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Directional gene transfer method of cabbage type rape C chromosome set

A Brassica napus and transgenic technology is applied in the field of ecological safety research of transgenic plants, and can solve the problems of transfer of exogenous target genes, difficulty of transgenic Brassica napus, identification of exogenous genes, etc.

Inactive Publication Date: 2006-12-27
INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] At present, all transgenic methods (such as: gene gun-mediated method, Agrobacterium-mediated method, pollen tube passage method, etc.) cannot accurately transfer the exogenous target gene to the specific chromosome of Brassica napus, and the existing transgenic methods When transgenic, the integration of the exogenous gene into the genome (chromosome) of the recipient rapeseed is random, and there is no quick and effective method to identify whether the exogenous gene is transferred to the A chromosome or to the C chromosome
Applying existing transgenic methods, it is very difficult to obtain transgenic Brassica napus in which the target gene is accurately inserted into the C chromosome

Method used

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  • Directional gene transfer method of cabbage type rape C chromosome set
  • Directional gene transfer method of cabbage type rape C chromosome set
  • Directional gene transfer method of cabbage type rape C chromosome set

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Transgenic bark kale (B.oleracea var.alboglabra)

[0061] 1 Genetic transformation of kale

[0062] 1.1 Agrobacterium preparation

[0063] The Agrobacterium strain LBA4404 containing the bar gene expression vector pCAMBIA3300 plasmid was streak-inoculated on the YEB plate medium supplemented with 50mg / L kanamycin, and cultured upside down at 28°C for 2-3 days. After the colony grows, pick a single colony from the YEB plate, inoculate it in 5ml YEB (containing 50 mg / L kanamycin) liquid medium, place it on a shaker at 200 rpm, and cultivate overnight at 28°C. Take the bacterial solution and inoculate it on the YEB plate supplemented with 50 mg / L kanamycin, and incubate it upside down at 28°C for 2 days. For each experiment, take a single colony and inoculate it on the YEB plate medium supplemented with 50mg / L kanamycin, culture it at 28°C for 1-2 days, and wash the bacteria with 1 / 2MS liquid medium (pH5.4) , and dilute the bacterial solution to OD600=0.08 as the bacter...

Embodiment 2

[0074] Synthesis of Brassica napus with C Chromosome Transgenic Bar Gene

[0075] 1.1 Artificial synthesis of Brassica napus as female parent

[0076]Seven Chinese cabbage varieties including Heiyebai, Rongyouai Kangqing, Tezao 50 cabbage moss, golden cabbage, red cabbage, Chaoshan sweet cabbage and double-low cabbage rape were used as female parents, and the bar gene transgenic Chinese kale was used as crops. The male parent is interspecifically crossed. 4-7 days after pollination, the fertilized ovary was taken for disinfection, and the ovary was cultured, and the medium was MS medium containing 500 mg / L hydrolyzed casein. After the ovary was cultured for 40 days, the seeds were peeled off from the mature pods and transferred to MS medium for germination. When seedlings germinate from the seeds, the seedlings are excised and transplanted on MS medium containing 0.01% colchicine for 7-10 days to perform chromosome doubling. Then, the seedlings were transferred to the mediu...

Embodiment 3

[0084] Molecular verification of bar gene located on chromosome C of Brassica napus

[0085] Genome Southern blotting analysis: Take about 20 μg of total genomic DNA from transgenic kale, C chromosome transgenic Brassica napus and non-transgenic rapeseed, digest them completely with endonuclease EcoR I, and run them on 0.8% agarose gel For electrophoresis separation, the DNA of plasmid pCAMBIA3300 was used as a positive control. The procedures of prehybridization, hybridization and membrane washing were carried out according to the method reported by Sharpe et al. (1995). The probe used was a 0.5 kb fragment of the bar gene amplified by plasmid PCR, and the detection was performed using a Digoxigenin labeling kit (Roche Diagnostics, Swiss).

[0086] Randomly select three PCR and Basta  Genomic Southern blot analysis of the positive synthetic rapeseed line (same transgenic male parent) and its male parent showed that the exogenous bar gene had been integrated into the genome...

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Abstract

The invention discloses a directional genetically modified method of wild cabbage rape C genome, and the steps include: A. breeding and conversion mediated by wild cabbage agrobacterium tumefaciens: first preparing agrobacterium tumefaciens bacterial solution, next preparing explant, thirdly laying the explant into the agrobacterium tumefaciens bacterial solution for breeding and conversion, fourthly checking resistance of herbicide, and fifthly checking PCR molecule; B. artificially synthesizing genetically modified wild cabbage rape, first artificially synthesizing cabbage cellular genetically modified wild cabbage rape, next artificially synthesizing wild cabbage cellular genetically modified wild cabbage rape, finally performing colorant layer duplication to artificially synthesized haploid rape; C. rating the genetically modified rape. The invention is of simple operation, the accidental purpose gene can be transferred on wild cabbage rape C chromosome with high accuracy, and the obtained genetically modified rape is of small production risk when the environment deactivates in accidentally genetically modified diffusion.

Description

technical field [0001] The invention relates to the field of research on the ecological safety of transgenic plants. Specifically, the invention relates to a gene-directed transformation method. The transgenic rapeseed obtained by applying the method has little ecological risk of exogenous transgene diffusion when released in the environment. Background technique [0002] Rapeseed is a general term for the cultivated Brassica plants of the genus Brassica for the purpose of harvesting seeds for oil extraction, mainly including: Brassica napus L., B. campestris L. and Brassica napus (B. . juncea Czern. et Coss.). The rapeseed that is planted in various countries in the world today is mainly Brassica napus, and the planting area of ​​other types of rapeseed is very small. The rape mentioned now refers to Brassica napus unless otherwise specified. [0003] Rapeseed is an important oil crop, ranking second only to soybean in the production of oi...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N15/82A01H4/00A01H1/02C12Q1/68
Inventor 方小平李均王转罗莉霞李俊
Owner INST OF OIL CROPS RES CHINESE ACAD OF AGRI SCI
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