Insect resistant cotton plants and methods for identifying same

Abstract

The invention provides specific transgenic cotton plants, plant material and seeds, characterized in that these products harbor a specific transformation event at a specific location in the cotton genome. Tools are also provided which allow rapid and unequivocal identification of the event in biological samples.

1 . A method for identifying elite event EE-GH6 in biological samples, which method comprises detection of an EE-GH6 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of EE-GH6. 
     
     
         2 . The method of  claim 1 , said method comprising amplifying a DNA fragment of between 100 and 500 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least two primers, one of said primers recognizing the 5′ flanking region of EE-GH6, said 5′ flanking region having the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 140 or the nucleotide sequence of SEQ ID No 11 from position 1 to position 463 or the 3′ flanking region of EE-GH6, said 3′ flanking region having the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 113 to nucleotide 438, the other primer of said primers recognizing a sequence within the foreign DNA having the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 141 to nucleotide 192 or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 112. 
     
     
         3 . The method of  claim 2 , wherein said primer recognizing the 5′ flanking region consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 140 140 or the nucleotide sequence of SEQ ID No 11 from position 1 to position 463 or said primer recognizing the 3′ flanking region of EE-GH6 consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 113 to nucleotide 438, and said primer recognizing a sequence within the foreign DNA consists of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 141 to nucleotide 192 or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 112. 
     
     
         4 . The method of  claim 2 , wherein said primer recognizing the 5′ flanking region comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 140 or said primer recognizing the 3′ flanking region of EE-GH6 comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 113 to nucleotide 438, and said primer recognizing a sequence within the foreign DNA comprises at its 3′ end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 141 to nucleotide 192 or the nucleotide sequence of SEQ ID No 2 from nucleotide 113 to nucleotide 438. 
     
     
         5 . The method of  claim 4 , wherein said primers comprise the sequence of SEQ ID No. 3 and SEQ ID No. 4, respectively or the sequence of SEQ ID No 5 and SEQ ID No. 7 respectively. 
     
     
         6 . The method of  claim 5 , which method comprises amplifying a fragment of about 189 or 197 bp using the EE-GH6 PCR identification protocol. 
     
     
         7 . A kit for identifying elite event EE-GH6 in biological samples, said kit comprising one primer recognizing the 5′ flanking region of EE-GH6, said 5′ flanking region having the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 140 or the nucleotide sequence of SEQ ID No 11 from position 1 to position 463 or one primer recognizing the 3′ flanking region of EE-GH6, said 3′ flanking region having the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 113 to nucleotide 438, and one primer recognizing a sequence within the foreign DNA, said foreign DNA having the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 141 to nucleotide 192 or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 112. 
     
     
         8 . The kit of  claim 7 , wherein said primer recognizing the 5′ flanking region consist of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 140 140 or the nucleotide sequence of SEQ ID No 11 from position 1 to position 463 or said primer recognizing the 3′ flanking region of EE-GH6 consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 113 to nucleotide 438, and said primer recognizing a sequence within the foreign DNA consists of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 141 to nucleotide 192 or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 112. 
     
     
         9 . The kit of  claim 7 , wherein said primer recognizing the 5′ flanking region comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 140 or the nucleotide sequence of SEQ ID No 11 from position 1 to position 463 or said primer recognizing the 3′ flanking region of EE-GH6 comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 113 to nucleotide 438, and said primer recognizing a sequence within the foreign DNA comprises at its 3′ end at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 141 to nucleotide 192 or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 112. 
     
     
         10 . The kit of  claim 7 , comprising a primer consisting of the sequence of SEQ ID No. 4 and a primer consisting of the sequence of SEQ ID No. 3 or comprising a primer consisting of the sequence of SEQ ID No. 5 and a primer consisting of the sequence of SEQ ID No. 7. 
     
     
         11 . A primer suitable for use in an EE-GH6 specific detection, having a sequence which, under optimized detection conditions specifically recognizes a sequence within the 5′ or 3′ flanking region of EE-GH6, said 5′ flanking region having the nucleotide sequence of SEQ ID No 0.1 from nucleotide 1 to nucleotide 140 or the nucleotide sequence of SEQ ID No 11 from position 1 to position 463 and said 3′ flanking region having the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 113 to nucleotide 438. 
     
