CN101058814A - 源于稻瘟病菌的真菌致病性基因mnh6及其用途 - Google Patents
源于稻瘟病菌的真菌致病性基因mnh6及其用途 Download PDFInfo
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- CN101058814A CN101058814A CN 200710067275 CN200710067275A CN101058814A CN 101058814 A CN101058814 A CN 101058814A CN 200710067275 CN200710067275 CN 200710067275 CN 200710067275 A CN200710067275 A CN 200710067275A CN 101058814 A CN101058814 A CN 101058814A
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Abstract
本发明公开了一种源于稻瘟病菌的真菌致病性基因MNH6,该基因的核苷酸序列或其互补链的核苷酸序列为SEQ ID NO:1。该基因MNH6编码的cDNA序列具有SEQ ID NO:2所示的核苷酸序列。该基因MNH6编码的蛋白质具有SEQ ID NO:3所示的氨基酸序列。该基因MNH6的启动子具有SEQ ID NO:4所示的核苷酸序列。本发明还公开了利用上述基因MNH6的表达作为靶标,在设计和筛选抗真菌药物中应用。本发明还公开了利用上述蛋白质的表达和修饰作为靶标,在设计和筛选抗真菌药物中应用。本发明的基因MNH6对稻瘟病菌等真菌的菌丝生长、分生孢子产生、分生孢子发芽、附着孢形成和侵染栓形成以及致病性有重要影响。
Description
技术领域
本发明属于植物病理学、农药学和微生物基因工程领域,提供了一个来源于稻瘟病菌的、在营养生长、孢子产生、附着胞形成与致病性中起重要作用的新基因MNH6的启动子与编码区的核苷酸序列及其编码蛋白质的氨基酸序列。
背景技术
由稻瘟病菌(Magnaporthe grisea)引起的稻瘟病是世界性的一种水稻上的毁灭性病害。世界上每年由稻瘟病造成的水稻产量损失在10~30%之间。我国几乎每年都有稻瘟病菌的流行爆发。最近的2005年,四川省和重庆市遭受到10年来最严重的稻瘟病,四川省共有20个市、127个县的230万亩稻田发生稻瘟病,重庆市共有100多万亩稻田发生稻瘟病菌;这次稻瘟病流行迅速、危害程度重、发病水稻品种多、对水稻产量影响严重。除了水稻外,稻瘟病菌也能侵染其它50多种禾本科植物,也对小米(Eleusine coracana)、大麦(Hordeum vulgare)、小麦(Triticum aestivum)等重要农作物造成严重的危害。稻瘟病菌也是一种研究植物病原真菌—寄主植物相互作用的模式生物,具有许多病原真菌共有的植物致病侵染循环,包括孢子产生、萌发、附着胞形成、侵染栓形成、侵入菌丝生长等致病过程;许多发达国家正在进行研究其致病分子机制,期望通过此类研究设计出新型农药筛选的药靶,从而开发、设计新型抗真菌药物。
稻瘟病菌的生活史包括有性阶段和无性阶段。有性阶段由2个不同交配型的菌株交配后产生子囊和子囊孢子。稻瘟病菌的交配型由交配基因MAT1-1和MAT1-2控制。无性阶段为营养菌丝分化产生分生孢子梗和梨形分生孢子。在自然界,稻瘟病菌主要通过无性阶段完成生活史,而分生孢子是植物的主要侵入源。
稻瘟病菌无性孢子落到水稻叶片表面后,侵染过程开始。当孢子与水相遇后,孢子顶端分隔内的粘胶释放出来,使孢子紧紧粘附在水稻叶片表面。水稻叶片表面具有一层蜡质角质膜,该膜具有很大的疏水性,而粘胶使孢子粘附在这一疏水的排斥性表面。粘附后2小时内,孢子萌发并产生芽管。芽管通常从孢子的一个顶端细胞伸出并生长。芽管顶端也分泌一种粘胶物质,使芽管紧紧粘附在植物叶片表面,防止被水滴冲走。4小时内,芽管生长停止,顶端钩状体形成并膨大形成附着胞。基质的硬度是芽管分化的一个重要因子,如稻瘟病菌孢子仅在硬的固体表面形成附着胞,而不能在液体表面或软基质表面形成附着胞。而基质疏水表面也通过类似于水稻叶片表面的蜡质的作用激发附着胞的形成。在孢子萌发后不久,产生芽管的细胞进行一次有丝***。一个细胞核留在孢子内,而另一个细胞核通过芽管移动,与孢子和芽管的细胞质内容物一起转移到形成中的附着胞。这些细胞质内容物包括储存在正在发育的附着胞中央大液泡的脂滴。然后,在附着胞和芽管之间形成隔膜。随着附着胞成熟,附着胞细胞壁出现黑色素沉积,最终形成黑色素层。黑色素层允许水分子自由通过,但溶解于水的物质不能自由通过,从而让附着胞建立并维持一个很大的细胞内膨压。附着胞的黑色素化过程完成后,附着胞产生一根狭小的菌丝--侵染栓。侵染栓对水稻叶片的穿透由于附着胞产生巨大的膨压而得以完成。膨压只要稍微降低就会阻止附着胞在人工表面或水稻叶片表面穿透。测量结果表明附着胞膨压达到8.0Mpa,相当于40倍汽车轮胎的压力。附着胞黑色素层的作用在于限制了细胞壁的渗透性,有利于细胞内渗透物质的积累和膨压的产生。甘油是一种可溶性物质,产生的渗透势足以使附着胞产生巨大的膨压。在膨压产生过程中,甘油在细胞内累积浓度超过3M。
侵入后,侵染栓分化成侵染菌丝,迅速在水稻叶片内生长并侵染其它细胞。侵入72小时后,病原菌生物量已经达到感染叶片的10%。5~7天后,分生孢子梗上分化出大量新的分生孢子,并从病斑释放出来。这些新形成的孢子被潮湿的空气带到邻近的植物开始新的侵染过程。在冬天,稻瘟病菌以菌丝和分生孢子形式在稻草和稻谷上越冬,完成侵染循环。
稻瘟病菌的致病过程是一个复杂的分子过程。目前已经克隆了许多与稻瘟病菌致病性相关的基因,如:MPG1、CPKA、PMK1、MAGB、PLS1、SMO1、PDE1、MPS1、PTH11、CBP1、ICL1、BUF1、ALB1、ACR1、RSY1、HEX1。其中多数与附着胞形成有关,如PMK1、MAGB、MAC1等;部分参与黑色素的形成和积累(ALB1、RSY1、BUF1等)以及甘油合成相关(ICL1等);少数与稻瘟病菌的穿透相关(MPS1、PLS1、PDE1等)。稻瘟病菌芽管分泌的一种疏水蛋白,由MPG1基因编码,参与菌丝和水稻叶表的互作,为形成正常的附着胞所必须(Talbot,1999)。MAGB基因编码的异源三聚G蛋白的α亚单位和MAC1编码的cAMP环化酶也是形成附着胞所必需的(Liu and Dean,1997;Choiand Dean,1997;Kulkarni and Dean,2004)。CPKA基因编码的cAMP依赖性蛋白激酶A的催化亚单位PKA-c是附着胞穿透所必须的(Mitchell & Hamer,1995;Xu et al,1997),而正在分化的芽管中的PKA活性对于形成正常的附着胞也非常关键(Adachi & Hamer1998)。同样,有丝***原激活的蛋白激酶(Mitogen-activated protein kinase,MAPK)基因PMK1是附着胞形成所需的(Xu & Hamer 1996)。黑色素合成系列基因也证明是附着胞穿透所不可缺少的(Chumley and Valent,1990;Motoyama等,1998)。在各种突变子库中也发现了一些突变子缺失致病性,如:编码一种与ATP运输体(ATP-bindingcassette transporters)相关的蛋白的ABC1基因(Urban等,1999)、编码P-型ATP酶的PDE1基因(Balhadere等,1999,2001)和编码tetraspannin样蛋白的PLS1基因(Clergeot等,2001)。尽管如此,稻瘟病菌的分子致病机制还是不清楚。
鉴定、克隆植物病原真菌的致病性基因,尤其是与侵入过程相关的基因,可以为设计、筛选抗真菌药物提供有用的靶标位点。目前已经在包括稻瘟病菌在内的一些真菌中证明了一些药物的靶点,如三环唑是一种很重要的稻瘟病菌杀菌剂,其作用是抑制黑色素的合成,靶点是稻瘟病菌的三羟萘还原酶;多肽杀菌剂sorphen A的作用靶点是青霉的脱甲基酶(CYP51)。因此,利用分子生物学技术鉴定、克隆新的在稻瘟病菌的致病过程中具有重要功能的致病性基因,可以为设计、筛选新的抗真菌药物提供有用的药物靶点。
发明内容
本发明要解决的技术问题是提供一种新的、对稻瘟病菌等真菌的菌丝生长、分生孢子产生、分生孢子发芽、附着孢形成和侵染栓形成以及致病性有重要影响的基因MNH6及该基因的用途。
