CN105274130B - 一种利用基因操作提高球孢白僵菌分生孢子产量和毒力的方法 - Google Patents
一种利用基因操作提高球孢白僵菌分生孢子产量和毒力的方法 Download PDFInfo
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Abstract
本发明涉及一种提高球孢白僵菌分生孢子产量和毒力的方法，通过破坏球孢白僵菌Pmt1基因编码的MIR结构域，并保留完成的PMT结构域，获得球孢白僵菌突变菌株，所述球孢白僵菌突变菌株具有提高的分生孢子产量和毒力。
Description
技术领域
本发明属于基因工程领域，涉及利用基因工程方法改良真菌性状。
背景技术
昆虫病原真菌是一类重要的昆虫病原微生物，是自然界控制昆虫种群的一类重要自然控制因子(Clarkson et al.,1996,Trends Microbiol.,4:197–203；Feng et al.,1994,Biocontrol Sci.Technol.,4:3–34；Roberts et al.,2004,Adv.Appl.Microbiol.,54:1–70)。其中白僵菌(Beauveria)、绿僵菌(Metarhizium)、棒束孢(Isaria)、轮枝孢(Verticillium)等丝孢类真菌由于其显著的流行潜力和生产的便利性，被广泛用于农、林以及卫生害虫的生物防治。而且，与细菌和病毒类昆虫病原微生物通过消化道侵染方式不同，昆虫病原真菌是唯一直接穿透体壁侵染昆虫的微生物，对于控制将口针直接插入植物韧皮部吸食汁液的刺吸式口器害虫如蚜虫、叶蝉、飞虱等具有独到优势(Hajek et al.,1994,Annu.Rev.Entomol.,39:293–322；St.Leger et al.,1996,Proc.Natl.Acad.Sci.USA,93:6349–6354)。因此，开发和应用真菌杀虫剂受到国内外的广泛关注，目前全球注册登记的真菌制剂多达170余种，用于防治农、林以及卫生害虫(deFaria et al.,2007,Biol Control,43:237-256)。
分生孢子是球孢白僵菌等昆虫病原真菌侵染昆虫的主要单元，产孢量是生产真菌杀虫剂重要参数。目前，生产上提高产孢量的方法主要是通过培养基及培养条件优化，尚无利用基因操作提高菌株分生孢子产量的报道。毒力或侵染速度是真菌制剂应用效果的重要指标，增强病原真菌毒力是提升其应用效果的重要环节。
细胞壁糖蛋白与真菌发育分化、侵染致病、细胞粘着和在宿主细胞附着、以及逆境胁迫反应密切相关(Gaur and Klotz,1997,Infect.Immun.,65:5289–529；Buck andAndrewa,1999,Appl.Environ.Microbiol.,65:465–471；Frieman et al.,2002,Mol.Microbiol.,46:479–492；et al.,2009,Plant Cell,21:3397–3412；Wang and St.Leger,2007,Eukaryot.Cell,6:808–816)。其中，O-甘露糖基化蛋白修饰是O-糖基化蛋白修饰的一种重要类型，该类修饰从酵母到人类具有进化上的保守性，主要是将甘露糖基添加到修饰的靶标蛋白(Gentzsc and Tanner,1996,EMBO J.,15:5752–5759；Strahl-Bolsinger et al.,1999,J.Biol.Chem.,274:9068–9075；Willer et al.,2003,Curr.Opin.Struct.Biol.,13:621–630)。在真菌中，蛋白质的O-甘露糖化由蛋白质O-甘露糖化转移酶(protein-O-mannosyltransferases(PMTs))起始。PMTs是一类定位于内质网的膜蛋白，将第一个甘露糖基加到修饰蛋白的丝氨酸(Ser)或苏氨酸(Thr)残基上。模式真菌酿酒酵母(Saccharomyces cerevisiae)含有七个PMT蛋白(ScPmt1-7)，分属于三个不同的亚家族，即PMT1亚家族(包括Pmt1、Pmt5和Pmt7)、PMT2亚家族(包括Pmt2、Pmt3和Pmt6)和PMT4亚家族(包括Pmt4)(Gentzsch and Tanner,1996,EMBO J.,15:5752–5759；1997,Glycobiology,4:481-486)。白色念珠菌(Candida albicans)和汉逊酵母菌(Hansenulapolymorpha)均含有五个PMT蛋白，包括两个PMT1亚家族蛋白、两个PMT2亚家族蛋白和一个PMT4亚家族蛋白(Rouabhia et al.,2005,Infect.Immun.,73:4571–4580；Kim et al.,2013,Fungal Genet.Biol.,58-59:10–24)。但是，裂殖酵母(Schizosaccharomycespombe)、隐球酵母(Cryptococcus neoformans)和丝状真菌如构巢曲霉(Aspergillusnidulans)、烟曲霉(A.fumigatus)、灰葡萄孢(Botrytis cinerea)、玉米黑粉菌(Ustilagomaydis)、球孢白僵菌(B.bassiana)等，每个PMT亚家族仅有一个成员(Willer et al.,2005,Mol.Microbiol.,57:156–170；Willger et al.,2009,PLoS One,4:e6321；Kriangkripipat and Momany,2009,Eukaryot.Cell,8:1475–1485；Zhou et al.,2007,Eukaryot.Cell,6:2260–2268；González et al.,2013,PLoS One,8:e65924；et al.,2009,Plant Cell,21:3397–3412；Wang et al.,2014,Glycobiology,24:638-648)。在酿酒酵母(S.cerevisiae)和白色念珠菌(C.albicans)中，PMT1和PMT2亚家族成员形成具有酶活形式的异源二聚体，PMT4则形成具有酶活形式的同源二聚体(Gentzsch et al.,1995,FEBS Lett.,377:128–130；Girrbach et al.,2003,J.Biol.Chem.,278:12554–12562)。在裂殖酵母中也发现相似的二聚体活性形式(Willeret al.,2005,Mol.Microbiol.,57:156–170)。
