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A method for obtaining a single chromosome of peony
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A single chromosome and chromosome technology, applied in the direction of plant cells, etc., can solve the problems of not widely used, different separation operations, expensive laser microscopic operating system, etc., and achieve the effect of reducing the chance of pollution, reducing the degree of overlap, and easy to distinguish
Active Publication Date: 2017-11-03
SHANGHAI CHENSHAN BOTANICAL GARDEN
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However, due to the high cost of the laser microscope operating system, it is not widely used
[0006] At present, Peng Renhai et al. (Isolation of Asian Cotton No. 7 Chromosome and Library Construction, Acta Cotton Sinica, 2012, 24(5): 379-385), Yang Hongyan (Study on Laser Microseparation Poplar Chromosome Technology, Beijing Forestry University, 2010), Ma Youzhi et al. (Micro-laser separation of wheat chromosomes, Acta Genetics, 26(l): 43-48, 1999), Gao Jurong et al. (Research on the cutting of single chromosomes in cotton by laser microdissection technology, experimental technology and management, 2009) Several chromosomes or single chromosomes in a single cell were obtained by laser microdissection. However, due to the different chromosome preparation, chromosome separation and collection of each species, there is no Chromosome Chromosome Microdissection in the prior art. Official Report on Separation Technology
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Embodiment 1
[0043] Example 1 The method of obtaining Fengdan single chromosome by microdissection technique
[0046] Reagent: carbo fuchsin, 45% acetic acid (volume ratio), 70% alcohol (volume ratio), 1% TE solution (10mmol / L Tris-HCl (pH 8.0), 1mmol / L EDTA (pH 8.0)).
[0047] 2. Sample Preparation
[0048] 1) Take out the bright yellow peony buds preserved in alcohol, with a diameter of 12-16 mm, use tweezers to tear out a stamen, and absorb the alcohol on absorbent paper.
[0049] 2) Then drop a small drop of 45% acetic acid on the ordinary glass slide, place the selected stamens in acetic acid to soften for 1-3 seconds, cut out a section of 1-2...
Embodiment 2
[0066] Example 2 Single Chromosome Amplification Experiment
[0067] Three peony single chromosomes excised from Example 1 were selected and amplified using a single-cell whole-genome amplification kit, followed by agarose gel electrophoresis, and then using a nanodrop (ultramicro spectrophotometer) to measure the DNA concentration.
[0068] see Figure 5 , is the agarose gel electrophoresis image of the target chromosomal DNA, the second hole from the left is DL2000DNAmarker, the 3rd, 4th, and 5th holes are the diffuse bands amplified from the three selected single chromosomes, and the length of the amplified fragments is 300 ~2000bp, 1% TE solution was used as blank control (CK group), and the measured DNA concentration is shown in Table 1:
[0069] Table 1 DNA concentration
[0070] Sample serial number
DNA concentration (ng / μl)
ck
0.1
1
862.2
2
914.4
3
756.9
[0071] It can be seen from Table 1 that the amplified co...
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Abstract
The invention discloses a method for obtaining a single chromosome of peony, which is obtained by micro-cutting technology. The method selects peony cells in the meiosis stage, and undergoes steps such as sample liquid preparation, microscopic examination, membrane transfer, and pressing to disperse peony chromosomes. , to obtain the sample; and then use a high-end laser microdissection system to successfully obtain a complete single line of peony pollen mother cells by preparing collection tubes, loading samples, finding and selecting target chromosomes, adjusting laser parameters, marking target chromosomes, and laser cutting Chromosomes, and there is no mutual contamination between chromosomes, paving the way for downstream research.
Description
technical field [0001] The invention belongs to the technical field of microscopic separation of cell biology, and in particular relates to a method for obtaining a single chromosome of tree peony, in particular to a method for obtaining a single chromosome of tree tree peony through laser microdissection separation technology. Background technique [0002] Chromosome microdissection separation technology is a bridge technology from cell biology to molecular biology. In recent years, this technology has been used in the construction of high-resolution genetic maps and physical maps of genes, the rapid and effective acquisition of chromosome zone-specific sequence tag sites (STS) and expressed sequence tags (EST), the construction of clonal linkage groups, and the homologous Gene positioning and cloning have shown great advantages. Since the technology came out in the early 1980s, the biggest breakthrough has been the combination with PCR technology. Later, by combining with...
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