生物技术通报 ›› 2020, Vol. 36 ›› Issue (11): 230-237.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0426
收稿日期:
2020-04-05
出版日期:
2020-11-26
发布日期:
2020-11-20
作者简介:
陈林,男,硕士研究生,研究方向:大豆遗传育种;E-mail: 基金资助:
CHEN Lin1,2(), PAN Zhen-zhi1, DAI Yi1, SONG Li1()
Received:
2020-04-05
Published:
2020-11-26
Online:
2020-11-20
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。
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.
核解离液 | 配制方法和储存使用 |
---|---|
Galbraith’s[ | 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[ | 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[ | 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[ | 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[ | 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[ | 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℃。 |
表1 核解离缓冲液配方
核解离液 | 配制方法和储存使用 |
---|---|
Galbraith’s[ | 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[ | 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[ | 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[ | 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[ | 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[ | 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℃。 |
图4 不同大豆品种在PEG模拟干旱胁迫前后细胞周期的分析 A、C、E、G:不同大豆品种在正常生长状态时根尖细胞核的DNA相对含量;B、D、F、H不同大豆品种在20% PEG 6000模拟干旱胁迫处理后根尖细胞核的DNA相对含量。(A和B)Holladay,P<0.001;(C和D)PI 567611,P<0.05;(E和F)PI 567651,P<0.001;(G和H)Fiskeby III,P<0.001
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