CN112195184A - OsMAPK6基因在改良水稻抗病性中的应用 - Google Patents
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Abstract
本发明属于植物基因工程技术领域,涉及OsMAPK6基因在改良水稻抗病性中的应用。本发明通过OsMAPK6基因超量表达研究,发现OsMAPK6基因能够影响水稻对白叶枯病的抵抗能力,超量表达OsMAPK6基因提高水稻对白叶枯病的抵抗能力。本发明克隆的OsMAPK6基因的核苷酸序列如序列表SEQ ID NO:1所示。本发明的基因可应用于水稻抗病改良中。
Description
技术领域
本发明属于植物基因工程技术领域,具体涉及OsMAPK6基因在改良水稻抗病性中的应用。所述基因可用于增强广谱抗白叶枯病水稻品种的培育。
背景技术
水稻是世界上最重要的粮食作物之一,全球大约一半以上的人口以稻米为主要粮食。我国又是世界上重要的水稻生产大国,水稻的产量和品质严重制约着我国经济社会的可持续发展。水稻病害是严重影响水稻产量和品质的关键因素之一,其中由革兰氏阴性菌黄单胞杆菌水稻变种-白叶枯病菌引发的水稻白叶枯病是水稻生产上最重要的细菌性病害。生产上防治白叶枯病主要有两种措施:一是利用化学药物抑制白叶枯病原菌的生长和繁殖;另一种是培育抗病水稻品种。化学药物防治虽然可以有效的控制白叶枯病害,但同时也污染了环境,破坏了生态,因此挖掘抗病基因,培育抗病水稻品种才是防治白叶枯病害的最有效途径。
水稻抗病基因主要可以分为主效抗病基因和抗病相关基因两大类。经过20多年的研究,水稻中已经鉴定到了很多参与调控对白叶枯病菌防卫反应的主效抗病基因(也被称为R基因)和抗病相关基因。如克隆和鉴定的Xa3/Xa26,Xa4,xa13,xa25和Xa1/Xa2/Xa14等,属于主效抗白叶枯病基因;如OsWRKY45,OsEDR1,OsMPK6,OsPAD4等,属于抗白叶枯病相关基因。目前,生产中,常常利用R基因来培育抗白叶枯病的水稻品种。但R基因所介导的白叶枯病抗性常常抗谱窄,且抗性易丧失等缺点;抗白叶枯病相关基因可以很好的克服R基因的这些缺陷。因此挖掘更多的抗白叶枯病相关基因对于改良水稻对白叶枯病菌的抗性具有重要意义。
丝裂原活化蛋白激酶级联反应(MAPK cascade)是真核生物中非常保守的信号传导方式,尤其在植物的免疫反应中发挥着关键的作用。水稻中有75个MAPKKKs,8个MAPKKs和17个MAPKs蛋白。经过10多年的研究,水稻中多个MAPK级联路径中的蛋白被鉴定到可以参与调控水稻对多种病原菌的免疫反应。如OsMPK5(也被称为OsMAPK3)可以负调控水稻对稻瘟病的抗性;OsMAPK15可以负调控水稻对白叶枯病和稻瘟病的抗性;OsMAPKK4-OsMAPK6级联反应可以参与调控对几丁质的识别和对稻瘟病的抗性;OsMAPKK10.2-OsMAPK6级联反应正调控水稻对细菌性条斑病的抗性;OsMAPKK3-OsMAPK7级联反应正调控水稻对白叶枯病的抗性。
本发明发现超量表达水稻OsMAPK6基因可以增强水稻对白叶枯病菌的广谱抗性。本发明对于改良水稻对白叶枯病的抗性,培育抗白叶枯病新品种方面具有重要的意义。
发明内容
本发明的目的在于克服现有技术存在的缺陷,通过对OsMAPK6基因的生物学功能的研究,发现OsMAPK6基因在水稻抗白叶枯病上具有重要调控作用,通过调节该基因表达水平影响水稻对白叶枯病的广谱抗病性。因此,本发明对于重要粮食作物水稻在抗白叶枯病的改良上具有重要的意义。
本发明的技术方案如下所述:
本发明证实在水稻中超量表达OsMAPK6基因使水稻对多种不同白叶枯病菌表现出抗性增强的能力。通过***研究,申请人发现OsMAPK6基因在水稻抗白叶枯病方面有着重要调控功能。
经过生物学功能验证证明本发明所提供的水稻OsMAPK6基因具有以下特性:
1、OsMAPK6基因的核苷酸序列如SEQ ID NO:1所示。
SEQ ID NO:1所示的核苷酸序列由水稻OsMAPK6基因及其上下游非编码序列共6462个脱氧核糖核苷酸组成。SEQ ID NO:1所示序列中第1位至154位的脱氧核糖核苷酸为OsMAPK6基因的上游非编码序列;第155位至399位的脱氧核糖核苷酸为OsMAPK6基因的第一个外显子序列;第400位至486位的脱氧核糖核苷酸为OsMAPK6基因的第一个内含子序列;第487位至616位的脱氧核糖核苷酸为OsMAPK6基因的第二个外显子序列;第617位至3173位的脱氧核糖核苷酸为OsMAPK6基因的第二个内含子序列;第3174位至3311位的脱氧核糖核苷酸为OsMAPK6基因的第三个外显子序列;第3312位至4370位的脱氧核糖核苷酸为OsMAPK6基因的第三个内含子序列;第4371位至4703位的脱氧核糖核苷酸为OsMAPK6基因的第四个外显子序列;第4704位至5034位的脱氧核糖核苷酸为OsMAPK6基因的第四个内含子序列;第5035位至5215位的脱氧核糖核苷酸为OsMAPK6基因的第五个外显子序列;第5216位至5828位的脱氧核糖核苷酸为OsMAPK6基因的第五个内含子子序列,第5829位至5998位的脱氧核糖核苷酸为OsMAPK6基因的第六个外显子序列,第5999位至6462位的脱氧核糖核苷酸为OsMAPK6基因的下游非编码序列。
