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

Inactive Publication Date: 2008-06-25
ZHEJIANG UNIV
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  • Application Information

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Problems solved by technology

[0004] There is currently no way to sel...

Method used

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  • Method for acquiring transgenic gramineae farm crop capable of being selectively eliminated

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Experimental program
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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

genetically modified rice

100% survival

0% survival

100% survival

Non-GMO Rice

...

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Abstract

The invention provides an acquisition method for transgenic gramineous crops capable of being selectively perished, comprising the following steps: when target genes are expressed, expression of genes participating in herbicide detoxification in the transgenic gramineous crops is inhibited by utilization of antisense RNA or RNAi means, and the transgenic gramineous crops capable of being selectively perished are obtained. The invention mainly has the advantages that: the transgenic gramineous crops acquired by utilization of the method can be selectively perished by Bentazon or sulphuryl carbamide herbicide as required, thereby transmission of the transgenic gramineous crops and interfusion to non-transgenic gramineous crops are prevented, and the method has great significance in transgenic crop research later on.

Description

(1) Technical field [0001] The invention relates to a method for obtaining selectively eliminated transgenic grass crops, as well as used genes and plasmids, and belongs to the field of plant genetic engineering. (2) Background technology [0002] Genetically modified crops have been extensively planted in many countries around the world. For example, genetically modified insect-resistant and herbicide-resistant corn has been widely promoted in North America, and genetically modified cotton has also been widely planted in China. Further, the use of transgenic crops as bioreactors to produce pharmaceutical proteins and industrial enzymes is also being extensively researched and developed (Larrick and Thomas, Current Opinion in Biotechnology, 2001, 12: 411-418). An important concern with growing these GM crops is their environmental spread and the mixing of inappropriate GM crops into our food. Therefore, controlling their spread and preventing them from being mixed into non...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/82A01H1/00C12N15/113
CPCY02A40/146
Inventor 沈志成徐晓丽方军林朝阳
Owner ZHEJIANG UNIV
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