CN106480073A - 云南疣粒野生稻MeXB3基因及其应用 - Google Patents
云南疣粒野生稻MeXB3基因及其应用 Download PDFInfo
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- CN106480073A CN106480073A CN201610900935.4A CN201610900935A CN106480073A CN 106480073 A CN106480073 A CN 106480073A CN 201610900935 A CN201610900935 A CN 201610900935A CN 106480073 A CN106480073 A CN 106480073A
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Abstract
本发明涉及一种云南疣粒野生稻MeXB3基因及其应用，属于分子生物学技术领域。云南疣粒野生稻MeXB3基因，其cDNA序列是SEQ ID NO.1所示的DNA序列，由云南疣粒野生稻中克隆和鉴定。编码云南疣粒野生稻MeXB3基因，由EQ ID NO.2所示的氨基酸序列组成。云南疣粒野生稻MeXB3基因在抗水稻白叶枯病中的应用。本发明的有益效果在于：克隆并鉴定了云南疣粒野生稻的MeXB3基因，并获得转MeXB3基因植株。该基因在疣粒野生稻中属于诱导型表达，对获得的转基因材料进行鉴定后发现，该基因能增强水稻对部分白叶枯病菌小种的抗性，从而提高水稻抗白叶枯病能力，为水稻抗病育种提供基因资源，对培育高抗白叶枯病水稻具有重要的理论及实际意义。
Description
技术领域
本发明涉及一种云南疣粒野生稻MeXB3基因及其应用，属于分子生物学技术领域。
背景技术
由革兰氏阴性菌黄单孢水稻变种(Xanthomonas oryzae pv.oryzae,Xoo)引起的细菌性病害是严重的全球性水稻病害之一。水稻是亚洲许多国家的主要粮食，因此水稻白叶枯病对亚洲国家粮食生产的威胁尤为严重(Singh et al.2001),该病害也严重影响了我国水稻的高产、稳产，在发病较重的年份和地区该病可使水稻减产50％甚至绝收(章琦等，2007)。过量使用化学药物防治白叶枯病可能导致环境破坏，选育带有主效抗病基因的品种才是控制水稻白叶枯病行之有效的方法。但是由于白叶枯病菌生理小种变异频繁，新的抗性品种在使用几年后抗性下降或消失(Hulbert et al.2001)。因此，不断挖掘利用显得抗病基因资源，研究相关抗病机理，提升和延展抗病基因功能，已成为水稻抗病育种的重要研究内容。
在长期的自然选择下，野生稻对环境的适应性远远高于栽培稻，具很多优良性状，如抗病、抗虫、耐旱等。因此，野生稻已成为水稻育种中基因资源发掘的宝库。云南疣粒野生稻(Oryza meyeriana Baill)具有较强的抗逆性，特别是高抗甚至是免疫白叶枯病(钱韦等，2001)，在稻属植物中表现突出，已成为抗病育种的重要种质资源，但云南疣粒野生稻的抗病机理还不清楚。目前已发掘的水稻白叶枯病抗性基因共38个，其中7个来自于野生稻，即来自于普通野生稻的Xa21、Xa23、Xa30(Song et al.1995；Zhang et al.1996；Jin etal.2007)，小粒野生稻Xa27(Wu et al.2008)，药用野生稻Xa29(Tan et al.2004)，澳洲野生稻Xa32(苗丽丽等，2010)，仅有一个隐性基因xa32来源于疣粒野生稻的(阮辉辉等，2008)。但xa32基因为隐性，且与SSR标记距离较远，使得该基因很难被用于水稻育种。因此，从云南疣粒野生稻中发掘新的抗病基因资源，已成为疣粒野生稻研究的重要内容。
