CN110487615B - 一种鉴定十字花科植物根肿病的复合荧光染色方法 - Google Patents
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Abstract
本发明公开一种鉴定十字花科植物根肿病的复合荧光染色方法，包括以下操作步骤：(1)在白菜播种两周后，接种根肿菌；(2)接种根肿菌后2天内控水，之后恢复正常管理及时浇水并补充营养元素；(3)接菌三周后取白菜根制作石蜡切片，先用苯胺蓝染色2‑4min，然后依次用ddH2O、70％的酒精、100％酒精清洗干净，再用尼罗红染色50‑70s，最后用100％酒精清洗30秒，晾干，在荧光显微镜紫外激发光下观察。本发明提供的一种鉴定十子花科植物根肿病的复合荧光染色方法，操作简单，绿色环保，成本低，可特异的将白菜根部细胞的细胞壁染成红色，淀粉粒染成亮绿色或者蓝绿色，而根肿菌被染成蓝色。
Description
技术领域
本发明属于荧光复染色技术领域，具体涉及一种鉴定十字花科植物根肿病的复合 荧光染色方法。
背景技术
十字花科根肿病是由芸薹根肿菌(Plasmodiophora brassicae Woron.)侵染引起的一种土传病害，严重危害十字花科植物的安全生产。芸薹根肿菌在侵染植株的过程中分为两个阶段，初侵染阶段(或根毛侵染，即土壤中休眠孢子变成初级游动孢子到达根毛表面后，穿过细胞壁侵入根毛内部，这个阶段被称为初侵染阶段)和次级侵染阶段(或皮层侵染，即病原菌在根毛内部形成次生原生质团，一部分细胞核裂解形成多核的游动孢子囊，每个游动孢子囊产生次游动孢子，侵入主根皮层，这个过程叫做次级侵染阶段)，整个侵染过程在寄主植物根部进行。世界上对根肿菌的研究已有很长的历史，但是至今我们对根肿菌的生活史了解尚不充分。根肿菌的初级侵染及次级侵染之间的关系，根肿菌游动孢子的释放等尚不明确。因此，观察受侵染作物根部的细胞学结构特征对根肿菌的侵染过程及其防治具有重要的研究意义，而染色技术是观察细胞学结构特征最重要环节之一。
目前，植物石蜡切片的染色方法常见的有苏木精、碱性品红、伊红、番红、固绿、苯胺蓝、甲苯胺蓝和台盼蓝等；复合染色方法有苏木精和伊红复染、番红和固绿复合染色。荧光染色剂主要有尼罗红、罗丹明、DNA染料包括碘化丙啶(PI)、Hoechst 33342等，RNA染料有噻唑橙、吖啶橙等。荧光染料，由于灵敏度高，操作方便，常被用于染色体分析、细胞周期、细胞凋亡等相关研究。另有很多核酸染料在多色染色系统中是非常有用的复染剂，可作为背景对照，标记细胞核使细胞内结构的空间关系一目了然。虽然有众多的染色方法，但是对于根肿菌侵染过程常用的染色方法仅限于苯胺蓝，碱性品红、亚甲蓝、甲苯胺蓝、藏红、阿斯特拉蓝、尼罗红几个染色剂且特异性较差，不能够很好的区分植物细胞及细胞内的根肿菌，不利于根肿菌侵染寄主的细胞学观察。因此有必要开发一个能够很好地区分寄主细胞与根肿菌的染色方法，为根肿菌的侵染机制研究及其防治奠定基础。
发明内容
为了解决上述存在的技术问题，本发明提供一种广泛应用于根肿菌侵染十字花科植物根部细胞学特征的观察方法。
本发明是通过以下技术方案实现的。
一种鉴定十字花科植物根肿病的复合荧光染色方法，包括以下操作步骤：
(1)在白菜播种两周后，接种根肿菌；
(2)接种根肿菌后2天内控水，之后恢复正常管理及时浇水并补充营养元素；
(3)接菌三周后取白菜根制作石蜡切片，先用苯胺蓝(Sigma，质量分数为0.025％)染色2-4min，然后依次用ddH2O、70％的酒精、100％酒精清洗干净，再用尼罗红(Sigma，0.5mg/ml)染色50-70s，最后用100％酒精清洗30秒，晾干，在荧光显微镜紫外激发光下观察。