为了解决上述技术问题,本发明提供了一种源于稻瘟病菌的真菌致病性基因MNH6,该基因的核苷酸序列或其互补链的核苷酸序列为SEQ ID NO:1。
本发明同时提供了上述基因MNH6编码的cDNA序列,该cDNA序列具有SEQ ID NO:2所示的核苷酸序列。
本发明同时提供了上述基因MNH6编码的蛋白质,该蛋白质具有SEQ ID NO:3所示的氨基酸序列。
本发明同时提供了上述基因MNH6的启动子,该启动子具有SEQ ID NO:4所示的核苷酸序列。
本发明还提供了利用上述基因MNH6的表达作为靶标,在设计和筛选抗真菌药物中的应用。
本发明还提供了利用上述蛋白质的表达与修饰作为靶标,在设计和筛选抗真菌药物中的应用。
本发明还提供了结合利用上述蛋白质以及启动子作为靶标,在设计和筛选抗真菌药物中的应用。
本发明的基因MNH6及该基因编码的蛋白质序列,以其全长核苷酸为探针,通过杂交分离源于其它病原真菌的具有相同功能的基因;或以序列设计引物,通过PCR分离源于其它病原真菌的具有相同功能的基因;或以其编码的蛋白质,杂交分离源于其它病原真菌的具有相同功能的蛋白。上述真菌包括:灰葡萄孢菌(Botrytis cinerea)、禾谷镰刀菌(Fusarium graminearum)、壳多胞菌(Stagonospora nodorum)或玉米黑粉病菌(Ustilagomaydis)。
本发明所称的抗真菌药物包括抗稻瘟病菌(Magnaporthe grisea)、灰葡萄孢菌(Botrytis cinerea)、禾谷镰刀菌(Fusarium graminearum)、壳多胞菌(Stagonosporanodorum)或玉米黑粉病菌(Ustilago maydis)的药物。
本发明利用基因敲除和基因互补证明了稻瘟病菌中一个对稻瘟病菌附着胞形成、附着胞穿透过程以及致病力具有关键性作用的基因MNH6。该基因的启动子、编码蛋白的表达和修饰可以作为新农药设计和筛选的药物靶点。
本发明的基因MNH6是通过cDNA差减文库从稻瘟病菌中克隆的。具体过程包括:通过筛选稻瘟病菌cDNA的SSH差减文库获得在菌丝或附着孢中差异表达的EST序列。通过RT-PCR验证目的基因在稻瘟病菌菌丝、孢子、附着孢中的存在情况。通过高保真长距离PCR从稻瘟病菌基因组DNA中获得目的基因。通过PCR或以该基因的EST序列为探针从稻瘟病菌cDNA文库验证和分离目的基因的完整的cDNA。目的基因的功能验证和注释通过目的基因的敲除和互补恢复来完成。目的基因在稻瘟病菌内的作用位点通过目的基因与报告基因GFP的融合蛋白在细胞的位置来确定。基因启动子的测定是将启动子与报告基因GFP连接,通过观察GFP在启动子指导下在稻瘟病菌的各个发育阶段的表达来进行的。
本发明所涉及的稻瘟病菌SSH差减文库是指利用SSH技术(Suppression subtractivehybridization,SSH)构建的、含有若干独立克隆的稻瘟病菌cDNA文库。
本发明所涉及的RT-PCR分析是指根据cDNA序列设计引物,对基因缺失突变子和互补恢复突变子的mRNA逆转录形成的cDNA进行PCR扩增,分别验证基因缺失突变子和基因互补恢复突变子中基因缺失和恢复后的转录本等情况。
本发明所涉及的目的基因克隆是指根据SSH文库中获得的EST序列,以此设计探针从cDNA文库中筛选cDNA克隆,对获得的cDNA克隆进行测序;并利用cDNA序列比对基因组数据库中DNA序列,以此设计引物从稻瘟病菌基因组DNA扩增获得基因的DNA序列,克隆到T载体中,进行测序;根据获得的DNA序列利用程序分析其转录序列,并以此设计引物从稻瘟病菌cDNA文库中扩增获得基因的cDNA序列,连接到T载体中,进行测序分析。
本发明所涉及的Shouthern杂交分析是指利用基因侧翼的DNA序列作为探针,对所获得的基因敲除转化子和基因恢复转化子分别进行Southern杂交,以分析基因敲除转化子的基因缺失情况和敲除载体的***拷贝数,以及分析基因互补恢复转化子基因***拷贝数等情况。
本发明所涉及的目的基因的功能确定是通过MNH6的敲除和恢复进行的。具体的MNH6基因的敲除过程包括敲除载体的构建,将敲除载体转入稻瘟病菌,得到基因敲除突变子;MNH6的基因敲除突变子的互补恢复过程包括互补载体的构建,互补载体导入基因敲除突变子菌株,得到基因恢复的转化子。基因敲除载体的构建是指将待敲除基因的两侧翼的一段DNA片段各自连接到含潮霉素抗性基因载体的潮霉素抗性基因的两侧。基因互补载体的构建是指将含有目标基因的全长的DNA片段与带有一个不同于潮霉素抗性基因的筛选标记(本发明中是草胺磷抗性基因)的载体连接。在本发明中将外源载体导入稻瘟病菌的优选方法是原生质体转化,实验者也可以根据自己的情况选用其它方法如ATMA法、基因枪法等。本发明中,基因敲除载体所导入的稻瘟病菌菌株是野生型菌株Guy-11,实验者也可以选用其它稻瘟病菌野生型菌株,基因缺失使突变子的表型发生不同于原来野生型的变化,对水稻等植物的致病性丧失。本发明中,基因互补载体所导入的菌株是本发明所得到的基因缺失突变子,互补载体在该基因缺失突变子基因组中的异位整合使该突变子的表型恢复正常。
本发明所提供的MNH6的cDNA克隆过程包括:从SSH差减文库中筛选含有目标基因MNH6的cDNA克隆,获得其EST序列,利用其EST序列从稻瘟病菌基因组数据库中获得相对应的一段DNA序列,再根据其序列设计引物从稻瘟病菌基因组DNA中扩增到目标基因的DNA片段,测序获得其DNA序列,利用GenScan(http://genes.mit.edu/genscan.html)进行启动子、编码区和加尾点的预测。根据预测的转录本序列设计引物,并通过PCR从稻瘟病菌cDNA文库中扩增获得,其序列如SEQ IDNO:2所示。
本发明还提供了MNH6编码的蛋白质,其具有如SEQ ID NO:3所示氨基酸序列或与SEQ ID NO:2所示的序列具有35%以上的序列一致性的并具有相似功能的氨基酸序列。该蛋白可以源于稻瘟病菌,也可以源于其它病原真菌。本发明提供了:灰葡萄孢菌(Botrytiscinerea)、禾谷镰刀菌(Fusarium graminearum)、壳多胞菌(Stagonospora nodorum)或玉米黑粉病菌(Ustilago maydis)的中具有与MNH6蛋白具有35%以上氨基酸序列一致性的同源蛋白的氨基酸序列。
本发明所涉及的基因因为同源重组造成的基因缺失突变导致稻瘟病菌生长速度减慢,产孢量下降、附着胞形成率下降、附着胞膨压降低和侵入栓形成率降低,并且在感病水稻品种上的致病力缺失。因此,本发明最重要的用途是:应用上述成果,设计和筛选能够破坏该基因的表达、剪切及其编码蛋白的表达的化合物,或设计和筛选能对该蛋白的氨基酸序列进行修饰的化合物,从而开发出新的抗真菌药物。此外,本发明的用途还包括利用该基因的DNA或cDNA序列作为探针在其它真菌中分离与该基因具有一定序列同源性的序列。
以上述所克隆基因编码蛋白的表达、修饰为靶标,设计、筛选新型抗真菌药物,或者以具有如SEQ ID No:3所示序列或与SEQ ID No:3具有35%以上序列一致性的并具有相似功能的蛋白中任一区域的氨基酸序列设计多肽,并制备抗体用于检测在化合物处理状况下蛋白的表达,均属于本发明的保护范围之内。
以上述所克隆基因的启动子为靶标,设计和筛选新型抗真菌药物,也属于本发明的保护范围之内。
因此,本发明具有以下优点:
1、附着胞是稻瘟病菌侵入寄主植物的重要结构,附着胞膨压是稻瘟病菌穿透寄主植物表皮角质层的主要机械力量,MNH6基因参与稻瘟病菌附着胞形成和附着胞膨压的产生,MNH6基因与稻瘟病菌的致病性密切相关,MNH6失活或缺失,导致稻瘟病菌附着胞形成率和附着胞膨压下降,致病性丧失。因此MNH6基因在稻瘟病菌致病过程中作用重大。
2、MNH6是一种真菌DNA结合蛋白,调控数百种基因的表达,对于稻瘟病菌的生长、发育和致病具有重要的作用,是一种适宜于开发新的抗真菌药物的特异的药物作用靶点。针对MNH6作为药物开发的靶标药靶,通过对大量化合物进行筛选,可以找到能够与药靶起作用的物质,这些物质可以被用做阻止真菌侵染。由于药靶的特异性,筛选到的抗真菌药物一般对于动物和植物的毒性较小。
综上所述,本发明提供的核苷酸序列和氨基酸序列可以作为靶标应用于抗真菌药剂的筛选与设计中,也可以利用该核苷酸序列的某一区段作为探针或利用该蛋白制备的抗体应用于稻瘟病菌及其他真菌的与该序列具有一定同源性的基因的克隆中。
附图说明