本发明所述的云南疣粒野生稻抗白叶枯病基因MeXB3属于蛋白泛素化降解过程的关键酶之一的E3泛素连接酶(ubiquitin ligase，E3)的编码基因，是水稻XB3基因的同源基因，与XBOS31(LOC4324295)有60％相似性。E3是一个超大的蛋白家族，可以分为HECT(homologous to the E6-AP carboxyl terminus)型、RING(Really Interesting NewGene)/U-box型、Cullin-RING型、APC/C型等(Vierstra RD et al.2009；Smalle etal.2004)。其中Ring型的E3的编码基因可以根据亚结构域分为30个亚家族，这些亚结构域包括BRCT、Ankyrin重复和vWA结构域(Wendy J et al.2012)。XB3基因是一个含有RING指结构域(Ring Finger Domain，RING)和锚蛋白重复序列区(Ankyrin Repeat Domain,ANKY)的泛素连接酶E3。水稻XB3基因所编码的蛋白是广谱抗病基因Xa21的锚定蛋白，XB3基因的缺失突变体中，Xa21水稻对白叶枯病的抗性降低。此外，XB3的表达量减少也导致了Xa21蛋白含量的减少。说明XB3对于Xa21的累积是必须的而且和Xa21调节的抗性相关(Wang etal.2006；Cheng et al.2010)。
目前还未见疣粒野生稻XB3基因研究的公开报道。因此，发明将为水稻抗病育种提供新的基因资源，为研究云南疣粒野生稻抗病机理奠定基础。
发明内容
本发明的目的在于克服云南疣粒野生稻基因发掘的不足，而提供一种云南疣粒野生稻MeXB3基因及其应用。
本发明的技术方案如下：
云南疣粒野生稻的MeXB3基因，其全长cDNA序列是SEQ ID NO.1所示的DNA序列。MeXB3基因全长cDNA序列长2133bp(碱基对)，编码437个氨基酸组成的蛋白，其氨基酸序列是SEQ ID NO.2所示的氨基酸序列。
本发明将云南疣粒野生稻MeXB3基因转入水稻，在抗水稻白叶枯病中的应用，
本发明的有益效果为：
本发明首次克隆并鉴定了云南疣粒野生稻的MeXB3基因，并获得转MeXB3基因植株。该基因在疣粒野生稻中属于诱导型表达，对获得的转基因材料进行鉴定后发现，该基因能增强水稻对部分白叶枯病菌小种的抗性，从而提高水稻抗白叶枯病能力，为水稻抗病育种提供基因资源，对培育高抗白叶枯病水稻具有重要的理论及实际意义。
附图说明
图1.qRT-PCR法进行MeXB3基因表达谱。
图2.MeXB3基因结构图。MeXB3基因具N端和C端分别有1个跨膜结构域，含有5个Ankryin保守结构域、1个Ring保守结构域。
图3.转MeXB3基因水稻植株的PCR检测。
图4.转MeXB3基因水稻植株抗白叶枯病的抗病性鉴定。
具体实施方式
下面结合具体实施实例，进一步阐述本发明。各实施例中无特殊说明的均为常规方法。
试验材料：云南疣粒野生稻(Oryza meyeriana Baill)，白叶枯病病原菌Y8，白叶枯病病原菌Y8为云南省典型的白叶枯病致病菌株，该菌株使用是利用其对水稻的侵染能力的共性从而导致水稻叶片形成白叶枯病病斑，因此，使用其它具有同样致病能力的各种白叶枯病菌菌株都能达到本发明的效果。以下各实施例所用试剂均为市售。
实施例1云南疣粒野生稻抗白叶枯病相关基因MeXB3的克隆及分析
(1)材料的培养及处理
云南疣粒野生稻(Oryza meyeriana Baill)栽于温室内，直至开花期。用NA培养基活化白叶枯病病原菌Y8，28℃培养2-3d，用无菌蒸馏水洗下菌苔，配制成OD600为0.6的菌液。在温室温度升至28℃时以剪叶法接种云南疣粒野生稻叶片，对照组用无菌水模拟接种，接菌后分别于2h、4h、8h、24h、48h、72h等量取样，对照即无菌水剪叶的云南疣粒野生稻也同时等量取样，取样后立即把样品放入液氮中速冻，并放-80℃冰箱中保存备用。
(2)总RNA的提取、定量及检测
分别取接菌后2h、4h、8h、24h、48h、72h的云南疣粒野生稻叶片100mg采用E.Z.N.A.Plant RNA Kit(美国OMEGA公司)试剂盒提取总RNA，操作方法按公司提供的试剂盒说明书进行。抽提完后，取0.5μL总RNA用Nanodrop2000分光光度计(美国Thermo FisherScientific公司)测定总RNA浓度，并用OD260/OD280的比值确认RNA纯度。取3μl总RNA在1％琼脂糖凝胶上分离总RNA，检测总RNA的完整性。
