Fertility restored, Herbicide tolerant canola

The modified canola (Brassica napus) was created through crossing of three modified canola lines for tolerance to herbicides. For tolerance to glufinosate, the modified canola expresses Streptomyces hygroscopicus phosphinothricin N-acetyltransferase, which catalyzes the acetylation of phosphinothricin to prevent glutamine synthetase inhibition. For tolerance to glyphosate, the modified canola expresses Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase, which does not bind glyphosate and allows for the continued biosynthesis of essential aromatic amino acids via the shikimate pathway. The modified canola additionally contains the engineered restoration of male fertility from a parental hybrid line. The canola expresses Bacillus amyloliquefaciens barnase, an RNase, in the tapetum cells of the pollen sac during anther development and causes male sterility by interfering with RNA production. However, the canola also expresses B. amyloliquefaciens barstar, which inhibits barnase by forming a stable complex with the enzyme. Thus, barnase activity is prevented in pollen tissues and male fertility is restored.

Please note the developer has not been confirmed.

pTCO113; pTHW118; PV-BNHT2672

DNA insert from MS11 vector pTCO113
The DNA insert from pTCO113 contains three gene cassettes: Streptomyces hygroscopicus phosphinothricin N-acetyltransferase (bar); Bacillus amyloliquefaciens barnase and B. amyloliquefaciens barstar.

Transcription of bar commences from the Arabidopsis thaliana ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) promoter  and terminates at the Agrobacterium tumefaciens transcript 7 3' untranslated region.

Transcription of barnase starts from the Nicotiana tabacum pTA29 promoter and ends at the A. tumefaciens nopaline synthase (nos) terminator. The transcript additionally contains the 3' untranslated region of Barnase. The pTA29 promoter is pollen specific and transcription is expected to occur only within pollen tissues.

Transcription of barstar commences from the nos promoter and terminates at a transcript 7 3' untranslated region.

Note:
- DNA sequencing and southern blot analyses indicated that the parental genome contain a single, intact T-DNA insert

DNA insert from RF3 vector pTHW118
The DNA insert from the pTHW118  contains two gene cassettes: barstar and bar.

Transcription of barstar starts from the pTA29 promoter and ends at a nos terminator. The transcript additionally contains the barstar gene terminator, which is not expected to be translated into protein.The pTA29 promoter is pollen specific and transcription is expected to occur only within pollen tissues.

Transcription of bar commences from the rbcS promoter and terminates at the transcript 7 3' untranslated region.

Note:
- Two codons on the N-terminus of bar have been substituted for the codons ATG and GAC.
- Southern blot and PCR anaylses indicated that the parental genome contains one complete copy of the transformation cassette and an inverted partial copy containing a portion of the pTA29 promoter, barstar and the nos terminator adjacent to the left T-DNA boundary.

DNA insert from MON88302 vector PV-BNHT2672
The DNA insert from PV-BNHT2672 contains a single gene cassette for expression of Agrobacterium tumefaciens 5-enolpyruvylshikimate-3-phosphate synthase (epsps). Transcription of epsps is under control of a chimeric promoter, containing the Figwort mosaic virus 35S enhancer and the Arabidopsis thaliana elongation factor 1-alpha (EF-1α), and Pisum sativum ribulose-1,5-bisphosphate carboxylase small subunit E9 terminator (rbcS-E9). The transcript initially contains an A. thaliana EF-1α leader, an EF-1α intron 1 and an A. thaliana chloroplast transit peptide 2 prior (5') to the epsps coding sequence. High levels of transcription are expected from the chimeric promoter. The EF-1α leader and intron additionally enhance expression. The chloroplast transit peptide targets EPSPS for accumulation within the chloroplast.

Note:
- Southern blot analysis indicated that the parental genome contains a single T-DNA insertion without backbone integration.

For more information, kindly refer to the parental LMO records.

Barnase ribonuclease is secreted by B. amyloliquefaciens and is lethal due its RNase activity. In the bacterium, the inhibitor, barstar, is also synthesized and binds to barnase after synthesis to prevent damage due to degradation of the cellular RNA. The inhibitor is removed upon secretion.

Glyphosate binds the endogenous 5-enolpyruvulshikimate-3-phosphate synthase enzyme, inactivating the enzyme and preventing the essential biosynthesis of aromatic amino acids (tyrosine, phenylalanine and tryptophan) and folates via the shikimate pathway. The introduction of a bacterial epsps coding sequence allows for the production of aromatic compounds in the presence of glyphosate as the bacterial EPSPS protein will not bind the compound.