图1是野生型菌株Guy11与MNH6基因缺失突变子Δmnh6及MNH6互补转化子在水稻感病品种CO39上的致病性比较图(孢子喷雾接种测定)。Guy11:为野生型菌株;Δmnh6:为MNH6基因缺失突变子;互补转化子:为MNH6基因互补恢复转化子;明胶:为0.2%(w/v)的明胶对照溶液。喷雾接种的孢子浓度为1×105/ml,接种后7天拍照。
图2是野生型菌株Guy11与MNH6基因缺失突变子Δmnh6及MNH6互补转化子在大麦上的致病性比较图(孢子喷雾接种测定)。Guy11:为野生型菌株;Δmnh6:为MNH6基因缺失突变子;互补转化子:为MNH6基因互补恢复转化子;明胶:为0.2%(w/v)的明胶对照溶液。喷雾接种的孢子浓度为1×105/ml,接种后4天拍照。
图3是野生型菌株Guy11与MNH6基因缺失突变子Δmnh6及MNH6互补转化子在大麦表皮上侵入栓形成比较图。Guy11:为野生型菌株;Δmnh6:为MNH6基因缺失突变子;24h、48h、72h、96h为接种后拍照时间。大麦离体叶片点接种的孢子浓度为1×105/ml。
图4是MNH6基因敲除载体构建示意图。
图5是MNH6基因互补载体构建示意图。
图6是MNH6基因敲除位置和过程示意图。A:Afl I;B:BamH I;H:Hind III;S:Sal I;Sm:Sma I。
图7是野生型菌株Guy11与MNH6基因缺失突变子Δmnh6及MNH6互补转化子在完全培养基(CM)上生长特征比较。培养基时间为9天。
图8是野生型菌株Guy11与MNH6基因缺失突变子Δmnh6及MNH6互补转化子的无性孢子形态特征比较。
图9是野生型菌株Guy11与MNH6基因缺失突变子Δmnh6菌丝对细胞壁降解酶的敏感性比较。用Glucanex细胞壁降解酶处理30分钟后拍照。
图10是MNH6基因编码蛋白在孢子和菌丝中的细胞核定位图。MNH6-GFP融合蛋白分布于细胞核中,左侧(A)为明视场下拍摄,右侧(B)为暗视场下拍摄。
图11是采用PAUP*4.0b10程序绘制的几种病原真菌中MNH6同源蛋白的序列比较图。图示说明:MNH6、bcMNH6、fgMNH6、snMNH6和umMNH6分别为来自稻瘟病菌(Magnaporthe grisea)、灰葡萄孢菌(Botrytis cinerea)、禾谷镰刀菌(Fusariumgraminearum)、壳多胞菌(Stagonospora nodorum)或玉米黑粉病菌(Ustilago maydis)。
具体实施方式
下面结合附图对本发明的具体实施方式作进一步详细说明。
实施例1:与稻瘟病菌致病性相关的基因的克隆:
1、SSH文库筛选:稻瘟病菌附着胞cDNA与产孢菌丝cDNA差减后获得的差减cDNA,克隆到T载体,测序获得cDNA序列,将获得的序列与稻瘟病菌数据库比较,确定获得的差异表达基因,并用RT-PCR、qRT-PCR或Northern杂交等验证差异表达特异性。
2、MNH6 cDNA的克隆:取保存在-70℃的稻瘟病菌Guy11菌株的附着胞cDNA文库,根据SSH文库筛选获得的cDNA序列设计合成PCR引物,利用5’-RACE和3’-RACE的方法扩增包含整个基因编码区在内的基因cDNA片段,克隆到T载体,测序获得MNH6cDNA的序列,其序列如SEQ ID:2所示。
3、MNH6基因组DNA的克隆:取保存在-20℃的稻瘟病菌Guy11菌株的基因组DNA,根据预测的基因DNA序列设计合成PCR引物,采用长片段高保真PCR法扩增包含整个全长基因的DNA片段,克隆到T载体,测序获得MNH6基因组DNA的序列,其序列如SEQ ID:1所示。
实施例2:MNH6基因缺失突变子的获得和鉴定
1、基因敲除载体的构建:将MNH6基因上游和下游的长约500~2000bp的DNA片段分别***到含有潮霉素抗性基因的载体中潮霉素抗性基因的两侧,如图4和图6所示。具体如下:采用高保真PCR的方法分别从稻瘟病菌DNA基因组中扩增MNH6基因上游和下游的长约500~2000bp的DNA片段,先连接到T载体上;然后上游DNA片段用限制性内切酶KpnI和SalI从T载体上切下,***到含有潮霉素抗性基因的pBSHPH1载体的同样酶切位点上;接着下游DNA片段也从T载体上用限制性内切酶SpeI和SacI从T载体上切下,***到上述已经***上游DNA片段的pBSHPH1载体的SpeI和SacI位点上,即获得MNH6基因敲除载体。含有潮霉素抗性基因的pBSHPH1载体的构建过程:先采用高保真PCR的方法从pCB1003质粒中扩增到潮霉素抗性基因(HPH或HygBr),连接到T载体上;然后用限制性内切酶HindIII和SalI切下,并***到pBS质粒的同样酶切位点中,即获得pBSHPH1载体。
2、稻瘟病菌原生质载体的制备和DNA转化:稻瘟病菌菌株先在CM平板上生长6天左右。切取两个直径约3cm的菌落,置于150mL CM液体培养基中,用捣碎器将其捣碎;然后均匀分装到两瓶200mL CM液体培养基中,28℃,125rpm,培养48小时。四层纱布过滤收集菌丝,用无菌水冲洗两遍,将水分压干。菌丝在酶解液(Glucanex,酶液浓度7.5mg/mL,0.7M NaCl溶液)酶解2-3hr中30℃,80rpm。取出酶解液,三层滤纸过滤,0.7M NaCL洗涤,除去残渣,滤液收集到50mL的离心管中。4℃,3000rpm,离心10min。用10-20mL STC(1.2M Sorbitol,10mM Tris-HCL PH7.5,20mM CaCL2)悬浮沉淀。4℃,3000rpm,离心10min;重复两次。取适量的STC悬浮沉淀,使终浓度为0.5~1.0×108个/mL。每个50ml离心管分装150μl原生质体,加入2μg线性化DNA,室温下放置25min。逐滴加入1mL PTC(60%PEG4000,10mM Tris-HCL PH7.5,20mMCaCL2),混匀,室温放置25min。缓慢加入5mL OCM液体培养基(CM液体培养基中加入1.2M Sorbitol/1M Sucrose),轻轻摇匀,置于28℃,100rpm,摇过夜。每管过夜培养的原生质体中加入含有200μg/mL潮霉素的固体OCM Top培养基(CM液体培养基中加入20%Sucrose,琼脂粉1g),混匀,倒入已铺有OCM Bottom培养基(CM液体培养基中加入20%Sucrose,琼脂粉1.5g)的平板,每管铺三个平板。黑暗,28℃,培养约一周左右,挑取转化子,接种到含有200μg/mL潮霉素的固体CM上。
3、MNH6基因缺失突变子的鉴定:提取具有潮霉素抗性的转化子DNA,用PCR、Southern杂交等方法验证转化子。
实施例3、MNH6基因缺失突变子的互补恢复:
将MNH6基因的全长DNA序列连接到带有草胺磷抗性基因的载体中,如图5所示。具体为:采用高保真长片段PCR方法(LD PCR)从稻瘟病菌基因组DNA中扩增得到含有MNH6基因全长编码区、启动子区域和终止区区域的DNA片段,先***到pCR-XL-TOPO载体中,然后再用限制性内切酶NotI和BamHI从TOPO载体中切下该DNA片段,***到含有草胺磷抗性基因的pBARKS1载体的NotI和BamHI位点,即构建成MNH6基因的互补恢复载体——pS121-HB载体。在进行DNA的原生质转化前,pBARATG1载体在用限制性内切酶NotI酶切线性化。
然后按照实施例2的方法,将线性化的此MNH6基因的互补载体转化MNH6基因缺失菌株原生质体。挑取具有草胺磷抗性的转化子接种到含有草胺磷的固体CM上。提取具有草胺磷抗性的转化子DNA和RNA,用RT-PCR、Southern杂交等方法验证转化子。
通过以下方法比较了基因敲除和重新互补恢复后表型的变化状况:在完全培养基上比较了MNH6基因突变子、互补恢复转化子和野生型原始菌株GUY11的生长变化(见图7);在完全培养基上比较MNH6基因突变子、互补恢复转化子和野生型原始菌株GUY11的产孢量变化(见表1);比较了MNH6基因突变子、互补恢复转化子和野生型原始菌株GUY11的孢子形态的变化(见图8、表1);比较了MNH6基因突变子和野生型原始菌株GUY11的菌丝对细胞壁降解酶敏感性的变化(见图9);比较了MNH6基因突变子、互补恢复转化子和野生型原始菌株GUY11孢子的萌发变化(见表2);比较了MNH6基因突变子、互补恢复转化子和野生型原始菌株GUY11孢子的附着胞形成率和附着胞个体大小的变化(见表2);比较了MNH6基因突变子和野生型原始菌株GUY11形成的附着孢的膨压变化(见表2);互补转化子致病性的恢复见图1和图2。
以下对表1和表2进行具体说明:
表1是野生型菌株Guy11与MNH6基因缺失突变子Δmnh6及MNH6互补转化子s121hb1的产孢量和孢子大小比较。