(3)Real time PCR(qRT-PCR)法分析MeXB3基因表达普
取接菌后不同时间的1ug总RNA做反转录合成cDNA第一链，具体方法按照PrimeScriptTM RT reagent Kit with gDNA Eraser反转录试剂盒(宝生物工程大连有限公司)说明书操作。根据MeXB3的EST序列设计qRT-PCR扩增引物，以水稻β-Actin基因作为内参基因，按照Premix Ex TaqTM II试剂盒(宝生物工程大连有限公司)进行qRT-PCR扩增，分析云南疣粒野生稻受白叶枯病菌胁迫后不同时期的表达情况。
MeXB3的EST序列上游引物MeXB3-QF的碱基序列如序列表中SEQ ID NO.3所示，MeXB3的EST序列下游引物MeXB3-QR的碱基序列如序列表中SEQ ID NO.4所示。
图1是MeXB3基因的qRT-PCR结果，结果显示MeXB3基因在疣粒野生稻中受白叶枯病菌表达，且在8h内随着接菌时间的增长表达量也随着增加，这说明MeXB3与疣粒野生稻抗白叶枯病有重要的关联。
(4)MeXB3基因全长cDNA的克隆
本发明中MeXB3基因全长cDNA的获得是通过3’RACE和5’RACE的方法实现的。3’RACE以(2)中提取的总RNA为模板，按照RACE 5’/3’Kit试剂盒(宝生物工程大连有限公司)说明书进行操作。5’RACE引物MeXB3-GSPS5序列如SEQ ID NO.5所示3’RACE引物MeXB3-GSPS3序列如SEQ ID NO.6所示。最后将MeXB3基因的5’端序列、3’端序列进行融合，拼接成一个完整的MeXB3基因的cDNA全长序列如序列表中SEQ ID NO.1所示。
云南疣粒野生稻抗白叶枯病基因MeXB3的EST序列如序列表中SEQ ID NO：7所示。
MeXB3基因EST序列的获得是通过对受白叶枯病菌诱导云南疣粒野生稻转录组测序的数据库中获得的，转录组测序是在北京百迈客生物技术有限公司进行。得到的序列在在NCBI(http://www.ncbi.nlm.gov)上进行Blast比对分析，得到与水稻XB3基因同源性达到94％的EST序列MeXB3。
(5)MeXB3基因结构分析
将(4)中获得的MeXB3基因全长cDNA核苷酸序列(如SEQ ID NO：1所示)用BioXM2.6翻译为氨基酸序列后提交到SMART(http://smart.embl-heidelberg.de/smart)上进行结构域分析。经分析MeXB3基因含有5个Ankryin保守结构域、1个Ring保守结构域和2个跨膜结构域，如图2所示。
实施例2云南疣粒野生稻MeXB3基因增强对水稻白叶枯病抗性的运用。
(1)MeXB3基因的表达载体构建
根据云南疣粒野生稻抗白叶枯病基因MeXB3的cDNA全长序列设计包含完整开放阅读框的引物，设计时加入限制性内切酶NcoI和BaglⅡ的酶切位点，用于将MeXB3基因重组到表达载体pCAMBIA2301中。MeXB3的ORF序列上游引物MeXB3-OF的碱基序列如序列表中SEQID NO.8所示，MeXB3的ORF序列下游引物MeXB3-OR的碱基序列如序列表中SEQ ID NO.9所示。
(2)转化农杆菌
取-80℃保存的LBA4404根癌农杆菌感受态，置冰上融解。取1μL含有MeXB3-F基因的表达载体pCAMBIA2301-MeXB3的质粒加入到100μL感受态中，混匀，加入到处理好的电击杯中。设置220V电压，点击转化。电击完成加入900μl的液体YEP培养基，28℃，200rpm摇床培养1.5h，菌液涂布在含50μg/mL卡那霉素(Kan)和100μg/mL利福平(Rif)平板上，28℃培养至形成单菌落。
(3)阳性克隆的鉴定与保存
