CN105145491A - 一种红麻根结线虫分离鉴定方法 - Google Patents
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Abstract
本发明提供一种红麻根结线虫分离鉴定方法，属于微生物技术领域。本发明采用的技术方案是：（1）经典形态学鉴定；（2）同工酶分析鉴定：（3）分子生物学鉴定：以引物5-GGTCAATGTTCAGAAATTTGTGG-3和5-TACCTTTGACCAATCACGCT-3进行PCR鉴定，扩增其位于线粒体DNA中的COll基因和lrRNA基因，将序列进行比对分析鉴定。进而明确根结线虫的生理小种。本发明集分离、纯化、鉴定为一体，采用“形态学+同工酶+分子生物学”相结合的方法进行鉴定与验证，结果表明分子生物学鉴定具有效率高、准确性强，适用于田间采样的快速分离，鉴定出虫体野生型种群或生理小种的生物学特性。
Description
技术领域
本发明涉及一种红麻根结线虫分离鉴定方法，属于微生物技术领域。
背景技术
红麻(kenaf)为锦葵科木槿属一年生纤维作物，是世界上仅次于棉花的第二大天然纤维作物之一，广泛栽培于印度、孟加拉、中国、泰国等国家。红麻纤维具有产量高、吸湿性强、散热性好、易降解等优良特性，因此引起了世界各国的重视。近年来，其作为造纸、纺织及生物质能、麻塑、麻碳的优良原料而备受企业关注。
随着研究的不断深入，红麻被广泛应用于纺织、建筑、家居、汽车工业等各个领域。为了满足人们对于天然纤维需求的增长，红麻的种植面积也在逐年递增，但是由于旱作物和人为重茬，以及育种单位很少涉及抗根结线虫病的育种选择，使得红麻根结线虫病害逐渐加重，降低了红麻的产量与纤维品质。红麻根结线虫病成为了影响我国红麻生产的主要病害，它在全国麻区普遍发生，尤其在温暖湿润的地区发病尤为严重。通过调查发现，根结线虫主要危害红麻根部，形成根结、根瘤，进而直接影响到红麻的产量与品质，成为国内外红麻生产推广的一大瓶颈。本技术方法通过对红麻根结线虫不同种群进行分离、纯化和鉴定，可以明确红麻根结线虫的形态和种类，从而为红麻根结线虫病的研究与防治提供理论依据和科学的技术方法。
发明内容
本发明的目的在于对生产上根结线虫发生动态变化提供一种简便、快速、准确的红麻根结线虫分离鉴定方法。对红麻根结线虫进行分离、纯化和鉴定并提供后续防控利用，提供鉴定和明确红麻根结线虫的形态、性质和种类，为红麻根结线虫的研究和防治提供科学依据，对红麻抗病虫新品种的选育和红麻生产水平的提高具有重要意义。本发明集红麻根结线虫分离、纯化和鉴定为一体，分离的病虫纯度高，可为红麻根结线虫的分离鉴定提供效率高、准确性强的技术方法。
本发明的目的通过如下技术方案实现：
一种红麻根结线虫分离鉴定方法，所述的黄红麻根结线虫分离鉴定方法具有分离的病虫纯度高、准确性强、效率高等优点，可用于红麻田间根结线虫病样本的及时分离鉴定和后续防控应用。技术流程由取样、培养纯化、会阴花纹形态观察、同工酶电泳分析和线粒体DNA扩增而成。
所述的一种红麻根结线虫分离鉴定方法按如下步骤进行：
（1）形态学鉴定：选取典型红麻根结线虫病病株，采用五点取样法采集寄主根系及周围5-20cm深的耕作层土壤500g。温室条件下将病根和病土接种到预先用灭菌土培植45d的盆栽番茄上进行纯化培养；在体视显微镜下，用解剖针从接种发病的番茄根中，挑取雌虫和卵块，将卵块放于盛有蒸馏水的培养皿中孵化4-5d即可得到根结线虫，用贝曼漏斗法分离土壤中的雄虫；获得虫体后，制作线虫玻片进行会阴花纹的形态鉴定；
（2）同工酶分析鉴定：运用常规聚丙烯酰胺凝胶电泳技术，对纯化的根结线虫样本进行酯酶和苹果酸脱氢酶电泳分析。挑取20头雌虫于1.5ml离心管中研磨提取酶样品，取20μL样品加入聚丙烯酰胺胶孔中，以爪哇根结线虫为对照进行电泳（60V1.5h;80V3.0h）。待电泳结束后，取出胶片，用蒸馏水清洗两次，加入MDH染色液于暗室内进行苹果酸脱氢酶染色20min，用蒸馏水漂洗后加入酯酶染色液(EST染色液)，于37℃水浴30min，完全显色后用蒸馏水漂洗3次，于固定液中固定2h后，用蒸馏水漂洗，拍照；
