CN105567726B - 一种高产脱氧雪腐镰刀菌烯醇的敲除突变体菌株的构建方法 - Google Patents
一种高产脱氧雪腐镰刀菌烯醇的敲除突变体菌株的构建方法 Download PDFInfo
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Abstract
一种高产脱氧雪腐镰刀菌烯醇的单敲除突变体菌株的构建方法，包括以下步骤：(1)利用split‑PCR方法，将禾谷镰刀菌脱氧雪腐镰刀菌烯醇合成过程中的负调控因子高亲和性磷酸二酯酶基因FgPDE2从禾谷镰刀菌PH‑1的基因组中敲除，获得单敲除突变体；FgPDE2的基因及上下游序列如SEQ ID NO：1所示；(2)采用PEG‑介导的原生质体转化到禾谷镰刀菌中，然后采用抗性筛选标记筛选敲除转化子；最后以禾谷镰刀菌的基因组为参照，利用5F和6R引物验证敲除转化子的目的基因被新霉素抗性基因所替代。本发明单敲除突变体菌株产毒量大幅提高；而双敲除突变体菌株产毒量更高，且该基因改造突变体丧失了对小麦穗的侵染能力，大大降低了双敲除突变体菌株可能的扩散造成的不良后果。
Description
技术领域
本发明一种高产脱氧雪腐镰刀菌烯醇的敲除突变体菌株的构建方法，具体涉及一种高产禾谷镰刀菌脱氧雪腐镰刀菌烯醇的敲除突变体菌株的构建方法。
背景技术
禾谷镰刀菌是引起小麦赤霉病(Fusarium head blight or scab)的病原真菌，不仅严重影响作物产量，而且可以产生多种对人畜有害的真菌毒素脱氧雪腐镰刀菌烯醇(DON)，使粮食或饲料的品质降低。脱氧雪腐镰刀菌烯醇(deoxynivalenol，DON)是一种倍半萜烯化合物，不易降解、稳定性高，在自然界中广泛存在，被认为是主要由镰刀菌在缺乏营养物质时，合成并作为主要天然毒素存在于赤霉病发病小麦中。DON毒素是污染率最高的真菌毒素之一，主要污染小麦、玉米等谷类作物，也污染粮食制品，如面包、饼干、麦制点心等；另外，在动物的奶、蛋中也有DON残留的报道。该毒素可抑制真核生物细胞蛋白质合成，破坏人和动物的免疫系统，可随食品、饲料进入食物链，严重威胁人畜健康。
我国是世界上受DON危害最严重的国家之一。因此，对谷物类制品中DON毒素的筛查及危害评估势在必行，对DON毒素形成机理的研究也同样需要大规模、大范围的开展起来。事实上，我国检验检疫部门一直十分重视这种毒素的检测监控研究，相关的科学研究也一直是赤霉病科研工作者的工作重点。然而，这些工作由于需要用到大量的DON毒素标准品，这些毒素标准品的价格又非常昂贵(1mg价格约为1000元人民币)，而且多是从国外出口，由此这些因素严重限制了相关工作的开展。由于真菌毒素涉及食品安全、环保、医疗等领域，需求广泛。因此，相关标准物制备已成为世界上各国研究的热点。
我国在真菌毒素标准物质制备方面的工作起步较晚，当前，合成真菌毒素标准品主要利用化学合成的方法，通过菌株培养、发酵、提取、纯化等制备工艺获得，虽然经历了不断的优化和工艺的完善，提取效率相对上世纪已有明显提高，但是真菌毒素得率仍相当有限，且纯化成本高昂。因此，真菌毒素提取制备工艺流程优化空间越来越小，研究人员已开始从筛选高产毒菌株入手寻求突破。菌株作为毒素制备的核心材料，其产毒能力决定了制备物质的生产效率、纯度和制备成本。目前，禾谷镰刀菌是DON主要的产毒菌种，我国研究人员主要通过对不同产地，不同来源的产毒真菌进行分离及毒素的测定，进而筛选适合工业生产的产毒菌株。然而，自然界中获取的禾谷镰刀菌产毒能力差别不大，很难筛选出高产毒量的菌株应用于规模生产。
现今，基因改造技术日益发展，而且在很多物种中已经有了基因改造获得良种的尝试。因此，开发一种高产脱氧雪腐镰刀菌烯醇的单敲除突变体菌株的构建方法，可大规模应用于工业生产，已经是菌种资源研究的当务之急。
