Method for acquiring transgenic gramineae farm crop capable of being selectively eliminated
A crop and genetically modified technology, applied in the field of plant genetic engineering, can solve the problem that there is no method to selectively eliminate genetically modified crops, and achieve the effect of preventing transmission
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Embodiment 1
[0036] Embodiment 1: the construction of rice transformation T-DNA vector
[0037] 1) Construction of rice T-DNA transformation vector pCAMB1300Rice-GlyR-450i:
[0038] The rice cytochrome P450 gene CYP81A6 (SEQ ID NO: 1, Pan et al., Plant Molecular Biology, 61: 933-943) fragment was amplified from the total genomic DNA of rice (Oryza sativajaponica L.) by PCR.
[0039] One of the 207bp fragments——R450FR, was obtained by PCR primer 450F (5' CTCGAG CAG TGC ACC AGA GTC ACA GAA ACA CAT CAC AC, the underline is the XHoI site) and 450R (5' AGA CTC CT TCT TGA CGA GGT GGA GGT GT, the underline is the BglII site), which is a 1-207bp fragment of the 5' end of the cDNA of the gene CYP81A6;
[0040] Another fragment——R450FR2, with a length of 327bp, was obtained by using primer 450F (5' CTCGAG CAGTGC ACC AGA GTC ACA GAA ACA CAT CAC AC, the underline is the XhoI site) and 450R2 (5' AGA TCT CGG TGA AGC ACT CCC TGG CGC AC, the underline is the BglII site) obtained by PCR. It is ...
Embodiment 2
[0043] Example 2: Obtaining glyphosate-resistant transgenic rice sensitive to bendazone and sulfonylurea herbicides
[0044] The method of obtaining transgenic rice is to adopt the existing technology ( Lu Xiongbin Gong Zuxun 1998 Life Science 10: 125-131; Liu Fan et al., 2003 molecular plant breeding 1:108-115). The mature and plump "Xiushui 110" seeds were dehulled, and callus was induced as transformation materials. T-DNA vector pCAMB1300Rice-GlyR-450i was introduced into Agrobacterium LBA4404 by electric shock method. Take the Agrobacterium plate containing the T-DNA vector pCAMB1300Rice-GlyR-450i, and pick a single colony to inoculate the Agrobacterium for transformation. The callus to be transformed is put into the Agrobacterium liquid (containing acetosyringone, 40mg / L) that OD660 is 0.6, allows Agrobacterium to bind to the callus surface, then the callus is transferred to the co-culture medium, Co-cultivate for 2 to 3 days. The transformed calli were rinsed w...
Embodiment 3
[0046] Example 3: Positive and negative selection of transgenic rice using herbicides
[0047] The obtained T0 transgenic rice plants transformed with pCAMB1300Rice-GlyR-450i and the non-transformed rice plants were cultured individually in the culture solution. These plants were divided into 3 treatments, each treatment consisted of 30 plants each of the two transgenes and 10 non-transgenic plants from different transformation events. Treatment 1: Spray 10mM glyphosate; Treatment 2: Spray 2500mg / L bentazone; Treatment 3: Spray water. Each treatment was sprayed with 80mL / square meter, and the survival rate of the plants was recorded 10 days after the treatment. The results are shown in Table 1 below.
[0048] Table 1: Sensitivity of transgenic rice to glyphosate and bentazone.
[0049] glyphosate
bentazone
water
pCAMB1300Rice-GlyR-450i
100% survival
0% survival
100% survival
Non-GMO Rice
...
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