CN111534501A - 一种水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk及其应用 - Google Patents
一种水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk及其应用 Download PDFInfo
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- CN111534501A CN111534501A CN202010323333.3A CN202010323333A CN111534501A CN 111534501 A CN111534501 A CN 111534501A CN 202010323333 A CN202010323333 A CN 202010323333A CN 111534501 A CN111534501 A CN 111534501A
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- sheath blight
- rice sheath
- protein kinase
- rsmapk
- kinase gene
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Abstract
本发明公开了一种水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk及其应用。本发明提供了水稻纹枯病菌MAPK蛋白激酶基因以及其靶标片段Rsmapk,MAPK蛋白激酶基因在水稻纹枯病菌侵染水稻的前期过程中被显著诱导表达;根据该水稻纹枯病菌MAPK蛋白激酶基因片段Rsmapk合成的dsRNA,能够体外处理使得水稻纹枯病菌MAPK蛋白激酶基因沉默,进而显著降低水稻纹枯病菌的致病力;所述dsRNA能够用于制备水稻纹枯病菌防治制剂,且将包含靶标片段Rsmapk的重组载体转化入植株后,能够得到抗水稻纹枯病菌转基因植物;因此,该水稻纹枯病菌MAPK蛋白激酶基因或其片段Rsmapk在防治水稻纹枯病菌、制备水稻纹枯病菌防治制剂、构建抗水稻纹枯病菌转基因植物中均具有广泛的应用前景。
Description
技术领域
本发明属于生物防治技术领域。更具体地,涉及一种水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk及其应用。
背景技术
水稻纹枯病(Rice sheath blight)是水稻的重要病害之一,危害程度仅次于稻瘟病,在种植密集和髙产稻区危害最严重,导致水稻的减产约在10%~30%,严重时可达到50%。然而,针对水稻纹枯病的发病率高且广的问题,至今未获得高抗性水稻品种,而且人们对水稻抗纹枯病病菌的机理知之甚少。
RNAi是一种在小干扰RNA(RNA small interfering RNAs,si RNAs)的指导下进行的一种同源mRNA配对并发生降解的、非常保守的调节机制。RNAi目前已经被广泛运用于植物保护领域,如植物抗病毒和害虫防治。病毒诱导基因沉默技术(Virus-induced genesilencing)简称VIGS技术,基于同源RNA互补结合降解的RNAi原理,是利用植物对病毒的天然防御机制发展的一种瞬时、快速鉴定植物基因功能的技术,是用基因序列验证基因功能的一种反向遗传学方法。
丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号通路广泛存在于真核生物体中,是一类进化上十分保守的信号传导途径。在大多数生物细胞内,受到胞外刺激后,MAPK将信号依次级联放大并传递至细胞及核内,调控各种细胞生物学过程,如细胞增殖、分化、转化及凋亡等。在真菌中,MAPK信号通路主要参与调节真菌的菌丝生长、致病性和交配过程等,模式真菌酿酒酵母(Saccharomyces cerevisiae),玉米瘤黑粉菌(Ustilago maydis)和稻瘟菌(Magnaporche moryzae)的MAPK信号通路研究较为深入。目前,针对水稻纹枯病菌(Rhizoctonia solani)的MAPK蛋白激酶基因,杨系玲等研究了其生物信息学,鉴定了该病原菌的MAPK级联信号途径基因,并预测了MAPK级联信号途径图;但没有对水稻纹枯病菌MAPK蛋白激酶基因的功能和应用等方面进行研究(水稻纹枯病菌MAPK级联信号途径分析及信号通路模型预测,中国农业科技导报,2017)。因此,筛选克隆水稻纹枯病的致病相关基因,对于研究水稻与纹枯病菌互作和开发抗纹枯病的水稻新品种具有重要意义。
发明内容
本发明要解决的技术问题是克服现有技术中对于水稻纹枯病的致病相关基因研究的缺陷和不足,提供一种水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk及其应用。
本发明的目的是提供一种水稻纹枯病菌MAPK蛋白激酶基因。
本发明另一目的是提供一种水稻纹枯病菌MAPK蛋白激酶基因编码的蛋白质。
本发明又一目的是提供一种水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk。
本发明又一目的是提供所述水稻纹枯病菌MAPK蛋白激酶基因或所述靶标片段Rsmapk在防治水稻纹枯病菌或制备水稻纹枯病菌防治制剂中的应用。
本发明又一目的是提供所述水稻纹枯病菌MAPK蛋白激酶基因或所述靶标片段Rsmapk在构建抗水稻纹枯病菌转基因植物中的应用。
