The flanking sequence of the foreign insert fragment of the transgenic rice line 134bt, its amplification primer and its application
A technology of transgenic rice and flanking sequences, which is applied in the field of plant biology and can solve the problems such as the PCR detection method of the 134Bt line that has not yet been established with an insect-resistant gene.
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Embodiment 1
[0043] Example 1 Obtained by Sequencing the Side Sequence of the Foreign Insertion Vector of the Transgenic Rice Line 134Bt
[0044] According to the known sequence of the pZTRT-Bt plasmid, design 3 outward specific nested primers TL1, TL2 and TL3 from the region of 100-200 bp within the left border terminator, and design 5 random degenerate primers AD1- AD5 (Table 2).
[0045] Perform TAIL-PCR, including 3 reactions. The first round reaction (20μl) system contains 10×buffer (including Mg 2+ ) 2 μl; dNTP, 0.2 mM; specific primer TL1, 0.5 μM; 3 μM degenerate primers AD1-AD5; Taq polymerase, 1.25 U; transgenic rice line 134Bt genomic DNA 1 μl. The template of the second round of reaction is the 40-fold dilution of the first round of PCR reaction product, and the template of the third round of reaction is the 10-fold dilution of the product of the second round of reaction. For the 3-round PCR reaction procedure, refer to Huang Haiqing’s paper (Huang Haiqing, 2004, Localizati...
Embodiment 2
[0052] Example 2 Qualitative PCR detection of flanking sequence of insert fragment in transgenic rice 134Bt
[0053] 1. Experimental materials
[0054] Transgenic rice: transgenic rice line 134Bt,
[0055] Conventional non-transgenic rice: Xiushui 134.
[0056] 2. Experimental method and process
[0057] 1. CTAB method to extract plant genomic DNA.
[0058] Use a small amount of CTAB method to extract DNA. The specific method is as follows: Take a small amount of leaves in a mortar (about 0.1g), add 700μl 1.5×CTAB DNA extraction solution, grind thoroughly, pour into a 1.5mL Eppendorf tube, and place in a 56°C water bath Shake continuously for 20 minutes; cool to room temperature after water bath, add 600 μL chloroform, shake vigorously to mix well, let stand for 5 minutes; centrifuge at 12000 rpm for 5 minutes, pipette 400 μL supernatant into a new 1.5 mL Eppendorf tube; add 800 μL pure alcohol , mix up and down, and place at -20°C for 10 minutes; centrifuge at 12,000 rpm ...
Embodiment 3
[0065] Example 3 Transgenic Rice Line 134Bt Specific Qualitative PCR Detection Kit
[0066] The detection kit contains the primers shown in Table 3.
[0067] Qualitative PCR sensitivity amplification of rice was carried out with primers LBF / LBR and RBF / RBR, and the results showed that rice lines containing components derived from transgenic rice line 134Bt could be detected with high sensitivity and specificity.
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