O-甘露糖化的生理学功能主要是通过基因敲除等手段进行研究的。在模式真菌酿酒酵母(S.cerevisiae)中，单个Pmt基因对菌株并不是必需的，但所有的Pmt参与细胞壁完整性、细胞完整性、细胞极化和形态发生等生物学过程(Girrbach et al.,2000,J.Biol.Chem.,275:19288–19296)。在白色念珠菌(C.albicans)中，Pmt亚基对菌株生长、形态发生、杀真菌剂抗性和毒力具有重要影响，但不同的Pmt亚基的作用存在差异或作用专化性(Timpel et al.,1998,J.Biol.Chem.,273:20837-20846；Prill et al.,2005,Mol.Microbiol.,55:546–560；Timpel et al.,2000,J.Bacteriol.,182:3063–3071；Rouabhia et al.,2005,Infect.Immun.,73:4571–4580)。单个敲除裂殖酵母(S.pombe)Pmt1或Pmt4基因均降低了菌株O-甘露糖基化水平，导致菌株细胞壁和分隔形成异常，严重影响了细胞形态和细胞的分裂(Willer et al.,2005,Mol.Microbiol.,57:156–170)。隐球酵母(C.neoformans)Pmt1和Pmt4对菌株形态发生和毒力具有关键作用，双敲除Pmt1和Pmt4对菌株是致死的(Olson et al.,2006；Eukaryot.Cell,6:222–234；Willger et al.,2009；PLoS One,4:e6321)。构巢曲霉(A.nidulans)所有单个Pmt亚基对菌株存活并不是必需的，但单个Pmt亚基突变株在高温下的生长明显受抑制，菌体形态异常(Kriangkripipat andMomany,2009,Eukaryot.Cell,8:1475–1485)。烟曲霉(A.fumigates)中，敲除Pmt1亚基导致菌株产生温度敏感表型(Zhou et al.,2007,Eukaryot.Cell,6:2260–2268)，然而敲除Pmt4导致菌丝形态异常，分生孢子产生极少以及出现不正常及极性生长(Mouyna et al.,2010,Mol.Microbiol.,76:1205–1221)，而降低Pmt2表达水平则导致菌株生长延滞、细胞壁缺陷、极性生长异常以及降低分生孢子产生等(Fang et al.,2010,Glycobiology,20:542–552)。植物病原真菌玉米黑粉菌(U.maydis)中，缺失Pmt1或Pmt2对菌株生长和配接无明显影响，但Pmt4参与保持细胞壁结构完整性，而且对侵染结构附着胞形成和穿透宿主植物体壁是必需的(et al.,2009,Plant Cell,21:3397–3412)。灰葡萄孢(B.cinerea)Pmt亚基(特别是Pmt2亚基)对维持细胞壁稳定性、菌丝形态、产孢以及胞外基质的产生等起了关键作用。而且，每个Pmt亚基均介导毒力和对寄主植物的穿透(Gonzálezet al.,2013,PLoS ONE,8:e65924)。生防真菌里氏木霉(Trichoderma reesei)中，敲除一个与Pmt2功能相似的Pmt亚基基因PmtI，引起菌株对高渗胁迫和细胞壁合成抑制剂荧光增白剂(calcofluor white)敏感性增强，细胞隔膜形成缺陷(et al.,2008,ActaBiochim.Pol.55:251–259)。另外，在白色念珠菌(C.albicans)、裂殖酵母(S.pombe)、隐球酵母(C.neoformans)、烟曲霉(A.fumigates)和玉米黑粉菌(U.maydis)中，敲除Pmt2同源基因导致菌株致死，表明Pmt2是这些真菌存活所必需的(Pill et al.,2005,Mol.Microbiol.55:546–560；Willer et al.,2005,Mol.Microbiol.57:156–170；Willgeret al.,2009,PLoS One,4:e6321；Fang et al.,2010,Glycobiology,20:542–552；et al.,2009,Plant Cell,21:3397–3412)。然而，在其它一些真菌包括A.nidulans、B.cinerea和T.reesei中，Pmt2并不是存活所必需的(Kriangkripipat andMomany,2009,Eukaryot.Cell,8:1475–1485；González et al.,2013,PLoS One 8:e65924；et al.,2008,Acta Biochim.Pol.,55:251–259)。由此表明，蛋白质O-甘露糖基化活性在真菌发育和生长过程中扮演了重要角色，但在不同的真菌中明显存在功能上的分化。
所有真核细胞Pmt1均含有一个PMT(dolichyl-phosphate-mannose-proteinmannosyltransferase)结构域和三个MIR(mannosyl transferase,inositoltriphosphate-and ryanodine receptor)结构域(Ponting,2000)。在酿酒酵母中， MIR结构域在体内或体外对Pmt1酶活(甘露糖基转移酶活)均是必需的(Girrbach et al.,2000,J.Biol.Chem.,275:19288–19296)，但删除MIR结构域不影响Pmt1对一个光敏肽底物的结合(Lommel et al.,2011,J.Biol.Chem.,286:39768–39775)。然而，在丝状真菌中，MIR结构域对Pmt1酶活的贡献以及功能，尚不明确。昆虫病原真菌球孢白僵菌中存在三个Pmt亚基Pmt1、Pmt2和Pmt4。利用基因敲除(Pmt1和Pmt4)和反义抑制(Pmt2)方法研究发现，这些Pmt亚基正调节球孢白僵菌生长、分生孢子产生、细胞壁完整性、毒力以及细胞对营养、细胞壁合成抑制剂、高渗、氧化、高温以及紫外辐射的敏感性。即敲除Pmt1或Pmt4和抑制Pmt2表达，均不同程度的导致菌株生长受阻、分生孢子产生减少、对细胞壁合成抑制剂、高渗、氧化、高温以及紫外辐射的敏感性增强(Wang et al.,2014,Glycobiology,24:638-648)。本研究以Pmt1为研究对象，采用基因敲除法破坏Pmt1的MIR结构域，保留完整的PMT结构域，体内、体外研究了删除MIR结构域对Pmt1酶活性及其功能的影响。研究结果发现，删除MIR结构域对Pmt1体内、体外的O-甘露糖基转移酶活性无明显影响，但限制了Pmt1介导分生孢子产生和毒力。即与敲出完整的Pmt1的结果相反，删除MIR结构域显著提升了分生孢子产量和毒力，并改变了分生孢子产生相关基因的转录或表达。
发明内容
本发明的一个目的是提供一种提高球孢白僵菌分生孢子产量和毒力的方法。
本发明的另一个目的是提供一种通过基因工程方法构建的球孢白僵菌突变菌株。
本发明的再一个目的是提供一种真菌杀虫剂。
本发明也提供Pmt1基因在制备真菌杀虫剂中的用途。
