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Method for screening transgenic paddy rice seed based on antibiotic

A technology of genetically modified rice and antibiotics, applied in biological testing, material inspection products, etc., can solve the problems of time-consuming, high cost, and large energy consumption, and achieve the effect of clear identification, simple operation and high reliability

Inactive Publication Date: 2008-08-13
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It takes a long time to screen and identify with this method, because the materials can only be obtained after the seedlings grow to a certain size, because it is not known whether these seedlings are positive or negative, and a lot of seedlings need to be planted, so it takes a lot of energy. DNA extraction, PCR, and electrophoresis are also a series of tedious tasks, which are time-consuming, laborious, and costly!

Method used

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  • Method for screening transgenic paddy rice seed based on antibiotic
  • Method for screening transgenic paddy rice seed based on antibiotic
  • Method for screening transgenic paddy rice seed based on antibiotic

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1. Genetically modified materials and antibiotics

[0038] Transgenic seeds and non-transgenic seeds, hygromycin (concentration 2.5mg / ml).

[0039] Description of transgenic materials: Zhonghua 11 was transfected with CA (carbonic anhydrase gene) gene, the vector used was pCAMBIA1301 (purchased by CAMBIA Research Center in Australia), the resistance selection marker was hygromycin, mediated by Agrobacterium EHA101 (purchased by takara company) Law.

[0040] 2. Condition determination:

[0041]Use six 9cm petri dishes, add 10ml of hygromycin solution to each dish, the concentration is 0.0, 100, 200, 400, 600, 800ul / L (the concentration of the mother solution is 2.5ug / ul), the size of the bedding under the dish is appropriate Two layers of filter paper, put about 30 seeds of Zhonghua 11 (non-transgenic) genes in each petri dish. Place them evenly. Cultivate in the dark at 28 degrees for 3 days. Observe the results. (such as: figure 1 ) From the results, it can be seen ...

Embodiment 2

[0066] 1. Genetically modified materials and antibiotics

[0067] GMO and non-GMO seeds, kanamycin

[0068] Description of transgenic materials: Zhonghua 11 was transfected with sbpase gene (1,7-2 phosphatase sedoheptulose gene derived from cyanobacteria), the vector used was pBI121 (genebank accession number: AY781296), and the resistance selection marker was kanamycin The element was transgenic by the Agrobacterium-mediated method, and the Agrobacterium strain EHA101 was purchased from Takara Company.

[0069] 2 conditions are determined:

[0070] Use six 9cm petri dishes, add 10ml of kanamycin solution to each dish, the concentration is 0.0mg / L, 100mg / L, 200mg / L, 400mg / L, 600mg / L, 800mg / L, and lay a bedding under the dish Two layers of filter paper of appropriate size, put about 30 recipient (non-transgenic) seeds in each petri dish. Place them evenly. Cultivate in the dark at 28 degrees for 3 days. The results show that the concentration of kanamycin is 0mg / L, 100mg / L ,...

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Abstract

The present invention discloses a method based on antibiotic for screening transgenic rice seeds, which comprises of: A) screening transgenic rice seeds by antibiotic, firstly, determining concentration of antibiotic and setting up screen conditions and breeding conditions, and last determining the result of screen; B) determining method of genotype homozygosis and heterozygosis of transgenic plant: comparing non-transformed receptor parent of target gene added antibiotic and not added antibiotic, transgenic plant not added antibiotic, screening transgenic plant seeds by antibiotic, comparing and analyzing the results of two comparison test and transgenic plant not added antibiotic to determine genotype of transgenic plant. The method of present invention is easy, can scale analyze, gain exogenous gene high expression plant and has merits of high efficiency, low cost, can save research resource.

Description

technical field [0001] The invention belongs to the technical field of rice transgenics, and more specifically relates to a method for screening transgenic seeds based on antibiotics, especially suitable for screening rice seeds. technical background: [0002] At present, the positive plants of rice transgenic seeds are screened by PCR method, and the further identification is by Southern blot. The process of the PCR screening method is as follows: 1. It takes about 40 days to germinate seedlings with rice seeds until they grow to the size of 6 leaves; 2. Cut about 0.2 grams of leaves. The genome was extracted by CTAB method; 3, PCR verification; 4, electrophoresis detection. It takes a long time to screen and identify with this method, because the materials can only be obtained after the seedlings grow to a certain size, because it is not known whether these seedlings are positive or negative, and a lot of seedlings need to be planted, so it takes a lot of energy. DNA ext...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/48
Inventor 李阳李阳生
Owner WUHAN UNIV
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