Methods for conditional transgene expression and trait removal in plants

Abstract

This invention relates to constructs for the conditional or regulated expression or excision of transgenes in plants using site-specific recombinase systems. The constructs comprise a variety of constitutive, inducible, tissue specific or developmental stage-specific promoters operably linked to either a transgene or the elements of one or more site-specific recombinase system. By matching promoters, responsive to various inducers, plant tissues or plant developmental states with the recombinase systems, stop fragments and transgenes, virtually any trait may be expressed or excised at any plant development stage or in any plant generation.

1 - 54 . (canceled)  
     
     
         55 . A method for transgene removal comprising: 
 1) providing a construct comprising: 
 a) a first recombinase element having the general structure P1-R1;  
 b) a second recombinase element having the general structure RS1-P2-TG-RS1;  
   wherein: 
 (i) R1 is a first recombinase coding sequence and 3′ region;  
 (ii) RS1 is a first recombinase site responsive to a first recombinase;  
 (iii) TG is transgene sequence and 3′ region encoding either a trait or a transformation marker;  
 (iv) P1 is a first promoter that expresses R1 in the germline of the first generation;  
 (vi) P2 is a second promoter, wherein P2 is activated before or at the same as P1;  
   wherein P1, and P2 are operably linked to their down stream elements;    2) providing a first plant comprising the first recombinase element;    3) providing a second plant comprising the second recombinase element;    4) crossing the first and second plants to create a first generation plant wherein the first promoter (P2) is activated, expressing the transgene;    5) activating the first promoter (P1) wherein the recombinase is expressed, resulting in the removal of the transgene from the chromosome.    
     
     
         56 . A method according to  claim 55  wherein P1 is a common germline selected from the group consisting of  Apetala  3 , Pistillata , Bcp I,  Apetala  1, Leafy, and  Agamous  genes.  
     
     
         57 . A method according to  claim 55  wherein P1 is a male germline promoter which excises the transgene from the pollen.  
     
     
         58 . A method according to  claim 55  wherein P1 is a floral common germline promoter that expresses the recombinase to excises the transgene from the progeny seed.  
     
     
         59 . A method according to  claim 57  wherein P1 is a promoter derived from  Arabidopsis  selected from the group consisting of  Apetala  3 (AP3),  Pistillata  (PI), Bcp I genes or a synthetic anther promoter.  
     
     
         60 . A method according to  claim 58  wherein P1 is a promoter derived from  Arabidopsis  selected from the group consisting of  Apetala  3 (AP3),  Pistillata  (PI), Leafy,  Agamous, Apetala  1.  
     
     
         61 . A method for transformation marker excision comprising: 
 1) providing a construct comprising a recombinase element having the general structure RS1-P1-R1-P2-TG1-RS1 P3-TG2;    wherein: 
 (i) R1 is a recombinase coding sequence and 3′ region;  
 (ii) RS1 is a recombinase site responsive to recombinase R1;  
 (iii) TG1 is transgene sequence and 3′ region encoding a transformation marker and 3′ region;  
 (iv) TG2 is trait transgene sequence and 3′ region;  
 (v) P1 is a first promoter that is activated in the germline of the primary transformant;  
 (vi) P2 is a second promoter that is activated in the primary transformant;  
 (vii) P3 is a third promoter;  
   wherein P1, P2 and P3 are operably linked to their down stream elements;    2) providing a transgenic plant comprising the recombinase element;    3) activating the second promoter (P2) whereby the transgene encoding a marker (TG1) expressed;    4) activating the first promoter (P1) whereby the recombinase (R1) is expressed resulting in the excision of the transgene encoding a marker (TG1); and    5) activating the third promoter (P3) whereby the trait transgene (TG2) is expressed.    
     
     
         62 . A method according to  claim 61 , wherein P1 is a common germline promoter that is inducible.  
     
     
         63 . A method according to  claim 61 , wherein P1 is a common germline promoter from  Arabidopsis  heat shock promoter.  
     
     
         64 . A method according to  claim 61 , wherein P1 is a common germline promoter from safener inducible promoter IN-2.  
     
     
         65 . A method according to  claim 61 , wherein P1 is a common germline promoter from developmentally regulated genes of shoot apical meristem, floral germline, or male germline cells.  
     
     
         66 . A method according to  claim 61 , wherein P1 is a common germline promoter derived from vegetative and floral shoot apical meristem, flower, or male germline wherein the vegetative and floral shoot apical meristem promoter is selected from the group consisting of Leafy,  Apetala  1 , Apetala  3 (AP3),  Pistillata  (PI) or their orthologs from other species, the flower promoter is selected from the group consisting of synthetic anther promoter and Bcp I genes or their orthologs from other species, and the male germline promoter is selected from the group consisting of  Apetala  3 (AP3),  Pistillata  (PI) or their orthologs from other species.  
     
     
         67 . A method according to  claim 57 , wherein the transformation marker is a morphological trait, hormone biosynthetic gene, selectable gene.  
     
     
         68 - 80 . (canceled)  
     
     
         81 . The method according to  claim 61  wherein the transgene is excised in pollen.  
     
     
         82 . A method for transgene removal comprising: 
 1) providing a transgenic plant comprising: 
 a) a first recombinase element having the general structure P1-R1;  
 b) a second recombinase element having the general structure RS1-P2 TG-RS1;  
   wherein: 
 (i) R1 is a first recombinase coding sequence and 3′ region;  
 (ii) RS1 is a first recombinase site responsive to a first recombinase;  
 (iii) TG is transgene sequence and 3′ region encoding either a trait or a transformation marker;  
 (iv) P1 is a first promoter that is activated in the germline of the primary transformant;  
 (vi) P2 is a second promoter, wherein P2 is activated before or at the same as P1;  
   wherein P1, and P2 are operably linked to their downstream elements;    2) activating the first promoter (P1), wherein the recombinase is expressed, resulting in the removal of the transgene from the chromosome.    
     
     
         83 . The method according to  claim 82 , wherein the transgene is excised in pollen.  
     
     
         84 . The method according to  claim 55 , wherein the first plant and the second plant are selected from the group consisting of tobacco,  Brassica , sunflower, cotton,  Arabidopsis , alfalfa, soybean, rice, corn, and rye.  
     
     
         85 . The method according to  claim 61 , wherein the plant is selected from the group consisting of tobacco,  Brassica , sunflower, cotton,  Arabidopsis , alfalfa, soybean, rice, corn, and rye.  
     
     
         86 . The method according to  claim 82 , wherein the plant is selected from the group consisting of tobacco,  Brassica , sunflower, cotton,  Arabidopsis , alfalfa, soybean, rice, corn, and rye.