the cry1Ac expression cassette: 2XOCS:35S promoter, adh1 intron VI, cry1A(c), potato pinII 3'-end. The vector also contained coding sequences for lac, fl(-)ori, bla, and colEl ori. Southern blot analysis showed integration of two intact copies of the cry1A(c) gene.

DPG165 - contains the bar expression cassette: 35S promoter, bar, Tr7 3'- end. The vector also contained coding sequences for lac, bla, and col El - origin. Southern blot analysis indicated that one intact copy of the bar gene and one rearranged copy of the bar gene had integrated into the host genome.

DPG320 - contains the protease inhibitor II gene from potato (pinII) transformation cassette.  The potato (pinII) gene in pDPG320 is under control of a 420 fragment of the Cauliflower Mosaic Virus promoter in combination with adh 1 intron 1 from maize and is terminated by the potato pinII 3'-end. Southern analysis indicated that one partial rearranged copy of the pinII gene was integrated into the host genome. PCR analysis analysis indicated that the pin II gene was rearranged in such a way that the coding sequence is non-functional cannot be expressed. This was confirmed by protein expression analysis

Vector Backbone Southern ananlysis indicated that there are four intact and one partial copy of the beta lactamase gene and four intact copies of the Col E1 origin of replication. The beta lactamase (bla) genes are not expressed as indicated by protein expression analysis.

Maize line DBT418 was genetically modified to contain two novel genes, cry1Ac and bar, for insect and herbicide tolerance respectively. Both genes were introduced into a maize line by particle acceleration (biolistic) transformation.

The transgenic maize line DBT418 was developed to resist ECB by producing its own insecticide. This event was genetically engineered by introducing the cry1Ac gene, isolated from the common soil bacterium Bacillus thuringiensis (Bt), into the maize line AT824. The cry1Ac gene produces the insect control protein Cry1Ac, a delta-endotoxin. Cry proteins, of which Cry1Ac is only one, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species. Following binding, pores are formed that disrupt midgut ion flow, causing gut paralysis and eventual death due to bacterial sepsis. Cry1Ac is lethal only when eaten by the larvae of lepidopteran insects (moths and butterflies), and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for the delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

In addition to the cry1Ac gene, DBT418 was developed to allow for the use of glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Rely®, Liberty®, and Finale®), as a weed control option, and as a breeding tool for selecting plants containing the cry1Ac gene. DBT418 contains the bar gene isolated from a common soil actinomycete, Streptomyces hygroscopicus. This gene allows for the production of the enzyme phosphinothricin N-acetyltransferase (PAT) which confers tolerance to glufosinate.

The PAT enzyme in maize line DBT418 converts L-phosphinothricin (PPT), the active ingredient in glufosinate ammonium, to an inactive form, thereby conferring resistance to the herbicide. In the absence of PAT, application of glufosinate leads to reduced production of the amino acid glutamine and increased ammonia levels in the plant tissues, resulting in the death of the plant. The PAT enzyme is not known to have any toxic properties.

Expression of the Cry1Ac protein and PAT occurred in most, but not all, tissues of the maize plant with levels highest in leaves, and lower levels in roots, prop roots, stalk, tassel, cob, husk, and kernels. No Cry1Ac or PAT proteins were detected in silk or pollen.