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Cultivating method for transgenic lepidoptera pest resistant rice

A transgenic rice, transgenic technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve problems such as finding resistance gene resources, and achieve the effect of protecting the environment

Inactive Publication Date: 2013-12-04
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Third, through large-scale screening, the resistance gene resources that effectively control insect pests have not been found in rice

Method used

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  • Cultivating method for transgenic lepidoptera pest resistant rice
  • Cultivating method for transgenic lepidoptera pest resistant rice
  • Cultivating method for transgenic lepidoptera pest resistant rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Embodiment 1: Construction of expression vector

[0063] Firstly, pCAMBIA1300 plasmid (gifted by the Center for the Application of Molecular Biology in International Agriculture, Australia) was digested with Xho I restriction endonuclease, the large fragment was recovered, and blunt-ended was cut with nuclease. At the same time, the pTW-a plasmid was digested with SmaI enzyme, and a small fragment (bar gene) was recovered. After ligating the recovered large and small fragments, Escherichia coli was transformed, positive clones containing the bar gene were selected, and the obtained vector was named intermediate vector pBar13.

[0064] Then cut the Ubi promoter from the pUBC plasmid with HindIII / BamHI double enzyme digestion (the direction of action of the Ubi promoter is SacI site → BamHI site), and insert it into the multiple cloning site of the pBar13 vector to form a pBar-Ubi intermediate carrier. At the same time, use BamHI / Sac I to completely excise cry1C (gene e...

Embodiment 2

[0065] Example 2: Agrobacterium-mediated genetic transformation

[0066] The Agrobacterium-mediated genetic transformation method refers to the method shown in the "Agrobacterium-mediated Genetic Transformation Operation Manual" published by the Weijia Key Laboratory of Crop Genetic Improvement of Huazhong Agricultural University and the article published by Professor Lin Yongjun (Lin and Zhang 2005). The transformation recipient was the embryogenic callus induced from mature seeds of rice variety "Minghui 63" (gifted by Fujian Academy of Agricultural Sciences).

[0067] After mature seeds were induced in the dark for 7 days, pale yellow embryogenic callus grew between the embryo and endosperm. After 40 days of culture, the isolated embryogenic callus proliferated on the subculture medium. Then, infect the embryogenic callus with the engineered bacteria containing the transformation vector pBar13-Cry1C, co-cultivate it at 28°C for 3 days and then culture it on the selection me...

Embodiment 3

[0130] Example 3: PCR detection of transgenic positive plants

[0131] Extraction of T by a small amount of genomic DNA extraction 0 The DNA of transgenic leaves was analyzed by PCR, and the primers for amplifying the cry1C* gene were F: 5'-ttctactggggaggacatcg-3' (SEQ ID NO: 6), R: 5'-cggtatctttgggtgattgg-3' (SEQ ID NO: 7) , the amplified product is 602bp.

[0132] PCR reaction system: total reaction system is 20μl, DNA template 30-50ng, 10×buffer2.0μl, 2mM dNTP1.5μl, 25mM MgCl 2 2.0 μl, 0.4 μl each of 10 μM primers (F / R), 1 U of Taq enzyme, plus sterilized ddH 2 0 to 20 μl.

[0133] PCR reaction program: 94°C, denaturation 3min; 94°C, denaturation 1min, 57°C annealing 1min, 72°C extension 1.5min, 35 cycles; 72°C extension 10min.

[0134] Detection of PCR products: the amplified products were electrophoresed on 0.8% agarose gel, stained with EB, observed and photographed by an ultraviolet analyzer.

[0135]Extraction method of a small amount of genomic DNA from leaves: T...

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Abstract

The invention belongs to the technical field of rice genetic engineering, and particularly relates to a cultivating method for transgenic lepidoptera pest resistant rice. The method comprises the following steps: using an artificially synthesized novel pesticidal gene cry1C as a target gene, wherein the nucleotide sequence is shown as SEQ ID NO: 1; using a bar gene as a selective marker gene and adopting an agrobacterium mediated genetic transformation method to introduce the target gene into rice excellent restorer minghui 63 to acquire a novel transgenic rice material T1C-19 highly resisting rice lepidoptera pests. Besides, a pest-resistant gene of T19-1 can be recombined into a new rice germplasm through sexual hybridization and somatic hybridization technologies to cultivate a new rice pest resistant product. The invention discloses the verification tests of processes of transformation vecter construction, rice genetic transformation, molecular identification of transformed plants, insertion site sequence analysis, indoor and field pest resistant experiments and the like.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a method for using an agrobacterium-mediated genetic transformation technology to introduce foreign genes into rice recipient varieties so as to obtain transgenic insect-resistant rice. Background technique [0002] Bacillus thuringiensis (Bt for short) is a Gram-positive bacterium that widely exists in soil, dust, water, deserts, plants, and insect corpses (Jia Shirong et al., 2001). In 1901, Japanese scholar Ishiwata isolated Bt bacteria from the body fluid of infected silkworm for the first time, and proved that some Bt bacteria had insecticidal activity against Lepidoptera insects. In the 1950s, it was found that the insecticidal activity of Bt bacteria was related to parasporal crystals (Hannay1953), and it was confirmed that the parasporal crystals were composed of proteins (Hannay and Fitz-James1955). Such proteins are called delta-endotoxins...

Claims

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Application Information

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IPC IPC(8): C12N15/84A01H5/00C12N15/11C12Q1/68
Inventor 林拥军唐微
Owner HUAZHONG AGRI UNIV
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