表2是野生型菌株Guy11与MNH6基因缺失突变子Δmnh6及MNH6互补转化子s121hb1的致病相关性特征比较,包括孢子萌发率,附着胞大小,附着胞膨压和附着胞形成率。
表1突变子Δmnh6的产孢量和孢子大小分析
菌株 | 产孢量a | 孢子大小b | ||
L | W | L/W | ||
Guy11 | 3.9±1.5 | 21.0±2.0c | 8.3±0.6 | 3.1±0.4 |
Δmnh6 | 2.2±0.7 | 24.1±2.9 | 7.6±0.9 | 4.0±0.8 |
s121hb1 | 5.1±2.2 | 22.1±2.0 | 8.5±0.5 | 3.3±0.4 |
注:
Guy11:为野生型菌株;Δmnh6:为MNH6基因缺失突变子;s121hb1,基因互补恢复突变子。
a,产孢量单位为×105个孢子/每个7mm直径的菌丝块;b,孢子大小单位μm。L,孢子长度;W,孢子宽度;L/W,孢子的长/宽比例。
表2突变子Δmnh6的致病相关性特征分析
菌株 | 致病相关性特征① | |||
孢子萌发率② | 附着胞大小③ | 附着胞膨压④ | ||
2h | 4h | 24h | 24h | |
Guy11 | 93.78±4.17a | 99.07±0.64a | 9.7±0.8a | 22.0±7.6b |
Δmnh6 | 92.97±3.01a | 99.00±0.15a | 8.9±0.9b | 57.8±6.7a |
s121hb1 | 91.59±1.69a | 98.84±0.30a | N/R | N/R |
附着胞形成率⑤ | ||||
6h | 12h | 24h | 48h | |
Guy11 | 86.64±5.16a | 94.47±2.54a | 98.52±0.75a | 98.78±0.96a |
Δmnh6 | 65.32±4.58b | 72.48±4.84b | 74.44±0.55b | 77.36±4.52b |
s121hb1 | 87.34±3.18a | 92.06±0.75a | 96.33±4.01a | 98.22±1.92a |
注:
Guy11:为野生型菌株;Δmnh6:为MNH6基因缺失突变子;s121hb1,基因互补恢复突变子。
①.Duncan′s新复极差测验,不同小写字母为差异达显著水平(p≤0.05)。
②.20μl浓度为1×105/ml的孢子接种塑料盖玻片,2h和4h后统计孢子的发芽率(%)。
③.附着胞直径单位:μm。
④.用2M甘油处理附着胞(孢子诱导24小时)后,附着胞的塌陷率(%)。
⑤.孢子在塑料盖玻片上诱导不同时间后,附着胞的形成率(%)。
实施例4:MNH6基因缺失突变子和互补突变子的致病性测定:
1、喷雾接种:大麦或水稻CO39种子种于花钵泥土中,培养8天(大麦)或14天(3~4叶期的水稻CO39植株)。从培养12天的CM平板上洗取稻瘟病菌野生型菌株、突变子和互补转变子的孢子,孢子浓度用0.2%(wt/v)的明胶稀释至1×105/ml。用微型的油漆喷雾器将孢子悬浮液喷雾接种到植株叶片表面。同时,以0.2%的明胶作为阴性对照喷雾接种。接种植物放在一个潮湿箱子内,于25℃黑暗环境中保湿培养24小时(大麦)或48小时(水稻)。然后接种植物放入人工气候箱(25℃,80%以上湿度,12小时光照周期)内继续培养至第4天(大麦)或第7天、第14天(水稻),使植物病症充分显示。拍照记录植物叶片的稻瘟病症状,并根据Bonman等(1986)报道的方法记录和评价植物的病症严重程度。同时,从每株受试植物中挑选一片受感染最严重的叶片,记录叶片上5cm长的一段区域内的稻瘟病菌病斑。结果见图1。
2、离体接种:大麦种子种于花钵泥土中,培养7~10天。从培养12天的CM平板上洗取稻瘟病菌野生型菌株、突变子和互补转变子的孢子,稀释至孢子浓度为5×104/ml或1×105/ml。孢子液20μl一滴点接种于离体大麦叶片上表面。在人工气候箱内保湿光照培养(光照/黑暗,12h∶12h),25℃。分别在培养24h,48h,72h和96h后,取出部分叶片观察。先观察病斑形成情况,并拍照记录。然后剪取部分病斑放入甲醇溶液中固定并脱去叶绿素。病斑叶片在甲醇溶液中固定24小时(室温)后,移入乳酚油固定透明液(乳酚油∶95%乙醇=1∶2)室温固定1小时。乳酚油配方为:乳酸20ml、苯酚20ml、20%甘油40ml、蒸馏水20ml。病斑叶片再用苔酚蓝/苯胺蓝(0.01%苔酚蓝/0.06%苯胺蓝,乳酚油)染色5~10分钟。染色叶片在乳酚油中脱色。在显微镜下观察病斑发展情况,统计24h和48h的附着胞穿透率、以及侵入菌丝的生长和次生孢子的产生情况,并拍照记录。结果见图2。
实施例5:MNH6的亚细胞定位:
将GFP基因的完整编码区序列连接到不带终止密码子的MNH6基因编码区的下游,构建成融合基因并连接到带有潮霉素抗性基因的载体中。按照实施例2的方法,将线性化的此载体转化野生型Guy11菌株原生质体。挑取具有潮霉素抗性的转化子在荧光显微镜下观察MNH6-GFP融合蛋白在稻瘟病菌细胞中的分布。结果表明MNH6定位于细胞质中。结果见图10。
实施例6:MNH6启动子的界定和表达分析:
1)将GFP基因的完整编码区序列连接到MNH6基因编码区上游(不含编码区序列)的一段DNA序列后,构建成融合基因并连接到带有潮霉素抗性基因的载体中。按照实施例2的方法,将线性化的此载体转化野生型Guy11菌株原生质体。挑取具有潮霉素抗性的转化子在荧光显微镜下观察MNH6启动子指导下的GFP蛋白在稻瘟病菌不同发育阶段的表达情况。或者2)将潮霉素抗性基因(HPH)的完整编码区序列连接到MNH6基因编码区上游(不含编码区序列)的一段DNA序列后,构建成融合基因并连接到带有草胺磷抗性基因的载体中。按照实施例2的方法,将线性化的此载体转化野生型Guy11菌株原生质体。挑取具有草胺磷抗性的转化子在具有不同浓度潮霉素的培养基中观察MNH6启动子指导下的HPH蛋白的表达以及菌落的生长情况。MNH6启动子序列见SEQ ID NO:4所示。
实施例7:真菌MNH6蛋白的生物信息学分析:
为了明确MNH6蛋白在其他病原真菌中是否保守,针对下列病原真菌:灰葡萄孢菌(Botrytis cinerea)、禾谷镰刀菌(Fusarium graminearum)、壳多胞菌(Stagonosporanodorum)或玉米黑粉病菌(Ustilago maydis),将MNH6蛋白的氨基酸序列对这些真菌的蛋白序列进行Blastp检索,获得上述病原真菌的同源蛋白的氨基酸序列,其序列分别如SEQ ID 5、6、7和8所示,并将这些蛋白分别命名为bcMNH6、fgMNH6、snMNH6和umMNH6。序列分析表明上述真菌的MNH6同源蛋白在氨基酸序列上与MNH6蛋白的一致性都在35%以上。利用PAUP*4.0b10程序绘制的上述真菌的ATG1蛋白的同源性关系图见图11。
实施例8:利用MNH6蛋白的表达或修饰作为靶标,筛选抗真菌的药物:
把受MNH6基因调控的下游基因(如稻瘟病菌中GAL1和SUC2基因的同源基因)的启动子与荧光蛋白GFP构建成融合蛋白载体,通过实施例2的方法转入到稻瘟病菌中,然后把在下游基因的启动子调控下的GFP表达的稻瘟病菌转化子菌株在液体完全培养基中大量培养,收集菌丝后分成若干小份,每一份中加入待筛选的化合物或候选药物继续在完全培养基培养数小时至数天,取菌丝在荧光显微镜下观察菌丝的GFP的绿色荧光表达情况。如果MNH6蛋白被候选药物抑制而失去活性,则失去对下游基因的启动子表达的调控能力,GFP蛋白不表达从而菌丝失去绿色荧光,说明该侯选药物具有抑制MNH6蛋白的作用。获得的具有抑制MNH6蛋白功能的候选药物再利用实施例4的方法,在接种的孢子液中加入侯选药物,测定野生型稻瘟病菌在这种侯选药物存在的条件下对水稻的致病性有没有减弱,进一步确定化合物的抗真菌效果。当测定结果为野生型稻瘟病菌株对水稻的致病性减弱时,说明候选药物具有明显的抗真菌效果,可以作为抗真菌药物的来源;当测定结果为野生型稻瘟病菌株对水稻的致病性没有变化或增强时,说明侯选药物没有抗真菌效果,不能用作抗真菌药物。
实施例9:利用MNH6的表达作为靶标,筛选抗真菌的药物。
把稻瘟病菌在液体完全培养基中大量培养,收集菌丝后分成若干小份,每一份中加入待筛选的化合物或候选药物继续在液体完全培养基培养数小时,提取菌丝的RNA,采用实时定量PCR的方法检测MNH6的表达状况。如果MNH6的表达被候选药物抑制,则菌丝细胞内没有MNH6的转录本或减少。获得的化合物再利用实施例4的方法,测定野生型稻瘟病菌在这种化合物存在的条件下对水稻的致病性有没有减弱,进一步确定化合物的抗真菌效果。
