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Improved method for detecting and identifying transgenic wheat with high sensitivity and high efficiency

A transgenic wheat, sensitive technology, applied in the field of cell biology and molecular biology research, can solve the problems of pollen mother cells, long pachytene chromosomes, difficult to trace and identify individual chromosomes, and limit the application of pachytene FISH, etc. To achieve the effect of overcoming the unsatisfactory detection effect, good advanced nature and strong signal

Inactive Publication Date: 2013-06-19
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

However, it is very difficult to select a suitable pollen mother cell for the preparation of pachytene chromosomes, and the pachytene chromosomes are very long, and it is difficult to trace and identify individual chromosomes. These unfavorable factors limit the application of pachytene FISH

Method used

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  • Improved method for detecting and identifying transgenic wheat with high sensitivity and high efficiency
  • Improved method for detecting and identifying transgenic wheat with high sensitivity and high efficiency
  • Improved method for detecting and identifying transgenic wheat with high sensitivity and high efficiency

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Identification of Ta DREB3 transgenic wheat and control material Jimai 19 by fluorescence in situ hybridization

[0063] 1. Preparation of Transgenic Wheat Mitotic Metaphase

[0064] The Ta DREB3 transgenic wheat used and the reference material Jimai 19 were provided by Ma Youzhi Laboratory of the Chinese Academy of Agricultural Sciences (XiaoYan H, Ming C, HuiJun X, Shiqing G, XianGuo C, LianCheng L, LiPu D, XinGuo Y, YouZhi M( 2005) Obtaining of transgenic wheats with GH-DREB gene and their physiological index analysis on drought tolerance. Southwest China Journal of Agricultural Sciences 18: 616-620).

[0065] 1)N 2 O treatment of root tip

[0066]When the root tip grows to 2-3cm, cut it off, put it in a wet centrifuge tube, cover it and put it in a N2O air chamber for 1-3h, the pressure is 10ATM (1.01Mpa);

[0067] 2) root tip fixation

[0068] Fix with 90% acetic acid for 5-10 minutes (no more than 1 hour), and wash with distilled water twice. The f...

Embodiment 2

[0091] Example 2. Preparation of metaphase chromosomes of different plants

[0092] 1. Seed Germination

[0093] (1) The seeds of various plants are respectively placed in petri dishes lined with moist filter paper, and germinated in an incubator at 21-23°C.

[0094] 2. Plant chromosome specimen preparation

[0095] 1) With embodiment 1.1. When the root tip grows to 2-3cm, cut it off, put it in a humid centrifuge tube, and treat it with nitrous oxide for 30 minutes to 5 hours. Depending on the plant species, the genome size is different, and the time of nitrous oxide treatment is different, such as peanuts, soybeans, etc. Plant root tips are treated for 30-40 minutes, wheat, corn and other plant root tips are treated for 2 hours, and for plants with large genomes such as lilies, the treatment time can reach 4-5 hours.

[0096] 2) With embodiment 1.2.

[0097] 3) With embodiment 1.3.

[0098] 4) With embodiment 1.4. Use filter paper to blot the water on the root tip sligh...

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Abstract

The invention discloses an improved method for detecting and identifying transgenic wheat with high sensitivity and high efficiency. The improved method comprises the steps of: (1) carrying out pretreatment and enzymolysis on tissue with vigorous cell division in a meristematic zone at a root tip of wheat through N2O gas to obtain a metaphase mitotic phase specimen with clear image and good dispersion of chromosomes; (2) marking a fluorescent probe of a target fragment to be detected (for example, a transgenic fragment carried by a transgenic carrier) by utilizing a nick translation method; (3) carrying out fluorescent in-situ hybridization on the metaphase mitotic phase chromosomes of the transgenic wheat obtained in the step (1) by utilizing the fluorescent probe of the target fragment of the transgenic carrier constructed in the step (2); and (4) analyzing fluorescent in-situ hybridization results obtained in the step (3), and comparing the fluorescent in-situ hybridization results with a contrast material to determine the distribution of fluorescent signals in the transgenic wheat to be detected. The improved method provided by the inventions can be applied to chromosomal localization of exogenous transgenic wheat with the sensitivity reaching 2-3kb, and also can be applied in the identification of transgenic signals of other crops.

Description

technical field [0001] The invention relates to a highly sensitive and efficient method for detecting transgenic wheat chromosomes, and also relates to a fast and efficient method for obtaining metaphase plant chromosomes in good shape, belonging to the research fields of cell biology and molecular biology. Background technique [0002] Wheat is one of the most important food crops in the world, which is closely related to social and economic development, food security supply and human nutrition and health. The narrow genetic base has become a key factor restricting the further improvement of important agronomic traits such as yield level, resistance and adaptability of crop varieties (Ye Xingguo et al. 2011). Genetic engineering breeding, as the main supplementary method of hybrid breeding, has achieved great success in crops such as soybeans, corn, cotton, and rapeseed. Genetic engineering has endowed these crops with some excellent traits such as herbicide resistance and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64
Inventor 韩方普吕振玲符书兰
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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