     
         12 . The primer of  claim 11 , wherein said primer consists of a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 140 or the nucleotide sequence of SEQ ID No 11 from position 1 to position 4630r a nucleotide sequence of 17 to 200 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 113 to nucleotide 438. 
     
     
         13 . The primer of  claim 11 , wherein said primer comprises at its extreme 3′ end a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 140 or the nucleotide sequence of SEQ ID No 11 from position 1 to position 463 or a nucleotide sequence of at least 17 consecutive nucleotides selected from the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 113 to nucleotide 438. 
     
     
         14 . A primer comprising at its extreme 3′ end the sequence of SEQ ID No. 3. 
     
     
         15 . A primer comprising at its extreme 3′ end the sequence of SEQ ID No. 7. 
     
     
         16 . The method of  claim 1 , which method comprises hybridizing a nucleic acid of biological samples with a specific probe for EE-GH6. 
     
     
         17 . The method of  claim 16 , wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5′ flanking sequence or the 3′ flanking sequence of EE-GH6 and the sequence of the foreign DNA contiguous therewith. 
     
     
         18 . The method of  claim 17 , wherein the sequence of said specific probe has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 120 to 161 or SEQ ID No. 2 from nucleotide 92 to 123, or the complement of said sequences. 
     
     
         19 . A kit for identifying elite event EE-GH6 in biological samples, said kit comprising a specific probe, capable of hybridizing specifically to a specific region of EE-GH6. 
     
     
         20 . The kit of  claim 19 , wherein the sequence of said specific probe has at least 80% sequence identity with a sequence comprising part of the 5′ flanking sequence or the 3′ flanking sequence of EE-GH6 and the sequence of the foreign DNA contiguous therewith. 
     
     
         21 . The kit of  claim 20 , wherein the sequence of said specific probe comprises a nucleotide sequence having at least 80% sequence identity with SEQ ID No. 1 from nucleotide 130 to 151 or SEQ ID No. 2 from nucleotide 102 to 123, or the complement of said sequences. 
     
     
         22 . A specific probe for the identification of elite event EE-GH6 in biological samples. 
     
     
         23 . The probe of  claim 22 , which comprises a nucleotide sequence having at least 80% sequence identity with a sequence comprising part of the 5′ flanking sequence or the 3′ flanking sequence of EE-GH6 and the sequence of the foreign DNA contiguous therewith, or the complement thereof. 
     
     
         24 . The probe of  claim 23  which has at least 80% sequence identity with SEQ ID No. 1 from nucleotide 120 to 161 or SEQ ID No. 2 from nucleotide 92 to 123, or the complement of said sequences. 
     
     
         25 . A specific probe for the identification of elite event EE-GH6 in biological samples, the sequence thereof comprising a nucleotide sequence being essentially similar to SEQ ID No. 1 from nucleotide 120 to 161 or SEQ ID No. 2 from nucleotide 92 to 123, or the complement of said sequences. 
     
     
         26 . A method for confirming seed purity, which method comprises detection of an EE-GH6 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of EE-GH6, in seed samples. 
     
     
         27 . A method for screening seeds for the presence of EE-GH6, which method comprises detection of an EE-GH6 specific region with a specific primer or probe which specifically recognizes the 5′ or 3′ flanking region of EE-GH6, in samples of seed lots. 
     
     
         28 . A method for determining the zygosity status of a plant, plant material or seed comprising elite event EE-GH6, said method comprising amplifying DNA fragments of between 100 and 500 bp from a nucleic acid present in said biological samples using a polymerase chain reaction with at least three primers, two of said primers specifically recognizing pre-insertion plant DNA, such as the 5′ flanking region of EE-GH6, said 5′ flanking region having the nucleotide sequence of SEQ ID No 1 from nucleotide 1 to nucleotide 140, or the nucleotide sequence of SEQ ID No 11 from position 1 to position 463 or the 3′ flanking region of EE-GH6, said 3′ flanking region having the nucleotide sequence of the complement of SEQ ID No 2 from nucleotide 113 to nucleotide 438 or the pre-insertion DNA having the nucleotide sequence of SEQ ID No 10 or the complement thereof, the third of said primers recognizing a sequence within the foreign DNA having the nucleotide sequence of the complement of SEQ ID No. 1 from nucleotide 141 to nucleotide 192 or the nucleotide sequence of SEQ ID No 2 from nucleotide 1 to nucleotide 112. 
     