发明内容
本发明所要解决的技术问题是，提供一种高产脱氧雪腐镰刀菌烯醇的单敲除突变体菌株的构建方法。
本发明解决其技术问题采用的技术方案是，一种高产脱氧雪腐镰刀菌烯醇的敲除突变体菌株的构建方法，包括以下步骤：
(1)构建基因敲除载体：利用split-PCR方法，将禾谷镰刀菌脱氧雪腐镰刀菌烯醇合成过程中的负调控因子高亲和性磷酸二酯酶基因FgPDE2从禾谷镰刀菌PH-1的基因组中敲除，获得单敲除突变体；FgPDE2的基因及上下游序列如SEQ ID NO：1所示；
(2)转化、筛选、鉴定：采用PEG-介导的原生质体转化到禾谷镰刀菌(优选禾谷镰刀菌野生型PH-1菌株)中，然后采用新霉素作为抗性筛选标记筛选敲除转化子；最后以禾谷镰刀菌(优选禾谷镰刀菌野生型PH-1菌株)的基因组为参照，利用5F和6R引物验证敲除转化子的目的基因被新霉素抗性基因所替代，即成。
进一步，步骤(1)中，将禾谷镰刀菌中的低亲和性磷酸二酯酶基因FgPDE1同时进行敲除，获得双敲除突变体，FgPDE1的基因及上下游序列如SEQ ID NO：2所示。
进一步，所述敲除FgPDE1所用引物序列PDE1-1F+PDE1-2R；PDE1-3F+PDE1-4R；YG/F+HY/R；HYG/F+HYG/R，如SEQ ID NO：3～10所示。
进一步，步骤(1)中，所述敲除FgPDE2所用引物序列PDE2-1F+PDE2-2R，PDE2-3F+PDE2-4R，EN/F+GE/R、GEN/F+GEN/R，如SEQ ID NO：11～18所示。
进一步，步骤(2)中，所述5F和6R引物为PDE2-5F，如SEQ ID NO：19所示，PDE2-6F，如SEQ ID NO：20所示；PDE1-5F，如SEQ ID NO：21所示，PDE1-6R，如SEQ ID NO：22所示。
本发明通过对禾谷镰刀菌脱氧雪腐镰刀菌烯醇DON合成过程中的负调控因子高亲和性磷酸二酯酶(phosphodiesterase)基因(FgPDE2，FGSG_06914)，利用split-PCR和PEG-介导的原生质体转化方法将该基因从禾谷镰刀菌PH-1的基因组中敲除，获得脱氧雪腐镰刀菌烯醇合成量大幅提高的单敲除突变体(基因改造菌株)；而将禾谷镰刀菌中的低亲和性磷酸二酯酶(phosphodiesterase)基因(FgPDE1，FGSG_06633)同时进行敲除，获得的双敲除突变体产毒量更高，且该基因改造突变体丧失了对小麦穗的侵染能力，大大降低了双敲除突变体菌株可能的扩散造成的不良后果。
实验证明，相较于野生型，Fgpde2单敲除突变体产毒量可提高67.5倍，而Fgpde1Fgpde2双敲除突变体则可提高257倍之多。
本发明之高产脱氧雪腐镰刀菌烯醇的敲除突变体菌株的构建方法可达到在工业生产中利用少量原材料菌丝大量合成脱氧雪腐镰刀菌烯醇的目的，是一个非常适合在实验室条件下用于制备DON的菌株。
附图说明
图1为本发明中基因改造菌株在液体培养条件下产毒量与野生型的对比结果。
图2为本发明中基因改造菌株在大米培养基中产毒量与野生型的对比结果。
图3为本发明中基因改造菌株对致病力的影响。
具体实施方式
以下结合实施例和附图对本发明作进一步说明。
从禾谷镰刀菌野生型菌株PH-1的基因组数据库(http://www.broadinstitute.org/annotation/genome/fusarium_graminearum/MultiHome.html)中搜索到高亲和性磷酸二酯酶(phosphodiesterase)基因(FgPDE2，FGSG_06914)F及上下游序列，FgPDE2的DNA序列如SEQ ID NO：1所示，高亮部分为基因的编码框。低亲和性磷酸二酯酶(phosphodiesterase)基因(FgPDE1，FGSG_06633)及上下游序列，FgPDE1的DNA序列如SEQID NO：2所示，高亮部分为基因的编码框。
