CN-122012368-A - Method for separating and dehydrating cotton boll cell nucleus in mature period

CN122012368ACN 122012368 ACN122012368 ACN 122012368ACN-122012368-A

Abstract

The application provides a method for separating cotton boll nuclei in a dehydrated mature period, which comprises the steps of taking cotton boll tissues in the dehydrated mature period, grinding the cotton boll tissues into fine powder under the protection of liquid nitrogen, wherein the tissues must contain seed coat parts, filtering a first separating liquid through a first pore-size filter, collecting filtrate, carrying out low-temperature centrifugation on the filtrate, discarding supernatant, adding a second separating liquid containing a nonionic detergent, carrying out resuspension precipitation on the resuspended precipitate, carrying out low-temperature centrifugation again, discarding supernatant, washing the precipitate for a plurality of times by using a high-salt washing liquid, resuspension of the washed precipitate by using a third separating liquid containing a higher osmotic pressure component, absorbing a nuclear enrichment interface layer after centrifugation, diluting the interface layer solution by using the third separating liquid, carrying out secondary filtration through a second pore-size filter, and carrying out low-temperature centrifugation on the filtered solution, thus obtaining the separated cotton boll nuclei.

Inventors

WAN DA
SUN ZIKUI
LIU YANYAN

Assignees

南京派森诺基因科技有限公司

Dates

Publication Date: 20260512
Application Date: 20260123

Claims (7)

1. A method for separating cotton boll nuclei in the dehydrated mature period is characterized by comprising the steps of taking cotton boll tissues in the dehydrated mature period and grinding the cotton boll tissues into fine powder under the protection of liquid nitrogen, wherein the tissues must contain seed coat parts, adding the fine powder tissues into a pre-cooled first separating liquid containing specific components, carrying out short homogenization and ice bath standing treatment, filtering the first separating liquid through a first pore-size filter, collecting filtrate, carrying out low-temperature centrifugation on the filtrate to obtain supernatant, adding a second separating liquid containing a nonionic detergent to carry out heavy suspension precipitation, carrying out low-temperature centrifugation on the heavy suspension to obtain supernatant again, carrying out repeated washing on the precipitate by using a high-salt washing liquid, carrying out density gradient centrifugation on the washed precipitate by using a third separating liquid containing a higher osmotic pressure component, absorbing cell nuclei after centrifugation to obtain enriched interface layer, diluting the interface layer solution with the third separating liquid, carrying out secondary filtration on the second pore-size filter on the filtrate, and carrying out low-temperature filtration on the obtained precipitate to obtain the cotton boll nuclei after centrifugation solution.
2. The method of claim 1, wherein the dehydrated mature cotton boll tissue is taken and ground to a fine powder under the protection of liquid nitrogen, wherein the tissue must contain a seed coat portion, the method comprises the steps of controlling the weight of the selected cotton boll tissue to be in a very small amount range, ensuring that the selected tissue contains the seed coat portion during sampling, and carrying out rapid grinding treatment on the cotton boll tissue containing the seed coat in the liquid nitrogen until the cotton boll tissue containing the seed coat is uniformly ground to form fine powder.
3. The method of claim 1, wherein filtering the first separated liquid through a first pore size filter to collect filtrate comprises first coarse filtering the separated liquid containing primarily dispersed tissue with a first filter having a pore size of 40 μm, wherein the first filter is used to retain a majority of large-particle cotton fibers and insufficiently broken tissue pieces, and collecting the filtrate after passing through the first filter as a starting material for subsequent processing.
4. The method of claim 1, wherein adding the second separation solution containing the nonionic detergent to resuspend the precipitate comprises adding Triton X-100 to the second separation solution based on the first separation solution, controlling the concentration range of Triton X-100 to be lower to achieve mild lysis, and fully and uniformly mixing the precipitate with the second separation solution by a slow blowing method.
5. The method of claim 1, wherein the step of overlaying the suspension onto a pre-applied solution of two different concentrations of Percoll density gradient and performing density gradient centrifugation comprises pre-applying the solution of two different concentrations of Percoll to the bottom of a centrifuge tube in sequence to form a discontinuous density gradient, wherein the first layer of Percoll solution comprises a higher proportion of the Percoll component, wherein the second layer of Percoll solution comprises a highest proportion of the Percoll component, wherein the suspension comprising nuclei is carefully overlaid on the uppermost layer of the density gradient, and wherein the different density components are effectively separated in the gradient by performing a longer medium speed low temperature centrifugation.
6. The method of claim 1, wherein the step of sucking up the cell nucleus enriched interface layer and subsequent treatment after centrifugation comprises observing a distinct cell nucleus enriched zone at the interface of the two Percoll density layers after centrifugation, carefully sucking up the cell nucleus enriched zone into a new container, adding the sucked up cell nucleus enriched solution into a third separation solution for dilution, and finely filtering the diluted solution by a second filter with a smaller pore size, wherein the pore size of the second filter is 20 μm.
7. The method of claim 1, wherein the first separation solution, the second separation solution, and the wash solution comprise a common component comprising Tris-HCl buffer system, a common component comprising spermidine, and the spermidine is used to stabilize the nuclear structure.

