Two similar but distinct second intron fragments from tobacco AGAMOUS homologs confer identical floral organ-specific expression sufficient for generating complete sterility in plants
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The carpel- and stamen-specific AtAGIP promoter derived from the Arabidopsis AGAMOUS (AG) second intron/enhancer is ideal for engineering complete sterility but it is highly host-specific. To ascertain whether a chimeric promoter with similar tissue specificity can be created for species other than Arabidopsis, we isolated two similar but distinct AG second intron/enhancers from tobacco (NtAGI-1 and NtAGI-2) and analyzed their ability to drive floral organ-specific expression in plants through the creation of forward- and reverse-oriented chimeric promoters, fNtAGIP1, rNtAGIP1, fNtAGIP2 and rNtAGIP2. Analyses of transgenic plants bearing each respective promoter fused to the β-glucuronidase (GUS) reporter gene showed that all four promoters are able, like the AtAGIP, to drive very similar carpel- and stamen-specific expression without any leaky activity in vegetative tissues. These results indicate that unlike their counterparts in rice and maize, the tobacco NtAGI-1 and NtAGI-2 enhancers share a highly conserved regulatory function. Interestingly, all four promoters display additional tissue specificity in petals, and their activity is influenced by the orientation of the incorporated enhancer, with reverse-oriented enhancers exhibiting approximately double the effectiveness of forward-oriented enhancers. These properties are novel and have not been observed with the AtAGIP promoter in Arabidopsis. As expected, these highly specific promoters can also direct the expression of the DT-A cytotoxic gene exclusively in carpels, stamens and petals, resulting in complete sterility through the precise ablation of targeted floral organs. Further analyses demonstrated that the resulting trait is mitotically stable, which is critical for the long-term containment of seed-, pollen- and fruit-mediated gene flow in field conditions.
KeywordsAGAMOUS Enhancers Tissue specificity Transgenic plants Sterility Gene containment
Promoter derived from the second intron of AG from Arabidopsis thaliana
Cauliflower mosaic virus 35S promoter
Cassava vein mosaic virus promoter
Diphtheria toxin A
AG homolog from Nicotiana tabacum
Second intron of the NAG gene from N. tabacum
Promoter derived from the second intron of the NAG gene from N. tabacum
Polymerase chain reaction
Reverse transcription polymerase chain reaction
We thank Mr. Dennis Bennett for his excellent technical assistance. This study was funded by the United States Department of Agriculture (USDA)—Agricultural Research Service Headquarter 2007 classes of postdoctoral grants and the Biotechnology Risk Assessment Program Grant (2006-03701) from the USDA National Institute of Food and Agriculture.
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