适合流式细胞仪分析的大豆细胞核解离液的筛选与应用

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

摘要/Abstract

摘要：

利用大豆Williams 82等多个品种为材料,比较6种不同细胞核解离液的核分离效果,以期制备适合流式细胞仪分析的大豆不同组织部位的高纯度细胞核悬液,并检测其在大豆细胞周期调控中的应用。将新鲜幼嫩的大豆叶和根组织在不同的细胞核解离液中进行机械切割后,细胞核从组织释放游离到核解离液,过400目滤网,离心富集及DAPI染色,流式细胞仪检测分析细胞核解离液中生物颗粒特性和DNA含量。结果表明：Galbraith’s和LB01 核解离液适用于大豆叶和根中细胞核的解离,其变异系数CV值较低,分别是2.78%和2.96%。其中LB01 核解离液可检测到显著的G2/M 期峰,在细胞周期及遗传倍性的研究中应用相对更好,且核解离液在不同大豆品种上具有一定的适用性,可成功检测PEG模拟的干旱胁迫对不同大豆根尖细胞周期的调控。

关键词: 流式细胞术, 细胞核解离液, 细胞周期, 大豆, 干旱

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

陈林, 潘贞志, 戴毅, 宋丽. 适合流式细胞仪分析的大豆细胞核解离液的筛选与应用[J]. 生物技术通报, 2020, 36(11): 230-237.

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.

图/表 5

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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℃。