实施例10:利用MNH6的启动子作为靶标,筛选抗真菌的药物。
把MNH6的启动子与荧光蛋白GFP构建成融合蛋白载体,或把MNH6的启动子与荧光蛋白GFP以及MNH6蛋白一起构建成融合蛋白载体,通过实施例2的方法转入到稻瘟病菌中,然后把在MNH6的启动子调控下的GFP表达的稻瘟病菌转化子菌株在液体完全培养基中大量培养,收集菌丝后分成若干小份,每一份中加入待筛选的化合物或候选药物继续在完全培养基培养数小时至数天,取菌丝在荧光显微镜下观察菌丝的GFP的绿色荧光表达情况。如果MNH6的启动子的被化合物抑制而失去活性,则GFP蛋白不表达从而菌丝失去绿色荧光。获得的化合物再利用实施例4的方法,测定野生型稻瘟病菌在这种化合物存在的条件下对水稻的致病性有没有减弱,进一步确定化合物的抗真菌效果。
最后,还需要注意的是,以上列举的仅是本发明的若干个具体实施例。显然,本发明不限于以上实施例,还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形,均应认为是本发明的保护范围。
序列表
SEQ ID NO:1
TTTACCCAGC CGGCTTTCTT TCTCCACAAT TATTCCCATC AATGCTCTCA GGTGGCCACA 60
CAAGTGGGGC TTCGTCTTGA ATTTCTTACT TTGTGTCAAG GTGTGTACAG TACGGTTTTT 120
TACCGGAGTA CCTCGCCTCG CACCACACCC ACCACCACGA CCCGTGTCCC TATCGCCCGA 180
GTTTCTGACA TGCCTGGATA CTTCGGCTCC CTAGCATGAA TTGATCATAC TTGCGTGTTG 240
ATAAGACGCC TAACAGCATG GGTTCAAAAT TGGAGTAGGG AAACCCTGGC TGAGGAGGAG 300
CATCTAGCAG TAGCTACCCA ATAATTGATG CCAGACTCAC ATTTACCAAG GTGTCAAGCT 360
ACCGGTACAG TAACCTTAGT TGCTTACGTA CGGGGCTAAA CAAGTTGTGC TGTGGGGAGA 420
GGATACCCCG GCTTCCGATG TACCGGTACG CGGCCAAACA CGCAGTGGTG TACAGTAACA 480
ATAGTCTGTC CTCCTGCACA AGTACCTCCA CACCAAGACA ACAAGCGCGA TTTGTCTGGA 540
CTTGGGATTT GCTTGGGGCA GGAACTTTTA CGCATTCCTT GTTAACCTGC AAACTCTGTT 600
GCCTTGCCAA TTCCGAGTAT TTGAGGGTGG CTCGAATCCA TCGAGTCAGC GACAAACGGT 660
ACGATATTAC AGAGCACGTG AGCTGAGACT GTTTCTAGCT ATTCGCACAA GTTACCTGAG 720
GTAGGTAGGT AGGTAGGTAG GTAGGTACCT TATGTATGCT ATGCTACCTA CCGGTATGCT 780
ACCTCCCTAC TTCGTAGTTA CAGGGCTCCC AGTGTTAGAT CACAACCAAC CGCAATGGCT 840
CAACATACCA GGTTGCCGTA AACTGACCTG ATTGGCGCGC ACTTGTTTTC TAATGCAAGT 900
GTTGATCTGC TATGTCGAAC GATTGAGGAA CCAGATAATA ATGAGGAAGG GGGCAGAATG 960
TGGGAACTGG TTGAGTTGAG CTCTGCTTGT CTGTCAAACA CGGCTGTCTG TACACAGGTA 1020
CAAAGGAACC TTGGTCAACC GAGAAAGTCT GCGAATGAAA TTCAGCGCCC TCAAACCTCC 1080
CGTCTCACAT AGCGGCGGAT ACTTCCCCAA CGGGCTGTGA GTGAGTGACA TTTTCCCGCT 1140
TGCTTCTCTT GCGGGCTCGA GCCGCCGCAA AGCCGTAAGA GGAGAGTGAG ATTAAAATCA 1200
CCTAAGAGGG ATTGCCGGGA TGGGATGGCC TCCACAGCAA CGACGGATTT CTGATTGACA 1260
TTGATGGAAT GGATGGCAGC CAAGGCAGGG TGATGATAGG CAGAAGCAAG CCAAAAGGTG 1320
AGCGGGTGGG CAATCGAGTC CCCGCTACTG GCTCTCCTAG GAAAAGTGCC AAAAGTCTCG 1380
TTTGAAAAAA TGAGGGGAGA TCAAAAGAAG TGGTGGCCCG GGAGCCAGGG ATGTACCCGT 1440
ACCTGGAACG TAGCGGTACG TTATGTGTCC GTTCTACTTG CCTGAGTAAA GTGAGCGGGC 1500
AAAAGCCGGC GTTGCCTTGG TGTCGCAGCA CCGTGCAAAT CCTTGGGTGA AGGTACCTTG 1560
CTTGCTTGCC TTTTGGTCGT CACATAATCG CCCCCCATCC CTATTTTGTA CTTACTGATT 1620
TGTAGGAAGA AGCCACCGCA GGTACCTGAC CTTCCGTGAG GGACACCATG TAGGTAATTT 1680
AGGTGCATAC CTTACCTACA GTACCTATCG TGCAACATGA GCAACCTATC TAAAAAGTTG 1740
ATAAAGTATA CTTTGGTTTA GGTATTGTAC TAGAAATAAA GATTAAATCT AGGAAGATTT 1800
TTTTTTAATA AAAAAAAGTC TTTAATGTGC ACCTAGTAGC AATTGCATGC GCAAGCAATT 1860
GGCTACAAAT TATGCATCGA TGTGTTGCGC CGTCTGGGTT GTACTGTAGG TAAGTAAGGT 1920
ACCTTAGCTT TCAGACAAAA GAGGGAAAGT ACGAGAGGAA AATCGAATTG AACTGGCACG 1980
GAAGGGAAGG GAAGGGAGTG CGAGAGACGG AGACTCAAGC TCGAACCAGC CAGCCCCCAG 2040
CCAGCTGCCC CTCCCTCTTT GGTGGCCTGC CTGCCAGGTG CGTGTGGGCG CGCGCGCGCT 2100
CTTCTCTTGC TTTGGTGTGG GCGAGGGTAC CAGCGCGAGG CAAGCTCCCC TCCCAAATTG 2160
GCCCAGTCCC TCACAATCAG CCGGCCCATA GTAAAAAGGC GCTCCCTCTA ACCTCCCCCC 2220
ATACAATTTT CCTCTGCCTC TCACTCTTTC TACACAGCCA AATCTCCCAT ACAAAATCCG 2280
CACCCAATTC TCATCGCAAC GCAGTGAGTA CCTCCGAATC CGCCTGCACA CACACCAAAA 2340
CACAAGTACC TCCCTACTTC CACTCTACCC ATCACCGCAA TCATCGCAGC AGCCAGCCAG 2400
CCCAATTGCC CTCTGCACCT ATTCATACAA GCCGCGCATT GAGCATCATT GGAACGCCCG 2460
TTGCCCGCGC ATCGTACCAG CGCAGTGGTG CGTTCCTTTG AGGACTTTCG CGTCGTCGCC 2520
GTTGCCAAAA GTCCAAAAAA TCTCAAACGG ATTTCACAAC CGCTTCGGTC GCATGCAGAG 2580
CTTTTAAACC ATCTGCACCT GCCTTCATCT TGTTCTTCCT GCACCTCAAC TACAACCCCT 2640