挑取转化的农杆菌单菌落接种于含50μg/mL Kan和100μg/mL Rif的液体培养基中，28℃，200rpm摇床培养16h，取1μL菌液进行PCR检测，检测引物为MeXB3-OF和MeXB3-OR。取检测结果为阳性的菌液，混合30％甘油，置于离心管中，于-80℃超低温冰箱中保存，备用。
(4)农杆菌介导的水稻遗传转化
本发明是利用水稻模式品种日本晴的幼胚进行的遗传转化，具体操作过程如下：
①水稻愈伤的诱导和继代
取栽培稻日本晴幼胚去壳后用75％乙醇消毒30sec，无菌蒸馏水洗涤2-3次后，用15％的NaClO并加入一滴吐温20灭菌20min，消毒完之后用无菌蒸馏水漂洗5-6次，从无菌蒸馏水中取出幼胚置于无菌滤纸上吸干水分，然后接种于诱导培养基上，每皿20-30粒。28℃恒温培养箱暗培养两周后，将长出来的愈伤组织切下接到继代培养基上，继代一周以后用于农杆菌的转化。所述的诱导培养基的配方是：MS+蔗糖30g/L+2,4-D 3.0mg/L+KT 0.4mg/L，pH＝5.8；所述的继代培养基的配方是：MS+蔗糖30g/L+2,4-D 2.0mg/L+KT 0.4mg/L，pH＝5.8。
②含目的MeXB3基因的表达载体pCAMBIA2301-MeXB3的农杆菌悬浮菌液的制备。
取出保存于-80℃的含有目的MeXB3基因的表达载体pCAMBIA2301-MeXB3的根癌农杆菌重组菌株LBA4404，划线接种于添加了50μg/ml Kan和25μg/ml Rif的LB平板上，在28℃下倒置暗培养2-3d。从平板上挑取单菌落，接种于30ml添加了50μg/ml Kan和25μg/ml RifYEP液体培养基中，28℃，180rpm震荡培养16-20h进行活化。活化后得到含目的MeXB3基因的表达载体pCAMBIA2301-MeXB3的农杆菌悬浮菌液(以下简称：悬浮菌液)，用于浸泡栽培稻日本晴的愈伤组织。
③水稻愈伤组织与农杆菌的共培养
将继代的质量较好的栽培稻日本晴愈伤组织切割成直径2-5mm的小块并转入共培养基，然后将悬浮菌液注入共培养基直至使每个愈伤组织都能被菌液充分浸泡，1min后将多余菌液吸出，28℃恒温培养箱暗培养2-3天。所述的共培养基的配方是：MS+蔗糖30g/L+2，4-D 2.0mg/L+乙酰丁香酮(As)20mg/L，pH＝5.2。
④抗性水稻愈伤组织的筛选
首先将共培养的愈伤组织用加有250mg/L头孢拉定的无菌蒸馏水清洗5-6次，每次2-3min，再用MS液体培养基+500mg/L头孢拉定震荡清洗并过夜，重复2-3次，直至洗液清澈。将愈伤组织置于无菌滤纸上，在超净台将其吹干。吹干的愈伤组织转入筛选培养基，28℃暗培养两周，一些褐化的愈伤组织边缘长出乳白色的新的抗性愈伤组织，将这些新长出的愈伤组织转到新鲜配制的筛选培养基上继续筛选15-20d，之后继续生长的乳黄色质地紧密的愈伤组织即为抗性愈伤。所述的筛选培养基的配方是：MS+蔗糖20g/L+葡萄糖10g/L+2,4-D2.0mg/L+头孢拉定250mg/L+50mg/L潮霉素，pH＝5.8。
⑤抗性水稻愈伤组织的分化、生根及炼苗
从筛选培养基上挑选抗性愈伤组织转至分化培养基上，置于28℃，16h/d的光照培养箱中培养，经过10-15d有绿点出现，20-40d进一步分化出小苗。
待幼苗在分化培养基长至2-3cm后，将其转入生根培养基，置于28℃，16h/d的光照培养箱中培养，待幼苗根系比较发达苗高约10cm时进行炼苗。
将已长出根的幼苗根部的培养基清洗干净后放入大试管(30mm×180mm)中，加入自来水，放在光照培养架上，每2-3天换一次水。在室内炼苗7-8d后移栽至温室土壤环境条件下，每2d浇一次水，水面以不淹没小苗为度，如果天晴，需要遮荫到小苗成活(以吐水为准)即得到转基因再生植株。
所述的分化培养基的配方是：MS+蔗糖30g/L+6-BA 2.0mg/L+NAA 0.5mg/L+KT1.5mg/L+头孢拉定150mg/L，pH＝5.8。所述的生根培养基的配方是：1/2MS+蔗糖15g/L+NAA0.5mg/L，pH＝5.8。
(5)转基因再生植株的分子生物学检测
①PCR检测
