SU1701747A1 - Method for isolation of plant cell nuclei - Google Patents

Abstract

The invention relates to the physiology and biochemistry of plants and can be applied in the analysis of the mechanisms of molecular biology of development, namely in the study of the relationship of the structural and functional elements of cell nuclei in the process of unfolding genetic programs. The purpose of the invention is to increase the yield of purified cellular nuclei with simultaneous preservation of the intra-nuclear enzymatic activity and the physiological properties of the structural and functional elements of the cellular nuclei. To achieve the goal, plant tissues were initially preserved in 80-90% glycerol, followed by homogenization in 20% glycerol, buffered, to isolate the cell nuclei, which are cleaned through a five-layer (50, 60, 70, 80, 90 may.% / volume) buffered glycerol gradient.

Description

The invention relates to the physiology and biochemistry of plants and can be applied in the analysis of the mechanisms of molecular biology of development, namely in the study of the relationship of the structural and functional elements of cell nuclei in the process of unfolding genetic programs.

The purpose of the invention is to increase the yield of purified cellular nuclei while maintaining their native nature.

An embodiment of the invention.

The work was carried out on resting, 1–2 day old seedlings, as well as 3–4 day old tissues of coleoptiles, leaves, and wheat roots of the Mironovskaya winter and winter, Moscow – 35, rye Chishminskaya winter, and the Chulpan variety. Sprouts and tissues were preserved at t - 25 ° C in 80-90% glycerol.

Kernels from plant tissues were isolated by three-step homogenization of tissue for 7 seconds, 15 seconds, 45 seconds at 15,000 rpm in 20% glycerol containing 0.02 M triethanolamine (TEA). HCI pH 6.8; 0.005 M MgCl; 0.025 M KCI; 0.003 CaCIa; 0.005 M NaCl; 0.004 M n-octal alcohol and 0.004 M / -mermer-captoethanol. The plant material, filtered through 4 layers of capron with a pore size of 70 microns, was centrifuged at 500 rpm for 5 minutes in order to free it from coarse, intact tissues and whole cells, then the supernatant was centrifuged at 2700 rpm for 20 minutes. The precipitate was cleaned through a five-layer glycerin baseline (50, 60, 70, 80, 90 May / volume) prepared on TEA HCI pH 6.8 with all the listed components of the homogenizing stage VI

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dy, excluding 20% glycerol. Gradient centrifugation was performed at 2000 rpm for 1 h. The passage of the cores through the five-layer glycerol gradient was monitored microscopically. The pellet was washed with 0.5% Triton X-100 on a homogenizing medium, without glycerol, followed by centrifugation at 2J700 rpm for 15 minutes, then washed three times in medium with the following composition: 0.005 M MgCl2.0.025 M KCI; 0.003 H / l CaCl2; 0.005 M NaCl; 0.01 M Tris-HCl pH 6.8, followed by centrifugation under the indicated conditions. The degree of purity of the isolated cell dermal preparations was determined microscopically, spectrophotometrically by protein / DNA, RNA / DNA component composition.

Claims (1)

Invention Formula The method of isolation of plant cell dermis, including conservation plant tissues at a temperature of minus 25 ° C in the presence of glycerol, homogenization, filtration and low-speed centrifugation of the homogenate and subsequent purification of the nuclear fraction using gradient centrifugation, characterized in that 80 -90% glycerol, homogenization is carried out in a buffer containing 20% glycerol, and the cores are cleaned in a density gradient of glycerol of 50, 60, 70, 80, 90% by weight.