CN114891714A - 一种蜜蜂病原真菌脱去病毒的方法 - Google Patents
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Abstract
本发明公开了一种蜜蜂病原真菌脱去病毒的方法，方法包括：步骤S1，病原菌在铺有玻璃纸并含有利巴韦林的PDA培养基上，30℃培养2‑3d；步骤S2，取边缘菌丝2cm×2cm的玻璃纸置于装有4mL混合酶解液的50mL离心管中，在30℃、150rpm震荡酶解2‑3h；步骤S3，四层擦镜纸过滤酶解液，并于4000rpm离心5min，用4mL渗透压稳定剂洗涤2次，重悬于2mL渗透压稳定剂中；步骤S4，对获得的原生质体悬液进行稀释等。本发明所提供的脱毒方法更方便、快速且效果好，为后续病毒的存在对宿主致病性的研究提供基础，具有重要的理论和实际意义。
Description
技术领域
本发明属于病原真菌脱病毒的方法，更具体地，涉及一种蜜蜂病原真菌脱去蜜蜂病毒的方法及其应用。
背景技术
真菌病毒(mycovirus或fungal virus)是一类感染真菌随后在宿主中复制稳定繁殖的病毒，根据基因组分类包括双链RNA(double-strand RNA,dsRNA)病毒、单链RNA(single-strand RNA,ssRNA)病毒,双链DNA病毒(double-strand DNA，dsDNA)和单链DNA(single-strand DNA,ssDNA)病毒。目前在食用和药用真菌、植物病原真菌以及在昆虫病原菌中均检测到真菌病毒。
虽然多数真菌病毒的侵染不能引起寄主形态或其它变化，但部分真菌病毒能导致宿主色素形成异常、生长速度降低、产孢量减少甚至不产孢子或宿主致病力降低等，此类病毒称为低毒真菌病毒(hypovirulence)具有作为绿色杀菌剂的潜力。典型的有CHV1(Cryphonectria hypovirus 1)能有效控制板栗疫病菌(Cryphonectria parasitica)引起的板栗树疫病，SsHADV-l(Sclerotinia sclerotiorum hypovirulence-associated DNAvirus 1)防治核盘菌(Sclerotinia sclerotiorum)引起的油菜菌核病。
塔宾曲霉(Aspergillus tubingensis)是诱导蜜蜂幼虫患病的一种病原真菌，该菌株属于曲霉属(Aspergillus spp.)。目前已经发现塔宾曲霉能促进球囊菌产孢子，可能是蜜蜂白垩病的爆发的重要促进因子。此外，在塔宾曲霉中发现多种蜜蜂病毒，对于蜜蜂病毒是否在其中发挥作用，本脱毒实验对蜜蜂病毒对蜜蜂病原真菌致病性研究具有重要的理论和实际意义。
鉴于此，特提出本发明。
发明内容
针对蜜蜂病原真菌脱毒实验的空白，本发明提供了一种蜜蜂病原真菌脱去病毒方法及其应用。
为实现上述目的，本发明的技术方案如下：
本发明提供一种蜜蜂病原真菌脱去病毒的方法，包括：
步骤S1，病原菌在铺有玻璃纸并含有利巴韦林的PDA培养基上，30℃培养2-3d；
步骤S2，取边缘菌丝2cm×2cm的玻璃纸置于装有4mL混合酶解液的50mL离心管中，在30℃、150rpm震荡酶解2-3h；
步骤S3，四层擦镜纸过滤酶解液，并于4000rpm离心5min，用4mL渗透压稳定剂洗涤2次，重悬于2mL渗透压稳定剂中；
步骤S4，对获得的原生质体悬液进行稀释，涂于含有利巴韦林0-200mg/L再生培养基，于30℃培养6-8d；
步骤S5，根据真菌RNA提取试剂盒，cDNA合成试剂盒，PCR反应检测蜜蜂病毒。
在可能的一个设计中，步骤S1中、利巴韦林浓度为0-200mg/L。
在可能的一个设计中，步骤S2中，混合酶解液中含质量浓度0-1％纤维素酶、质量浓度0-1％蜗牛酶。
在可能的一个设计中，步骤S3中渗透压稳定剂为0-1mol/LNaCl溶液。
在可能的一个设计中，步骤S4中的再生培养基组分，以质量份数计，包括酵母浸出粉2份、蛋白胨5份、NaCl 2份、可溶性淀粉1份、CaCl21份、MgSO4·5H2O 1份、琼脂18份，加水定容至1L。