根据本发明的一方面，一种提高球孢白僵菌分生孢子产量和毒力的方法，通过破坏球孢白僵菌Pmt1基因编码的MIR结构域，并保留完成的PMT结构域，获得球孢白僵菌突变菌株，所述球孢白僵菌突变菌株具有提高的分生孢子产量和毒力。
本发明优选的方法是通过同源重组破坏球孢白僵菌Pmt1基因编码区的部分序列，保留包括起始密码ATG在内的完整的PMT活性结构域，获得Pmt1部分序列破坏的突变体，所述突变体中PMT结构域编码区正常转录。
具体地，破坏球孢白僵菌Pmt1基因编码的MIR结构域优选地是以除草剂抗性基因bar置换Pmt1基因的MIR结构域部分编码区，构建同源重组表达载体。
根据本发明的另一方面，制备球孢白僵菌突变菌株的方法，包括下述步骤：
1)以基因工程方法破坏球孢白僵菌Pmt1基因的部分编码序列，获得重组表达载体；
2)将步骤1)获得的重组表达载体转入球孢白僵菌野生型菌株，破坏球孢白僵菌Pmt1基因中PMT活性结构域下游的MIR结构编码区，获得球孢白僵菌突变菌株，所述球孢白僵菌突变菌株具有提高的分生孢子产量和毒力。
本发明优选地用于制备球孢白僵菌突变菌株的重组载体为同源重组表达载体，具有图2B所述载体结构。
本发明利用同源重组破坏Pmt1基因的部分编码区，获得的突变菌株中PMT活性结构域编码区可正常转录，并且提高了球孢白僵菌分生孢子产量和毒力。可用于制备真菌杀虫剂。
附图说明
图1：pK2-gusT载体图谱。其中，PtrpC和TtrpC为来源于构巢曲霉色氨酸合成酶基因启动子和终止子；GUS为β-葡萄糖苷酸酶基因。
图2：同源重组破坏Pmt1基因的MIR编码区、载体图谱及基因破坏突变体筛选、Pmt1回复互补策略、载体图谱及回复互补转化子筛选。其中，A是同源重组破坏Pmt1基因的MIR部分编码区策略；小箭头及字母和数字表示引物及引物位置；“ATG”和“TAA”分别表示翻译起始密码和终止密码子；B是同源重组表达载体pΔMIR图谱；C是Pmt1回复互补策略；小箭头及字母和数字表示引物及引物位置；大箭头表示基因从5'-端到3'-端的方向；D是Pmt1回复互补载体pCB-Pmt1载体图谱；E是PCR筛选转化子；其中，M:Ladder指DNA Marker(MBIFermentas)；WT:野生菌株；Control:pΔMIR随机插入转化子；ΔMIR：Pmt1基因的MIR编码区破坏突变体；ΔMIR::Pmt1：MIR回复互补转化子；F是以actin基因为参比基因，RT-PCR扩增验证基因破坏部分的转录；WT:野生菌株；Control:pΔMIR随机插入转化子；ΔMIR：MIR编码区破坏突变体；G中左图是以bar基因片段制备探针与ΔMIR(MIR编码区破坏突变体)和Control(pΔMIR随机插入转化子)的Southern杂交结果，右图是以 sur基因片段制备的探针与ΔMIR::Pmt1(MIR回复互补转化子)的Southern杂交结果；H是PCR和RT-PCR扩增验证PMT活性结构域编码区；DNA是指以PCR扩增验证结果；mRNA是指RT-PCR扩增验证结果。
图3：Pmt1的蛋白特征、预测的穿膜结构及O-甘露糖基转移酶活性；其中，
A是Pmt1的蛋白质组成及结构域示意图，PMT为多萜基磷酸甘露糖蛋白甘露糖基转移酶(Dolichyl-phosphate-mannose-protein mannosyltransferase)结构域，位于第52-298氨基酸；MIR为利阿诺定和肌醇三羟甲基氨基甲烷磷酸受体及蛋白甘露糖基转移酶结构域(Domain in ryanodine and inositol trisphosphate receptors and protein O-mannosyltransferases)，分别位于第329-379、404-463和478-529氨基酸；B是预测的Pmt1在内质网膜的穿膜结构；cytosol：细胞质；ER lumen：内质网膜内腔；C是破坏Pmt1部分序列后残存序列编码蛋白的组成及结构域示意图；bar：草甘膦除草剂抗性基因编码蛋白(磷化麦黄酮乙酰转移酶)；D是破坏Pmt1部分序列后残存序列编码蛋白在内质网膜的穿膜结构预测；E是利用甲醇诱导型毕赤酵母表达的Pmt1全蛋白(902个氨基酸)和PMT domain(310个氨基酸)经纯化后的SDS-PAGE电泳检测结果。结果显示，纯化的蛋白为电泳纯度；F是纯化后Pmt1全蛋白和PMT domain的O-甘露糖基转移酶活性结果；G图是ΔMIR突变体和野生菌株的O-甘露糖基转移酶活性检测结果。
图4：破坏Pmt1的MIR结构域编码区影响菌落生长
菌株在PDA、Czapek agar以及不同氮源和碳源培养基于26℃生长15天的菌落形态。其中，WT：球孢白僵菌野生型菌株；ΔMIR：破坏Pmt1的MIR结构域编码区的突变体；ΔMIR::Pmt1：MIR回复互补转化子。
图5：破坏Pmt1的MIR结构域编码区影响分生孢子产量、孢子大小、芽生孢子产量及生物量积累。其中，A是菌株在Czapek agar和1/4SDAY培养基上的分生孢子产量；B是分生孢子宽度和长度；C是液体培养3天和4天的菌体生物量；D是液体培养条件下3天和4天产生芽生孢子数量。
图6：破坏Pmt1的MIR结构域编码区影响产孢相关基因的表达。在固体培养基上培养4、5、10和20天，在野生菌株和破坏Pmt1的MIR结构域编码区突变体中产孢相关基因的表达模式。
图7：破坏Pmt1的MIR结构域编码区改变了分生孢子表面碳源表位、疏水性及在固体表面的附着性。其中，A是利用分生孢子与荧光标记的凝集素反应强度；ConA：识别ɑ-葡糖糖和甘露糖以及ɑ-N-乙酰氨基葡萄糖胺(GlcNAc)残基的凝集素；GNL：特异识别甘露糖残基的凝集素；WGA：结合β-GlcNAc和唾液酸残基的凝集素；B是分生孢子疏水性，即分生孢子在水相中分布的比例；C是分生孢子在疏水性、亲水性以及弱极性固体表面的附着性；hydrophobic：疏水性固体表面；weakly polar：弱极性固体表面；hydrophilic：亲水性固体表面。
图8：破坏Pmt1的MIR结构域编码区提高球孢白僵菌毒力。其中，A为通过“经典”的体壁接种方式接种浓度为2×107/ml的分生孢子后试虫的存活率趋势；B为微量注射2μl浓度为1×107/ml的分生孢子到试虫血腔后，试虫的存活率趋势；C为体壁接种后虫菌体(在虫体内繁殖的菌体)在不同时间的繁殖量；D为注射接种后虫菌体繁殖量；试虫为3龄大蜡螟幼虫；试验重复3次。其中，WT:野生菌株；ΔMIR：破坏Pmt1的MIR结构域编码区的突变体；ΔMIR::Pmt1：MIR回复互补转化子；control：用0.05％Tween-80做相同处理的试虫。
图9：Pmt1的MIR结构域影响虫菌体分化。A是利用“经典”体壁接种(2×107conidiaml-1)96h的虫菌体形态；B是微量注射接种(2μl 107conidia ml-1)54h的虫菌体形态；箭头所指为虫菌体；试虫为3龄大蜡螟幼虫；试验重复3次。其中，WT:野生菌株；ΔMIR：破坏Pmt1的MIR结构域编码区的突变体；Bar＝50μm.