Description

Method for separating and dehydrating cotton boll cell nucleus in mature period Technical Field The invention relates to the technical field of information, in particular to a method for separating and dehydrating cotton boll nuclei in the mature period. Background Cotton genetics and molecular biology research are core directions in the field of agricultural science, and have important significance for improving cotton quality, yield and stress resistance. Research in this area has been of great concern worldwide, not only in relation to the sustainable development of agricultural production, but also directly in relation to the raw material supply of the textile industry. By deeply researching genetic information of cotton cells, important basis can be provided for breeding and gene improvement. However, current methods face challenges during cotton nucleus extraction, especially when dealing with dehydrated mature cotton boll tissue, which is often not satisfactory. Many traditional techniques are difficult to deal with the characteristics of high hardness of cotton boll tissues and high fiber content, so that the integrity of cell nuclei is easily damaged in operation, and meanwhile, the purity of the extracted cell nuclei is low, so that the requirement of subsequent research is difficult to meet. These problems have often limited researchers in obtaining reliable experimental materials. In a further level, the extraction of nuclei from cotton boll tissue faces a key technical difficulty, namely how to ensure complete release and efficient separation of nuclei while avoiding mechanical damage. The cotton boll tissue is very tough in the dehydration maturity stage, the internal fiber is tightly wound, and if the conventional grinding or separation mode is directly adopted, the cell nucleus is easily broken due to the extrusion of external force. Worse, the seed coat portion, although containing abundant nuclear resources, is firmly packed and difficult to be gently released due to its special tissue characteristics. This situation results in that even if much time and effort are put into it, the extracted nuclei tend to be insufficient in number or poor in quality. In practical operation, for example, when grinding cotton boll tissue, if the force is too large, the cell nucleus is directly destroyed, and if the force is too small, the tissue cannot be broken sufficiently, so that the cell nucleus cannot be separated from the seed coat. Still further, even if a part of the nuclei is released, the purification process is extremely difficult due to the interference of fiber impurities and cell debris in the subsequent separation step, and the obtained nuclei are often mixed with other substances and cannot be used for precise genetic analysis. Therefore, how to efficiently release and purify high quality nuclei from dehydrated mature cotton boll tissue that is hard and high in fiber content, while protecting the integrity of the nuclei, is a key issue in current research. Disclosure of Invention The invention provides a method for separating and dehydrating cotton boll nuclei in mature period, which mainly comprises the following steps: Taking dehydrated mature cotton boll tissue and grinding the cotton boll tissue into fine powder under the protection of liquid nitrogen, wherein the tissue must contain seed coat part, adding the fine powder tissue into a pre-cooled first separating liquid containing specific components, carrying out short homogenization and ice bath standing treatment, filtering the first separating liquid through a first pore size filter, collecting filtrate, carrying out low-temperature centrifugation on the filtrate, adding a second separating liquid containing nonionic detergent to carry out resuspension precipitation, carrying out low-temperature centrifugation on the re-suspended precipitate again, discarding the supernatant, washing the precipitate for multiple times by using a high-salt washing liquid, overlapping the washed precipitate with a third separating liquid containing higher osmotic pressure components, carrying out density gradient centrifugation on the re-suspension onto two pre-paved Percoll density gradient solutions, sucking a cell nucleus enrichment interface layer after centrifugation, diluting the interface layer solution with the third separating liquid, carrying out secondary filtration through a second pore size filter, and carrying out low-temperature centrifugation on the filtered solution to obtain the separated cotton boll cell nuclei. Further, the cotton boll tissue in the dewatering mature period is taken and ground into fine powder under the protection of liquid nitrogen, and the tissue must contain seed coat parts, wherein the weight of the selected cotton boll tissue is controlled in a very small range, the selected cotton boll tissue is ensured to contain the seed coat parts during sampling, and the cotton boll tissue containing the seed coat is su