设计用于扩增FgPDE2上下游同源序列以及新霉素抗性基因的引物，用于基因敲除，它们的序列如SEQ ID NO：3-10所示；设计用于扩增FgPDE1上下游同源序列以及潮霉素抗性基因的引物，用于基因敲除。它们的序列如SEQ ID NO：11-18所示；设计用于检测阳性敲除体的PCR引物，DNA序列如SEQ ID NO：19-22所示。
实施例1：高产脱氧雪腐镰刀菌烯醇的敲除突变体菌株的构建
(1)以禾谷镰刀菌野生型PH-1菌株的DNA为模板，利用引物PDE2-1F+PDE2-2R，PDE2-3F+PDE2-4R扩增基因起始密码子上游U和终止密码子下游序列D；以含新霉素抗性基因的质粒PFL2为模板，利用引物GEN/F+GE/R、FN/F+GEN/R扩增新霉素抗性基因序列G1和G2；其中，PCR扩增反应体系：在50微升PCR反应液中，含有50纳克模板DNA，10微升5X Pfu缓冲液，1微升10毫摩尔dNTP，0.5微升引物P1(10微摩尔)，0.5微升引物P2(10微摩尔)，0.4微升FastPfu DNA聚合酶(5个活性单位/微升)；PCR反应条件：94℃2分钟；94℃20秒，55℃20秒，72℃40秒，32个循环；及终延伸72℃5分钟；反应完成后，在1％琼脂糖凝胶上电泳，凝胶成像系统中紫外灯下照相并对条带进行切割回收回收PCR产物；通过Spilt PCR方法将U和G1片段，D和G2片段分别连接，通过异丙醇沉淀浓缩方法浓缩上述连接产物。
(2)收集CMC液体培养基中的PH-1孢子，在YEPD液体培养基中培养12个小时，收集0.5克左右菌丝，30℃酶解(2.5％崩溃酶+0.5％溶壁酶)3小时，将获得的原生质体稀释为4x107个/毫升，通过PEG介导的方法将步骤(1)得到回收产物转化进原生质体中，并用TB3液体培养基对原生质体进行复苏。
(3)待原生质体复苏生长16h后，转入固体培养基中，用300毫克/升终浓度的新霉素筛选，待转化子长出固体培养基后，挑取并转入新的培养基，提取转化子DNA并利用5F和6R引物检测对转化子进行进一步的确认。
在获得的基因改造菌株的基础上，用同样的方法进一步对FgPDF1进行敲除，采用潮霉素作为抗性筛选标记，获得双敲除突变体。
实施例2：评价基因改造突变体的毒素合成量
1、液体培养产毒法：获取野生型和突变体的孢子，加入到液体产毒培养基(液体产毒培养基(1升)：30克蔗糖、1克硝酸钠、1克磷酸二氢铵、0.5克七水硫酸镁、0.5克氯化钾、10毫克七水硫酸亚铁、0.03％植物凝胶以及200微升微量元素混合液(微量元素混合液(100毫升)：5g柠檬酸、5克七水硫酸锌、0.25克五水硫酸铜、50毫克一水硫酸锰、50毫克硼酸、50毫克二水钼酸钠。)，pH值用氢氧化钠调至6.5。)至终浓度104个孢子/毫升，黑暗静置培养7天，对培养基中的毒素进行测定。毒素测定利用Beacon公司生产的ELISA试剂盒，具体操作为：吸取培养基，离心去除菌丝，获得样品；向试剂盒中配备的反应杯中依次加入酶、样品、抗体，混合均匀，静置反应10分钟，弃反应液，用试剂盒中的清洗液，洗反应杯5次，加入底物反应5分钟，用终止液终止反应；在酶标仪中测OD450数值并与标样所作的标准曲线进行对比，算出具体数值。
实验结果见图1，由图1可知，相较于野生型，Fgpde2单敲除突变体产毒量提高了67.5倍，而Fgpde1 Fgpde2双敲除突变体则提高了257倍之多。