待步骤(4)所获得的转基因再生植株生长旺盛时，取其嫩叶，提取叶片中的DNA。DNA的提取采用AxyPrep基因组DNA小量制备试剂盒(美国AXYGEN公司)按照说明书进行提取。
以提取的水稻总DNA为模板，非转基因植株为阴性对照(NC)，MeXB3基因的表达载体pCAMBIA2301-MeXB3为阳性对照(C)，以MeXB3-OF和MeXB3-OR为引物，进行PCR检测。检测结果如图3所示，其中标记为S1、S2、S3、S4、S7、S8、S9、S10的泳道处存在与阳性对照相同大小的基因片段，表明这几株再生植株为阳性转基因植株。
②MeXB3基因的亚细胞定位
由于MeXB3基因的表达载体pCAMBIA2301-MeXB3带有绿色荧光蛋白(GFP)，因此取①中鉴定为阳性转基因植株的根尖1-2mm置于载玻片上加一滴清水再盖上盖玻片，轻轻压碎根尖，在莱卡(Leica SP8)激光共聚焦显微镜下观察亚细胞定位情况，根GFP的激发波长将观察波长设置为480nm-550nm。观察结果显示MeXB3定位于细胞膜上。
③转基因再生植株抗白叶枯病鉴定
白叶枯病菌株Y8(本实验室保存)在NA培养基上28℃培养2-3天，配制成OD600处值为0.6的菌液。在孕穗期使用剪叶法对PCR鉴定的阳性转基因植株接种白叶枯病菌Y8生理小种，以同期栽培的非转基因日本晴为对照(共接种10株)，每株剪5片完全伸展的叶子的叶尖1-3cm。接种15天后，当病斑长度明显且稳定时调查病情，分别测量每片叶片的病斑长度，并统计每株植株病斑长度的平均值，根据方中达提出的抗性分级标准进行如下分级：
0级(I)：病斑长度0-0.2cm；
1级(HR)：病斑长度0.2-1.5cm；
3级(MR)：病斑长度1.5-3.0cm；
5级(MS)：病斑长度3.0-5.0cm；
7级(S)：病斑长度5.0-10.0cm；
9级(HS)：病斑长度大于10.0cm。
基因MeXB3转化植株对白叶枯病菌的抗病性鉴定结果见图4，表明本发明云南疣粒野生稻基因MeXB3转化植株对白叶枯病菌的抗性较非转基因植株有显著提升，表明本发明基因MeXB3为抗白叶枯病的新基因。
SEQUENCE LISTING 1
<110> 云南省农业科学院生物技术与种质资源研究所
<120> 云南疣粒野生稻MeXB3基因及其应用
<130> 云南省农业科学院生物技术与种质资源研究所
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 2133
<212> DNA
<213> Oryza meyeriana Baill
<220>
<221> 1-2133
<222> (1)..(2133)
<400> 1
cctttgctac ttgtgtatgt acaaaaggca cgcttggttg tacttgtaca gccagctttg 60
ggagaacagt taagagtagt tgaagaagaa aacgaagtag actttgaaga gaaataaaaa 120
ggatgcatgg aaaaagtaag gaaagggact gtaaagtagt agtatactat atctaaggat 180
catcgatatt gaaatgcggt actactaaca acaataaaac catctgttta aaattatttg 240
tcattttagt tttttctcag gtcctttaaa tatttgtatc tttataggat ggaaatgaaa 300
aaattagagt ggagaaatag aagagctttg aatgaaaaaa gaacaacgca agtggtttga 360
ggatgaaaga aacatccaat cgaccaatcc aatggcaatg ctctttcgtg tgtgtgtgta 420
tataaacaga gagagagaga cccaagtgtc ccgtcctctg ccgcctcctt gatcaccggc 480