2、本发明的OsMAPK6基因序列可用于在作物,特别是水稻的抗病育种、转基因株系和转基因新品种中的应用上。
更详细的技术方案参见《具体实施方式》。
与现有技术相比,本发明的有益效果在于:
OsMAPK6基因正向调控水稻对白叶枯病菌的广谱抗病性。
附图说明
图1:本发明鉴定和分离克隆水稻抗病相关基因OsMAPK6基因以及验证OsMAPK6基因功能的技术流程图。
图2:本发明所用遗传转化载体pU1301-OsMAPK6的载体图谱。附图标记说明:RB和LB分别表示T-DNA的右边界和左边界,GUS表示β-葡糖苷酸酶基因,Hpt表示潮霉素磷酸转移酶基因,PUbi表示玉米泛素基因启动子,TEVL表示烟草蚀刻病毒翻译前导肽序列,NOS表示胭脂碱合成酶基因的多聚腺苷酸化信号。
图3:OsMAPK6基因超量表达水稻株系中OsMAPK6基因表达量鉴定。附图标记说明:图3中的A图表示qRT-PCR显示OsMAPK6基因超量表达水稻家系9阳性单株的表达量明显高于野生型和分离出的阴性;图3中的B图表示qRT-PCR显示OsMAPK6基因超量表达水稻家系55阳性单株的表达量明显高于野生型和分离出的阴性。
图4:OsMAPK6基因超量表达水稻株系接种白叶枯病菌PXO347后的病斑长度。附图标记说明:图4中的A图表示OsMAPK6基因超量表达家系9接种白叶枯病菌PXO347的14天后的发病病斑长度与野生型植株和转基因阴性植株相比显著性变短;图4中的B图表示OsMAPK6基因超量表达家系55接种白叶枯病菌PXO347的14天后的发病病斑长度与野生型植株和转基因阴性植株相比显著性变短。结果表明OsMAPK6基因超量表达水稻株系增强了对白叶枯病菌PXO347的抗病性。
图5:相对于野生型,OsMAPK6基因超量表达水稻株系对不同白叶枯病菌(PXO99、PXO71、PXO341、PXO347、T1、Zhe173)具有广谱抗病性。
图6:OsMAPK6基因超量表达水稻株系接种白叶枯病菌PXO341后叶片中白叶枯病菌生长量。相对于野生型对照,OsMAPK6基因超量表达水稻株系中白叶枯病菌数量显著降低。
具体实施方式
对序列表的说明:
序列表SEQ ID NO:1是OsMAPK6基因的核苷酸序列,序列长度为6462bp。
下面结合具体的实施例进一步阐述本发明。图1描述了鉴定和分离克隆OsMAPK6基因以及验证OsMAPK6基因功能的流程。需要说明的是,这些实施例仅仅是为了说明本发明,而不能以任何方式构成对本发明权利要求范围的限制。
下述实施例中所用方法如无特别说明均为常规方法,具体步骤可参考:《Molecular Cloning:A Laboratory Manual》(Sambrook,J.,Russell,David W.,Molecular Cloning:A Laboratory Manual,3rd edition,2001,NY,Cold Spring Harbor)或相关产品。所用试剂或仪器未注明生产厂商者,均为可以通过市场采购的常规产品。
实施例1:OsMAPK6基因超量表达材料的获得
(1)OsMAPK6基因超量表达载体的构建
本实施例是关于pU1301-OsMAPK6载体构建的一个通用说明。
以水稻品种中花11(又称ZH11,来源于中国农业科学院作物科学研究所一个常用水稻品种)的cDNA为模板,设计引物MPK1F(5’-GGGGTACCATGGACGCCGGGGCGC-3’)和MPK1R(5’-GGGGATCCCTACTGGTAATCAGGGTTGAACGC-3’)利用高保真DNA聚合酶PCR扩增得到OsMAPK6基因的全长cDNA片段(SEQ ID NO:1所示序列中第155位至399位脱氧核糖核苷酸,第487位至616位脱氧核糖核苷酸,第3174位至3311位脱氧核糖核苷酸,第4371位至4703位脱氧核糖核苷酸,第5035位至5215位脱氧核糖核苷酸,第5829位至5998位脱氧核糖核苷酸,计1197个脱氧核糖核苷酸)。电泳回收PCR产物,用限制性内切酶KpnI和BamHI酶切PCR产物,电泳回收酶切产物,同时将载体pU1301用KpnI和BamHI酶切过夜并回收。将回收的酶切产物与载体片段以摩尔比约3:1的量进行连接(T4 Ligase)过夜。第二天将连接产物转化大肠杆菌DH5α,并于37℃培养过夜,可获得单克隆。选取单克隆在3ml含卡那霉素抗生素的LB培养基中过夜培养,第二天提取质粒,然后酶切质粒,对切出外源片段的单克隆进一步测序验证。从而获得植物转化载体(pU1301-OsMAPK6)。植物转化载体的图谱见图2。
(2)OsMAPK6基因超量表达水稻株系的获得及其鉴定
申请人将含有强启动子PUbi驱动OsMAPK6基因全长cDNA的pU1301-OsMAPK6载体通过农杆菌介导的遗传转化方法导入水稻品种中花11,获得了多个独立的转基因家系,选取家系9和家系55,利用qRT-PCR技术来检测OsMAPK6基因的表达水平。