ACACCGTGAC AAAGCAACGG GCCCATCAGC AACTCCATCG ATACCCGCCA TTCGGCATCA 2700
TCATCATCTA CAACAACTGA CCGGACAACC AAACAGGCAA TTGCACAATT AAACACCACA 2760
TTTCGACTTT TCTCAAAAAC CTCTTAAAGG TACGTAACGT GGGCTCAGTC AACCGATTCC 2820
AAGCTTCGAA TCCTTATTTC TACACTCAAT CGGCTTCAAT TGAGATTGCG ACAACGTCCA 2880
CTTTGCTCTG CGCTCAATTC AACGCCAGCC GAGGGACGCG TCAAGCTCCG TCACTCCCAA 2940
ATCACTCTCA CCTCACGACG CGTCAATCAG GCACCAGACC ACATCCCACT TCCACCCACT 3000
TTATCGCACC TCACATTAGC CCCAAAAACC CACCACCTAA CAACACCAAC CCATTCACAC 3060
CAATCATACA TTTGACAGAC AGAATCTAAC AACCACACAG TCAAAGCCAT TCACCATGCC 3120
TAAGGCCGCT ACTAAGAAGA GCGCCAAGCC TGAGAAGCGC CGCGCCAAGA AGGACCCTAT 3180
GGCCCCCAAG CGTGGTCTCT CCGCATACAT GTTCTTCGCC AACGAGCAGC GTGACAACGT 3240
CAGGGAGGAG AACCCCGGTG TCACATTCGG CCAAGTTGGC AAGATCCTGG GTGAGCGCTG 3300
GAAGGCTCTG AGCGACAAGC AGAGGGCGCC ATACGACGCC AAGGCTGCTG CCGACAAGAA 3360
GAGATACGAG GACGAGAAGG CTGCTTACCA GGTGAGTTCC TTTTGACTTT GGTCCCTCAA 3420
ATTCAGAAAG GCAATCCGCT AACCTCATCA ATCTAGGCTG GCGGCGACGA GGATGAGTCC 3480
TCTTGATTCT CGCCTGATCG TTTGCCTGCG CGAACCATTC TCCAGGGCTG AAAGACCATT 3540
GAGATGATCA AATCATGTCT CCAGCTCGTC AGATCATGAA CCACCATTCT GATCGGGCGG 3600
AATGCCACTG GCGCGCTATT TTCTTTCATG TCACTTTGGG TTTGGTCAGG TCAGGTGTTG 3660
AGTGTTAGCC ACGGACTGGG TTCTCTTTTT TCGGCATTTG TTATCGTAGC TTTTTCGATC 3720
TGTTCTACTT TTTTTTTCTC TTGATGAAGG GGGGAATACC CCTATGGCCG GAGGGGAATG 3780
GGGTAAGGGA AAGGATTCCC ACTTCATCGG GCTTGGACGA TGGTATTATG ATTTGTTTTT 3840
CCAGAGATAC CACACCATCA TCTTACTCCA CCGTCAGGAA GTGAGAATAA GACTTTGGGA 3900
AAACGGCAGC CCAAGCCATC ACACAAACTT CGATGAGCTG TATCACTAGA TTAAAAGGCG 3960
ACGCCGAACG GCTTTTCCTT GCTAAATACA GTGAAAACCG TTTCTGAGCA ACGAACTTTT 4020
TATCACATGC AATGGCTAGA GTTCCTCTCA CGCAATACCC AACAAGACCC AAAATACATG 4080
GGTAGTCGTG AATTCTCCCT TCATCCTGTC GGTGCTTGGT GTTGGTTCTT GCATACTCTT 4140
AGTCAGCACT AATACCCAAA GAGGCTCATG TTGCATCATT ACCTTGGAGA GCAAAATTAG 4200
TCTGCACGTC GTCATCTGTA ATAAGAATAA ACTCGACTAT AACTTTAAAA TTTAGGGAGG 4260
CCCTGTCCGT CCCATGTACA TTCCATGTGT GGGTATCAAA AGTAACTAAT AAACTGACAT 4320
GTCATATTCC GTTAAAGTGT ACAGTAGTCA GAAAGGAGTT AAGTAAACAG CGCTGATGAT 4380
TTCTATCCTG GACAAAGTAT AAATTGAGAA TTAGGGCCCC ATAGCACCTC CCTCTTACGT 4440
AGTGTTGGTA CCTTGGCGAG GCGGCGGGCT GTCGCCAGGC AATTCGTGGA TTACGTGCGG 4500
ATGGTATAGC TCTGCCGGAG CCACATGTGG TCGATATGGG GAGTGAGCTG ATTCGCAGCG 4560
GTGGCGTTAG TAGTCTTGAC CAAACAGAAA ATTGCAAAAC AGGACTACTT GATCAAGCAA 4620
AGAGAAATGA TACCAATAGA AAAATTCATG GTGCCACGCA TACCTTGTTG CATAGGTGTC 4680
GTCGACTGCG GAGTGCCATG CACGCTACTA TGGGTGGAGT GAGGGTACCA GCCCTGTTTT 4740
TGCGTCTGCT GCTGCTGCTG CTGCTGCTCC ACGCTAAAAG GTGTATTGGT ATCTGCCGGG 4800
CT 4802
SEQ ID NO:2
CCAAATCTCC CATACAAAAT CCGCACCCAA TTCTCATCGC AACGCAGCAA TTGCACAATT 60
AAACACCACA TTTCGACTTT TCTCAAAAAC CTCTTAAAGT CAAAGCCATT CACCATGCCT 120
AAGGCCGCTA CTAAGAAGAG CGCCAAGCCT GAGAAGCGCC GCGCCAAGAA GGACCCTATG 180
GCCCCCAAGC GTGGTCTCTC CGCATACATG TTCTTCGCCA ACGAGCAGCG TGACAACGTC 240
AGGGAGGAGA ACCCCGGTGT CACATTCGGC CAAGTTGGCA AGATCCTGGG TGAGCGCTGG 300
AAGGCTCTGA GCGACAAGCA GAGGGCGCCA TACGACGCCA AGGCTGCTGC CGACAAGAAG 360
AGATACGAGG ACGAGAAGGC TGCTTACCAG GCTGGCGGCG ACGAGGATGA GTCCTCTTGA 420
TTCTCGCCTG ATCGTTTGCC TGCGCGAACC ATTCTCCAGG GCTGAAAGAC CATTGAGATG 480
ATCAAATCAT GTCTCCAGCT CGTCAGATCA TGAACCACCA TTCTGATCGG GCGGAATGCC 540
ACTGGCGCGC TATTTTCTTT CATGTCACTT TGGGTTTGGT CAGGTCAGGT GTTGAGTGTT 600
AGCCACGGAC TGGGTTCTCT TTTTTCGGCA TTTGTTATCG TAGCTTTTTC GATCTGTTCT 660
ACTTTTTTTT TCTCTTGATG AAGGGGGGAA TACCCCTATG GCCGGAGGGG AATGGGGTAA 720
GGGAAAGGAT TCCCACTTCA TCGGGCTTGG ACGATGGTAT TATGATTTGT TTTTCCAGAG 780
ATACCACACC ATCATCTTAC TCCACCGTCA GGAAGTGAGA ATAAGACTTT GGGAAAACGG 840
CAGCCCAAGC CATCACACAA ACTTCGATGA GCTGTATCAC TAGATTAA 888
SEQ ID NO:3