ggccatcgcc tacctagcgg tggctatggg gcacggcctc agctgcagcc gcgacaccga 540
cgagcaccac ttgttccgcg cggcgcaggt gggcgacatc tacgcactgg gcgacctcct 600
cgccgccgac cccgcgctcg ctcgccgcgc aaccatatac ggccgcttca ccgctctgca 660
catcgccgcc ggcaatggca gcctccaggt ggtgtccatg ttgctggatc gcggtgttat 720
gccggacgtg ttgaaccgga agaggcagac gccgctgatg ctggcggcca tgcatggcaa 780
caccgactgc gtgctcgtcc tcctccacgc cggcgccaac atcctcacct tcgactcgcc 840
gcgcgccagg acgtgcctcc accacgccgc ctactgcggc cacgccgact gcctgcaggc 900
catcctcgcc gcctcctggg gcttcgcgcg cttcgtcaac gtcagggacg agcagggcgc 960
cacgccgctg catctcgccg ccaggcaggg ccgcccggcc tgcgtgcgcc tgctgctcga 1020
caagggcgcc atcgtctccg ccgccaccgg cgcctacggc ttccccggga gcacgccact 1080
gcacctggcc gcccgcgcgg gcaacctgga ctgcatccgg gagctgctcg cctggggggc 1140
ggaccgcctc cagagggact cggccggcag gatcgcgtac ggtgtcgcgc tcaggcgggg 1200
ccatcgcgcg tgcgccgcgc tgctcaaccc tgcggcggcg gagcccattg tgtggccgtc 1260
cccgctcaag ttcatcgccg agctcgaggc ggacgccaag gctctgctgg aggcggcgct 1320
catggaggcc aacagggaga gggagaagag gatcctccta ctgaaggagg gaagcgggta 1380
ctgtccgccg tcaccggctc actccgccgg cgcggacgag gaggaagctt gcaacatctg 1440
cttcgagcag gcgtgcagca tggaggtgaa ggagtgcggg caccagatgt gcgcggcgtg 1500
cacgctggcg ctctgctgcc acagcaagcc caaccccaag acactcctcc tgcacccgcc 1560
ggcctgcccc ttctgccgca ccaccatctc ccgcctcgtc gtcgccacca aggccgccgc 1620
cgacaggccg gcgtcaccgc gcctaagccg gcggcggtcg aggcgatctc gcgatgggag 1680
cagcagcttc aaggggctct cgtcggccat ggggtcattt tccaagatag ggcgcggctc 1740
cggccgattg gttgacggag gcgagctcgc ggacaagcct gatcacgacc tctcttctgt 1800
cgtcgtcggt gtcatgtgat ttgaaagcca aaaacccaga gacgcctgcc tgcattgcca 1860
ttaatttaat cttgttagag cttgggtaat caatcctcca gcaagctcaa ccaaggttga 1920
ctaattacca ttagcagcgt tactagtata tatacaccta ccatgttaat tgatacttga 1980