（3）分子生物学鉴定：分别以纯化后的根结线虫种群为样本，采用SDS法提取其DNA，以引物5-GGTCAATGTTCAGAAATTTGTGG-3和5-TACCTTTGACCAATCACGCT-3进行PCR，扩增其位于线粒体DNA中的COll基因和lrRNA基因。反应体系为：0.25μLTaq，2.5μL10×Buffer,2μLdNTPMixture,引物各1μL，1.0ng模板，加水至25μL。扩增条件为：94℃预变性5min，94℃变性1min，51℃退火1min，72℃延伸2min,35个循环，72℃延伸10min。电泳、回收纯化并测序，然后将序列进行比对分析鉴定。
本发明的显著优点：与其它病虫分离方法相比，本发明提供的方法具有以下显著优点：集红麻根结线虫分离、纯化与鉴定为一体，分离病虫的效率高、准确性强；弱化了红麻根结线虫中其他病虫的干扰，分离病虫的纯度高，操作程序十分简便；适用于红麻田间根结线虫病样本的及时分离，保持了病虫野生型特性。总之，本发明提供的方法具有操作简单、效率高、准确性强等科学技术优点，是红麻根结线虫准确分离、纯化和鉴定的有效方法，在红麻根结线虫的分离鉴定研究上具有重要的应用价值和科学意义。
附图说明
图1：根结线虫雌虫会阴花纹图。A南方根结线虫，B象耳豆根结线虫，C爪哇根结线虫，D花生根结线虫。
图2酯酶和苹果酸脱氢酶电泳分析。A图：15为对照爪哇根结线虫；1为象耳豆根结线虫；7和9为爪哇根结线虫，其余为南方根结线虫；B图：1为对照爪哇根结线虫；3和10为象耳豆根结线虫；8为爪哇根结线虫；其余为南方根结线虫。
图3mtDNA序列扩增。M为maker；1和2为爪哇根结线虫（1.7kb）；3为象耳豆根结线虫（0.7kb）；4和6为南方根结线虫（1.7kb）；5为花生根结线虫（1.1kb）。
具体实施方式
下面通过实施例对本发明作进一步的说明，其目的仅在于更好理解本发明的内容，而非限制本发明的保护范围：
实施例1：
（1）形态学鉴定：选取典型红麻根结线虫病病株，采用五点取样法采集寄主根系及周围5-20cm深的耕作层土壤约500g。温室条件下将病根和病土接种到预先用灭菌土培植45d的盆栽番茄上进行纯化培养；在体视显微镜下，用解剖针从接种发病的番茄根中，挑取雌虫和卵块，将卵块放于盛有蒸馏水的培养皿中孵化4-5d即可得到J2，用贝曼漏斗法分离土壤中的雄虫；获得虫体后，制作线虫玻片进行会阴花纹的形态观察。如图1所示：A，B，C，D分别为南方根结线虫，象耳豆根结线虫，爪哇根结线虫和花生根结线虫的会阴花纹图；
（2）同工酶分析鉴定：运用常规聚丙烯酞胺凝胶电泳技术，对纯化的根结线虫样本进行酯酶和苹果酸脱氢酶电泳分析。挑取20头雌虫于1.5ml离心管中研磨提取酶样品，取20μL样品加入聚丙烯酰胺胶孔中，以爪哇根结线虫为对照进行电泳（60V1.5h;80V3.0h）。待电泳结束后，取出胶片，用蒸馏水清洗两次，加入MDH染色液于暗室内进行苹果酸脱氢酶染色20min，用蒸馏水漂洗后加入EST染色液，于37℃水浴30min，完全显色后用蒸馏水漂洗3次，于固定液中固定2h后，用蒸馏水漂洗，拍照。如图2所示：A图是以15号爪哇根结线虫为对照；以MDH带型可以判定1号为象耳豆根结线虫；7号、9号的MDH和EST带型与对照组相似，判定为爪哇根结线虫；其余一致的判定为南方根结线虫。B图以1号爪哇根结线虫为对照；判定3号、10号为象耳豆根结线虫；8号为爪哇根结线虫；其余为南方根结线虫。
（3）分子生物学鉴定：分别以纯化后的根结线虫种群为样本，提取其DNA，以引物5-GGTCAATGTTCAGAAATTTGTGG-3和5-TACCTTTGACCAATCACGCT-3进行PCR，扩增其位于线粒体DNA中的COll基因和lrRNA基因。反应体系为：0.25μLTaq，2.5μL10×Buffer,2μLdNTPMixture,引物各1μL，1.0ng模板，加水至25μL。扩增条件为：94℃预变性5min，94℃变性1min，51℃退火1min，72℃延伸2min,35个循环，72℃延伸10min。电泳、回收纯化并测序，然后将序列进行比对分析鉴定。如图3所示：以1号爪哇根结线虫为对照，从条带大小可以看出2，4，6号与1号大小相似（1.7kb），初步判定为爪哇根结线虫；3号（0.7kb），5号（1.1kb）各为一组。经测序后，与NCBI数据库中已知序列比较后，判定1、2号为爪哇根结线虫；3号为象耳豆根结线虫；4、6号为南方根结线虫；5号为花生根结线虫。
