CN110793834A - 一种苹果果肉细胞Ca2+荧光染色方法 - Google Patents
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Abstract
本发明涉及一种苹果果肉细胞Ca2+荧光染色方法,属于化学检测领域。本苹果果肉细胞Ca2+荧光染色方法,包括:提取原生质体、FDA检测原生质体活性和Fluo‑8/am对苹果果肉原生质体钙离子染色共三个步骤。与现有技术相比,本发明采用酶解的方法获取有活性的果肉细胞原生质体,然后再采用Fluo‑8/am荧光染剂进行钙离子荧光染色。经本发明实验发现:使用Fluo‑8/am荧光染剂37℃避光染色30分钟,可成功染色苹果果肉原生质体钙离子,且无区室化现象。
Description
技术领域
本发明涉及一种苹果果肉细胞Ca2+荧光染色方法,属于化学检测领域。
背景技术
苹果(Malus domestica)作为落叶果树主栽树种是世界上栽培面积较广、产量较大的树种之一。2019年苹果已成为我国第一大水果,目前为止全国苹果种植面积超过4500万亩,年产量达4800多万吨。
钙是植物所必需的大量元素,在植物细胞质中,作为重要的胞内信使参与信号转导,起重要调节作用,同时也影响着植物的生长发育、品质调控等方方面面。细胞质中的钙离子水平常呈现出“尖峰形成”和细胞内“钙波”形式的变化,认为是细胞间触发信息传递的方式,参与植物生长发育过程中的各种代谢反应及信号转导,具有重要研究价值。在细胞内钙离子分布呈现出复杂的时空动态变化,由钙离子含量失调引起的病害比比皆是,如苹果苦痘病、梨褐斑病等,果实品质受损,造成严重经济损失。钙离子荧光呈像是最直观的观察细胞质中的钙离子动态方式。
果肉细胞中有中央大液泡,把细胞质挤压到细胞四周,形成一层很薄的细胞质,当直接对果肉细胞进行染色时,产生的钙离子荧光无法区分细胞质和细胞壁中钙荧光。另外当徒手切片时,果肉细胞排列有多层,造成荧光呈像叠加,对分析细胞质中的钙离子有很大的影响。
发明内容
本发明为了解决上述技术问题,提供一种苹果果肉细胞Ca2+荧光染色方法。
本发明解决上述技术问题的技术方案如下:一种苹果果肉细胞Ca2+荧光染色方法,包括:
1)提取原生质体
配置含20mmol/L CaCl2·2H2O、5mmol/L MES(脂肪酸甲酯磺酸盐)和0.4-0.8mol/LD-山梨醇的溶液,Tris(三羟甲基氨基甲烷)调溶液pH=5.8,过0.22μm水溶性滤膜,所得滤液即为钙浴液;
向上述钙浴液中加入0.3%(mg/ml)离析酶和0.5%(mg/ml)纤维素酶,得含有离析酶和纤维素酶的钙浴液;
取无病的苹果果实表皮下1-2cm的果肉,切成长0.5cm宽0.8cm厚0.1cm的薄片,取一片薄片放入到0.5mL含有离析酶和纤维素酶的钙浴液中,设置摇床温度为28℃,转速为50rpm,全程避光酶解1小时,然后用钙浴液清洗薄片两次,洗去酶液,终止反应,得原生质体悬浮液;
2)FDA(荧光素二乙酸)检测原生质体活性
原生质体失活严重影响内含物的准确含量,在此基础上的一切研究就毫无意义。因此,通过FDA来检测所提原生质体的活性,是确保后续荧光探针染钙离子实验准确性的基础。FDA自身本不发荧光,渗入到活细胞后释放荧光素,在蓝光激发下呈现绿色荧光;而死细胞不会呈现绿色荧光。
取1)所得原生质体悬浮液99μl,加1μl浓度1wt%FDA的PBS溶液,使FDA最终工作浓度为0.01wt%,充分混匀后室温避光5分钟,用荧光显微镜GFP通道观察原生质体荧光现象,只要有绿色荧光即可进入下一步;
3)Fluo-8/am对苹果果肉原生质体钙离子染色