本发明再一目的是提供一种水稻纹枯病菌防治制剂。
本发明上述目的通过以下技术方案实现:
本发明提供了一种水稻纹枯病菌MAPK蛋白激酶基因,所述基因的全长cDNA序列如SEQ ID NO:1所示,全长cDNA序列的长度为708bp。
所述基因的全长DNA序列如SEQ ID NO:2所示,全长DNA序列的长度为993bp,包含6个内含子,分别位于第49~102位、464~527位、677~732位、801~851位、915~974位、956~1010位;其中,第49~102位、464~527位、677~732位、801~851位剪切位置符合"GT-AG法则"。
所述基因的编码序列如SEQ ID NO:3所示,编码序列的长度为708bp,其编码235个氨基酸。
本发明还提供了一种水稻纹枯病菌MAPK蛋白激酶基因编码的蛋白质,所述蛋白质的氨基酸序列如SEQ ID NO:4所示,由235个氨基酸组成,蛋白质的分子量为26.96kDa。
本发明研究发现水稻纹枯病菌MAPK蛋白激酶基因是水稻纹枯病菌与致病力有关的基因,该基因的靶标片段Rsmapk在水稻纹枯病菌侵染水稻的前期过程中被诱导,表达量持续升高;因此,以下应用均应在本发明的保护范围之内:
所述水稻纹枯病菌MAPK蛋白激酶基因或所述靶标片段Rsmapk在防治水稻纹枯病菌或制备水稻纹枯病菌防治制剂中的应用。
所述水稻纹枯病菌MAPK蛋白激酶基因或所述靶标片段Rsmapk在构建抗水稻纹枯病菌转基因植物中的应用。
本发明还提供了一种水稻纹枯病菌防治制剂,含有能够抑制所述水稻纹枯病菌MAPK蛋白激酶基因表达的物质。
优选地,所述物质为包含靶标片段Rsmapk的dsRNA、重组载体或重组菌。
优选地,所述dsRNA的核酸序列如SEQ ID NO:6所示。
另外,本发明还提供了一种构建抗水稻纹枯病菌转基因植物的方法,体外合成含有水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk的dsRNA,用于体外处理并沉默水稻纹枯病菌MAPK蛋白激酶基因;
或者将水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk构建到TRV2载体中,用注射法将该载体注射到植株中,获得表达水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk的转基因植株;
或者利用转基因植株中产生dsRNA,用于沉默接种的水稻纹枯病菌MAPK蛋白激酶基因,降低水稻纹枯病菌在植株上的致病力。
根据本发明提供的水稻纹枯病菌MAPK蛋白激酶基因或所述靶标片段Rsmapk的序列信息,本领域技术人员可以通过以下方法容易地获得与靶标片段Rsmapk等同的片段:
(1)通过数据库检索获得;(2)以靶标片段Rsmapk为探针筛选其它丝核菌基因组文库或cDNA文库获得;(3)根据靶标片段Rsmapk序列信息设计寡核苷酸引物,用PCR扩增的方法从丝核菌或其它近缘真菌的基因组、mRNA和cDNA中获取;(4)在靶标片段Rsmapk的基础上用基因工程方法改造获得;(5)用化学合成的方法获得该靶标片段Rsmapk。
本发明具有以下有益效果:
本发明获得了一种水稻纹枯病菌MAPK蛋白激酶基因,该基因是水稻纹枯病菌(土传真菌病害)的致病力关键基因,该基因在水稻纹枯病菌侵染水稻的前期过程中被显著诱导表达,根据该水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk合成的dsRNA,能够体外处理使得水稻纹枯病菌MAPK蛋白激酶基因沉默,进而显著降低水稻纹枯病菌的致病力;
本发明还提供了一种水稻纹枯病菌防治制剂,含有能够抑制所述水稻纹枯病菌MAPK蛋白激酶基因表达的物质,所述物质为包含靶标片段Rsmapk的dsRNA、重组载体或重组菌;将高效沉默水稻纹枯病菌MAPK蛋白激酶基因的重组载体转化入植株,得到抗水稻纹枯病菌转基因植物,该植物对水稻纹枯病菌具有显著的抗病性,有效地解决了生产上由纹枯病菌引起的土传真菌病害的难题,有效地防控土传真菌病害;
另外,该方法只是在植物中表达一个小片段的基因,不产生蛋白质等产物,不会产生转基因食品带来的潜在风险;因此,该水稻纹枯病菌MAPK蛋白激酶基因或其靶标片段Rsmapk在防治水稻纹枯病菌、制备水稻纹枯病菌防治制剂、以及构建抗水稻纹枯病菌转基因植物中均具有广泛的应用前景。
附图说明
图1是水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk的扩增结果图;其中,M:DNA分子量标准;1:PCR产物。
图2是MAPK蛋白激酶基因在水稻纹枯病菌侵染过程的基因表达分析结果图。
图3是dsRNA体外处理水稻纹枯病菌24、48、72和96h的RNA提取结果图;其中,M:DNA分子量标准;1:24h提取的RNA;2:48h提取的RNA;3:72h提取的RNA;4:96h提取的RNA。
图4是dsRNA体外处理水稻纹枯病菌0、24、48、72和96h的转录水平结果图。
图5是VIGS载体信息图。
图6是本发明构建得到的TRV2-Rsmapk沉默载体的图谱。
图7是本发明构建得到的TRV2-Rsmapk沉默载体转化至根癌农杆菌GV3101的菌落验证结果图;其中,M:DNA分子量标准;1、2:菌落。
图8接种水稻纹枯病菌6d后的本氏烟草的表型图。
图9是接种水稻纹枯病菌6d后的本氏烟草的真菌生物量和靶基因转录水平分析结果图;其中,(A)图为接种水稻纹枯病菌6d后的本氏烟草的真菌生物量分析结果图;(B)图为接种水稻纹枯病菌6d后的本氏烟草的靶基因转录水平分析结果图。
具体实施方式