以上所述仅为本发明的较佳实施例，凡依本发明申请专利范围所做的均等变化与修饰，皆应属本发明的涵盖范围。
SEQUENCELISTING
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<211>20
<212>DNA
<213>人工序列
<400>2
tacctttgaccaatcacgct20
Claims (4)
1.一种红麻根结线虫分离鉴定方法，其特征在于：所述方法包括形态学鉴定、同工酶分析鉴定、分子生物学鉴定。
2.根据权利要求1所述的一种红麻根结线虫分离鉴定方法，其特征在于：该方法按如下步骤进行：
（1）形态学鉴定：选取典型红麻根结线虫病病株，采用五点取样法采集寄主根系及周围5-20cm深的耕作层土壤500g，温室条件下将病根和病土接种到预先用灭菌土培植45d的盆栽番茄上进行纯化培养；在体视显微镜下，用解剖针从接种发病的番茄根中，挑取雌虫和卵块，将卵块放于盛有蒸馏水的培养皿中孵化4-5d即可得到根结线虫，用贝曼漏斗法分离土壤中的雄虫；获得虫体后，制作线虫玻片进行会阴花纹的形态鉴定；
（2）同工酶分析鉴定：运用聚丙烯酰胺凝胶电泳技术，对纯化的根结线虫样本进行酯酶和苹果酸脱氢酶电泳分析；
（3）分子生物学鉴定：以纯化后的不同根结线虫种群为样本，提取其DNA，以引物5-GGTCAATGTTCAGAAATTTGTGG-3和5-TACCTTTGACCAATCACGCT-3进行PCR，扩增其位于线粒体DNA中的COll基因和lrRNA基因，将序列进行比对分析鉴定。
3.根据权利要求2所述的一种红麻根结线虫分离鉴定方法，其特征在于：步骤（2）具体方法为：挑取20头雌虫于1.5ml离心管中研磨提取酶样品，取20μL样品加入聚丙烯酰胺胶孔中，以爪哇根结线虫样品为对照进行电泳，待电泳结束后，取出胶片，用蒸馏水清洗两次，加入MDH染色液于暗室内进行苹果酸脱氢酶染色20min，用蒸馏水漂洗后加入酯酶染色液，于37℃水浴30min，完全显色后用蒸馏水漂洗3次，于固定液中固定2h后，用蒸馏水漂洗，拍照。
4.根据权利要求2所述的一种红麻根结线虫分离鉴定方法，其特征在于：步骤（3）中PCR反应体系为：0.25μLTaq，2.5μL10×Buffer,2μLdNTPMixture,引物各1μL，1.0ng模板，加水至25μL，扩增条件为：94℃预变性5min，94℃变性1min，51℃退火1min，72℃延伸2min,35个循环，72℃延伸10min。
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| CN106048047A (zh) * | 2016-07-15 | 2016-10-26 | 聊城大学 | 利用变形梯度凝胶电泳鉴定海洋线虫的方法 |
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| CN1529164A (zh) * | 2003-10-21 | 2004-09-15 | 南京农业大学 | 松材线虫检测试剂盒及其检测方法 |
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| CN1529164A (zh) * | 2003-10-21 | 2004-09-15 | 南京农业大学 | 松材线虫检测试剂盒及其检测方法 |
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