具体实施方式
通过以下实施例可以进一步理解本发明的优点和特点，不应该理解为是对本发明范围的限制。
以下实施例中所用仪器和试剂，除特殊说明以外均为普通市售。
【实施例1】
1.利用同源重组破坏球孢白僵菌Pmt1的MIR结构域编码区
构建球孢白僵菌Pmt1的MIR结构域编码区同源重组表达载体策略如下：利用bar基因的表达元件(SEQ ID NO.42)置换Pmt1的MIR结构域部分编码区。即在bar表达元件两端衔接Pmt1的侧翼序列构成同源重组表达载体，经遗传转化导入球孢白僵菌，通过载体两侧衔接的Pmt1侧翼序列与球孢白僵菌基因组中的同源序列进行双交换，置换Pmt1的MIR结构域的部分编码区(349bp)，达到破坏目的结构域编码区的目的(如图2A所示) 。
具体操作如下：
根据Pmt1序列和草甘膦抗性基因bar表达元件序列设计引物L1、L2、R1、R2、B1和B2，分别扩增Pmt15'端和3'端序列和bar基因表达元件序列，采用重叠PCR技术融合上述元件，克隆到利用gus基因置换潮霉素抗性基因 hyg的pPk2(Fungal Genetics StockCenter；McCluskey,2003,Adv.Appl.Microbiol.,52:245–262)载体上(PK2-gusT，如图1所示)。即以球孢白僵菌基因组DNA为模板，利用引物L1/L2和R1/R2扩增Pmt15'端(984bp)和3'端(991bp)序列，在L1引物5'-端引入BamHI位点，在L2引物5'-端引入部分bar基因元件部分上游序列，在R1引物5'端引入bar元件部分下游序列，在R2引物5'端引入SpeI位点。
然后分别以Pmt1基因5'端序列(L)、bar元件和Pmt1基因3'端序列(R)互为引物扩增，融合L::bar::R元件。扩增体系如下：10×LA Taq PCR Buffer(含Mg2+)2.5μl，2.5mmol·L-1dNTP 2μl，L、bar和R片段各200ng，LA Taq 0.7U(热启动时加入)，用水补足至25μl体系。扩增程序为：94℃5min；94℃30s，56℃45s，72℃3min，20个循环；72℃延伸10min。然后以1μl扩增产物为模板，利用引物L1和R2扩增L::bar::R融合片段。扩增体系如下：10×LA TaqPCR Buffer(含Mg2+)2.5μl，2.5mmol·L-1dNTP 2μl，5μmol·L-1引物L1和R2各1μl，上述扩增产物1μl，LA Taq 0.7U(热启动时加入)，用水补足至25μl体系。扩增程序如下：94℃5min；94℃30s，55℃30s，72℃2min，35个循环；72℃延伸10min。扩增产物在1.0％(w/v)的琼脂糖凝胶电泳，回收扩增片段测序验证。然后用BamHI和SpeI酶切融合片段，连接于用相同酶切后的PK2-gusT，形成同源重组表达载体pΔMIR(图2B)。
将表达载体pΔMIR转入根癌农杆菌AGL-1，转化参照Fang et al.方法(Fang etal.,2004,J.Invertebr.Pathol.,85:18-24)。然后利用根癌农杆菌介导法转化球孢白僵菌分生孢子(Fang et al.,2004,J.Invertebr.Pathol.,85:18-24)。在含60ppm除草剂草甘膦(glufosinate)的察氏培养基(Czapek-Dox agar)平板上筛选抗性菌落。提取抗性转化子DNA，利用引物S1/S2扩增筛选基因破坏突变体，若随机插入转化子则扩增出部分同源重组元件(1057bp)和野生型基因部分片段(407bp)两条带，而基因破坏转化子只扩增出部分同源重组元件片段(1057bp)一条带，而野生菌株则只存在野生型带(407bp)。根据该方案，筛选到基因破坏突变体ΔMIR(图2E)。
提取基因破坏突变体基因组DNA，利用EcoRI酶切后于1.0％的琼脂糖凝胶电泳分离，通过高盐转膜法转移到尼龙膜(HybondTM-N nylon membrane，Amersham Biosciences,USA)，然后用引物B3/B4扩增bar基因片段(411bp)(SEQ ID NO.40)为探针，用地高辛标记探针进行杂交，杂交为单拷贝插入的转化子为进一步确认的基因破坏转化子(图2G)，以随机插入整合转化子DNA为对照(Control，图2G)。具体操作参照地高辛标记试剂盒(DIG-HighPrime DNA labeling and detection starter kit I，Roche)。
接种野生菌株(WT)和破坏Pmt1部分编码区突变体(ΔMIR)于添加0.5％ (w/v)蛋白胨的Czapek-Dox broth，26℃培养12h，提取RNA。RNA提取按照EASYspin植物RNA快速提取试剂盒(北京艾德生物科技有限公司)方法进行。紫外分光光度计定量RNA。取2μg RNA利用oligo(dT)引物反转录成cDNA第一链，反转录参照试剂盒(oligo(dT)-primed cDNAsynthesis kit(MBI Fermentas)说明书。将合成的cDNA第一链稀释成10ng/μl，以actin(Gen-Bank ID:HQ232398)为参比基因进行RT-PCR检测MIR编码区和5'端包含起始密码子“ATG”在内的PMT结构域编码区在ΔMIR中的转录。扩增体系如下：10×EX Taq PCR Buffer(含Mg2+)2.5μl，2.5mmol·L-1dNTP 2μl，5μmol·L-1引物各1μl，稀释的cDNA一链模板1μl，EXTaq 0.7U，用水补足至25μl体系。扩增程序如下：94℃5min；94℃30s，55℃30s，72℃2min，25个循环；72℃延伸10min。扩增产物在1.0％(w/v)的琼脂糖凝胶电泳检测Pmt1的转录情况。扩增actin和MIR结构域编码区转录本的引物对分别为actin-F/actin-R和3'RT1/3'RT2，扩增5'端包含起始密码子“ATG”在内的PMT结构域转录本的引物为5'RT1/5'RT2，并利用DNA为模板扩增5'端包含起始密码子“ATG”在内的PMT结构域序列的基因组DNA为对照(如图2H)。引物序列附后。结果表明，MIR结构域在突变体中不能转录(如图2F所示)，而5'端包含起始密码子“ATG”在内的PMT结构域序列可正常转录(如图2H所示)。
L1：5'-CGGGATCCCGTACAAGTCGGATGGTGT-3'(SEQ ID NO.1)
L2：5'-TCAATGTCATCTTCTGTCGACTCATGAAGCCGTCGCCGTCA-3'(SEQ ID NO.2)
R1：5'-TGCCCGTCACCGAGATCTAATAGATGTTCGTCCTCCCGTCTCT-3'(SEQ ID NO.3)
R2：5'-GGACTAGTCCTCGTACTTGTCGTAGAAT-3'(SEQ ID NO.4)
B1：5'-GTCGACAGAAGATGACATTGA-3'(SEQ ID NO.5)
B2：5'-CTATTAGATCTCGGTGACGGGCA-3'(SEQ ID NO.6)
B3：5'-ACCTTCTTAAGTTCGCCCTT-3'(SEQ ID NO.7)
B4：5'-TGTAGAGCGTGGAGCCCAGT-3'(SEQ ID NO.8)
actin-F：5'-TTGGTGCGAAACTTCAGCGTCTAGTC-3'(SEQ ID NO.9)
actin-R：5'-TCCAGCAAATGTGGATCTCCAAGCAG-3'(SEQ ID NO.10)
3'RT1：5'-AGCGTCAGCTCTTCCTCCA-3'(SEQ ID NO.11)
3'RT2：5'-TATTCCTCGTACTTGTCAT-3'(SEQ ID NO.12)
5'RT1：5'-ATGGCCGGACCAAGCTCGGC-3'(SEQ ID NO.13)
5'RT2：5'-TCATGAAGCCGTCGCCGTCA-3'(SEQ ID NO.14)
2.回复互补MIR破坏突变体
