Micro-dissection method for exogenous chromosomes in trititrigia alien substitution line

A technique for isomorphism and chromosome of echinopsis, which is applied in the field of molecular cytogenetics, can solve problems such as hindering the preparation of well-distributed chromosome specimens, affecting DNA sequences, and high DNA content, and achieves the advantages of easy screening and identification research, simple methods, and simple production Effect

Active Publication Date: 2012-07-25
HENAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] (1) The existence of plant cell walls and the difficulty of synchronization hinder the preparation of well-distributed chromosome specimens and affect the cutting efficiency
[0004] (2) The ploidy of the plant genome is diverse, the chromosome structure varies greatly, and the sequence of genes is not conservative enough; the DNA content of plant chromosomes is high and the protein content is low, and most chromosomes do not show G, R or Q bands, which affects the DNA sequence. The prevailing C-band damages the chromosomal DNA too much, which is not conducive to the construction of a complete DNA library; while some plants have no obvious difference in chromosome size and similar band

Method used

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  • Micro-dissection method for exogenous chromosomes in trititrigia alien substitution line
  • Micro-dissection method for exogenous chromosomes in trititrigia alien substitution line
  • Micro-dissection method for exogenous chromosomes in trititrigia alien substitution line

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Experimental program
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preparation example Construction

[0058] Step 1. Preparation of Wheat Root Tip Cell Chromosome Specimen

[0059] 1) Take 0.5 parts of Chinese spring wheat seeds and 0.5 parts of Shannong wheat seeds according to the ratio of parts by weight, mix them evenly, and then put the mixed wheat seeds in 2 parts of water, soak until they are white, take out the wheat seeds, and put them under temperature Place it at 1-4°C for 24 hours, then cultivate it at 25°C to germinate the wheat seeds, continue to cultivate until the root length of the new wheat is 1.5-2.0cm, take the root tip of 0.5-0.8cm, spare;

[0060] 2) Take the wheat root tips in step 1) and place them in the ice-water mixture at 0°C, and after standing for 24 hours, take out the wheat root tips and put them in 2-3 portions of Carnot’s solution at a temperature of 1-4°C, and place them After 24 hours, take it out, put the wheat root tip in 70% alcohol for later use;

[0061] 3) Preparation of reagents:

[0062] ① Mixed enzyme liquid: take 1 part of cellu...

Embodiment 1

[0098] The micro-segregation technique of exogenous chromosomes in trititie heterosubstitution lines comprises the following steps:

[0099] Step 1. Setting of SLμCELLCUT system parameters

[0100] 1) Illumination intensity of common light source

[0101] Set the illumination intensity of the common light source to 120V / 100W.

[0102] 2) Ultraviolet laser parameter setting

[0103] Under the illumination intensity of the ordinary light source of 120V / 100W, the parameters of the ultraviolet laser were set as follows: 100×objective lens, laser speed of 10, focus of 49, laser energy of 60, 40×objective lens, laser speed of 51, focus of 50. Laser energy is 75, 20×objective lens, laser speed is 59, focus is 74, laser energy is 66, 10×objective lens, laser speed is 49, focus is 72, laser energy is 79, 4×objective lens, laser speed is 82, focus is 79, laser energy is 70;

[0104] 3) Selection of cutting samples

[0105] Use the circular tools listed in the software to draw, comp...

Embodiment 2

[0166] The micro-segregation technique of exogenous chromosomes in trititie heterosubstitution lines comprises the following steps:

[0167] Step 1. Setting of SLμCELLCUT system parameters

[0168] 1) Illumination intensity of common light source

[0169] Set the illumination intensity of the common light source to 120V / 100W.

[0170] 2) Ultraviolet laser parameter setting

[0171] Under the illumination intensity of the ordinary light source of 120V / 100W, the parameters of the ultraviolet laser were set as follows: 100×objective lens, laser speed of 10, focus of 49, laser energy of 60, 40×objective lens, laser speed of 51, focus of 50. Laser energy is 75, 20×objective lens, laser speed is 59, focus is 74, laser energy is 66, 10×objective lens, laser speed is 49, focus is 72, laser energy is 79, 4×objective lens, laser speed is 82, focus is 79, laser energy is 70;

[0172] 3) Selection of cutting samples

[0173] Use the circular tools listed in the software to draw, comp...

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Abstract

A micro-dissection method for exogenous chromosomes in trititrigia alien substitution line includes: 1) preparing chromosome specimens of root cells of wheat; 2) preparing chromosome specimens of pollen mother cells; 3) performing microscopic dissection of chromosomes of root cells of the wheat; 4) performing microscopic cutting of chromosomes of pollen mother cells; and 5) performing DOP-PCR (degenerate oligonucleotide primed-polymerase chain reaction) on the cut chromosomes of root cells of the wheat and chromosomes of pollen mother cells, detecting a PCR product I and a PCR product II by polyacrylamide native gel electrophoresis, determining that the PCR product II contains target genes of cut common wheat chromosomes in gel imaging system, and determining that the PCR product IV contains target genes of cut intermediate trititrigia exogenous chromosomes in the gel imaging system. The preparation method is simple, the identification method is simple, simple in analyst and convenient in cutting and recovering of the chromosomes and creates basis for subsequent identification and analyst of the cut chromosomes.

Description

technical field [0001] The invention relates to a method for cutting and identifying exogenous chromosomes, in particular to a method for micro-separating exogenous chromosomes in wheat-Thinopyrum intermedium heterosubstitution lines, belonging to the field of molecular cytogenetics. Background technique [0002] Chromosome microdissection and microcloning (chromosome microdissection and microcloning) technology is a new technology of molecular cytogenetics founded by Scalenghe in 1981. The technology first cuts the Drosophila chromosome, and successfully obtained 80 clones, which were then used to cut the chromosomes of mice and humans. With the development of PCR technology, micro-dissection and micro-cloning technology has been greatly developed. Since the use of chromosome micro-dissection and micro-cloning technology to obtain rye DNA clones, this technology has been used in the construction of plant chromosome libraries, specific probe screening, and anti-bacteria. Th...

Claims

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Application Information

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IPC IPC(8): C12N5/04C12Q1/68
Inventor 王黎明王春平董普辉袁建国袁瑛
Owner HENAN UNIV OF SCI & TECH
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