以下结合具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1 水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk的克隆
1、实验方法
水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk的克隆,包括以下步骤:
S1.水稻纹枯病菌RNA的提取
水稻纹枯病菌RNA的提取使用TaKaRa总RNA提取试剂盒(code No.9769)。称取0.1g水稻纹枯病菌菌丝于液氮中迅速研磨成粉末,加入到含有Buffer RL裂解液的1.5mL灭菌离心管中,将裂解液12,000rpm,4℃离心5min。将上清液小心吸取到新的1.5mL灭菌离心管中。加入样品裂解步骤中上清液1/2体积的无水乙醇,立即将混合液(含沉淀)全部转入到RNASpin Column(含2mL Collection Tube)中。12,000rpm,离心1min,弃滤液。依次将500μL的Buffer RWA,600μL的Buffer RWB,600μL的Buffer RWB加入至RNA Spin Column中,12,000rpm离心30秒钟,弃滤液。然后空管12,000rpm离心1min。将RNA Spin Column安置于1.5mL的RNase Free Collection Tube,在RNA Spin Column膜中央处加入125μL的RNaseFree ddH2O(65℃预热),室温静置5min。12,000rpm离心2min洗脱RNA,得到水稻纹枯病菌RNA。
S2.cDNA第一链的合成
以步骤S1得到的水稻纹枯病菌RNA为模板,利用TARAKA PrimeScript反转录试剂盒(code No.6210A)合成cDNA。在微量离心管中加入2000ng Total RNA、1μL Oligo dTPrimer、1μL dNTP Mixture(10mM each)、补充RNase Free ddH2O至10μL。轻轻混匀,离心数秒后,65℃保温5min,冰上迅速冷却。依次加4μL 5×PrimeScript II Buffer、0.5μL RNaseInhibitor(40U/μL)、1μL PrimeScript II RTase(200U/μL)、补充RNase Free ddH2O至20μL,缓慢混匀。42℃保温50min,95℃保温5min(酶失活),得到水稻纹枯病菌cDNA,冰上冷却后-20℃保存备用。
S3.靶标片段Rsmapk的克隆
靶标片段Rsmapk的克隆引物为4099F/R,4099F:ACGCTGCTGATGACGGAA,如SEQ IDNO:7所示,4099R:AGACGGCTAACGATGGGTAA,如SEQ ID NO:8所示。以步骤S2得到的水稻纹枯病菌cDNA为模板,使用擎科(code No.TP001)高保真聚合酶,进行PCR扩增反应。反应条件为:98℃,2min;98℃,10s,58℃,15s,40个循环;最后72℃,10min。使用TAE缓冲液制作1.0%的琼脂糖凝胶,将PCR产物进行琼脂糖凝胶电泳。使用Axygen PCR清洁试剂盒试剂盒进行PCR产物纯化。
2、实验结果
水稻纹枯病菌MAPK蛋白激酶基因的全长cDNA序列如SEQ ID NO:1所示,全长DNA序列如SEQ ID NO:2所示,编码序列如SEQ ID NO:3所示,其编码的蛋白质的氨基酸序列如SEQID NO:4所示。
水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk的核苷酸序列如SEQ ID NO:5所示。
水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk的扩增结果如图1所示,可以看出,本发明成功扩增得到水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk,大小为275bp。
实施例2 MAPK蛋白激酶基因在水稻纹枯病菌侵染水稻过程的表达分析
1、实验方法
MAPK蛋白激酶基因在水稻纹枯病菌侵染水稻过程的表达分析,包括以下步骤:
S1.接种喷雾的制备:首先使用PDA平板(培养皿直径90mm,8mL PDA培养基)将本实验室保存的水稻纹枯病菌(R.solani AG-1IA)GD-118菌株(舒灿伟等,一种优化的适用于双向电泳的水稻纹枯病菌蛋白质提取方法,华中农业大学学报,2017)活化,黑暗条件下,28℃培养2d。之后使用灭菌后的刀片将带有真菌的PDA平板切割成边长约为1mm大小的正方形菌丝块,转移至锥形瓶中(含200mL PDB),黑暗条件下,28℃,180r/min,震荡培养2d后,整瓶匀浆成液体,使OD600为1.0,即可用于接种。
S2.水稻的种植和接种:分别使用10%的次氯酸钠与30%的双氧水对水稻种子(水稻品种:日本晴)进行消毒,再将种子置于无菌培养皿内,使用无菌水浸泡3d以上直至发芽,然后将发芽的种子转移至含有10cm无菌土的方盆(长:80cm;宽:40cm;高:15cm)内。接种选择水稻三叶一心期,在盆内无水干燥条件下使用步骤S1得到的匀浆后的液体进行接种,每株水稻接种5mL,30℃,湿度90%以上利于发病。
S3.样品的实时荧光定量PCR分析:为了验证不同基因在水稻纹枯病菌侵染水稻过程中的表达量,选取了接种后的6个时间点,24h,48h,72h,96h,120h后收集整株水稻为样品。使用TaKaRa MiniBEST Plant RNA Extraction Kit(code No.9769)进行不同时间的水稻样品总RNA的提取,具体方法同“实施例1中的步骤S1”。