3.1)制备F-127储存液:
称取100mg Pluronic F-127粉末,加入0.5mL DMSO(二甲基亚砜),40-50℃水浴锅中加热30分钟,配制成20%(w/v)DMSO母液,即为F-127储存液,室温保存;
3.2)制备Fluo-8/am储存液:
取50μg Fluo-8/am粉末,离心后加入22.79μl DMSO,配制成2mmol/L Fluo-8/am储存液,分装成2μL/管,-20℃避光密封保存;
3.3)制备Fluo-8/am工作液:
用时提前取2μL 3.2)制备的Fluo-8/am储存液,室温回温,加2μL 3.1)制备的F-127储存液和4μL PBS(聚丁二酸丁二醇酯),配制成0.5mmol/L Fluo-8/am工作液,现用现配;
3.4)取99μL 1)所得原生质体悬浮液于离心管中,加1μL 3.3)制备的Fluo-8/am工作液,使Fluo-8/am荧光染剂最终加载浓度为5μmol/L,混匀;
3.5)离心管开盖放于37℃恒温培养箱中避光孵育30分钟;
3.6)微型离心机清洗2次,以去除多余探针;
3.7)制片,用荧光显微镜GFP通道观察。
在上述技术方案的基础上,本发明还可以做如下改进。
进一步,在1)中,所述D-山梨醇可用甘露醇替换。
采用上述进一步方案的有益效果是加山梨醇或者甘露醇都可以维持原生质体的渗透势。
进一步,在1)中,所述钙浴液分装-20℃保存,如常用则4℃保存。
采用上述进一步方案的有益效果是上述温度便于溶液长时间保存。
进一步,在1)中,所述苹果为新鲜成熟的烟富3号。
进一步,所述荧光显微镜选自美国ThermoFisher公司生产的EVOS Auto 2智能全自动荧光显微成像系统。
采用上述进一步方案的有益效果是快速成像,操作简单快捷。激光共聚焦也可以,但操作较麻烦。
本发明苹果果肉细胞Ca2+荧光染色方法的有益效果是:
与现有技术相比,本发明采用酶解的方法获取有活性的果肉细胞原生质体,然后再采用Fluo-8/am荧光染剂进行钙离子荧光染色。经本发明实验发现:使用Fluo-8/am荧光染剂37℃避光染色30分钟,可成功染色苹果果肉原生质体钙离子,且无区室化现象。
液泡作为“钙库”,其钙离子含量在亚微摩尔级,而胞质中钙离子含量较少,处于毫摩尔级,相差巨大。前有学者对化学荧光试剂颇有质疑,认为其染色产生的荧光是由于液泡中的钙离子所产生,并无法判断是否为胞质中的钙离子。通过本发明图2可以清楚看到,荧光发生在原生质体边缘的胞质中(图2中8个近似圆形结构均显示出绿色荧光),而中间大液泡并没有因为含有高钙离子而发荧光(由于液泡并未显示荧光,因此在图2中显示为黑色)。因此,推翻了化学荧光试剂(包括Fluo-8/am在内)存在区室化现象的说法。
Fluo-8/am是目前最亮的可见光激发波长钙离子荧光探针,与钙离子结合后的最大激发波长为490nm,最大发射波长为514nm,通过激光共聚焦、荧光显微镜等来检测细胞内钙离子水平的变化。迄今,还没有本领域技术人员将其用于检测苹果果肉细胞钙离子。
附图说明
图1为实施例1所得单个原生质体图;
图2为FDA检测37℃处理30分钟后原生质体活性(GFP通道)图;
图3为实施例1和对照组处理烟富3号健康苹果的荧光染色结果图。
具体实施方式
以下结合附图对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。
实施例1
1材料与器材
1.1材料
新鲜健康的烟富3号苹果,贮藏于4℃备用。
离析酶购自Yakult生产的离析酶R-10,牌号为(L0021)Macerozyme R-10。
纤维素酶购自Yakult生产的纤维素酶R-10,牌号为(L0012)Cellulase R-10。