从中国,湖北,武汉大田取孕穗期的剑叶叶片,根据TransZol(北京全式金生物技术有限公司)使用说明书抽提RNA。取5μg总RNA用DNaseI(美国Invitrogen公司)处理15分钟以去除基因组DNA污染,然后使用oligo(dT)15寡聚引物和M-MLV反转录酶(美国Promega公司)进行反转录。采用实时定量PCR分析试剂盒Green PCR Master Mix(大连Tokara公司),并根据试剂盒使用说明书,在ABI 7500Real-Time PCR system(美国AppliedBiosystems公司)仪器上进行实时定量PCR反应。用水稻内源肌动蛋白基因的表达量衡量、并均一化样品RNA含量。qRT-PCR分析中OsMAPK6基因特异PCR引物是real-MPK1-F(5′-GTGGTCTGTGGGCTGTATTT-3′)和real-MPK1-R(5′-GTTCCGATGAGCTCCATTAGTAG-3′),肌动蛋白基因PCR引物是actin-F(5′-TGCTATGTACGTCGCCATCCAG-3′)和actin-R(5′-AATGAGTAACCACGCTCCGTCA-3′)。
qRT-PCR结果显示,转基因家系中阳性单株中OsMAPK6基因的表达水平显著高于阴性单株和野生型对照中的表达水平,结果见图3。
实施例2:OsMAPK6基因超量表达水稻株系的相关分析与功能验证
(1)OsMAPK6基因超量表达水稻株系孕穗期抗白叶枯病表型分析
在中国,湖北,武汉的夏季田间对OsMAPK6基因超量表达水稻株系及野生型对照进行白叶枯病菌接种试验。结果显示,本发明的OsMAPK6基因超量表达水稻株系在孕穗期接种白叶枯病菌致病小种PXO347,与野生型(非转基因的水稻品种中花11,下同)相比,OsMAPK6基因超量表达水稻家系9和家系55阳性单株发病长度均显著性短于阴性单株和野生型对照(p<0.01)。见图4。以上结果表明,OsMAPK6基因超量表达水稻株系可以增强水稻对白叶枯病菌致病小种PXO347的抗性。
(2)OsMAPK6基因超量表达水稻株系对不同白叶枯病菌的广谱抗性分析
在中国,湖北,武汉夏季田间对OsMAPK6基因超量表达水稻株系及野生型对照进行白叶枯病菌接种试验。结果显示,OsMAPK6基因超量表达水稻株系在孕穗期接种不同白叶枯病菌致病小种(PXO99、PXO71、PXO341、PXO347、Zhe173、T1,上述所有白叶枯病菌由国际水稻研究所赠送),与野生型相比,OsMAPK6基因超量表达水稻家系9和家系55阳性单株发病长度均显著性变短(p<0.01)。见图5。以上结果表明,超量表达OsMAPK6基因可以增强水稻对不同白叶枯病菌的广谱抗病性。
(3)OsMAPK6基因超量表达水稻株系中白叶枯病菌数量分析
在中国,湖北,武汉夏季田间对OsMAPK6基因超量表达水稻株系在孕穗期接种白叶枯病菌致病小种PXO341后分析叶片中白叶枯病菌生长情况。在孕穗期于田间对OsMAPK6基因超量表达水稻株系和野生型对照分别接种白叶枯病菌致病小种PXO341(由国际水稻研究所赠送),接种后在不同时间取叶片材料(接种剪口下3cm的叶片),同一份材料取三片叶(代表三个试验重复)。根据已报道的方法处理叶片材料,分析细菌的生长数量。主要分析步骤如下:用75%酒精消毒叶片表面1分钟,晾干,置于研钵中加1ml灭菌蒸馏水研磨成匀浆,然后加倍灭菌水稀释成不同浓度梯度,每个浓度梯度重复涂三个PSA培养皿(马铃薯200g,琼脂20g,蔗糖20g,去离子水定容至1000ul),22~25℃黑暗下生长2-3天后统计细菌菌落数。以每片叶上白叶枯病菌菌落数的LOG值绘制细菌生长曲线。白叶枯病菌生长分析表明,在接种白叶枯病菌致病小种PXO341后,OsMAPK6基因超量表达水稻株系叶片中白叶枯病菌数量显著低于野生型对照。鉴定结果见图6。
序列表
<110> 华中农业大学
<120> OsMAPK6基因在改良水稻抗病性中的应用
<141> 2020-10-04
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6462
<212> DNA
<213> 水稻(oryza sativa)
<220>
<221> gene
<222> (1)..(6462)
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<221> 3'UTR
<222> (5999)..(6462)
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<222> (5829)..(5998)
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<221> Intron
<222> (5216)..(5828)
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<222> (3174)..(3311)
<220>
<221> Intron
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<220>
<221> Intron
<222> (487)..(616)
<220>
<221> exon
<222> (155)..(399)
<220>
<221> 5'UTR
<222> (1)..(154)
<400> 1
acacacaaaa aaaacacaca caaaaaaaaa agaggcgaga gctacaaaac gaggccgaaa 60