以球孢白僵菌基因组DNA为模板，利用引物pRC1/pRC2(分别在pRC1和pRC2的5'-端引入PstI和XbaI酶切位点，引物序列附后)扩增Pmt1基因(包括启动子序列、编码区和终止子序列，共5320bp)。用PstI和XbaI酶切扩增片段，克隆到用相同酶切的载体pCB1536(Fungal Genetics Stock Center)上，形成载体pCB-Pmt1(如图2C和图2D所示)，该载体携带除草剂氯嘧磺隆(chlorimuronethyl)抗性基因sur。利用高频电击介导法将载体转入MIR结构域编码区破坏突变体。白僵菌转化方法参照文献(Jin et al.,2008,BiotechnolLett,30:1379–1383)，转化后在含有10μg ml-1chlorimuronethyl的察氏培养基(Czapek-Dox agar)上筛选抗性菌落，提取DNA，利用引物S1/S2验证转化子。若将Pmt1成功导入MIR破坏突变体，则扩增出两条带，一条为含有bar基因的部分同源重组元件(1057bp)，另一条为野生型基因部分片段(407bp)(如图2E所示)。利用引物3'RT1/3'RT1和RT-PCR验证转化子中MIR结构域编码区是否回复正常转录。RT-PCR操作如下：接种分生孢子于1/4SDAY液体培养基中培养48小时，收集菌丝提取RNA，反转录后合成cDNA第一链。以野生菌株为对照进行RT-PCR表达分析。RNA提取按照EASYspin植物RNA快速提取试剂盒(北京艾德生物科技有限公司)方法进行。紫外分光光度计定量RNA。取2μg RNA利用oligo(dT)引物反转录成cDNA第一链，反转录采照试剂盒(oligo(dT)-primed cDNA synthesis kit(MBI Fermentas)说明书。将合成的cDNA第一链稀释成10ng/μl，以actin(Gen-Bank ID:HQ232398)为参比基因进行RT-PCR和引物3'RT1/3'RT1检测MIR结构域编码区在转化子中的转录。扩增体系如下：10×EX Taq PCR Buffer(含Mg2+)2.5μl，2.5mmol·L-1dNTP 2μl，5μmol·L-1引物各1μl，稀释的cDNA一链模板1μl，EX Taq 0.7U，用水补足至25μl体系。扩增程序如下：94℃ 5min；94℃30s，55℃ 30s，72℃ 2min，25个循环；72℃延伸10min。扩增产物在1.0％(w/v)的琼脂糖凝胶电泳检测MIR结构域编码区的转录情况。扩增actin和Pmt1的引物对分别为actin-F/actin-R和3'RT1/3'RT1，引物序列附后。结果表明，MIR结构域在回复互补转化子中能够正常转录(如图2F所示)。
提取回复互补转化子基因组DNA，利用EcoRI酶切后于1.0％的琼脂糖凝胶电泳分离，通过高盐转膜法转移到尼龙膜(HybondTM-N nylon membrane，Amersham Biosciences,USA)，然后利用引物Sur1/Sur2扩增sur片段(380bp)(SEQ ID NO.41)为探针，用地高辛标记进行杂交，杂交结果为单拷贝插入的转化子(图2G)。具体操作参照地高辛标记试剂盒(DIG-High Prime DNA labeling and detection starter kit I，Roche)。
pRC1：5'-AAACTGCAGATTGGTCGATTATGTGTTGG-3'(SEQ ID NO.15)
pRC2：5'-GCTCTAGAGAGCAAGGGTTCTAACATGA-3'(SEQ ID NO.16)
Sur1：5'-AGTGTGCTGAGGAGGGCTAT-3'(SEQ ID NO.17)
Sur2：5'-ACACGGTCATCGAAGCGGCCA-3'(SEQ ID NO.18)
【实施例2】
1.Pmt1蛋白的结构域分析
Pmt1蛋白结构域分析采用NCBI网站(http://www.ncbi.nlm.nih.gov/)中的blastp程序进行。利用TMAP和TMPRED软件和酵母Pmts的预测方法和分析数据预测Pmt1蛋白穿膜结构(Strahl-Bolsinger and Scheinost,1999，J.Biol.Chem.,274:9068–9075)。
结构域分析表明，Pmt1由902个氨基酸组成，包含一个PMT(Dolichyl-phosphate-mannose-protein mannosyltransferase)结构域和三个MIR(Domain in ryanodine andinositol trisphosphate receptors and protein O-mannosyltransferases)结构域。其中PMT结构域位于氨基端第52至298氨基酸，而三个MIR结构域分别位于第329-379氨基酸、第404-463氨基酸和第478-529氨基酸之间(图3A)。四个假定的N-糖基化位点(NX(S/T))分别位于第380-382氨基酸、第393-395氨基酸、第592-594氨基酸和第786-788氨基酸。一个保守的DE基序位于第73–74氨基酸(图3B)，该基序是受体结合和(或)催化位点的组成部分，在原核和真核细胞中高度保守，位于穿膜结构的第一个颈环(loop 1)区域中(Loibl andStrahl,2013，Biochimica et Biophysica Acta 1833:2438–2446)。
利用TMAP和TMPRED软件预测结果，结合分析DE基序和N-糖基化位点应该暴露于内质网腔，推测Pmt1包含七个穿膜结构，其中氨基端和羧基端分别位于细胞质和内质网腔(图3B)。而破坏Pmt1的MIR结构域编码区序列，通过PCR和RT-PCR验证表明，突变体保留了包括起始密码子“ATG”在内的完整的PMT结构域(图2H)。突变体中残留氨基酸的组成分析如图3C，预测的残留氨基酸保留了前面五个穿膜结构和保守的DE基序(图3D)。
2.酵母表达Pmt1和PMT结构域与蛋白纯化
利用反转录PCR(reverse transcription PCR，RT-PCR)扩增球孢白僵菌完整的Pmt1编码区(2709bp)和PMT结构域编码区(930bp)cDNA。扩增完整Pmt1和PMT结构域cDNA序列的引物分别为Com1/Com2和Pm1/Pm2，分别在完整Pmt1和PMT结构域cDNA的3'端引入NotI和XhoI 内切酶酶切位点。将RT-PCT产物克隆到PUCm-T载体(上海生工)。然后，将GST标签通过引物引入cDNA序列的5'端，即以克隆在PUCm-T的cDNA为模板，分别以引物GST1/Com2和GST1/Pm2扩增，引入GST标签，并在标签的5'端引入SnaBI酶切位点。利用相应的内切酶(序列中引入的内切酶位点)酶切PCR产物，然后克隆到相同内切酶酶切的载体pPIC9K(Invitrogen,Carlsbad,CA,USA)上。测序确认准确无误后，然后利用电转化法将质粒导入甲醇诱导型毕赤酵母(Pichia pastoris)GS115菌株(Invitrogen)，于选择培养基(1.34％[w/v]YNB[yeast nitrogen base][Gifco,KS,USA]、40mg mL-1biotin[Invitrogen],2％[w/v]glucose,1.5％[w/v]agar)上培养。将生长的重组菌株在YPD培养基(1％[w/v]yeastextract,2％[w/v]peptone,2％[w/v]dextrose,1.5％[w/v]agar)上连续继代培养三次。然后将转化子转接到含有1.5mg mL-1geneticin(G418)(TaKaRa,Dalian,China)的YPD培养基上筛选多拷贝转化子。筛选的转化子进一步利用引物P5'和P3'扩增验证。引物序列附后。
Com1：5'-ATGGCCGGACCAAGCTCGGC-3'(SEQ ID NO.19)
Com2：5'-ATAAGATTGCGGCCGCTTACAACTCGTCCTTGCGAG-3'(SEQ ID NO.20)
Pm1：5'-ATGGCCGGACCAAGCTCGGC-3'(SEQ ID NO.21)
Pm2：5'-CCGCTCGAGTTACATGAAGCCGTCGCCGT-3'(SEQ ID NO.22)