Met Pro Lys Ala Ala Thr Lys Lys Ser Ala Lys Pro Glu Lys Arg
1 5 10 15
Arg Ala Lys Lys Asp Pro Met Ala Pro Lys Arg Gly Leu Ser Ala
20 25 30
Tyr Met Phe Phe Ala Asn Glu Gln Arg Asp Asn Val Arg Glu Glu
35 40 45
Asn Pro Gly Val Thr Phe Gly Gln Val Gly Lys Ile Leu Gly Glu
50 55 60
Arg Trp Lys Ala Leu Ser Asp Lys Gln Arg Ala Pro Tyr Asp Ala
65 70 75
Lys Ala Ala Ala Asp Lys Lys Arg Tyr Glu Asp Glu Lys Ala Ala
80 85 90
Tyr Gln Ala Gly Gly Asp Glu Asp Glu Ser Ser
95 100
SEQ ID NO:4
GAGCCGCCGC AAAGCCGTAA GAGGAGAGTG AGATTAAAAT CACCTAAGAG GGATTGCCGG 60
GATGGGATGG CCTCCACAGC AACGACGGAT TTCTGATTGA CATTGATGGA ATGGATGGCA 120
GCCAAGGCAG GGTGATGATA GGCAGAAGCA AGCCAAAAGG TGAGCGGGTG GGCAATCGAG 180
TCCCCGCTAC TGGCTCTCCT AGGAAAAGTG CCAAAAGTCT CGTTTGAAAA AATGAGGGGA 240
GATCAAAAGA AGTGGTGGCC CGGGAGCCAG GGATGTACCC GTACCTGGAA CGTAGCGGTA 300
CGTTATGTGT CCGTTCTACT TGCCTGAGTA AAGTGAGCGG GCAAAAGCCG GCGTTGCCTT 360
GGTGTCGCAG CACCGTGCAA ATCCTTGGGT GAAGGTACCT TGCTTGCTTG CCTTTTGGTC 420
GTCACATAAT CGCCCCCCAT CCCTATTTTG TACTTACTGA TTTGTAGGAA GAAGCCACCG 480
CAGGTACCTG ACCTTCCGTG AGGGACACCA TGTAGGTAAT TTAGGTGCAT ACCTTACCTA 540
CAGTACCTAT CGTGCAACAT GAGCAACCTA TCTAAAAAGT TGATAAAGTA TACTTTGGTT 600
TAGGTATTGT ACTAGAAATA AAGATTAAAT CTAGGAAGAT TTTTTTTTAA TAAAAAAAAG 660
TCTTTAATGT GCACCTAGTA GCAATTGCAT GCGCAAGCAA TTGGCTACAA ATTATGCATC 720
GATGTGTTGC GCCGTCTGGG TTGTACTGTA GGTAAGTAAG GTACCTTAGC TTTCAGACAA 780
AAGAGGGAAA GTACGAGAGG AAAATCGAAT TGAACTGGCA CGGAAGGGAA GGGAAGGGAG 840
TGCGAGAGAC GGAGACTCAA GCTCGAACCA GCCAGCCCCC AGCCAGCTGC CCCTCCCTCT 900
TTGGTGGCCT GCCTGCCAGG TGCGTGTGGG CGCGCGCGCG CTCTTCTCTT GCTTTGGTGT 960
GGGCGAGGGT ACCAGCGCGA GGCAAGCTCC CCTCCCAAAT TGGCCCAGTC CCTCACAATC 1020
AGCCGGCCCA TAGTAAAAAG GCGCTCCCTC TAACCTCCCC CCATACAATT TTCCTCTGCC 1080
TCTCACTCTT TCTACACAGC CAAATCTCCC ATACAAAATC CGCACCCAAT TCTCATCGCA 1140
ACGCAGTGAG TACCTCCGAA TCCGCCTGCA CACACACCAA AACACAAGTA CCTCCCTACT 1200
TCCACTCTAC CCATCACCGC AATCATCGCA GCAGCCAGCC AGCCCAATTG CCCTCTGCAC 1260
CTATTCATAC AAGCCGCGCA TTGAGCATCA TTGGAACGCC CGTTGCCCGC GCATCGTACC 1320
AGCGCAGTGG TGCGTTCCTT TGAGGACTTT CGCGTCGTCG CCGTTGCCAA AAGTCCAAAA 1380
AATCTCAAAC GGATTTCACA ACCGCTTCGG TCGCATGCAG AGCTTTTAAA CCATCTGCAC 1440
CTGCCTTCAT CTTGTTCTTC CTGCACCTCA ACTACAACCC CTACACCGTG ACAAAGCAAC 1500
GGGCCCATCA GCAACTCCAT CGATACCCGC CATTCGGCAT CATCATCATC TACAACAACT 1560
GACCGGACAA CCAAACAGGC AATTGCACAA TTAAACACCA CATTTCGACT TTTCTCAAAA 1620
ACCTCTTAAA GGTACGTAAC GTGGGCTCAG TCAACCGATT CCAAGCTTCG AATCCTTATT 1680
TCTACACTCA ATCGGCTTCA ATTGAGATTG CGACAACGTC CACTTTGCTC TGCGCTCAAT 1740
TCAACGCCAG CCGAGGGACG CGTCAAGCTC CGTCACTCCC AAATCACTCT CACCTCACGA 1800
CGCGTCAATC AGGCACCAGA CCACATCCCA CTTCCACCCA CTTTATCGCA CCTCACATTA 1860
GCCCCAAAAA CCCACCACCT AACAACACCA ACCCATTCAC ACCAATCATA CATTTGACAG 1920
ACAGAATCTA ACAACCACAC AGTCAAAGCC ATTCACC 1957
SEQ ID NO:5
>bcMNH6 Botrytis cinerea灰葡萄孢菌
Met Asp Ala Asp Ser Ser Cys Ser Thr Asp Thr Ser Lys Ser Ala
1 5 10 15
Asn Leu Lys Asp His Ser Pro Thr Asn Ser Gln Ala Pro Ala Arg
20 25 30
Thr Glu Gln Thr Ser Thr Ser His Ile Thr Asn Arg Glu Asp Thr
35 40 45
Glu Glu Gln Ser Ile Pro Ser Asn Leu Ser Pro Ser Ser Ile Thr
50 55 60
Pro Pro Lys Ile Lys Met Arg Ile Thr Pro Thr Leu Ile Ser Gly
65 70 75
Pro Leu Asn His Glu Ile Pro Thr Arg Asn Ser Ser Ser Ile Pro
80 85 90
Ser Ile Ser Ala Ala Pro Thr Asp Pro Tyr Val His Gln Pro Gln
95 100 105
Leu Asn Leu Ser Glu Met Gln Asn Leu Val His Glu Arg Gln Val
110 115 120
Gln Thr Thr Met Pro Pro Asn Ile Ala Gln Ser Thr Pro Ser Asn
125 130 135
Leu Ala Arg His Phe Gln Arg Ile Ser Leu Gln Asn Lys Arg Lys
140 145 150
Asp Pro Ser Ala Pro Lys Arg Gly Ile Ser Ala Tyr Met Phe Tyr
155 160 165
Ala Asn Asp Gln His Asp Arg Val Arg Gln Glu Asn Pro Ala Leu
