Transgenic rice event17314 and methods of use thereof
An event, rice technology, applied in genetic engineering, botanical equipment and methods, biochemical equipment and methods, etc., can solve problems such as non-conformance mode
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Embodiment 1
[0090] Example 1: Transformation and event selection in rice
[0091] This example illustrates how to generate transgenic rice events and how to select event 17314. By using such as figure 1 The indicated transgenic DNA fragment (whose sequence is shown in SEQ ID NO: 5) was subjected to particle gun-mediated transformation of rice cells to generate transgenic glyphosate-tolerant event 17314. The transgenic DNA segment comprises a promoter (P-CaMV.e35S) molecule derived from cauliflower mosaic virus, an expression cassette comprising an enhancer for replication, operably linked to an internal gene derived from rice actin 1 Containing molecule (I-Os.Act1), operably linked to a DNA molecule encoding a chloroplast transit peptide (CTP2, Arabidopsis EPSPS), operably linked to a glyphosate-resistant EPSPS (AGRtu.EPSPS CP4) The DNA molecule of is operably linked to the 3' transcription termination region DNA molecule (T-AGRtu.nos) derived from the nopaline synthase gene of Agrobac...
Embodiment 2
[0100] Example 2: Isolation of rice chromosomal sequences adjacent to the inserted DNA
[0101] Rice genomic DNA for all PCR reactions was isolated using a rapid high pH / high salt lysis protocol. For this protocol, approximately 0.1 g of freeze-dried ground leaf tissue was mixed with 600 μl (microliter) of lysis buffer (100 mM Tris, 1 M KCl, 10 mM EDTA, pH 9.5) by shaking, followed by incubation at 65° C. for 45-60 minutes. Next, the test tube was shaken again, and 200 μl of precipitation buffer (5M potassium acetate, pH 7.0) was added, shaken again, and centrifuged. Transfer a 600 µl aliquot of the DNA solution to a clean tube and add 500 µl of ice-cold isopropanol to precipitate the DNA. After centrifugation, the DNA pellet was washed with 70% ethanol, air-dried, and the DNA was resuspended in 250 μl of water.
[0102] Extension of rice genomic DNA adjacent to the transgene insertion was obtained for the 17314 event using a TAIL-PCR protocol essentially as described by Liu...
Embodiment 3
[0115] Example 3: Event-specific endpoint TAQMAN analyze.
[0116] This example illustrates the event-specific endpoint TAQMAN developed to determine event 17314 in a sample thermal amplification method. Tables 7 and 8 describe examples of conditions for which this method can be used. The DNA molecules used in this method are, for example, primers SQ4183 (SEQ ID NO: 7) and SQ4191 (SEQ ID NO: 8) and 6FAM TM - Labeled oligonucleotide probe PB1491 (SEQ ID NO: 9). Additional probes and primers can be designed based on the sequences and / or flanking sequences of the transgene inserts provided herein. When used with PB1491 (SEQ ID NO:9) in these reaction methods, SQ4183 (SEQ ID NO:7) and SQ4191 (SEQ ID NO:8) produced DNA amplicons recognizable for event 17314 DNA. Controls for this analysis included a positive control from rice containing event 17314 DNA, a negative control from non-transgenic rice, and a negative control containing no template DNA.
[0117] These assays were...
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