GST1：5'-CCTACGTAATGTCCCCTATACTAGGTTA-3'(SEQ ID NO.23)
P5'：5'-GACTGGTTCCAATTGACAAGC-3'(SEQ ID NO.24)
P3'：5'-GGCAAATGGCATTCTGACATCCT-3'(SEQ ID NO.25)
接种筛选的重组酵母菌株于25mL BMGY培养基(2％[w/v]peptone,1％[w/v]yeastextract,100nmol L-1potassium phosphate[pH 6.0],biotin mL-140mg and 1％[v/v]glycerol)，在28℃、160rpm条件下摇瓶培养48h(OD600≈2.0)，放大接种于500mL用0.5％(v/v)甲醇(methanol)置换1％(v/v)甘油(glycerol)的BMGY培养诱导目标蛋白表达。诱导培养7天后，6000g离心收集上清液，然后用0.45-mm的滤膜除去酵母细胞。用80％(NH4)2SO4于4℃过夜沉淀滤液中蛋白质，于12000g、4℃离心5min收集沉淀的蛋白质。用5ml 0.2M PBS(pH7.5)溶解沉淀蛋白质，用脱盐柱HiprePTM 26/10Desalting column inPrime Pus(GE Healthcare Life Sciences)脱盐。脱盐后的样品用Magene GSTTM proteinpurification system(Promega)纯化目标蛋白。纯化蛋白用SDS-PAGE(12％(w/v)聚丙烯酰胺分离胶)电泳检测目标蛋白纯度。电泳结果显示，纯化的蛋白达到电泳纯(图3E)。以牛血清白蛋白(bovine serum albumi，BSA)为标准，用BCA(Bicinchoninic acid)proteinassay kits(Generay Biotech Co.,Ltd,Shanghai,China)测定纯化蛋白质浓度。
3.球孢白僵菌膜蛋白提取
球孢白僵菌膜蛋白提取参照Wang等方法(2014，Glycobiology 24:638-648)。接种分生孢子到萨氏培养基(Sabouraud dextrose broth)至终浓度为107孢子/mL，于25℃、160rpm摇瓶培养2天。过滤收集菌丝，用无菌水洗涤3次，然后用液氮速冻研磨成粉末，将粉末悬于10mL含有0.3mM MgCl2、1mM phenylmethylsulfonyl fluoride(PMSF)和10mM巯基乙醇(mercaptoethanol)的50mM Tris–HCl(pH 7.5)缓冲液中混匀，于12,000×g、4℃离心10min去除菌丝等碎片。取上清液于70,000×g、4℃离心1h，然后用含有0.3mM MgCl2、1mMPMSF和33％甘油(glycerol)的50mM Tris–HCl(pH 7.5)缓冲液溶解沉淀，即膜蛋白。以牛血清白蛋白(bovine serum albumi，BSA)为标准，用BCA(Bicinchoninic acid)proteinassay kits(Generay Biotech Co.,Ltd,Shanghai,China)测定提取膜蛋白质的浓度。
4.MIR结构域对Pmt1酶活的影响
O-甘露糖基转移酶活性测定参照Weston等方法(1993,Eur.J.Biochem.215：845-849)并略有改进。即50μL反应体系包括0.02μCi of Dol-P-[3H]mannose(AmericanRadiolabeled Chemicals)、3.5mM Ac-YATAV-NH2(acceptor peptide)、5μL of 20％Triton X-100、10μL of 0.2M HEPES(pH 7.5)和25μg提取的球孢白僵菌膜蛋白或10μg酵母GS115表达后纯化的蛋白。反应体系于25℃反应30min，然后加入1mL 60％氯仿(chloroform)和40％甲醇(methanol)混合物终止反应。反应混合物于13000g离心5min分离油相和水相，取沉淀蛋白和下层油相于液体闪烁仪测定总吸收的放射性活性。PMT酶活性定义为每克蛋白每小时转化的DPM(每分钟每分钟衰变数)。每次测定设三个样本，重复三次。
利用酵母表达并纯化的完整Pmt1(902个氨基酸)和PMT domain(310个氨基酸)蛋白进行体外酶活测定。结果表明，Pmt1和PMT domain从供体Dol-P-[3H]mannose转移[3H]mannose到合成的受体肽Ac-YATAV-NH2的活性分别为2.32(±0.14)×106dpm mg-1h-1和2.34(±0.08)×106dpm mg-1h-1，二者无显著差异(图3F)。
为进一步明确MIR结构域在体内对Pmt1酶活性的影响，我们提取了球孢白僵菌野生菌株和ΔMIR突变体(破坏Pmt1的MIR结构域编码区而残留完整PMT结构域区域)膜蛋白进行Pmt酶活测定。结果表明，野生菌株和突变体ΔMIR从供体Dol-P-[3H]mannose转移[3H]mannose到合成的受体肽Ac-YATAV-NH2活性分别为0.90(±0.08)×106dpm mg-1h-1和0.94(±0.18)×106dpm mg-1h-1，二者的无显著差异(P>0.05,图3G)。
由此表明，删除球孢白僵菌MIR结构域编码区不影响Pmt1的酶活性。
【实施例3】
1.破坏Pmt1的MIR结构域编码区对菌株生长的影响
为揭示Pmt1的MIR结构域与球孢白僵菌生长发育的关系，分别在完全培养基PDA、基本培养基察氏培养基(Czapek-Dox agar)和不同碳源、氮源培养基上比较研究的野生菌株、突变体ΔMIR和Pmt1回复互补转化子的菌落生长。结果表明，尽管球孢白僵菌不同的培养基或不同营养条件下的生长速率存在差异，但与野生菌株和回复互补转化子相比，突变体ΔMIR在大部分培养条件下的生长速率明显下降。在PDA、Czapek-Dox agar和除甘氨酸(Gly)以外的不同氮源培养基上，突变体ΔMIR的生长速率比野生菌株下降了5.28％-11.94％(P<0.05，图4)。在以甘露糖(mannitiol)、阿拉伯糖(arabitol)或N-乙酰氨基葡萄糖(GlcNAc)为唯一碳源的培养基上，突变体ΔMIR的生长速率与野生菌株无明显差异。然而，在以葡萄糖(glucose)、蔗糖(sucrose)、甘油(glycerol)、赤藓糖(erythritol)、木聚糖(xylitol)或海藻糖(trehalose)为唯一碳源的培养基上，突变体ΔMIR的生长速率比野生菌株下降了3.9％-29.3％(P<0.05，图4)。
2.破坏Pmt1的MIR结构域对产孢及生物量的影响
分生孢子产量参照Zhang等(2009,Appl.Environ.Microbiol.,75:3787–3795)的方法进行。具体操作如下：20ml Czapek-Dox agar(Czapek)和添加1％(wt/vol)酵母浸膏(yeast extract)的1:4稀释的Sabouraud’s dextrose agar培养基(1/4SDAY)冷却至45℃，然后加入50μl 1×107孢子/ml的分生孢子悬浮液混匀，倒入直径为90-mm的培养皿制备平板。混菌平板于26℃和15h/9h的光照与黑暗交替循环条件下培养15天，用0.05％(vol/vol)Tween 80收集分生孢子，然后用四层擦镜纸过滤除去菌丝碎片。用血球计数板于显微镜下计数分生孢子浓度，然后换算成培养基单位面积产生的分生孢子数。每个菌株设三个重复，每次试验重复四次。
分生孢子大小测定：在显微镜下采用软件Image-Pro Plus 6.0sorftware(Bio-rad,USA)测定分生孢子长径和宽径，每个菌株至少测定400个孢子。
检测结果发现，尽管破坏MIR结构域编码区降低了菌落生长速率，但显著提高了分生孢子产量。在Czapek agar和1/4SDAY培养基上，ΔMIR的分生孢子产量分别为5.81±0.33mm-2和21.81±0.52mm-2，比野生菌株分别提高了44.53％和115.3％(P<0.01，图5A)。分子孢子大小测定结果发现，ΔMIR分生孢子明显膨大，平均宽径和长径分别为2.94±0.02μm和3.35±0.11μm，显著大于野生菌株分生孢子的宽径(2.65±0.04μm)(P<0.01)和长径(2.91±0.10μm)(P<0.01)(图5B)。