本发明与现有技术相比，具有以下优点：
(1)本发明所采用的原生质体脱毒方法，耗时短，操作简便，无需高端昂贵的实验器材。由于病毒在真菌中的分布不均匀，即在部分细胞中不含有病毒，通过混合酶解液将细胞壁酶解以后，原生质体释放出来。
(2)本发明中渗透压稳定剂是保存原生质体，使其能稳定存在液体中而不失水或膨胀而导致失活。
(3)利巴韦林是一种RNA病毒复制抑制剂，通过抑制病毒的复制而减少病毒积累，从而达到增加不含病毒的真菌细胞数量；利巴韦林：又称病毒唑，是一种病毒抑制剂，进入被病毒感染的细胞后能迅速磷酸化，其产物作为病毒合成酶的竞争性抑制药，抑制肌苷单磷酸脱氢酶、流感病毒RNA多聚酶和mRNA鸟苷转移酶，从而引起细胞内三磷酸鸟苷的减少，损害病毒RNA和蛋白合成，使病毒的复制与传播受抑。
(4)混合酶：由蜗牛酶和纤维素酶组合构成，其中蜗牛酶含有纤维素酶、果胶酶、淀粉酶和蛋白酶等20多种酶；由于真菌细胞壁由几丁质、脱乙酰壳多糖、葡聚糖、纤维素、半乳聚糖等组成，因此采用混合酶将细胞壁降解，是真菌原生质体释放出来。
(5)渗透压稳定剂：由NaCl溶液组成，旨在维持原生质体活性，避免其吸水涨破。
(6)原生质体脱毒原理：病毒在每个细胞的分布不均匀，存在病毒含量少或不含病毒的细胞，因此通过原生质体再生方法可能获得不含病毒的后代。
附图说明
图1是本发明实施例1中以0mg/L利巴韦林试剂处理的菌样PCR扩增的蜜蜂病毒的电泳图(上样量均为5μL，M表示100bp DNA marker)；
图2是本发明实施例1中以100mg/L利巴韦林试剂处理的菌样PCR扩增的蜜蜂病毒的电泳图(上样量均为5μL，M表示100bp DNA marker)。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步详细说明。
应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例中，如无特别说明，所用手段均为本领域常规的手段。
本文中所用的术语“包含”、“包括”或其任何其它变形，意在覆盖非排它性的包括。例如，包含所列要素的组合物、步骤、方法、制品或装置不必仅限于那些要素，而是可以包括未明确列出的其它要素或此种组合物、步骤、方法、制品或装置所固有的要素。
此外，下面所描述的本发明各个实施方式中所涉及到的技术特征只要彼此之间未构成冲突就可以相互组合。
在本发明实施例中，所用的蜜蜂真菌为发明人所在实验室分离纯化的蜜蜂病原真菌塔宾曲霉。
在分离纯化前，将真菌菌株接种在铺有玻璃纸的PDA(马铃薯浸粉6g/L，葡萄糖20g/L，琼脂20g/L)培养基上，30℃黑暗条件下培养6-8d。
实施例1
本发明提供一种蜜蜂病原真菌脱去病毒的方法，包括：
S1、病原菌在铺有玻璃纸的含有100μmol/L利巴韦林的PDA培养基上，30℃培养2d。
S2、取带有菌丝2cm×2cm的玻璃纸置于装有4mL混合酶解液的50mL离心管中，在30℃、150rpm摇床中酶解2.5h，其中步骤S2中，混合酶解液中含质量浓度0.5％纤维素酶、质量浓度1％蜗牛酶。
S3、四层擦镜纸过滤酶解液，并于4000rpm离心5min，用4mL渗透压稳定剂洗涤2次，重悬于2mL渗透压稳定剂中，步骤S3中渗透压稳定剂为0.6mol/LNaCl溶液。
S4、对获得的原生质体悬液进行稀释，涂于再生平板上，于30℃培养6d，步骤S4中的再生培养基组分，以质量份数计，包括酵母浸出粉2份、蛋白胨5份、NaCl 2份、可溶性淀粉1份、CaCl21份、MgSO4·5H2O 1份、琼脂18份，加水定容至1L。
步骤S5，根据真菌RNA提取试剂盒，cDNA合成试剂盒，PCR反应检测蜜蜂病毒，所需引物见表1：
表1病毒中英文名对照
表2病毒检测所需引物
本发明与现有技术相比，具有以下优点：
(1)本发明所采用的原生质体脱毒方法，耗时短，操作简便，无需高端昂贵的实验器材。由于病毒在真菌中的分布不均匀，即在部分细胞中不含有病毒，通过混合酶解液将细胞壁酶解以后，原生质体释放出来。
