Fluorescence in situ hybridization method for metaphase chromosome of mulberry

A fluorescence in situ hybridization and chromosome technology, applied in the field of genetics, can solve problems such as the application of fluorescence in situ hybridization technology, chromosome swelling and deformation, chromosome production failure, etc., and achieve clear signal points, good shape, and stable number Effect

Active Publication Date: 2014-10-01
SOUTHWEST UNIVERSITY
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Problems solved by technology

However, in the process of FISH chromosome preparation, the pretreatment, the concentration of probes and antibodies, the temperature and time of co-denaturation of probes and chromosomes all affect the experimental results. For example, whether the pretreatment is appropriate is the most critical operation in chromosome preparation technology. The steps determine whether the chromosome length is moderate and whether it is conducive to hybridization; if hypotonicity and insufficient or excessive enzymatic hydrolysis will lead to the failure of chromosome preparation; and if the concentration of probes and antibodies is too high, it will easily lead to incomplete eluti

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  • Fluorescence in situ hybridization method for metaphase chromosome of mulberry
  • Fluorescence in situ hybridization method for metaphase chromosome of mulberry
  • Fluorescence in situ hybridization method for metaphase chromosome of mulberry

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[0025] Example 1

[0026] The fluorescence in situ hybridization method of mulberry metaphase chromosomes includes the following steps:

[0027] (1) Preparation of chromosome slide specimens: Using the young leaves of Sichuan mulberry as the material, the chromosome slide specimens were prepared by the wall-removing hypotonic flame drying method. The specific steps are as follows:

[0028] The young leaves of Sichuan mulberry were pretreated in a 0.002M 8-hydroxyquinoline aqueous solution at 25°C and protected from light for 3 hours, and then fixed at 4°C for more than 2 hours with a methanol:glacial acetic acid volume ratio of 3:1 fixing solution. Then rinse with distilled water 3 times; then, the fixed mulberry leaves were hypotonicized in 1 / 15M KCl aqueous solution at 25°C for 30 minutes, rinsed with distilled water twice, and then contained 5% (W / V) cellulase and 5% (W / V) Enzymatic hydrolysis in a mixed solution of pectinase at 25°C for 2 hours, then rinse with distilled water 3...

Example Embodiment

[0038] Example 2

[0039] The fluorescence in situ hybridization method of mulberry metaphase chromosomes includes the following steps:

[0040] (1) Preparation of chromosome slide specimens, the preparation method is the same as in Example 1;

[0041] (2) Preparation of 25S rDNA probe: According to the conservative region sequence of 25S rDNA genes, design primers to amplify 25S rDNA in the conservative region, as follows:

[0042] 25S rDNA forward primer: 5'-ccaaatgcctcgtcatctaa-3' (SEQ ID NO.3);

[0043] 25S rDNA reverse primer: 5'-gcgaatcaacggttcctct-3' (SEQ ID NO. 4);

[0044] Then the genomic DNA of Sichuan mulberry was used as a template, and SEQ ID NO.3 and SEQ ID NO.4 were used as primer pairs for PCR amplification to obtain the nucleotide sequence shown in SEQ ID NO.6, which was then combined with pMD19-T simple Connect vector to obtain pMD19-25S rDNA;

[0045] Then the obtained recombinant plasmid pMD19-25S rDNA was used as template, and 25S rDNA probe was prepared by PCR DIG ...

Example Embodiment

[0047] Example 3

[0048] Example 3 The method for fluorescence in situ hybridization of mulberry metaphase chromosomes is the same as Example 1, except that the probes added in the hybridization solution are a mixture of biotin-labeled 5S rDNA probes and digoxigenin-labeled 25S rDNA probes Probe: The preparation method of the biotin-labeled 5S rDNA probe is the same as the preparation method of the 5S rDNA probe in Example 1. The difference is that the PCR Biotin Probe Synthesis Kit (Roche) is used to prepare the probe, which contains Biotin-dUTP . When incubating the antibody, first incubate with a concentration of 2ng / μL of goat-derived fluorescein-labeled anti-digoxigenin antibody (Anti-digoxigenin-fluorescein, Fab fragments (from sheep)) for 1h, and then use a concentration of 10ng / μL Cy3-labeled anti-biotin antibody (Cy TM 3-Streptavidin Conjugate(ZyMAX TM Grade)) incubate for 1h, the result of microscopic examination is as image 3 Shown.

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Abstract

The invention discloses a fluorescence in situ hybridization method for metaphase chromosome of mulberry. The fluorescence in situ hybridization method comprises the following steps: the enzymolysis wall degaradation hypotonic flame drying method is adopted to prepare a chromosome slide sample, so that a high-quality mulberry chromosome flake can be obtained; then 5S rDNA and 25S rDNA probes are prepared, and the probes are in repetitive sequence, strong in fluorescence signal, easy to detect and high in repeatability; finally, fluorescence in situ hybridization of the chromosome is conducted to obtain the mulberry chromosome in complete form. The fluorescence in situ hybridization method can be used for mulberry chromosome identification and structure research, and provides a new way for evolution research and the like of mulberry.

Description

technical field [0001] The invention belongs to the field of genetics, and in particular relates to a fluorescence in situ hybridization method for metaphase chromosomes of mulberry trees. Background technique [0002] Mulberry (Morus alba L.) is a plant of the genus Moraceae. As an important economic tree, its leaves are the main feed for silkworms. Mulberry trees have been cultivated for more than 7,000 years. However, most common mulberry trees are cultivated species propagated by cuttings and grafting. The genetic background is relatively vague, which makes mulberry trees encounter great obstacles in the study of genetic breeding and evolutionary analysis. At present, conventional karyotype analysis of mulberry chromosomes has been reported, and mulberry trees have rich types of chromosome ploidy changes, for example, Sichuan mulberry (M.notabilis) has 14 chromosomes, Indian mulberry (M.indica), white mulberry (M.alba) There are 28 chromosomes in M. bombycis, 42 in M. b...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6841C12Q2563/131
Inventor 何宁佳李杨徐云敏向仲怀
Owner SOUTHWEST UNIVERSITY
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