芽生孢子的定量参照Ying等(2013,Environ.Microbiol.,15:2902–2921)等的方法进行。具体操作如下：用0.05％(vol/vol)Tween 80收集于添加1％(wt/vol)酵母浸膏(yeast extract)的Sabouraud’s dextrose agar培养基(SDAY)上培养的分生孢子，接种于50ml 1:4稀释的SDY(1/4SDY，即去除琼脂的1/4SDAY)液体培养基至终浓度为106孢子/ml，然后于25℃、180rpm摇瓶培养。培养3d和4d后，取样于血球计数板计数芽生孢子浓度，并换算成每50ml产生芽生孢子的数量。取样同时，收集每瓶培养的菌体，用无菌水洗涤3次，然后于60℃烘干成恒重，分析天平称量菌体生物量。根据芽生孢子数量和积累的生物量计算单位干重生物量产生的芽生孢子数量。每个菌株设三个样本，试验重复三次。
结果表明，ΔMIR突变体在液体培养条件下积累的生物量显著高于野生菌株。培养3d和4d，ΔMIR突变体在50ml 1/4SDY培养基中积累的生物量分别为0.333±0.023g和0.362±0.005g干重，显著高于野生菌株积累的生物量0.300±0.002g(P<0.05)和0.311±0.004g干重(P<0.01)(图5C)。尽管培养3d后野生菌株和ΔMIR突变体产生的芽生孢子数量无显著差异，但培养4d后ΔMIR突变体产生的芽生孢子数量([1.60±0.33]×109g-1干重)显著低于野生菌株([2.34±0.20]×109g-1干重)(P<0.05，图5D)。
3.破坏Pmt1的MIR结构域对产孢相关基因表达的影响
为探究Pmt1MIR结构域介导分生孢子产生可能的机制，我们利用Real-time qRT-PCR方法检测了产孢相关基因在野生菌株和ΔMIR突变体在产孢时期的转录模式。5个产孢相关的基因分别是：flbA：编码G-蛋白信号结构域蛋白的调控子(Lee and Adams,1994b,Mol.Microbiol.,14:323-334)；4个编码分生孢子产生相关转录因子：包括flbB(basic-zipper type)(Etxebeste et al.,2008,Eukaryot.Cell,7:38-48)、flbC(C2H2type)(Kwonet al.,2010,Mol.Microbiol.,77:1203-1219)和flbD(Myb-like)(Wieser and Adams,1995,Gene Dev.,9:491-502)；fluG：参与一个分生孢子发育信号的合成(Lee and Adams,1994a,Gene Dev.,8:641-651)，两个孢子壁疏水编码基因hyd1和hyd2(Zhang et al.,2011b,Mol.Microbiol.,80:1365–2958)。
具体操作如下：涂布接种分生孢子于SDAY平板，于26℃培养4d、5d、10d和20d，收集2g新鲜的气生菌丝和分生孢子，利用RNA提取试剂盒(RNeasy kit[Qiagen]提取总RNA。取2μg RNA，利用cDNA反转录试剂盒(oligo[dT]-primed cDNA synthesis kit[MBI Fermentas]反转录合成cDNA第一链，作为模板用于基因转录分析。Real-time RT-PCR操作采用定量PCR试剂盒(Bio-Rad)进行，以actin基因(Gen-Bank ID:HQ232398)为参比基因中和目标基因的表达水平。采用CFX Manager software(Bio-Rad,USA)计算靶标基因相对于参比基因的转录水平(Normalized fold expression)(Luo et al.,2012,Fungal Genet.Biol.,49:544–555)。扩增fluG、flbA、flbB、flbC、flbD、hyd1和hyd2的引物对分别为FluG1/2、FlbA1/2、FlbB1/2、FlbC1/2、FlbD1/2、hyd1f/r和hyd2f/r，引物序列附后。
研究结果表明，在所有检测时期(4-20d)，flbA在ΔMIR中的转录水平受到显著抑制(～1.3-4.1-fold,P<0.01)(图6A),而10d后flbC在突变体ΔMIR中的转录水平显著上调(图6C)。在产孢早期(4-5d)，flbB、flbD和fluG在突变体ΔMIR中的转录水平相对于野生菌株均有不同程度的变化，但在10d后这三个基因在突变体ΔMIR中转录水平均显著上调(～1.6-6.1-fold,P<0.01，图6B和D)。在第5d和20d，hyd1在突变体ΔMIR中的转录水平显著上调，但在第4d和10d的转录水平略有抑制(图6F)。hyd2的转录在第5天在突变体中受到抑制，但在第10d则显著上调(图6F)。由此表明，破坏MIR结构域编码区上调和干扰了产孢相关基因的表达，影响了分生孢子产生。
FluG1：5'-CCTCCCTAGTTTGGTCGCTTTCTC-3'(SEQ ID NO.26)
FluG2：5'-CGCTGTCGGTAATCTGCTCCTC-3'(SEQ ID NO.27)
FlbA1：5'-CCAATCCACTCGCCGCTCTC-3'(SEQ ID NO.28)
FlbA2：5'-CGGAGGAAAGAGAATCGGTAGAGG-3'(SEQ ID NO.29)
FlbB1：5'-GCACTGACACGCCGACAAGAGC-3'(SEQ ID NO.30)
FlbB2：5'-CCGCCGCCGAAGCCTGTTG-3'(SEQ ID NO.31)
FlbC1：5'-TCCATCTCCAACTTGCTGGGTCTC-3'(SEQ ID NO.32)
FlbC2：5'-GGCGGCGTAGGCGGAAGG-3'(SEQ ID NO.33)
FlbD1：5'-CGGCAAGCGATGGGCAGAGATTG-3'(SEQ ID NO.34)
FlbD 2：5'-ACGAGCAAGGTGACGGTAGAGGTG-3'(SEQ ID NO.35)
hyd1f：5'-ATCTACTGCTGCAACGAGAA-3'(SEQ ID NO.36)
hyd1r：5'-TACTGGATAAGACTGCCAAT-3'(SEQ ID NO.37)
hyd2f：5'-AGTGTCAAGACTGGCGACAT-3'(SEQ ID NO.38)
hyd2r：5'-ATCCGAGGACGGTGATGGGA-3'(SEQ ID NO.39)
4.破坏Pmt1的MIR结构域对分生孢子表面特性的影响
为探究破坏Pmt1的MIR结构域对细胞表面特性的影响，我们分别采用荧光标记的植物凝集素检测分生孢子表面的碳源表位、利用微生物结合碳氢化合物的试验(MATH)检测分生孢子疏水性以及分生孢子在固体基质的附着性。
凝集素结合试验参照Wanchoo等方法(2009,Microbiol-SGM,155:3121–3133)进行，选择的凝集素分别是Alexa Fluor 488-labeled lectins concanavalin A[ConA](识别ɑ-glucose、mannose和ɑ-N-acetylglucosamine[GlcNAc])(Molecular Probes-Invitrogen)、wheat germ agglutinin[WGA](结合β-GlcNAc和sialic acids)(MolecularProbes-Invitrogen)和fluorescein-labeled lectin Galanthus nivalis[GNL](特异性识别甘露糖残基[mannose residues](Vector Laboratories)。具体操作如下：参照具体凝集素(lectin)说明书配制结合缓冲液，将分生孢子悬浮于缓冲液，并添加凝集素在黑暗条件下反应1h，然后利用结合缓冲液清洗5次去除未结合的凝集素(lectin)。结合凝集素的分生孢子采用流式细胞仪检测，激发光波长为488nm，发射光波长为530nm，检测器为FACSCalibur，检测软件为CellQuest Pro(Becton Dickinson)。计算平均荧光强度。