具体地，上述步骤(1)中，白菜播种的基质在使用前经过灭菌处理。
具体地，上述步骤(1)中，接种根肿菌时，菌浓度为1×107个/ml，每棵白菜接种量为1ml。
具体地，上述步骤(3)中，石蜡切片的制作步骤包括取材、固定、洗涤和脱水、透明、浸蜡、包埋、切片和粘片、脱蜡。
具体地，上述步骤(3)中，第一次清洗时，ddH2O的清洗时间为1-5min，70％酒精的清洗时间为1-3min，100％酒精的清洗时间为1min。
由以上的技术方案可知，本发明的有益效果是：
本发明提供的一种鉴定十字花科植物根肿病的复合荧光染色方法，操作简单，绿色环保，成本低，可特异的将大白菜根部细胞的细胞壁染成红色，淀粉粒染成亮绿色或者蓝绿色，而根肿菌被染成蓝色。
附图说明
图1为苯胺蓝与尼罗红复合染色后荧光显微镜紫外激发光观察根肿菌侵染3周时易感病大白菜BJN3-2根横切面，A箭头指示为淀粉粒，B箭头指示为细胞壁，C箭头指示为根肿菌。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
一种鉴定十字花科植物根肿病的复合荧光染色方法，包括以下操作步骤：
(1)在白菜播种两周后，接种根肿菌，其中白菜播种的基质在使用前经过灭菌处理，接种根肿菌时的菌浓度为1×107个/ml，每棵白菜接种量为1ml；
(2)接种根肿菌后2天内控水，之后恢复正常管理及时浇水并补充营养元素；
(3)接菌三周后取白菜根制作石蜡切片，先用苯胺蓝(Sigma，质量分数为0.025％)染色3min，然后依次用ddH2O、70％的酒精、100％酒精清洗干净，ddH2O的清洗时间为3min，70％酒精的清洗时间为2min，100％酒精的清洗时间为1min；再用尼罗红(Sigma，0.5mg/ml)染色60s，最后用100％酒精清洗30秒，晾干，在荧光显微镜紫外激发光下观察，其中石蜡切片的制作步骤包括取材、固定、洗涤和脱水、透明、浸蜡、包埋、切片和粘片、脱蜡。
当然，上述说明并非是对本发明的限制，本发明也并不限于上述举例，本技术领域的普通技术人员，在本发明的实质范围内，作出的变化、改变、添加或替换，都应属于本发明的保护范围。
Claims (5)
1.一种鉴定十字花科植物根肿病的复合荧光染色方法，其特征在于，包括以下操作步骤：
（1）在白菜播种两周后，接种根肿菌；
（2）接种根肿菌后2天内控水，之后恢复正常管理及时浇水并补充营养元素；
（3）接菌三周后取白菜根制作石蜡切片，先用苯胺蓝染色2-4min，然后依次用ddH2O、70%的酒精、100%酒精清洗干净，再用尼罗红染色50-70s，最后用100%酒精清洗30秒，晾干，在荧光显微镜紫外激发光下观察。
2.根据权利要求1所述的一种鉴定十字 花科植物根肿病的复合荧光染色方法，其特征在于，步骤（1）中，白菜播种的基质在使用前经过灭菌处理。
3.根据权利要求1所述的一种鉴定十字 花科植物根肿病的复合荧光染色方法，其特征在于，步骤（1）中，接种根肿菌时，菌浓度为1×107个/ml，每棵白菜接种量为1ml。
4.根据权利要求1所述的一种鉴定十字 花科植物根肿病的复合荧光染色方法，其特征在于，步骤（3）中，石蜡切片的制作步骤包括取材、固定、洗涤和脱水、透明、浸蜡、包埋、切片和粘片、脱蜡。
5.根据权利要求1所述的一种鉴定十字 花科植物根肿病的复合荧光染色方法，其特征在于，步骤（3）中，第一次清洗时，ddH2O的清洗时间为1-5min，70%酒精的清洗时间为1-3min，100%酒精的清洗时间为1min。
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