Screening and Application of the Nuclear Dissociation Solutions of Soybeans Suitable for Flow Cytometry Analysis

doi:10.13560/j.cnki.biotech.bull.1985.2020-0426

Abstract

Abstract:

Using soybean Williams 82 and other varieties as research materials,the nuclear separation effects of 6 different kinds of nuclear dissociation solutions were compared,aiming to prepare the high-purity nucleus suspensions from different soybean tissues suitable for flow cytometry analysis,as well as to detect the application of them in the regulation of soybean cell cycle. The fresh young leaves and roots of soybean were mechanically cut in different nuclear dissociation solutions to release the nuclei from the tissues. After passing through 400 mesh sieves,enriched by centrifugation,and DAPI staining,the characteristics of biological particles and DNA content in the nuclear dissociation solution were detected and analyzed by flow cytometry. The results showed that Galbraith’s and LB01 dissociation solutions were suitable for the dissociation of nuclei from the soybean leaves and roots,and their coefficient of variation values were quite low at 2.78% and 2.96%,respectively. Among them,a distinct G2/M cell cycle peak was found in the LB01 nuclear dissociation buffer,which was more suitable for cell cycle and genetic ploidy research. Moreover,the nuclear dissociation solution can be successfully applied to analyze the regulation of drought stress simulated by PEG on the cell cycle of different soybean tips.

Key words: flow cytometry, nuclear dissociation solutions, cell cycle, soybean, drought

CHEN Lin, PAN Zhen-zhi, DAI Yi, SONG Li. Screening and Application of the Nuclear Dissociation Solutions of Soybeans Suitable for Flow Cytometry Analysis[J]. Biotechnology Bulletin, 2020, 36(11): 230-237.

Figures/Tables 5

References 28

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核解离液	配制方法和储存使用
Galbraith’s^[11]	45 mmol/L MgCl₂,20 mmol/L MOPS,30 mmol/L Sodium citrate,0.1%（V/V）Triton X-100。1 mol/L NaOH调pH至7.0,0.22 μm 过滤并分装贮存于-20℃。
mG^[25]	45 mmol/L MgCl₂,20 mmol/L MOPS,30 mmol/L Sodium citrate,10 mmol/L Na₂EDTA,1% PVP-40,0.2%（V/V）Triton X-100。1 mol/L NaOH调pH至7.0,0.22 μm过滤,按照20 μL/mL加入2-mercaptoethanol 并分装贮存于-20℃。
LB01^[13]	15 mmol/L Tris,2 mmol/L Na₂EDTA,0.5 mmol/L Spermine tetrahydrochloride,80 mmol/L KCl,20 mmol/L NaCl,0.1%（V/V）Triton X-100。1 mol/L NaOH调pH至7.5,0.22 μm过滤,加入2-mercaptoethanol使得终浓度为15 mmol/L并分装贮存于-20℃。
WPB^[16]	200 mmol/L Tris,4 mmol/L MgCl₂,2 mmol/L Na₂EDTA,86 mmol/L NaCl,10 mmol/L Sodium pyrosulfite,1%（W/V）PVP-10,1%（V/V）Triton X-100。HCl调pH至7.5,0.22 μm过滤并分装贮存于-20℃。
GPB^[16]	0.5 mmol/L Spermine tetrahydrochloride,30 mmol/L Sodium citrate,80 mmol/L KCl,20 mmol/L NaCl,20 mmol/L MOPS,0.5%（V/V）Triton X-100。HCl调pH至7.0,0.22 μm并分装贮存于-20℃。
Tris·MgCl₂^[4]	200 mmol/L Tris,4 mmol/L MgCl₂,0.5%（V/V）Triton X-100。HCl调pH至7.5,0.22 μm过滤并分装贮存于-20℃。

核解离液	配制方法和储存使用
Galbraith’s^[11]	45 mmol/L MgCl₂,20 mmol/L MOPS,30 mmol/L Sodium citrate,0.1%（V/V）Triton X-100。1 mol/L NaOH调pH至7.0,0.22 μm 过滤并分装贮存于-20℃。
mG^[25]	45 mmol/L MgCl₂,20 mmol/L MOPS,30 mmol/L Sodium citrate,10 mmol/L Na₂EDTA,1% PVP-40,0.2%（V/V）Triton X-100。1 mol/L NaOH调pH至7.0,0.22 μm过滤,按照20 μL/mL加入2-mercaptoethanol 并分装贮存于-20℃。
LB01^[13]	15 mmol/L Tris,2 mmol/L Na₂EDTA,0.5 mmol/L Spermine tetrahydrochloride,80 mmol/L KCl,20 mmol/L NaCl,0.1%（V/V）Triton X-100。1 mol/L NaOH调pH至7.5,0.22 μm过滤,加入2-mercaptoethanol使得终浓度为15 mmol/L并分装贮存于-20℃。
WPB^[16]	200 mmol/L Tris,4 mmol/L MgCl₂,2 mmol/L Na₂EDTA,86 mmol/L NaCl,10 mmol/L Sodium pyrosulfite,1%（W/V）PVP-10,1%（V/V）Triton X-100。HCl调pH至7.5,0.22 μm过滤并分装贮存于-20℃。
GPB^[16]	0.5 mmol/L Spermine tetrahydrochloride,30 mmol/L Sodium citrate,80 mmol/L KCl,20 mmol/L NaCl,20 mmol/L MOPS,0.5%（V/V）Triton X-100。HCl调pH至7.0,0.22 μm并分装贮存于-20℃。
Tris·MgCl₂^[4]	200 mmol/L Tris,4 mmol/L MgCl₂,0.5%（V/V）Triton X-100。HCl调pH至7.5,0.22 μm过滤并分装贮存于-20℃。