gcgaccaaat ctcgcgacga attccgcctc cactttccct tcccttcctc ctccacctcc 120
acctcctcgt cgcgatccaa atccgaatcc ggccatggac gccggggcgc agccgccgga 180
cacggagatg gcggaggccg gcggcgggca gcagccgcct gctgcggctg cggcggcggg 240
ggcgggggca ggggcgggga tgatggagaa catccaggcg acgctgagcc atggcgggag 300
gttcatccag tacaacatct tcgggaacgt gttcgaggtc accgccaagt acaagccccc 360
catcctcccc atcggcaagg gcgcctacgg catcgtctgg ttcgtttgct cccccctctc 420
tcccattccg ttggcggcgg cggcctgatt ttggtttgga ttttggattt tgggtttgga 480
tgacagctcg gcgctcaact cggagacggg ggagcaggtg gcgatcaaga agatcgccaa 540
cgcgttcgac aacaagatcg acgccaagcg cacgctcagg gagatcaagc tgctccgcca 600
catggaccac gagaatgtca gtgctcccga tccgctccat tgtttcgacc acggaattcc 660
cttccccccc acccaccctc gatttttagg cgcagatttt gagattctcg ttgaatgctc 720
ttgtctctgg ttagttaagt tggttgcatt aaatggggaa aggaggagga ggtgagggtt 780
aggttaccaa atgattgagt tcgtcactgc tcggcctaat ttggtagagc ttttccctcc 840
attcttgctg ctgatgatga gtgctgcgag cttcagaaat agaggtaact ttggagatgc 900
ctttaaagaa gccatctgcc ttttggggat gttgttattc attgtgttct tgaatagtgg 960
gggagcgagt gatgtgaaca gacctgacct ggagagtagc aacatcacat gattctcgta 1020
cgtgctgatg tcacaagcca caactagctt gaggaggagg aggaggagga ggaggaggag 1080
gaggaccaat ctggtgttac acctttttcc taagtcagtc aaatccagtc tgaatgcagt 1140
gggagtgact gccagtgggg agggaattag gggataggag tactaggaat tgtgttattg 1200
ttggaaagtt ttgagattga ctttgtgtgt gcatgttgga cttggacctt tgaaatttac 1260
caatttaaag actgactaat aggagtagcc cactgttgta tttcctgcgt aggcctgaag 1320
tatcattttc agcttaccag aggcaacata ttttttctat ggttagtggt agaaagacag 1380
ccacagggga agtgaattag agaataggcc atatcttcat agccagccct agaaaaactc 1440
agcttcttct gaagaggtga attaaatttg gatggtcata aatattccgt tataattaaa 1500
ctagtggatt ttcccatgca aatatgagag gagctagatt taaaattaga gttatatttt 1560
gtatagaatt tactggtcaa cctggaccgt tgtaatctag acccaccact ataataaacg 1620
ggcatatatt tatctttttt ccccgattaa tgtcgttatt ctaggcttct agattgaacg 1680
tggtggcacc cttttgcacc ctctttatta taacataata gatcaattga tacttcccta 1740
aaaaaggttc atacaaaaag ttgctcagat actgcttttg ctgaaccata gactggttga 1800
cacaggcata cacaaatgca tgctcatgcc tttttttgga agttttttca aactaaaact 1860
gatccatttt gtttgtatcc tactgcagaa agagaagtgt ttaatttcag gttttagtgc 1920
tccaattatg caggggaggc ttttgggagt tataccagtt acgggtgttt gtagaaaacg 1980