利用TARAKA PrimeScript反转录试剂盒(code No.6210A)进行RNA反转录,具体方法同“实施例1中的步骤S2”,将反转录所得的cDNA产物稀释10倍后用于实时荧光定量PCR。
利用Primer Premier 5.0软件设计靶标片段Rsmapk的实时荧光定量PCR引物4099qPCR F/R,4099qPCR F:CATCGAGTTATCCGAACGCAG,如SEQ ID NO:9所示;4099qPCR R:ATCAGCGCTGTGTAGTGC,如SEQ ID NO:10所示。引物的特异性通过凝胶电泳、测序和溶解曲线检验。实验使用Bio-Rad CFX real-time PCR system,反应体系为20μL,其中包含10μLMIX、0.2μM引物4099qPCR F/R、2μL cDNA模板和适量的纯水。每个样品进行三次技术重复。
水稻纹枯病菌的内参基因使用GAPDH进行样品间的基因表达的均一化处理,引物为GAPDH F/R,GAPDH F:GGTCGGCAAAGTCATACCAT,如SEQ ID NO:11所示;GAPDH R:TCTGCGTCCTTCTTGGAGATA,如SEQ ID NO:12所示。结果采用2-ΔΔCt法进行分析(苏晓峰,2014)。
2、实验结果
MAPK蛋白激酶基因在水稻纹枯病菌侵染过程的基因表达分析结果如图2所示,可以看出,MAPK蛋白激酶基因在水稻纹枯病菌侵染水稻的前期过程中被诱导,且随着时间延长表达量持续升高;说明该基因在水稻纹枯病菌与水稻的互作过程中可能起到关键作用。
实施例3 含有靶标片段Rsmapk的dsRNA的合成和体外沉默实验
1、实验方法
含有靶标片段Rsmapk的dsRNA的合成和体外沉默实验,包括以下步骤:
S1.含有靶标片段Rsmapk的dsRNA的体外合成:使用T7RNA Polymerase(ThermoFisher Scientific,Catalog number:EP011)合成含有靶标片段Rsmapk的dsRNA。通过在任一扩增引物的5'末端添加T7启动子序列,可以使用PCR将T7RNA聚合酶启动子添加到任何DNA序列中。
最小的T7RNA聚合酶启动子序列是:5'-TAATACGACTCACTATAGG-3'。在上下游引物5'末端分别添加T7启动子序列,dsRNA合成引物T7-4099F/R,即上游引物T7-4099F:TAATACGACTCACTATAGGACCCTCCAACCTGCTCCTAA,如SEQ ID NO:13所示;下游引物T7-4099R:TAATACGACTCACTATAGGCCTCAACAGTGATT CGCTTCTT,如SEQ ID NO:14所示。
以水稻纹枯病菌的cDNA为模板进行PCR扩增得到dsRNA的模板,经过转录纯化后得到含有MAPK蛋白激酶基因靶标片段Rsmapk的dsRNA。
S2.dsRNA对水稻纹枯病菌的体外沉默实验:从培养2d的水稻纹枯病菌PDA平板边缘取边长2mm正方形菌饼放入直径60mm的培养皿中(含有7mL PDB),静置培养48h后,以500ng/mL加入dsRNA,并在0、24、48、72和96h收集菌丝样品,并使用TaKaRa MiniBEST PlantRNA Extraction Kit(code No.9769)进行不同时间的水稻样品总RNA的提取,具体方法同“实施例1中的步骤S1”,每次处理重复三次,实验重复三次。
2、实验结果
dsRNA的核酸序列如SEQ ID NO:6所示。
dsRNA体外处理水稻纹枯病菌24、48、72和96h的RNA提取结果如图3所示,可以看出,各时间段样品提取出的RNA质量良好。
dsRNA体外处理水稻纹枯病菌0、24、48、72和96h的转录水平结果如图4所示,可以看出,MAPK蛋白激酶基因的转录水平在24hpi有显著降低,并随着处理时间的增加持续降低,96hpi的沉默效率高达80%以上;说明体外施用dsRNA处理水稻纹枯病菌沉默相关基因的表达是切实可行的。
实施例4 TRV2-Rsmapk沉默载体构建和转化烟草研究
1、实验方法
TRV2-Rsmapk沉默载体构建和转化烟草研究,包括以下步骤:
S1.载体构建及农杆菌转化:根据水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk的编码序列,设计引物两端包含EcoRI和BamI酶切位点的特异性载体构建引物VIGS-4099F/R,上游引物VIGS-4099F:CCGgaattcGTCCGTTGGTTGTATTCTC,如SEQ ID NO:15所示;下游引物VIGS-4099R:CGCggatccAACAGT GATTCGCTTCTTG,如SEQ ID NO:16所示。以水稻纹枯病菌cDNA为模板,利用擎科(code No.TP001)高保真酶进行PCR扩增,将扩增产物回收后,和载体pYL156同时在37℃下双酶切5h。将酶切后的目的片段和载体利用T4DNA连接酶,16℃过夜连接,将连接好的产物转化入E.coil DH5α感受态,测序确定无误后,扩繁提取质粒(TRV2-Rsmapk沉默载体),保存备用。其中,VIGS载体信息如图5所示。