170 175 180
Ser Phe Gly Gln Leu Gly Ile Leu Leu Gly Glu Glu Trp Arg Ala
185 190 195
Leu Ser Val Gly Gln Arg Ser Val Tyr Glu Glu Met Ala Thr Lys
200 205 210
Asp Leu Arg Arg Tyr Glu Glu Glu Leu Ala Arg Tyr Arg Gly
215 220
SEQ ID NO:6
>fgMNH6 Fusarium graminearum 禾谷镰刀菌
Met Pro Lys Ala Ala Ala Pro Ala Lys Arg Ala Thr Arg Thr Lys
1 5 10 15
Arg Ala Lys Lys Asp Pro Asn Ala Pro Lys Arg Gly Leu Ser Ala
20 25 30
Tyr Met Phe Phe Ala Asn Glu Gln Arg Glu Asn Val Arg Glu Glu
35 40 45
Asn Pro Gly Ile Ser Phe Gly Gln Val Gly Lys Leu Leu Gly Glu
50 55 60
Arg Trp Lys Ala Leu Asn Glu Lys Gln Arg Ala Pro Tyr Glu Ala
65 70 75
Lys Ala Ala Ala Asp Lys Lys Arg Tyr Glu Asp Glu Lys Gln Ala
80 85 90
Tyr Asn Ala Asp Gln Glu Glu Glu Glu Ser Ser
95 100
SEQ ID NO:7
>snMNH6 Stagonospora nodorum壳多胞菌
Met Lys Ser Lys Gln Arg Leu Ala Ser Ser Met Met Trp Ser Met
1 5 10 15
Cys Arg Glu Thr Ala Leu Arg Ser Arg Ala Glu Asp Glu Met Val
20 25 30
Arg Thr Arg Arg Glu His Thr Ala Ser Tyr Leu Ser Gln Pro Leu
35 40 45
Asp Phe Ile Ala Phe Leu Gln Leu Leu Pro Gln Leu His Pro His
50 55 60
Ile Asn Met Pro Lys Glu Lys Val Thr Arg Gly Lys Gly Arg Ala
65 70 75
Thr Lys Ala Asp Gly Gly Lys Lys Lys Lys Asp Pro Asn Ala Pro
80 85 90
Lys Arg Gly Leu Ser Ala Tyr Met Phe Phe Ala Asn Glu Gln Arg
95 100 105
Asp Lys Val Arg Glu Asp Asn Pro Gly Ile Lys Phe Gly Glu Val
110 115 120
Gly Lys Met Leu Gly Glu Lys Trp Lys Ala Leu Asn Glu Lys Gln
125 130 135
Arg Thr Pro Tyr Glu Ala Lys Ala Ala Ala Asp Lys Lys Arg Tyr
140 145 150
Glu Glu Glu Lys Ala Ala Tyr Gln Ala Gly Glu Glu Glu Glu Glu
155 160 165
Glu Ser Glu
SEQ ID NO:8
>umMNH6 Ustilago maydis玉米黑粉病菌
Met Pro Ser Ser Phe Leu Ser Thr Gln Ser Ser Phe Ala Ser Val
1 5 10 15
Leu Ile Val Ser Pro Ala Arg Ala Ile Val Lys Met Ala Lys Ala
20 25 30
Asp Thr Lys Thr Lys Ser Ser Thr Ser Thr Gln Lys Arg Thr Thr
35 40 45
Lys Ala Lys Lys Asp Pro Asp Ala Pro Lys Arg Pro Leu Ser Ala
50 55 60
Tyr Met Phe Phe Ser Gln Asp Gln Arg Glu Arg Val Lys Asn Ala
65 70 75
Asn Pro Glu Ala Gly Phe Gly Glu Val Gly Arg Leu Leu Gly Ala
80 85 90
Lys Trp Lys Glu Met Ser Glu Ala Glu Lys Lys Pro Tyr Asn Asp
95 100 105
Met Ala Asn Arg Asp Lys Ala Arg Ala Glu Ala Glu Lys Ala Ala
110 115 120
Tyr Asn Lys Arg Arg
125
Claims (6)
1、一种源于稻瘟病菌的真菌致病性基因MNH6,其特征是:该基因的核苷酸序列或其互补链的核苷酸序列为SEQ ID NO:1。
2、如权利要求1所述的基因MNH6编码的cDNA序列,其特征是:所述cDNA序列具有SEQID NO:2所示的核苷酸序列。
3、如权利要求1所述的基因MNH6编码的蛋白质,其特征是:所述蛋白质具有SEQ IDNO:3所示的氨基酸序列。
4、如权利要求1所述的基因MNH6的启动子,其特征是:所述启动子具有SEQ ID NO:4所示的核苷酸序列。
5、利用如权利要求1所述的基因MNH6的表达作为靶标,在设计和筛选抗真菌药物中应用。
6、利用如权利要求3所述的蛋白质的表达和修饰作为靶标,在设计和筛选抗真菌药物中应用。
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106047901A (zh) * | 2016-06-08 | 2016-10-26 | 浙江省农业科学院 | 源于稻瘟病菌的真菌致病性基因Moarrdc1及用途 |
CN108546708A (zh) * | 2018-04-19 | 2018-09-18 | 贵州大学 | 一种丝状真菌基因敲除突变体的筛选方法 |
CN110714030A (zh) * | 2019-12-03 | 2020-01-21 | 中国农业大学 | 一种长喙壳菌外源基因转化的方法 |
CN111534525A (zh) * | 2020-05-22 | 2020-08-14 | 山东农业大学 | 禾谷镰刀菌FgARL1基因及其应用 |
CN115043834A (zh) * | 2022-05-12 | 2022-09-13 | 中国农业大学 | 一种萘啶酰胺类化合物及其制备方法与应用 |
-
2007
- 2007-02-15 CN CN200710067275A patent/CN100591767C/zh not_active Expired - Fee Related
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106047901A (zh) * | 2016-06-08 | 2016-10-26 | 浙江省农业科学院 | 源于稻瘟病菌的真菌致病性基因Moarrdc1及用途 |
CN108546708A (zh) * | 2018-04-19 | 2018-09-18 | 贵州大学 | 一种丝状真菌基因敲除突变体的筛选方法 |
CN110714030A (zh) * | 2019-12-03 | 2020-01-21 | 中国农业大学 | 一种长喙壳菌外源基因转化的方法 |
CN111534525A (zh) * | 2020-05-22 | 2020-08-14 | 山东农业大学 | 禾谷镰刀菌FgARL1基因及其应用 |
CN115043834A (zh) * | 2022-05-12 | 2022-09-13 | 中国农业大学 | 一种萘啶酰胺类化合物及其制备方法与应用 |
CN115043834B (zh) * | 2022-05-12 | 2023-06-06 | 中国农业大学 | 一种萘啶酰胺类化合物及其制备方法与应用 |
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