气生分生孢子表面疏水性检测采用Rosenberg和Doyle(1990,Microbial CellSurface Hydrophobicity.Am Soc Microbiol,Washington,DC,pp.1–37)采用微生物细胞结合碳氢化合物(MATH)测验并略有改进(Luo et al.，2012,Fungal Genet.Biol.,49:544–555)。具体操作如下：收集Czapek-Dox agar plates培养的分生孢悬浮于0.05％Tween-80，然后用四层擦镜纸过滤除去菌丝等碎片，离心沉淀分生孢子。用灭菌水洗涤分生孢子3次，然后将分生孢子重悬于0.01％(v/v)Tween-80，并稀释至420nm的光密度为0.8左右。取750μl稀释的分生孢子悬液加入1.5ml的聚丙烯离心管中，然后加入250μl十六烷，于涡旋仪涡旋1min充分混匀，然后于4000rpm离心30s分离水相(下层)和油相(上层)。取200μl水相加入96孔酶标板，利用酶标仪测定420nm的光密度。通过比较水相中的分生孢子数(OD420nm)和最初在0.01％Tween-80的总孢子数，估算分生孢子疏水性。
分生孢子在固体基物的附着性测定参照Holder和Keyhani的方法(2005,Appl.Environ.Microbiol.,71:5260–5266)进行。具体操作如下：分生孢子悬浮于50mM的碳酸钙缓冲液(pH 9.2)，按每毫升分生孢子悬浮液加入1mg/ml Fluoresceinisothiocyanate(FITC)贮存液，在黑暗条件下标记20min。将荧光标记的分生孢子培育在不同黑色滴定板测验基质上，定量测定孢子附着性。取100μl 2×107cells ml-1真菌孢子悬液加入黑色滴定板孔中，于黑暗条件下25℃浮于4h，轻轻吸去未结合的细胞，然后用450μl TB缓冲液(50mM Tris-HCl,pH 8.0)洗涤3次。利用酶标仪在激发光为495nm、发射光为530nm和截止波长(cutoff wavelength)为515nm条件下测定荧光强度。对每一个试验，制定一个荧光强度对细胞数目(直接计数法)的标准曲线。通常，在每次洗涤前后测定荧光强度。弱极性滴定板(Weakly，Fluorotrac F200)和亲水性滴定板(hydrophilic，Fluorotrac F600)购自Greiner Biotech公司(Longwood,FL)。疏水性滴定板的制备如下：在Fluorotrac F200滴定板池中，加入Sigmacote(Sigma Corp.,St.Louis,Mo.)制备一层硅酮，即滴定板用Sigmacote处理三次，然后在通风橱过夜处理已确保溶剂挥发完全。
荧光标记的凝集素结合试验结果表明，突变体ΔMIR和野生菌株分生孢子结合ConA的能力无明显差异，但更多的GNL和WGA与ΔMIR分生孢子发生结合反应，平均荧光强度分别比野生菌株提高了0.76倍4.79倍(P<0.01，图7A)。由此表明，破坏Pmt1的MIR结构域改变了细胞表面的碳源表位。
疏水性检测结果表明，突变体ΔMIR分生孢子分布在水相中的比例为27.65％±0.68％，显著低于野生型分生孢子分布在水相中的比例34.02％±0.71％(P<0.01，图7B)。该结果表明，破坏Pmt1的MIR结构域显著提高了分生孢子的疏水性。
附着性检测结果发现，破坏Pmt1的MIR结构域增强了分生孢子在疏水性和弱极性基物表面的附着性，但显著降低了在亲水性基物表面的附着性。洗涤1次和3次后，突变体ΔMIR分别有74.68％和45.07％的孢子(总孢子数2×106)附着于疏水性表面、76.13％和28.86％的孢子(总孢子数2×106)附着于弱极性表面，而野生菌株附着于疏水表面的分生孢子分别占总孢子数(2×106)的53.09％和19.55％，附着于弱极性表面的分生孢子分别占总孢子数(2×106)的64.91％和16.45％。统计分析表明，突变体ΔMIR在疏水表面和弱极性表面的附着率均显著高于野生菌株(P<0.01，图7C)。在亲水性固体表面，洗涤1次和3次后，突变体ΔMIR附着的孢子仅占总孢子数(2×106)的31.94％和20.05％，均显著低于野生菌株的附着率(分别为48.72％and 28.58％)(P<0.01，图7C)。
5.破坏Pmt1的MIR结构域提高了菌株的毒力
为评价Pmt1的MIR结构域与球孢白僵菌毒力的关系，以大蜡螟(Galleriamellonella)3龄幼虫为是试虫，采用两种方法进行生物测定。第一种方式代表了典型的真菌孢子侵染方式，即体表接种；第二种方式则是克服体壁将孢子直接注射进昆虫体腔以检测是否存在逃避昆虫免疫识别缺陷或对昆虫存活的影响。两种生物测定结果均显示，突变体ΔMIR的毒力均高于野生菌株(图8)。典型的体表接种生测结果表明，在2×107孢子/ml浓度下，ΔMIR引起昆虫死亡的半致死时间LT50是103.3h±0.7h，显著少于相同剂量下野生菌株引起的半致死时间(107.3h±1.1h)(P<0.01，图8A)。将2μl 107孢子/ml的孢子悬浮液微量注射进虫体内进行生物测定，结果表明，注射ΔMIR菌株孢子引起昆虫的半致死时间LT50是65.7h±0.6h，显著少于相同剂量下野生菌株引起的半致死时间(67.6h±0.8h)(P<0.01，图8B)。而两种生物测定中，回复互补转化子ΔMIR::Pmt1与野生菌株无明显差异(图8A和B)。由此表明，MIR限制了Pmt1对球孢白僵菌毒力的控制，失活MIR结构域引起菌株毒力增强。
6.破坏Pmt1的MIR结构域影响虫菌体的分化
为揭示破坏Pmt1的MIR结构域对菌体侵染、侵染后在虫体内的繁殖及分化的影响，分别在两种方式接种后定时取虫体血淋巴，利用血球计数板在显微镜下统计虫菌体数量。结果表明，两种方式接种后，ΔMIR在虫体内的繁殖速度明显高于野生菌株(图8C和D)。经典的体表接种后96-114h，ΔMIR在虫体内的菌体繁殖数量比野生菌株提高了23.2％-82.9％(P<0.01，图8C)，而注射接种后48h-84h，ΔMIR在虫体内的菌体繁殖数量比野生菌株提高了111.1％-175.2％(P<0.01，图8D)。镜检过程中发现，相对于野生菌株在虫体内形成短的或呈棍棒形的芽孢子，ΔMIR在虫体内分化的菌体明显变长，有些菌体分化成分枝的菌丝体(图9A和B)。
Claims (6)
1.一种提高球孢白僵菌分生孢子产量和毒力的方法，其特征在于通过删除球孢白僵菌Pmt1基因编码第311-427位氨基酸的核苷酸序列，并保留完整的PMT结构域，获得球孢白僵菌突变菌株，所述球孢白僵菌突变菌株具有提高的分生孢子产量和毒力。
2.权利要求1所述的方法，其中通过同源重组删除球孢白僵菌Pmt1基因编码第311-427位氨基酸的核苷酸序列，保留包括起始密码ATG在内的完整的PMT活性结构域，获得Pmt1部分序列破坏的突变体，所述突变体中PMT结构域编码区正常转录。
3.权利要求1或2所述的方法，其中所述删除球孢白僵菌Pmt1基因编码第311-427位氨基酸的核苷酸序列是以除草剂抗性基因bar置换Pmt1基因的MIR结构域部分编码区，构建同源重组表达载体。
4.制备球孢白僵菌突变菌株的方法，包括下述步骤：
1)以基因工程方法删除球孢白僵菌Pmt1基因编码第311-427位氨基酸的核苷酸序列，获得重组表达载体；
2)将步骤1)获得的重组表达载体转入球孢白僵菌野生型菌株，删除球孢白僵菌Pmt1基因中PMT活性结构域下游的编码第311-427位氨基酸的核苷酸序列，获得球孢白僵菌突变菌株，所述球孢白僵菌突变菌株具有提高的分生孢子产量和毒力。
5.一种球孢白僵菌突变株，其特征在于用权利要求4所述的方法制备。
6.一种真菌杀虫剂，含有权利要求5所述的球孢白僵菌突变株。
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