S2.根癌农杆菌感受态的制备及电击转化:将根癌农杆菌GV3101单菌落接于4mL含Rif(25μg/mL)的LB液体培养基中,28℃,200r/min振荡培养2d。取培养液3mL的接种量转接至200mL LB培养液中,200r/min,28℃振荡培养至对数生长期(细胞浓度OD600为0.5-0.6)。离心弃去废液,收集菌体。用预冷的无菌双蒸水将菌液重悬洗涤3次,后用2mL无菌预冷的10%w/v甘油重悬菌体。取100μL菌悬液于1.5mL离心管,-70℃保存备用。加3μL质粒于菌液中,轻轻混匀并转移至无菌预冷的电击杯中,电击后迅速加入1mL LB液体培养基,混匀并转移细胞至1.5mL的离心管中,在28℃摇床中200r/min培养2-3h;取100μL菌液涂布于含Rif(25μg/m L)和Kan(50μg/mL)的LB平板中,倒置放于28℃培养箱中培养2天,观察转化子生长情况,取未质粒DNA的农杆菌在同样电击条件下的结果作为对照。挑取单菌落,使用pYL156载体验证引物(PYL156F/R)进行PCR验证,上游引物PYL156F:AATTCACTGGGAGATGATACGCTG,如SEQ ID NO:17所示;下游引物PYL156R:CCTATGGTAAGACAATGAGTCGGC,如SEQ ID NO:18所示。
S3.农杆菌培养和烟草瞬时转化:农杆菌单克隆加入5mL的液体LB培养基(25μg/mLRif和50μg/mL Kan)中,放于28℃摇床中进行200r/min过夜培养,将1mL菌液加入50mL LB(25μg/mL Rif和50μg/mL Kan)中,放于28℃摇床中进行200r/min振荡培养。当培养物OD600到0.5-0.6时,6000rpm,5min低温离心收集菌体,弃去废液。将菌体重悬于注射基质(10mMMES、10mM MgCl2、100μM乙酰丁香酮)中,使OD600为0.8-1.0。把两种农杆菌菌株(TRV1与TRV2+靶标基因片段)以1:1混合,室温静置3-5h,不要摇动。最后利用注射器将含有注射基质的农杆菌注射于最嫩的叶片中。
S4.致病力检测和生物量测定:从注射后的第10天起,本氏烟草的白化现象就非常明显,这表明了在本氏烟草体内VIGS载体已经产生出大量dsRNA,并发挥着干扰作用。因此,我们选择了从注射VIGS系列载体后的第10天,进行活体接种。对接菌后第6天(post-inoculation day,dpi)进行本氏烟草的病情指数统计,通过统计叶片病斑和凋萎叶片的数量进行致病力的检测。提取接种水稻纹枯病菌6d后本氏烟草(转基因植株和阴性对照)中部叶片的总DNA,以立枯丝核菌(R.solani)内部转录间隔区ITS序列为靶标片段,上游引物RsF:GCCTTTTCTACCTTAATTTGGCAG,如SEQ ID NO:19所示;下游引物Rs R:GTGTGTAAATTAAGTAGACAGCAAATG,如SEQ ID NO:20所示。同时以烟草actin基因为内参基因,上游引物EF1a:TGGTGTCCTCAAGCCTGGTAT,如SEQ ID NO:21所示;下游引物EF1b:ACGCTTGAGATCCTTAACCGC,如SEQ ID NO:22所示。具体方法同“实施例2中的步骤S3”,结果采用2-ΔΔCt法进行分析(苏晓峰,2014)。
2、实验结果
本发明构建得到的TRV2-Rsmapk沉默载体的图谱如图6所示,可以看出,所构载体全长9925bp,经对比与实际测序结果相同。
本发明构建得到的TRV2-Rsmapk沉默载体转化至根癌农杆菌GV3101的菌落验证结果如图7所示,条带大小为712bp,条带单一明亮且大小正确;说明沉默载体TRV2-Rsmapk已成功转进根癌农杆菌GV3101。
接种水稻纹枯病菌6d后的本氏烟草的表型如图8所示,可以看出,转基因烟草对水稻纹枯病菌的抗性明显提高。
接种水稻纹枯病菌6d后的本氏烟草的真菌生物量和靶基因转录水平分析结果如图9所示,可以看出,转基因阳性烟草的真菌生物量明显降低,仅为野生型的40%,转基因植物体内MAPK蛋白激酶基因的表达量下降了60%。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
序列表
<110> 华南农业大学
<120> 一种水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk及其应用
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 708
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgcatcgag ttatccgaac gcaggacctt agcgacgatc atgcccaata ttttatctac 60
caaacgcttc gcgccctcaa ggcactacac agcgctgatg tcatccaccg tgatctcaaa 120
ccctccaacc tgctcctaaa cgcgaattgc gatctcaagg tctgcgactt tggtcttgca 180
cggtccgttc ggacggccga gccttcgggc accgaaacag gcttcatgac ggaatacgtt 240
gcgacgagat ggtatcgcgc acccgaaatc atgcttacgt ttaagcagta caccaaggca 300
atcgacgtct ggtccgttgg ttgtattctc gcggagatgt taagcggaaa gcccttgttc 360
cctggacggg actaccatca ccagttgact ttgatcttgg acgtgctagg cacacccacg 420
ctagacgagt tctacgcgat caccacccga cgatcccgcg attacattcg tgcactccca 480
