Method for isolation of transcription termination sequences

Abstract

The invention relates to efficient, high-throughput methods, systems, and DNA constructs for identification and isolation of transcription termination sequences. The invention relates further to specific terminator sequences identified by said methods isolated from rice.

We claim: 
     
         1 . An expression construct comprising in 5′ to 3′ direction:
 a) a promoter sequence functional in plants; 
 b) a nucleic acid sequence of interest to be expressed operably linked to the promoter sequence of a); and 
 c) at least one sequence operably linked to the promoter sequence of a) and the nucleic acid sequence of b), wherein said at least one sequence is selected from the group consisting of:
 i) the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46; 
 ii) sequences having a homology of at least 60%, 80%, 90% or 95% with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; 
 iii) sequences hybridizing under high stringency conditions equivalent to hybridization at 68° C. in a solution consisting of 5×SSPE, 1% SDS, 5×Denhardt's reagent and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1×SSPE and 0.1% SDS at 68° C. with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; and 
 iv) a fragment of at least 50, 100, 250 or 500 consecutive base pairs of any of the sequences of i), ii) and iii), 
 
 wherein said at least one sequence of c) is heterologous to said promoter sequence of a) and/or said nucleic acid sequence of interest to be expressed of b), and is mediating termination of expression of the nucleic acid sequence of b) induced from said promoter sequence of a). 
 
     
     
         2 . A vector comprising the expression construct of  claim 1 . 
     
     
         3 . A transgenic host cell or non-human organism comprising:
 a) the expression construct of  claim 1 ; or   b) a vector comprising said expression construct.   
     
     
         4 . A transgenic plant comprising:
 a) the expression construct of  claim 1 ; or   b) a vector comprising said expression construct.   
     
     
         5 . A transgenic expression construct comprising at least two expression cassettes and having a structure comprising in 5′-3′-direction:
 a1) a first promoter sequence functional in plants; 
 b1) a first nucleic acid sequence of interest to be expressed operably linked to said promoter sequence of a1); 
 c) at least one sequence operably linked to the promoter sequence of a1) and the nucleic acid sequence of b1), wherein said at least one sequence is selected from the group consisting of:
 i) the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46; 
 ii) sequences having a homology of at least 60%, 80%, 90% or 95% with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; 
 iii) sequences hybridizing under high stringency conditions equivalent to hybridization at 68° C. in a solution consisting of 5×SSPE, 1% SDS, 5×Denhardt's reagent and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1×SSPE and 0.1% SDS at 68° C. with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; and 
 iv) a fragment of at least 50, 100, 250 or 500 consecutive base pairs of any of the sequences of i), ii) and iii), 
 
 b2) a second nucleic acid sequence of interest to be expressed; and 
 a2) a second promoter sequence functional in plants operably linked to said nucleic acid sequence of interest to be expressed of b2), 
 wherein said at least one sequence of c) is heterologous to at least one element selected from the promoter sequence of a1), the promoter sequence of a2), the nucleic acid sequence of interest to be expressed of b1), and the nucleic acid sequence of interest to be expressed of b2), and is mediating termination of expression induced from said promoter sequences of a1) and a2). 
 
     
     
         6 . A vector comprising the transgenic expression construct of  claim 5 . 
     
     
         7 . A transgenic host cell or non-human organism comprising:
 a) the transgenic expression construct of  claim 5 ; or   b) a vector comprising said transgenic expression construct.   
     
     
         8 . A transgenic plant comprising:
 a) the transgenic expression construct of  claim 5 ; or   b) a vector comprising said transgenic expression construct.   
     
     
         9 . A method for terminating transcription of a nucleic acid molecule in an expression construct, comprising operably linking a terminator sequence to a heterologous nucleic acid molecule, wherein said terminator sequence is selected from the group consisting of:
 i) the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46;   ii) sequences having a homology of at least 60%, 80%, 90% or 95% with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism;   iii) sequences hybridizing under high stringency conditions equivalent to hybridization at 68° C. in a solution consisting of 5×SSPE, 1% SDS, 5×Denhardt's reagent and 100 μg/mL denatured salmon sperm DNA followed by washing in a solution comprising 0.1×SSPE and 0.1% SDS at 68° C. with any of the sequences of SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46, and capable of terminating transcription in a plant cell or organism; and   iv) a fragment of at least 50, 100, 250 or 500 consecutive base pairs of any of the sequences of i), ii) and iii).