atcggattag gcaaattttg gagtagaagt ccttagataa tgcacctgct ttattgccat 2040
cagctaagat tgtgtatcaa gtcaaattct acttttcaaa tctggctata aattcaattg 2100
tatgtacgga aagttggaaa catgagctaa gatttgtaca tcaagtccaa ttctactttt 2160
aaaatatagc ttgcacaaaa aatggctatt ttgcattatt acataaaaca ttacttccaa 2220
attggcgatc ctgtattctg aaagatgtat tggaactttt tttttgtcat gtgcacgtgc 2280
tgcaggttgg ctgcagacag aaggcagcag cccgagtagc gatttctggc tgcagtatat 2340
tacttcaata ccaagttatc atttaattat gaagtgcact aagctgttgc tttctatgta 2400
catgtaagct tatctatgag ataaaaaaac aaacaaacaa aaaaagcccg tggttaggat 2460
ggcttgcggc attcagctaa gtcgagaaaa atgcccctga acgtggcacg tcactgggca 2520
tgcttaacct cacgcgggga gcactcttct accagcatta acgagggggg attttttttg 2580
tgcaaaacct gggcttgagc cttggtcggt gtcccctcta ccagagactc taccactgtg 2640
ctacacacac gtttgttcag cttatctatg agataaatta ttagaatcct ctacataagt 2700
tattagaatt atgcaaagtc tcctccattg cttccttcta ttgatctatt atatgtccat 2760
cttcgtctaa agccctttac tagtcactat tcaagtcatc attagcttgc tgttcatccc 2820
cattacacac tctaattttc attttaaggg aaatgcatgt tggtctgcct ccttaaaaaa 2880
ccaccatgtt tgagcgttta aatggcaaaa acacttgagc tataacatta agccatcatg 2940
aataagatag atctctttgt caagtcattt tcatatggta tttcctggtc catgcaatat 3000
gtttgttctt gctttggttg gtctggacat agttgattta cttattttat ctactcctat 3060
aagatatttg gagctggtct gttgtatttc acatatccag caagctcttg tgtttgcaca 3120
tcgatgctcc aaaaggcaat aacttgtctc aacttcctac ttatgtgatg cagattgttg 3180
ccataaggga tatcatacct cctccacaaa ggaattcatt caatgacgtt tatattgcat 3240
atgaattgat ggatactgat ctgcatcaaa ttattcgctc aaatcaagca ttgtcagagg 3300
agcactgcca ggtaccttct tcaaaagaaa aacacaactt tatttccact ttagcattta 3360
tttcatacaa atggcttttc tggtctgctt ggattctctt gcttgtctag gcctctgctc 3420
aaattgagtt ttcaagacct aaaaagtggg aagtattgaa gtatgtattg atggtttata 3480
aatgttttgt gtaagaacta taaggaattt ggtacaaaag gttgaatgta aagcttttat 3540
ggtaattttg tatcacattt taagaattct gcaggtatgc ataatctttg aaattaagaa 3600
acgaatttag cgacctacta atatacctga acgtaccttt gccctgcaaa gcatatatgc 3660
aaagtgctac aattaggtta tctcagtgca ccattgatca ttggatcaaa ctaacgtttt 3720
ctttggagat accatattat ttgctaaccg atataaaatc gaataatgag ttgattgcta 3780
tttatcagta gaccacatac tgttaggagc tggcggcaag gcaccgcagc tccgaaggtt 3840
gtagcagtgc ttgacgggag ggtgatgccg atgccctcgc ctcccacaaa gccatggtga 3900