ttccggaaac gtcgaccatt cgcccagtta ttcccgaatg cttctgcgct cgccgtggac 540
tttttgacga aaactttgac ctttgatccc aagaagcgaa tcactgttga ggacgcactc 600
tgccaccctt acctagaggc atacgtcatg atcccgatga tgagcccgtt gcccctcctc 660
tggaccccga ctttttcgag tttgatcgtt gcacaaggac gacattag 708
<210> 2
<211> 993
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgcatcgag ttatccgaac gcaggacctt agcgacgatc atgcccaagt aagtacctcc 60
cgccttgcac ccatgcgcca catctaactc gcatattttc agtattttat ctaccaaacg 120
cttcgcgccc tcaaggcact acacagcgct gatgtcatcc accgtgatct caaaccctcc 180
aacctgctcc taaacgcgaa ttgcgatctc aaggtctgcg actttggtct tgcacggtcc 240
gttcggacgg ccgagccttc gggcaccgaa acaggcttca tgacggaata cgttgcgacg 300
agatggtatc gcgcacccga aatcatgctt acgtttaagc agtacaccaa ggcaatcgac 360
gtctggtccg ttggttgtat tctcgcggag atgttaagcg gaaagccctt gttccctgga 420
cgggactacc atcaccagtt gactttgatc ttggacgtgc taggtaagca ctttccgggg 480
attctcccca ccttattctc tcgcagatcc tcacacatcg ttacaaggca cacccacgct 540
agacgagttc tacgcgatca ccacccgacg atcccgcgat tacattcgtg cactcccatt 600
ccggaaacgt cgaccattcg cccagttatt cccgaatgct tctgcgctcg ccgtggactt 660
tttgacgaaa actttggtac gagcacatgt ttgatatcta ggttcgcgcg tattctgacc 720
tggttcttct agacctttga tcccaagaag cgaatcactg ttgaggacgc actctgccac 780
ccttacctag aggcatacgt gcgtagacgt ccaaaaacca acgttagaac tcagctaacg 840
cgtctctata gcatgatccc gatgatgagc ccgttgcccc tcctctggac cccgactttt 900
tcgagtttga tcgtgcgttc attcccttcc ttacatatat acgttcatcg ctgactcaca 960
ctccttgtcc acagtgcaca aggacgacat tag 993
<210> 3
<211> 708
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgcatcgag ttatccgaac gcaggacctt agcgacgatc atgcccaata ttttatctac 60
caaacgcttc gcgccctcaa ggcactacac agcgctgatg tcatccaccg tgatctcaaa 120
ccctccaacc tgctcctaaa cgcgaattgc gatctcaagg tctgcgactt tggtcttgca 180
cggtccgttc ggacggccga gccttcgggc accgaaacag gcttcatgac ggaatacgtt 240
gcgacgagat ggtatcgcgc acccgaaatc atgcttacgt ttaagcagta caccaaggca 300
atcgacgtct ggtccgttgg ttgtattctc gcggagatgt taagcggaaa gcccttgttc 360
cctggacggg actaccatca ccagttgact ttgatcttgg acgtgctagg cacacccacg 420
ctagacgagt tctacgcgat caccacccga cgatcccgcg attacattcg tgcactccca 480
ttccggaaac gtcgaccatt cgcccagtta ttcccgaatg cttctgcgct cgccgtggac 540
tttttgacga aaactttgac ctttgatccc aagaagcgaa tcactgttga ggacgcactc 600
tgccaccctt acctagaggc atacgtcatg atcccgatga tgagcccgtt gcccctcctc 660
tggaccccga ctttttcgag tttgatcgtt gcacaaggac gacattag 708
