Genetic elements introduced from pTDL008 (derived from pGSV20):
Transcription of the Bacillus thuringiensis Cry1Ab gene begins at the Subterranean clover stunt virus promoter (Ps7s7) and terminates at the Flavera bidentis NADP-malic enzyme 1 (me1) terminator. A 5’ leader sequence from Oryza sativa GE1 was included to enhance transcription. The gene cassette is in the counter-clockwise orientation.

A second gene cassette contains a herbicide tolerance gene, Streptomyces hygroscopicus phosphinothricin N-acetyltransferase gene (bar), which is under transcriptional control of the Cauliflower mosaic virus (CMV) 35S promoter and the Agrobacterium tumefaciens nopaline synthase (nos) terminator.

Note:
Sequencing of the 9056 bp inserted transgenic construct and Southern blot analysis revealed an almost full copy of the T-DNA construct (with an incomplete 3`me1 terminator) was inserted into the T304-40 LM cotton line in addition to:
- a partial 3' me1 terminator;
- a partial copy of the cry1Ab gene cassette, with a truncated Ps7s7 promoter, in a tail-to-tail orientation, and
- a partial copy of the bar gene cassette in which the nos terminator is truncated.

pTEM12 (derived from pGSC1700):
Another bar gene cassette was integrated into the parental line in a counter-clockwise orientation. Transcription commences from the Cassava vein mosaic virus promoter and terminates the CMV 35S terminator.

A Cry2Ae gene cassette was also integrated into the parental line. Transcription of the B. thuringiensis Cry2Ae gene is controlled by the CaMV 35S promoter and terminator. In addition to the Cry2Ae coding sequence, the transcript contains the 5’ untranslated leader of Petunia hybrida chlorophyll a/b-binding protein and the Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase small subunit transit peptide for enhancing transcription and higher steady-state levels of the protein in the leaves, respectively.

Note:
Southern Blot analysis indicated that a single copy of the insert is present in the GHB119 line and a configuration that correspond to that in the original vector. No insertion of plasmid backbone sequences was detected.

pCOT-1:
Transcription of the Escherichia coli hygromycin B phosphotransferase is under control of the A. thaliana ubiquitin 3 promoter and the nos terminator. The gene cassette is present in the counterclockwise orientation. The hygromycin B phosphotransferase (aph4) gene is used for selection of transformants.

The expression of B. thuringiensis vegetative insecticidal protein 3A (vip3A) is under control of the A. thaliana actin 2 promoter and the nos terminator. Coding sequence of the Vip3A gene was altered for optimal expression in plant cells. Southern blot analysis confirmed the protein expression.

Note:
Southern blot analysis of the genomic DNA from COT102 revealed the incorporation of single intact copies of the vip3Aa and aph4, along with intact copies of their respective regulatory sequences. Results of these analyses also demonstrated that none of the vector backbone sequences, including the streptomycin adenyltransferase gene, were incorporated into the genomic DNA.

For more information, kindly review the records of the parental lines.