1.2器材
荧光显微镜(ThermoFisher,EVOS Auto 2,美国),恒温摇床,低速离心机,恒温培养箱;
2实验方法
2.1提取原生质体
制备钙浴液:配置含20mmol/L CaCl2·2H2O、5mmol/L MES(脂肪酸甲酯磺酸盐)和0.4mol/L D-山梨醇的溶液,Tris(三羟甲基氨基甲烷)调溶液pH=5.8,过0.22μm水溶性滤膜,所得滤液即为钙浴液,分装-20℃保存,常用4℃保存。
向上述钙浴液中加入0.3%(mg/ml)离析酶和0.5%(mg/ml)纤维素酶,得含有离析酶和纤维素酶的钙浴液;
取无病健康的烟富3号苹果果实表皮下1-2cm的果肉,切成长0.5cm宽0.8cm厚0.1cm的薄片,取一片薄片放入到0.5mL含有离析酶和纤维素酶的钙浴液中,设置摇床温度为28℃,转速为50rpm,全程避光酶解1小时,然后用钙浴液清洗薄片两次,洗去酶液,终止反应,得原生质体悬浮液。
为了展示上述酶解方法所得原生质体的质量和状态,将洗去酶液后的薄片,贴果片吸取得到单个原生质体,如图1所示,原生质体的质膜轮廓清晰,形态饱满圆润。
2.2FDA(荧光素二乙酸)检测原生质体活性
本领域公知,直接提取的原生质体肯定是有高度活性的。为了证明经过37℃处理30分钟后的原生质体仍然具有活性。因此,在FDA染色前,对2.1)所得原生质体悬浮液37℃处理30分钟后,再进行下面的步骤。
取处理后的原生质体悬浮液99μl,加1μl浓度1wt%FDA的PBS溶液,使FDA最终工作浓度为0.01wt%,充分混匀后室温避光5分钟,用荧光显微镜GFP通道观察原生质体荧光现象,显示所提取原生质体普遍具有绿色荧光,活性较强,具体检测结果如图2所示,进入下一步;
2.3Fluo-8/am对苹果果肉原生质体钙离子染色
(1)制备F-127储存液:
称取100mg Pluronic F-127粉末,加入0.5mL DMSO(二甲基亚砜),40-50℃水浴锅中加热30分钟,配制成20%(w/v)DMSO母液,即为F-127储存液,室温保存;
(2)制备Fluo-8/am储存液:
取50μg Fluo-8/am粉末,离心后加入22.79μl DMSO,配制成2mmol/L Fluo-8/am储存液,分装成2μL/管,-20℃避光密封保存;
(3)制备Fluo-8/am工作液:
用时提前取2μL(2)制备的Fluo-8/am储存液,室温回温,加2μL(1)制备的F-127储存液和4μL PBS(聚丁二酸丁二醇酯),配制成0.5mmol/L Fluo-8/am工作液,现用现配;
(4)取99μL 2.1所得原生质体悬浮液于离心管中,加1μL(3)制备的Fluo-8/am工作液,使Fluo-8/am荧光染剂最终加载浓度为5μmol/L,混匀;
(5)离心管开盖放于37℃恒温培养箱中避光孵育30分钟;
(6)微型离心机清洗2次,以去除多余探针。
(7)制片,用荧光显微镜GFP通道观察,结果如图3所示。
通过图3可以看出,使用Fluo-8/am染色的原生质体在GFP通道荧光明显,在复合场中也可看到绿色荧光(GFP通道和复合场图中的圆形结构显示绿色荧光),可有效观察到钙离子在原生质体内的分布,在细胞质中有荧光,在液泡内没有荧光(黑色部分是无荧光的液泡)。并结合相关处理软件,能够计算出原生质体内钙离子的含量。
图3中的对照组是做了相同的处理,具体指实施例1的步骤2.1和步骤2.3中的(5)-(7),但没有加Fluo-8/am荧光染剂的原生质体。加对照是为了说明不加Fluo-8/am荧光染剂,原生质体没有荧光。通过图3对照组可以看出,未加荧光染剂的原生质体在GFP通道、复合场和明场均无荧光,说明原生质体无自荧光现象。