2、大米诱导产毒法：将6克大米置于50毫升三角瓶内，加入2毫升无菌水浸泡2小时后121℃灭菌1小时；接种PDA培养基中活化好的菌株，每个瓶子内接3个5毫米直径的菌饼，置于25度培养21天；冷冻干燥样品过夜，用液氮充分研磨以后称取4克样品用于DON测定，置于50毫升离心管并加入16毫升的乙腈/水(84∶16)，摇床上水平震荡1小时；过滤样品，取滤液3毫升过柱(3毫升塑料小柱，填充500毫克C18/中性氧化铝＝1∶3)，收集滤液转移至离心管，50度干燥过夜；加入100微升的TMS(TMSI/TMCS＝100/1)硅烷化试剂，震荡10分钟；加入800微升的色谱级异辛烷，轻微上下颠倒使充分混匀，加入超纯水800微升，轻微上下颠倒使样品变澄清并分层；静置10分钟，将700微升上清全部转移到GC上样瓶中，在气相色谱质谱联用仪上测定毒素含量。
实验结果见图2，由图2可知，相较于野生型，Fgpde2单敲除突变体产毒量提高了9.8倍，而Fgpde1 Fgpde2双敲除突变体则提高了12.4倍。
此外，申请人还对基因改造突变体的致病力进行鉴定与分析，见图3。由图3可知，Fgpde2单敲除突变体致病力跟野生型差别不大，然而双敲除突变体的致病力几乎完全丧失。
综上，虽然该菌株能够大量产毒，但是无法侵染小麦穗。因此，该菌株对农作物无害，大大降低了由于可能的扩散带来的不稳定风险，是一个非常适合在实验室条件下用于制备DON的菌株。
Claims (1)
1.一种高产脱氧雪腐镰刀菌烯醇的敲除突变体菌株的构建方法，其特征在于，包括以下步骤：
（1）构建基因敲除载体：利用split-PCR方法，将禾谷镰刀菌（Fusarium graminearum）脱氧雪腐镰刀菌烯醇合成过程中的负调控因子高亲和性磷酸二酯酶基因FgPDE2从禾谷镰刀菌PH-1的基因组中敲除，获得单敲除突变体；FgPDE2的基因及上下游序列如SEQ ID NO：1所示；
（2）转化、筛选、鉴定：采用PEG-介导的原生质体转化到禾谷镰刀菌中，然后采用抗性筛选标记筛选敲除转化子；最后以禾谷镰刀菌的基因组为参照，利用5F和6R引物验证敲除转化子的目的基因被新霉素抗性基因所替代，即成；
步骤（1）中，将禾谷镰刀菌中的低亲和性磷酸二酯酶基因FgPDE1同时进行敲除，获得双敲除突变体，FgPDE1的基因及上下游序列如SEQ ID NO：2所示；
所述敲除FgPDE2所用引物序列PDE1-1F+PDE1-2R；PDE1-3F+PDE1-4R；YG/F+HY/R；HYG/F+HYG/R，如SEQ ID NO：3～10所示；
步骤（1）中，所述敲除FgPDE1所用引物序列PDE2-1F+ PDE2-2R，PDE2-3F+ PDE2-4R，EN/F+GE/R、GEN/F+GEN/R，如SEQ ID NO：11～18所示；
步骤（2）中，所述禾谷镰刀菌为禾谷镰刀菌野生型PH-1菌株；所述5F和6R引物为PDE2-5F，如SEQ ID NO:19所示，PDE2-6F，如SEQ ID NO:20所示；PDE1-5F，如SEQ ID NO:21所示，PDE1-6R，如SEQ ID NO:22所示；所述抗性筛选标记对于FgPDE2进行敲除，选用新霉素；对FgPDE1进行敲除，选用潮霉素。
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| The Fusarium graminearum Genome Reveals a Link Between Localized Polymorphism and Pathogen Specialization;Christina A. Cuomo等;《SCIENCE》;20070907;第317卷;全文 * |
| 禾谷镰刀菌中FgPDE1基因的敲除及其功能的研究;常玉梅 侯占铭;《中国生物工程杂志》;20151231;全文，特别是摘要、第59页右栏至60页左栏、第60页1.1.2引物设计、第61页1.2方法 * |
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