     
         29 . The method according to  claim 28 , wherein said first primers comprises the nucleotide sequence of SEQ ID No 7, said second primer comprises the sequence of SEQ ID No 6 and said third primer comprises the sequence of SEQ ID No 5 or SEQ ID No 4. 
     
     
         30 . A method of detecting the presence of elite event EE-GH6 in biological samples through hybridization with a substantially complementary labeled nucleic acid probe in which the probe:target nucleic acid ratio is amplified through recycling of the target nucleic acid sequence, said method comprising:
 a) hybridizing said target nucleic acid sequence to a first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 141 to nucleotide 158 or its complement or said first nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No 2 from nucleotide 95 to nucleotide 112 or its complement;   b) hybridizing said target nucleic acid sequence to a second nucleic acid oligonucleotide comprising the nucleotide sequence of SEQ ID No 1 from nucleotide 123 to nucleotide 140 or its complement or said labeled nucleic acid probe comprising the nucleotide sequence of SEQ ID No 2 from nucleotide 113 to nucleotide 130 or its complement, wherein said first and second oligonucleotide overlap by at least one nucleotide and wherein either said first or said second oligonucleotide is labeled to be said labeled nucleic acid probe;   c) cleaving only the labeled probe within the probe:target nucleic acid sequence duplex with an enzyme which causes selective probe cleavage resulting in duplex disassociation, leaving the target sequence intact;   d) recycling of the target nucleic acid sequence by repeating steps (a) to (c); and   e) detecting cleaved labeled probe, thereby determining the presence of said target nucleic acid sequence.   
     
     
         31 . A transgenic cotton plant, or cells, parts, seed or progeny thereof, each comprising elite event EE-GH6 in its genome, reference seed comprising said event having being deposited at the ATCC under deposit number PTA-8398. 
     
     
         32 . The transgenic cotton plant, seed, cells, parts or progeny of  claim 31 , the genomic DNA of which, when analyzed using the Elite event identification protocol for EE-GH6 with two primers comprising the nucleotide sequence of SEQ ID 3 and SEQ ID 4 respectively, yields a DNA fragment of about 189 bp. 
     
     
         33 . Seed comprising elite event EE-GH6 deposited at the ATCC under deposit number PTA-8398 or derivatives therefrom. 
     
     
         34 . A cotton plant or a part thereof, or seed therefrom obtained from the seed of  claim 33 . 
     
     
         35 . A cotton plant, or seed, cells or tissues thereof, each comprising elite event EE-GH6 in its genome, obtained by propagation of and/or breeding with a cotton plant grown from the seed deposited at the ATCC under deposit number PTA-8398. 
     
     
         36 . A cotton seed comprising elite event EE-GH6, reference seed comprising said event having been deposited at the ATCC under deposit number PTA-8398. 
     
     
         37 . A transgenic cotton plant, cell or tissue, comprising elite event EE-GH6, produced from the seed of  claim 36 . 
     
     
         38 . A method for producing a cotton plant or seed comprising elite event EE-GH6 comprising crossing a plant according to any one of  claims 31  to  37  with another cotton plant, and planting the seed obtained from said cross. 
     
     
         39 . Cotton genomic DNA comprising elite event EE-GH6 
     
     
         40 . Genomic DNA produced from a cotton plant, plant cell or seed according to any one of  claims 31  to  37 . 
     
     
         41 . An isolated nucleic acid molecule comprising a nucleotide sequence essentially similar to SEQ ID No. 1 from nucleotide 130 to nucleotide 151 or SEQ ID No. 2 from nucleotide 92 to 123, or the complement of said sequences. 
     
     
         42 . The isolated nucleic acid molecule of  claim 41  comprising a nucleotide sequence essentially similar to SEQ ID No. 1 from nucleotide 120 to nucleotide 161 or SEQ ID No. 2 from nucleotide 82 to 133, or the complement of said sequences.