本发明在用Fluo-8/am染色原生质体前,前后尝试了低温(4℃)染色法、低压渗透法、常温法和高温(37℃)染色法等。上述各个方法操作过程一致,只是改变了Fluo-8/am荧光试剂加载进入原生质体的环境条件,将环境条件与加载时间做正交实验,发现无论加载时间多少,荧光染色效果皆不佳,呈现微弱荧光或不荧光。综合比较,高温(37℃)染色法耗时最短,染色效果最佳。
以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (5)
1.一种苹果果肉细胞Ca2+荧光染色方法,其特征在于,包括:
1)提取原生质体
配制含20mmol/L CaCl2·2H2O、5mmol/L MES和0.4-0.8mol/L D-山梨醇的溶液,Tris调溶液pH=5.8,过0.22μm水溶性滤膜,所得滤液即为钙浴液;
向上述钙浴液中加入0.3%(mg/ml)离析酶和0.5%(mg/ml)纤维素酶,得含有离析酶和纤维素酶的钙浴液;
取无病的苹果果实表皮下1-2cm的果肉,切成长0.5cm宽0.8cm厚0.1cm的薄片,取一片薄片放入到0.5mL含有离析酶和纤维素酶的钙浴液中,设置摇床温度为28℃,转速为50rpm,全程避光酶解1小时,然后用钙浴液清洗薄片两次,洗去酶液,终止反应,得原生质体悬浮液;
2)FDA检测原生质体活性
取1)所得原生质体悬浮液99μl,加1μl浓度1wt%FDA的PBS溶液,使FDA最终工作浓度为0.01wt%,充分混匀后室温避光5分钟,用荧光显微镜GFP通道观察原生质体荧光现象,只要有绿色荧光即可进入下一步;
3)Fluo-8/am对苹果果肉原生质体钙离子染色
3.1)制备F-127储存液:
称取100mg Pluronic F-127粉末,加入0.5mL DMSO,40-50℃水浴锅中加热30分钟,配制成20%(w/v)DMSO母液,即为F-127储存液,室温保存;
3.2)制备Fluo-8/am储存液:
取50μg Fluo-8/am粉末,离心后加入22.79μlDMSO,配制成2mmol/L Fluo-8/am储存液,分装成2μL/管,-20℃避光密封保存;
3.3)制备Fluo-8/am工作液:
用时提前取2μL 3.2)制备的Fluo-8/am储存液,室温回温,加2μL 3.1)制备的F-127储存液和4μL PBS,配制成0.5mmol/L Fluo-8/am工作液,现用现配;
3.4)取99μL 1)所得原生质体悬浮液于离心管中,加1μL 3.3)制备的Fluo-8/am工作液,使Fluo-8/am荧光染剂最终加载浓度为5μmol/L,混匀;
3.5)离心管开盖放于37℃恒温培养箱中避光孵育30分钟;
3.6)微型离心机清洗2次,以去除多余探针;
3.7)制片,用荧光显微镜GFP通道观察。
2.根据权利要求1所述的一种苹果果肉细胞Ca2+荧光染色方法,其特征在于,在1)中,所述D-山梨醇可用甘露醇替换。
3.根据权利要求1所述的一种苹果果肉细胞Ca2+荧光染色方法,其特征在于,在1)中,所述钙浴液分装-20℃保存,如常用则4℃保存。
4.根据权利要求1所述的一种苹果果肉细胞Ca2+荧光染色方法,其特征在于,在1)中,所述苹果为新鲜成熟的烟富3号。
5.根据权利要求1所述的一种苹果果肉细胞Ca2+荧光染色方法,其特征在于,所述荧光显微镜选自美国ThermoFisher公司生产的EVOS Auto 2智能全自动荧光显微成像系统。
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