3D fluorescence in-situ hybridization method for poplar root tip based on frozen section

Abstract

The invention discloses a 3D fluorescence in-situ hybridization method for a poplar root tip based on a frozen section, belonging to the field of molecular cytogenetics. The method comprises the following steps: acquiring a poplar root tip, fixing the poplar root tip with a Carnoy's fixative, then performing washing with water, embedding the poplar root tip with an embedding agent, carrying out freezing, conducting slicing, adhering an obtained section onto a glass slide, carrying out FISH steps such as section baking, denaturation, hybridization, antibody detection and the like, and finally carrying out observing under a laser confocal microscope and capturing a 3D image. The method provided by the invention has the advantages that the complete tissue structure of the root tip can be obtained and a FISH signal is clear. Compared with a 2D FISH result obtained by a traditional section preparation method, the method of the invention has the advantages that the method can visually display real space occupation of chromosomes in cells at a three-dimensional level and accurately locate the space distribution of target sequences in the cells at the three-dimensional level, is applicableto the fields of chromosome space occupation and gene expression of poplars, analysis of Hi-C sequencing results and the like, and has wide application prospects.

Description

Technical field [0001] The invention belongs to the field of molecular cytogenetics, and specifically relates to a 3D fluorescent in situ hybridization (FISH) method of poplar root tip based on frozen section, which can be used for the research of molecular cytogenetics and epigenetics of poplar. Background technique [0002] Fluorescence in situ hybridization (FISH) is a molecular cytogenetics technology developed in the 1980s. It combines the nucleic acid probe directly or indirectly labeled with fluorescein with the nucleic acid sequence in the sample to be tested according to The principle of complementary base pairing is followed by hybridization and observation under a fluorescence microscope. According to the fluorescence signal, the DNA or RNA in the sample to be tested is qualitatively, quantitatively or locally analyzed. In the past 20 years, plant FISH technology has made great progress, especially in terms of probe types, from the commonly used rDNA and other repetiti...

AI Technical Summary

Problems solved by technology

However, most of the FISH techniques in plants are still at the 2D level, and cannot distinguish the real spatial occupancy of chromosomes in cells.

This is mainly because the production methods of plant FISH are mainly smears, pressed sheets, and drip sheets. Hybrid annealing of sequences, but these production methods change the original spatial position of chromosomes in cells, so they cannot visually display the real spatial occupancy of chromosomes on a three-dimensional level

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