ggagaaacag agaaaagagt ggccttatat aggcgattac aatctgctaa tgaatcagaa 3960
ctaactgaaa caaacctcaa atcttaaggg ataactctta tctaatgcaa gcaacagata 4020
acggcgagaa cgacactgtg gcgtgccatg cacgctcacc gaactcgtgt tcttgagaca 4080
ttcaaaggat tccccacata acacattcaa cgtgagaagg gttcaatgcc caatcacact 4140
ggagaggtct tgctgtattg cagcttgcaa gctcttatga tcaatgctac agatagatct 4200
ctccctttct tgagtggaac tctagaatgg ctgcctcacc tatctttcct tatccagata 4260
acttgattgt gttctcttgt attttccttg tgcatgcatc taccatgttc tgagaaataa 4320
tacatgtttg gtataactgt tcttgcgtaa ttctttacct ttttgtgcag tatttccttt 4380
atcagattct ccgtggcttg aagtatatac attcagcaaa tgtccttcac cgagacttga 4440
agcccagcaa cctacttttg aatgcaaatt gtgacctcaa aatttgtgat tttggacttg 4500
ctcgtaccac ctcagaaacc gattttatga ctgagtatgt tgtcacaaga tggtataggg 4560
caccggaact tctgttgaat tcctctgaat atactgcagc aattgatgtg tggtctgtgg 4620
gctgtatttt tatggaactc atggatcgta aacctttgtt tcctggaaga gatcatgtcc 4680
atcaattacg tctactaatg gaggttagaa cacactctta tcttttgaac tttattttat 4740
attgaattga tcaggttatg ttgtgtggtc atatatttag cataactgga gtactgtttt 4800
ggaaatatat aatatgttga ttatcctatg ggaatatgtt aaaactaagg aaaatgcaaa 4860
gaggagagaa tttatgttgc tccagaaggt cctggctttc tggctgatat gcaggatact 4920
gaattctgag tcatggctat taatacaaac gcatttctca ctagaggatt tatatactat 4980
ctgtttcaca ttattgtgtg tttgattttc ctatcataaa tttcttaatt gcagctcatc 5040
ggaacgccaa atgaggctga tctggatttt gtaaatgaaa atgcaagaag atacattcgc 5100
caacttccta gacatgcaag gcagtccttt cctgaaaaat ttccacatgt tcatccttta 5160
gcaattgatc tggttgaaaa gatgctgaca tttgatccta gacagagaat aacaggtcag 5220
tattggcata gtatgtttta gtgatttaaa gggcatttga atgttgtttt tgccatctta 5280
tctatcttgc caatagcaag taaaaaatag gcctttctcc taaagaaaga aaagagtgaa 5340
agaagccttt gaaagccctc ataaactatg gtttttttta acaccatgtc tacctttctt 5400
ttaatctgcc actggccatt tctagttagc gtttaccact tcctatctgt tttaagatag 5460
aaagtaacaa ctgcagtgga tacttaaata tatttgtaga gcatctgtgc atgacttcta 5520
tattactaaa tcacatgcaa acctaaatat tttttttaaa aaaaaaaact atgtcgatcc 5580
tttttacctc ttgatccacc ctaacctaat ccaggtggta taggtgtgtc atcactttat 5640
tcttaatgcg tgaattgagc atcattctta actcataggt gggaatgttg aaagagtcat 5700
tgccttgtgg tgttttctaa tgttgaaaat gcattgttcc ctgtgtcaaa acttttgacc 5760