(2)本发明中渗透压稳定剂是保存原生质体，使其能稳定存在液体中而不失水或膨胀而导致失活。
(3)利巴韦林是一种RNA病毒复制抑制剂，通过抑制病毒的复制而减少病毒积累，从而达到增加不含病毒的真菌细胞数量；利巴韦林：又称病毒唑，是一种病毒抑制剂，进入被病毒感染的细胞后能迅速磷酸化，其产物作为病毒合成酶的竞争性抑制药，抑制肌苷单磷酸脱氢酶、流感病毒RNA多聚酶和mRNA鸟苷转移酶，从而引起细胞内三磷酸鸟苷的减少，损害病毒RNA和蛋白合成，使病毒的复制与传播受抑。
(4)混合酶：由蜗牛酶和纤维素酶组合构成，其中蜗牛酶含有纤维素酶、果胶酶、淀粉酶和蛋白酶等20多种酶；由于真菌细胞壁由几丁质、脱乙酰壳多糖、葡聚糖、纤维素、半乳聚糖等组成，因此采用混合酶将细胞壁降解，是真菌原生质体释放出来。
(5)渗透压稳定剂：由NaCl溶液组成，旨在维持原生质体活性，避免其吸水涨破。
(6)原生质体脱毒原理：病毒在每个细胞的分布不均匀，存在病毒含量少或不含病毒的细胞，因此通过原生质体再生方法可能获得不含病毒的后代。
实施例2：
与实施例1的区别在于：
S1、病原菌在铺有玻璃纸的含有0μmol/L利巴韦林的PDA培养基上，30℃培养3d。
S2、取带有菌丝2cm×2cm的玻璃纸置于装有4mL混合酶解液的50mL离心管中，在30℃、150rpm摇床中酶解3h，其中步骤S2中，混合酶解液中含质量浓度0.5％纤维素酶、质量浓度1％蜗牛酶。
S3、四层擦镜纸过滤酶解液，并于4000rpm离心5min，用4mL渗透压稳定剂洗涤2次，重悬于2mL渗透压稳定剂中，步骤S3中渗透压稳定剂为0.6mol/LNaCl溶液。
S4、对获得的原生质体悬液进行稀释，涂于再生平板上，于30℃培养7d。
如图1-2所示，本发明的方法是能够用于对蜜蜂病毒对蜜蜂病原真菌致病性研究，其中序列号为：ABPV(accession number:OL321878),CBPV(accession number:OL321879),IAPV(accession number:OL321884),KV(accession number:OL321882)and DWV-A(accession number:OL321883)。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。
应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
Claims (5)
1.一种蜜蜂病原真菌脱去病毒的方法，其特征在于，包括：
步骤S1，病原菌在铺有玻璃纸并含有利巴韦林的PDA培养基上，30℃培养2-3d；
步骤S2，取边缘菌丝2cm×2cm的玻璃纸置于装有4mL混合酶解液的50mL离心管中，在30℃、150rpm震荡酶解2-3h；
步骤S3，四层擦镜纸过滤酶解液，并于4000rpm离心5min，用4mL渗透压稳定剂洗涤2次，重悬于2mL渗透压稳定剂中；
步骤S4，对获得的原生质体悬液进行稀释，涂于含有利巴韦林0-200mg/L再生培养基，于30℃培养6-8d；
步骤S5，根据真菌RNA提取试剂盒，cDNA合成试剂盒，PCR反应检测蜜蜂病毒。
2.根据权利要求1所述的一种蜜蜂病原真菌脱去病毒的方法，其特征在于，步骤S1中、利巴韦林浓度为0-200mg/L。
3.根据权利要求1或2所述的一种蜜蜂病原真菌脱去病毒的方法，其特征在于，
步骤S2中，混合酶解液中含质量浓度0-1％纤维素酶、质量浓度0-1％蜗牛酶。
4.根据权利要求1所述的一种蜜蜂病原真菌脱去病毒的方法，其特征在于，
步骤S3中渗透压稳定剂为0-1mol/LNaCl溶液。
5.根据权利要求1所述的蜜蜂病原真菌脱去病毒的方法，其特征在于，步骤S4中的再生培养基组分，以质量份数计，包括酵母浸出粉2份、蛋白胨5份、NaCl 2份、可溶性淀粉1份、CaCl21份、MgSO4·5H2O 1份、琼脂18份，加水定容至1L。
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