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A probe combination and method suitable for physical location of rDNA in plant chromosomes

A technology for physical positioning and chromosomes, which is applied in the field of probe combinations for physical positioning of plant chromosome rDNA, can solve the problems of high reagent cost, cumbersome process, inconsistent length, etc., and achieves the effects of convenient physical positioning, simple and convenient operation steps, and improved sensitivity.

Active Publication Date: 2019-06-18
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Disadvantages of this probe: probe labeling requires high DNA quality, and high reagent costs are required from obtaining high-quality DNA to probe labeling and size detection; and it is necessary to preserve the bacterial liquid, shake the bacteria to expand Escherichia coli, The process is cumbersome, time-consuming and labor-intensive; the probes labeled by the nick translation method are double-stranded and inconsistent in length, and the hybridization efficiency is low

Method used

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  • A probe combination and method suitable for physical location of rDNA in plant chromosomes
  • A probe combination and method suitable for physical location of rDNA in plant chromosomes
  • A probe combination and method suitable for physical location of rDNA in plant chromosomes

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Embodiment 1

[0047] This embodiment provides a probe combination for plant chromosome in situ hybridization, which includes a 5S rDNA probe and a 45S rDNA probe;

[0048] The nucleotide sequence of the 5S rDNA probe is shown in SEQ ID NO.1;

[0049] 45S rDNA probe consists of 5.8S rDNA probe, 18S rDNA probe and 25S rDNA probe.

[0050] The nucleotide sequence of the 5.8S rDNA probe is shown in SEQ ID NO.2, the nucleotide sequence of the 18S rDNA probe is shown in SEQ ID NO.3, and the nucleotide sequence of the 25S rDNA probe is shown in SEQ ID NO .4 shown.

[0051] Among them, both ends of the 5S rDNA probe are labeled with FAM fluorescent groups;

[0052] The 5.8S rDNA probe, 18S rDNA probe and 25S rDNA probe are all labeled with TAMRA fluorophore at both ends.

[0053] The nucleotide sequences and modifications of each rDNA oligonucleotide probe are shown in Table 1 below:

[0054] Table 1 Nucleotide sequence and modification of each rDNA probe

[0055]

[0056]

Embodiment 2

[0058] A kind of method for plant chromosome in situ hybridization that the present embodiment provides, it comprises the steps:

[0059] 1 Preparation of metaphase chromosomes

[0060] (1) Material collection: take the root tips (0.5-1.5 cm) of vigorously growing plants as materials.

[0061] (2) Pretreatment: pretreat the freshly obtained root tip with 0.7mM cycloacetamide at room temperature for 4-8 hours (the time required for different species is different, determined according to the actual situation), rinse with distilled water, and transfer to the card Nuo fixative (3:1 ethanol:acetic acid, v / v).

[0062] (3) Enzymolysis: place the fixed root tip in distilled water and rinse thoroughly, then cut the root tip meristem, and put it into an enzyme mixture containing 4% cellulose and 2% pectinase. The enzymatic hydrolysis is about 0.5-1.5h in a 37°C incubator (the time required for different species is different, and it is determined according to the actual situation).

...

Embodiment 3

[0082] In this example, Ili tulip (T.iliensis Regel) of the genus Tulip was used as a material, and fluorescence in situ hybridization was carried out according to the method in Example 2. The results were as follows: figure 1 shown.

[0083] From figure 1 It can be seen that the chromosomes are in good shape and the fluorescence signal is very clear. There are 5 5SrDNA loci (green) in Yili tulip, 4 of which are located in the near centromere region on the short arms of homologous chromosomes 4 and 10, and the other 1 One is located on the long arm of one of the homologous chromosomes 11. There are 15 45S rDNA loci (red) in Yili tulip, most of which are located at the end of the long arm, 2 of which are located at the end of the short arm of chromosome 11, and 12 loci are located at 5, 6, 8, 9, 10 and 12 end of the long arm of the homologous chromosome. One of the homologous chromosomes 7 has a 45S rDNA locus at the end of the long arm, and the other has no signal.

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Abstract

The invention discloses a probe combination and method suitable for the physical location of plant chromosome rDNA, and relates to the technical field of molecular cytogenetics. The probe combination includes the probes of SEQ ID NO.1-4, which are respectively 5S rDNA probe, 5.8S rDNA probe, 18S rDNA probe and 25S rDNA probe; the probe combination and method are used to carry out plant chromosome priming In situ hybridization, which can capture higher resolution chromosome and fluorescence in situ hybridization signals, and is suitable for chromosome in situ hybridization of various plants; and the probe combination is an oligonucleotide probe, which has the advantages of low production cost and convenient use , Simple operation and so on.

Description

technical field [0001] The invention relates to the technical field of molecular cytogenetics, in particular to a probe combination and method suitable for physical location of plant chromosome rDNA. Background technique [0002] Fluorescence in situ hybridization (FISH) uses a DNA fragment labeled with a fluorescent labeling substance such as biotin as a probe to perform molecular hybridization with chromosomal DNA, and directly detects the complementarity with the probe under a fluorescence microscope. The location of the DNA segment. [0003] The rDNA (Ribosomal DNA, ribosomal DNA) can be physically positioned on the chromosome by fluorescence in situ hybridization. At present, rDNA has been extensively physically located in the genomes of important model crops and commercial crops, providing direct information for chromosome identification, genome structure analysis, physical map construction, and species kinship research of these species. [0004] Common rDNA probe pr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/6841C12N15/11
CPCC12Q1/6841C12Q1/6895C12Q2563/107
Inventor 席梦利兰月辛昊阳赵乙琏倪润欣陈曦
Owner NANJING FORESTRY UNIV
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