tgattttctc tgcatgtttc atattactga atgccccctt atcttttctc ctatttcttc 5820
ttatccagtt gaaggtgccc ttgcacatcc ttacctggca tcactgcatg acataagtga 5880
tgagccagtc tgctcatcac ccttcagctt tgacttcgag cagcatgcat tgtccgagga 5940
acaaatgaag gatctaatct accaagaagg ccttgcgttc aaccctgatt accagtagct 6000
ggtgttctat ttcagccttg gattgattct attcatatgg agttttttcc tcctgcgcca 6060
caaaaggtcg ccgacagtga tcactagttg taaataattg cctcacctga aaaatcctcc 6120
ctggttcaaa gctgaaggtg ttgttctaag agtagaaatg tactttgtga tcaagttcct 6180
gggtagctgc tatgccattc ttatgcttat gtatgttgtt taatgtggga tttttttcca 6240
tcttaaatgt ttttagtccc ttttgtaaga agagttagtt catgaacgat gacggcctaa 6300
attctgcggt tatcatcaaa ttccccattt tcttgtcgat tcatggattt ctcatggttt 6360
tacttaatgc tccatgttgt aagacgtggt caatggaaga ggatatattg actcttgatt 6420
cagtggtggc agtttggagt tgatcgtaag actggaacat ta 6462
Claims (2)
1.水稻OsMAPK6基因在增强水稻对白叶枯病抗性中的应用,其特征在于,所述OsMAPK6基因的核苷酸序列如SEQ ID NO:1所示。
2.过量表达OsMAPK6基因在增强水稻对白叶枯病抗性中的应用,所述OsMAPK6基因的核苷酸序列如SEQ ID NO:1所示。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101386856A (zh) * | 2008-10-21 | 2009-03-18 | 华中农业大学 | 水稻抗病相关基因OsWRKY45-2和它在改良水稻抗病性中的应用 |
CN109112148A (zh) * | 2017-08-08 | 2019-01-01 | 华中农业大学 | 水稻OsMPK1基因在改良水稻抗病性中的应用 |
CN109207485A (zh) * | 2018-09-22 | 2019-01-15 | 华中农业大学 | OsAPS1基因在改良水稻抗病性中的应用 |
CN110904101A (zh) * | 2018-09-14 | 2020-03-24 | 华中农业大学 | miR395基因及其调控位点与应用 |
CN112266922A (zh) * | 2020-10-02 | 2021-01-26 | 华中农业大学 | OsMAPKK4基因在改良水稻抗病性中的应用 |
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Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101386856A (zh) * | 2008-10-21 | 2009-03-18 | 华中农业大学 | 水稻抗病相关基因OsWRKY45-2和它在改良水稻抗病性中的应用 |
CN109112148A (zh) * | 2017-08-08 | 2019-01-01 | 华中农业大学 | 水稻OsMPK1基因在改良水稻抗病性中的应用 |
CN110904101A (zh) * | 2018-09-14 | 2020-03-24 | 华中农业大学 | miR395基因及其调控位点与应用 |
CN109207485A (zh) * | 2018-09-22 | 2019-01-15 | 华中农业大学 | OsAPS1基因在改良水稻抗病性中的应用 |
CN112266922A (zh) * | 2020-10-02 | 2021-01-26 | 华中农业大学 | OsMAPKK4基因在改良水稻抗病性中的应用 |
Non-Patent Citations (11)
Cited By (2)
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---|---|---|---|---|
CN116042648A (zh) * | 2022-10-26 | 2023-05-02 | 华中农业大学 | OsGELP77基因在改良水稻抗病性中的应用 |
CN116042648B (zh) * | 2022-10-26 | 2024-02-02 | 华中农业大学 | OsGELP77基因在改良水稻抗病性中的应用 |
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