<210> 4
<211> 235
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met His Arg Val Ile Arg Thr Gln Asp Leu Ser Asp Asp His Ala Gln
1 5 10 15
Tyr Phe Ile Tyr Gln Thr Leu Arg Ala Leu Lys Ala Leu His Ser Ala
20 25 30
Asp Val Ile His Arg Asp Leu Lys Pro Ser Asn Leu Leu Leu Asn Ala
35 40 45
Asn Cys Asp Leu Lys Val Cys Asp Phe Gly Leu Ala Arg Ser Val Arg
50 55 60
Thr Ala Glu Pro Ser Gly Thr Glu Thr Gly Phe Met Thr Glu Tyr Val
65 70 75 80
Ala Thr Arg Trp Tyr Arg Ala Pro Glu Ile Met Leu Thr Phe Lys Gln
85 90 95
Tyr Thr Lys Ala Ile Asp Val Trp Ser Val Gly Cys Ile Leu Ala Glu
100 105 110
Met Leu Ser Gly Lys Pro Leu Phe Pro Gly Arg Asp Tyr His His Gln
115 120 125
Leu Thr Leu Ile Leu Asp Val Leu Gly Thr Pro Thr Leu Asp Glu Phe
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Tyr Ala Ile Thr Thr Arg Arg Ser Arg Asp Tyr Ile Arg Ala Leu Pro
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Phe Arg Lys Arg Arg Pro Phe Ala Gln Leu Phe Pro Asn Ala Ser Ala
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Leu Ala Val Asp Phe Leu Thr Lys Thr Leu Thr Phe Asp Pro Lys Lys
180 185 190
Arg Ile Thr Val Glu Asp Ala Leu Cys His Pro Tyr Leu Glu Ala Tyr
195 200 205
Val Met Ile Pro Met Met Ser Pro Leu Pro Leu Leu Trp Thr Pro Thr
210 215 220
Phe Ser Ser Leu Ile Val Ala Gln Gly Arg His
225 230 235
<210> 5
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<212> DNA
<213> 人工序列(Artificial Sequence)
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gtccgttggt tgtattctcg cggagatgtt aagcggaaag cccttgttcc ctggacggga 60
ctaccatcac cagttgactt tgatcttgga cgtgctaggc acacccacgc tagacgagtt 120
ctacgcgatc accacccgac gatcccgcga ttacattcgt gcactcccat tccggaaacg 180
tcgaccattc gcccagttat tcccgaatgc ttctgcgctc gccgtggact ttttgacgaa 240
aactttgacc tttgatccca agaagcgaat cactg 275
<210> 6
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<212> RNA
<213> 人工序列(Artificial Sequence)
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uaauacgacu cacuauagga cccuccaacc ugcuccuaaa cgcgaauugc gaucucaagg 60
ucugcgacuu uggucuugca cgguccguuc ggacggccga gccuucgggc accgaaacag 120
gcuucaugac ggaauacguu gcgacgagau gguaucgcgc acccgaaauc augcuuacgu 180
uuaagcagua caccaaggca aucgacgucu gguccguugg uuguauucuc gcggagaugu 240
uaagcggaaa gcccuuguuc ccuggacggg acuaccauca ccaguugacu uugaucuugg 300
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auuacauucg ugcacuccca uuccggaaac gucgaccauu cgcccaguua uucccgaaug 420
