Method for cultivating insect-resistant transgenic rice T1c-19

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

Inactive Publication Date: 2016-01-06
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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  • Method for cultivating insect-resistant transgenic rice T1c-19
  • Method for cultivating insect-resistant transgenic rice T1c-19
  • Method for cultivating insect-resistant transgenic rice T1c-19

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: Construction of expression vector

[0064] Firstly, pCAMBIA1300 plasmid (gifted by the Center for the Application of Molecular Biology in International Agriculture, Australia) was digested with XhoI restriction endonuclease, the large fragment was recovered, and cut into blunt ends 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.

[0065] 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 / SacI to completely excise cry1C (the dire...

Embodiment 2

[0066] Example 2: Agrobacterium-mediated genetic transformation

[0067] The Agrobacterium-mediated genetic transformation method refers to the method shown in the "Agrobacterium-mediated Genetic Transformation Operation Manual" published by the State 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).

[0068]After mature seeds were induced in the dark for 7 days, a pale yellow embryogenic callus grew between the embryo and endosperm. After 40 days of culture, the embryogenic callus was isolated and proliferated on the subculture medium. Then, the engineered bacteria containing the transformation vector pBar13-Cry1C were used to infect the embryogenic callus, and after co-cultured at 28°C for 3 days, they were cultur...

Embodiment 3

[0133] Example 3: PCR detection of transgenic positive plants

[0134] 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), and the amplified The product is 602bp.

[0135]PCR reaction system: total reaction system is 20μl, DNA template 30-50ng, 10×buffer2.0μl, 2mMdNTP1.5μl, 25mMMgCl 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.

[0136] PCR reaction program: 94°C, denaturation for 3min; 94°C, denaturation for 1min, annealing at 57°C for 1min, extension at 72°C for 1.5min, 35 cycles; extension at 72°C for 10min.

[0137] Detection of PCR products: the amplified products were electrophoresed on 0.8% agarose gel, stained with EB, observed and photographed by an ultraviolet analyzer.

[0138] Extraction method of a small am...

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Abstract

The invention belongs to the technical field of genetic engineering for rice, and particularly relates to a method for cultivating insect-resistant transgenic rice T1c-19. The method includes transferring target genes into excellent rice restorer lines minghui 63 by the aid of synthesized novel insecticidal genes cry1C, bar genes and agrobacterium tumefaciens mediated genetic transformation processes to obtain novel transgenic rice materials T1C-19 with high rice Lepidoptera insect resistance. The novel insecticidal genes cry1C are shown as SEQ ID NO:1 and are used as the target genes, and the bar genes are used as selectable marker genes for transferring the target genes into the excellent rice restorer lines minghui 63. The method has the advantages that the insect-resistant genes of T19-1 further can be recombined into novel rice germ plasma by the aid of sexual hybridization and somatic hybridization technologies, flanking sequences of insertion fragments of exogenous genes of transgenic plants of the rice are 5'-end flanking sequences shown as SEQ ID NO:3 and or 3'-end flanking sequences shown as SEQ ID NO:5, and the method for completely cultivating novel insect-resistant varieties (strains) of rice is provided; the T1C-19 is a novel genetic resource.

Description

[0001] The present invention is a divisional application of application number 2013101382916, and the filing date of the original application is April 19, 2013. technical field [0002] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a method for cultivating insect-resistant transgenic rice T1c-19. The invention uses the genetic transformation technology mediated by Agrobacterium to introduce foreign genes into rice recipient varieties to obtain transgenic insect-resistant rice Methods. Background technique [0003] Bacillus thuringiensis (Bt) 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 insectic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29A01H5/00
Inventor 林拥军唐微
Owner HUAZHONG AGRI UNIV
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