Fertility restored, Glufinosate tolerant canola

The modified canola (Brassica napus) was created through crossing of two modified canola lines. The modified canola expresses Streptomyces hygroscopicus phosphinothricin N-acetyltransferase, which catalyzes the acetylation of phosphinothricin to prevent glutamine synthetase inhibition and thus confers tolerance to glufinosate herbicides. The modified canola additionally contains the engineered restoration of male fertility. 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.

pTCO113; pTHW118

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.

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.