cuucugcgcu cgccguggac uuuuugacga aaacuuugac cuuugauccc aagaagcgaa 480
ucacuguuga ggccuauagu gagucguauu a 511
<210> 7
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<212> DNA
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acgctgctga tgacggaa 18
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
agacggctaa cgatgggtaa 20
<210> 9
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
catcgagtta tccgaacgca g 21
<210> 10
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atcagcgctg tgtagtgc 18
<210> 11
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggtcggcaaa gtcataccat 20
<210> 12
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tctgcgtcct tcttggagat a 21
<210> 13
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
taatacgact cactatagga ccctccaacc tgctcctaa 39
<210> 14
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
taatacgact cactataggc ctcaacagtg attcgcttct t 41
<210> 15
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ccggaattcg tccgttggtt gtattctc 28
<210> 16
<211> 28
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
cgcggatcca acagtgattc gcttcttg 28
<210> 17
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
aattcactgg gagatgatac gctg 24
<210> 18
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
cctatggtaa gacaatgagt cggc 24
<210> 19
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
gccttttcta ccttaatttg gcag 24
<210> 20
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<213> 人工序列(Artificial Sequence)
<400> 20
gtgtgtaaat taagtagaca gcaaatg 27
<210> 21
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
tggtgtcctc aagcctggta t 21
<210> 22
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
acgcttgaga tccttaaccg c 21
Claims (10)
1.一种水稻纹枯病菌MAPK蛋白激酶基因,其特征在于,所述基因的全长cDNA序列如SEQID NO:1所示。
2.一种水稻纹枯病菌MAPK蛋白激酶基因,其特征在于,所述基因的全长DNA序列如SEQID NO:2所示。
3.一种水稻纹枯病菌MAPK蛋白激酶基因,其特征在于,所述基因的编码序列如SEQ IDNO:3所示。
4.一种水稻纹枯病菌MAPK蛋白激酶基因编码的蛋白质,其特征在于,所述蛋白质的氨基酸序列如SEQ ID NO:4所示。
5.一种水稻纹枯病菌MAPK蛋白激酶基因靶标片段Rsmapk,其特征在于,其核苷酸序列如SEQ ID NO:5所示。
6.权利要求1~3任一所述水稻纹枯病菌MAPK蛋白激酶基因或权利要求5所述靶标片段Rsmapk在防治水稻纹枯病菌或制备水稻纹枯病菌防治制剂中的应用。
7.权利要求1~3任一所述水稻纹枯病菌MAPK蛋白激酶基因或权利要求5所述的靶标片段Rsmapk在构建抗水稻纹枯病菌转基因植物中的应用。
8.一种水稻纹枯病菌防治制剂,其特征在于,含有能够抑制权利要求1~3任一所述水稻纹枯病菌MAPK蛋白激酶基因表达的物质。
9.根据权利要求8所述的制剂,其特征在于,所述物质为包含靶标片段Rsmapk的dsRNA、重组载体或重组菌。
10.根据权利要求9所述的制剂,其特征在于,所述dsRNA的核酸序列如SEQ ID NO:6所示。
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