Method for extracting nucleuses of pear pollen tube

Abstract

The invention discloses a method for extracting nucleuses of a pear pollen tube. The method comprises the following steps: 1) collecting pollen, that is, collecting anther in a big bud stage and drying the anther; 2) culturing the pollen; 3) releasing nucleuses, that is, grinding the pollen with a single-cell screen and carrying out filtration by using a dual-cell screen so as to release the nucleuses; 4) purifying the nucleuses by using Percoll density gradient centrifugation; and 5) rinsing Percoll. The method does not relate to enzyme extraction of protoplast or conventional technologies of grinding and filtering, and has the advantages of simple and fast operation, low cost, an obvious purification effect, etc.; the purified nucleuses are hardly contaminated by cytoplasm, can not onlybe used for preparation of DNA fibers but also be used for proteomics research of nucleuses, and have important significance to research on growth, metabolism and self-incompatible response of pear pollen tubes.

Description

technical field [0001] The invention belongs to the field of plant biochemistry, and relates to a method for extracting pear pollen tube nuclei, in particular to a method for extracting and purifying cell nuclei from pear pollen tubes by utilizing cytochemical treatment and subcellular organelle classification and screening technology. Background technique [0002] The nucleus contains almost all genetic information and is extremely important for gene expression and regulation. With the development of genome sequencing, DNA fiber fluorescence in situ hybridization and the construction of large fragment genome libraries play an important role in promoting plant genome sequencing. The extracted and purified nuclei are the basis for making DNA fibers and constructing large fragment genome libraries. In addition to DNA and RNA, the nucleus also contains some proteins, and these proteins show dynamic changes with different intracellular and extracellular environments, thus parti...

AI Technical Summary

Problems solved by technology

Pear pollen tube is a special plant cell. After adding the extract, it is difficult to completely break the pollen tube wall by traditional mortar and pestle grinding methods, allowing the nucleus to be released with the cytoplasm

At the same time, the use of traditional liquid nitrogen grinding will damage the organelles, which is not suitable for cell nucleus separation

However, pre-extracting pollen tube protoplasts and then extracting cell nuclei by breaking the wall requires a large amount of cellulase, isolated enzyme, etc., and the cost is high and the steps are complicated.

The cell grinding solution is directly centrifuged after filtration, the purification effect is not good, and it often contains a lot of impurities

Density centrifugation has a better effect, but the traditional sucrose isodensity gradient is not easy to control, and the quality of purified nuclei is not good.

The current nuclear extraction technology has many deficiencies, and it is not suitable for pollen tube nucleus extraction. Therefore, the establishment of a set of pollen tube nucleus extraction and purification technology is of great significance for the study of pear pollen tube growth metabolism and self-incompatibility reaction.

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