A method for fluorescence in situ hybridization of mature pollen cells of Chinese cabbage

A fluorescence in situ hybridization and pollen cell technology, applied in the field of molecular cytogenetics, can solve the problems of pollen cell fluorescence in situ hybridization is still blank, pollen cells are difficult to tile, and probes are not easy to penetrate, so that it is easy to break and test The effect of simplifying the process and conveniently obtaining materials

Inactive Publication Date: 2020-09-18
HEBEI AGRICULTURAL UNIV.
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0004] The purpose of the present invention is to provide a method for fluorescence in situ hybridization of mature pollen cells of Chinese cabbage, aiming at that the pollen grains of Chinese cabbage contain hard pollen walls, the pollen cells are difficult to spread well on the glass slide, and the probes are not easy to penetrate into the pollen nucleus , Pollen cell fluorescence in situ hybridization is still blank and other issues, the invention can use the prepared pollen cell specimens for FISH hybridization to obtain a clear hybridization signal picture, reflecting the different hybridization patterns of the target sequence in vegetative cells and germ cells

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  • A method for fluorescence in situ hybridization of mature pollen cells of Chinese cabbage
  • A method for fluorescence in situ hybridization of mature pollen cells of Chinese cabbage
  • A method for fluorescence in situ hybridization of mature pollen cells of Chinese cabbage

Examples

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Embodiment 1

[0034] Example 1 The present invention is illustrated by taking the fluorescence in situ hybridization of 45S rDNA and 5S rDNA probes in Chinese cabbage 'B153' mature pollen cells as an example.

[0035] (1) Determination of flower buds at mature pollen stage

[0036] During the full flowering stage of Chinese cabbage 'B153', take flower buds of different sizes that can bloom for 1-5 days, peel off an anther from each flower bud, place it on a clean glass slide, add a drop of DAPI staining solution, and gently squeeze it with tweezers Press to release the pollen grains, remove the anther wall, cover the cover, press lightly with your thumb, check the development period of the pollen under a fluorescent microscope, and confirm that the pollen in the flower bud 1-2 days before flowering has developed into a mature trinuclear pollen grain(( figure 1 ).

[0037] (2) Preparation of mature pollen cell specimens

[0038]Tablet pressing: Take a large flower bud 1-2 days before flo...

Embodiment 2

[0052] Example 2 The present invention is illustrated by taking the fluorescence in situ hybridization of ribosomal 45S rDNA and 5S rDNA probes in mature pollen cells of Chinese cabbage 'yellow sarson' as an example.

[0053] (1) Preparation of slide specimens of mature pollen cells

[0054] During the full flowering stage of oily Chinese cabbage 'yellow sarson', the large flower buds 1-2 days before flowering were taken, and a fresh anther was peeled off and placed on a clean glass slide.

[0055] (2) Preparation and labeling of probes

[0056] Genomic DNA of oily cabbage 'yellow sarson' was extracted by CTAB method, and 45S rDNA and 5S rDNA fragments were amplified by PCR method using it as a template. The composition of PCR reaction system, reaction conditions, purification of amplification products and probe labeling were the same Example 1.

[0057] (3) Fluorescence in situ hybridization

[0058] ① The pretreatment of the specimen is the same as in Example 1.

[0059]...

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Abstract

The invention belongs to the technical field of crop molecular cytogenetics, and in particular relates to a method for fluorescent in situ hybridization of Chinese cabbage mature pollen cells. The method comprises steps of determining flower buds in a Chinese cabbage mature pollen stage, preparing a mature pollen cell sheet, conducting probe labeling, conducting in situ hybridization, conducting signal detection and the like. The method provided by the invention is characterized in that the flower buds before 1-2d of blossoming of Chinese cabbage are prepared, fresh pollen grains are separated from the flower buds in a 50% glacial acetic acid solution, and pollen walls are broken by virtue of vertical tabletting, so that complete vegetative cells and generative cells are released; cell samples, which are pre-processed, are denatured together with a denaturation probe at 80 DEG C for 3min, and the in situ hybridization is directly conducted, so that a fluorescent hybridization picture that signals are clear is obtained, showing a signal difference of target DNA in the vegetative cells and generative cells. The method provided by the invention fills a technical gap in fluorescent in situ hybridization of mature pollen cells of brassica plants; and the method is applicable to molecular cytogenetics study on pollen development of the Chinese cabbage.

Description

technical field [0001] The invention relates to a fluorescence in situ hybridization method of mature pollen cells of Chinese cabbage, which belongs to the technical field of molecular cytogenetics. Background technique [0002] Pollen is the executor of sexual reproduction in flowering plants. It is not only essential for the regulation of crop fertility and the utilization of heterosis, but also an excellent material for studying the mechanism of cell differentiation and development. The development of pollen includes microsporogenesis (microsporogenesis) and male gametogenesis (male gametogenesis). Microsporogenesis is the differentiation and development of sporogenous cells in plant anthers into pollen mother cells, and then undergoes meiosis to form haploid microspores; male gametophytes Occurrence is that microspores differentiate into a large and loose vegetative cell and a small and compact germ cell through an asymmetric mitosis first, and then the vegetative cell n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6841
CPCC12Q1/6841C12Q2563/107
Inventor 轩淑欣申书兴冯大领王彦华陈雪平
Owner HEBEI AGRICULTURAL UNIV.
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