ccatacaccg atagctaggg caaataggaa tcaaataata ttgtattgca ttgaatttgt 2040
gcttgtagca ttttgttaca gtgccacctc cctggctagt tatttttggc ttgtcatact 2100
catagtaaat aaatactcct ccctatttac tgc 2133
SEQUENCE LISTING 2
<110> 云南省农业科学院生物技术与种质资源研究所
<120> 云南疣粒野生稻MeXB3基因及其应用
<130> 云南省农业科学院生物技术与种质资源研究所
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 437
<212> PRT
<213> Oryza meyeriana Baill
<400> 1
Met Gly His Gly Leu Ser Cys Ser Arg Asp Thr Asp Glu His His Leu
1 5 10 15
Phe Arg Ala Ala Gln Val Gly Asp Ile Tyr Ala Leu Gly Asp Leu Leu
20 25 30
Ala Ala Asp Pro Ala Leu Ala Arg Arg Ala Thr Ile Tyr Gly Arg Phe
35 40 45
Thr Ala Leu His Ile Ala Ala Gly Asn Gly Ser Leu Gln Val Val Ser
50 55 60
Met Leu Leu Asp Arg Gly Val Met Pro Asp Val Leu Asn Arg Lys Arg
65 70 75 80
Gln Thr Pro Leu Met Leu Ala Ala Met His Gly Asn Thr Asp Cys Val
85 90 95
Leu Val Leu Leu His Ala Gly Ala Asn Ile Leu Thr Phe Asp Ser Pro
100 105 110
Arg Ala Arg Thr Cys Leu His His Ala Ala Tyr Cys Gly His Ala Asp
115 120 125
Cys Leu Gln Ala Ile Leu Ala Ala Ser Trp Gly Phe Ala Arg Phe Val
130 135 140
Asn Val Arg Asp Glu Gln Gly Ala Thr Pro Leu His Leu Ala Ala Arg
145 150 155 160
Gln Gly Arg Pro Ala Cys Val Arg Leu Leu Leu Asp Lys Gly Ala Ile
165 170 175
Val Ser Ala Ala Thr Gly Ala Tyr Gly Phe Pro Gly Ser Thr Pro Leu
180 185 190
His Leu Ala Ala Arg Ala Gly Asn Leu Asp Cys Ile Arg Glu Leu Leu
195 200 205
Ala Trp Gly Ala Asp Arg Leu Gln Arg Asp Ser Ala Gly Arg Ile Ala
210 215 220
Tyr Gly Val Ala Leu Arg Arg Gly His Arg Ala Cys Ala Ala Leu Leu
225 230 235 240
Asn Pro Ala Ala Ala Glu Pro Ile Val Trp Pro Ser Pro Leu Lys Phe
245 250 255
Ile Ala Glu Leu Glu Ala Asp Ala Lys Ala Leu Leu Glu Ala Ala Leu
260 265 270
Met Glu Ala Asn Arg Glu Arg Glu Lys Arg Ile Leu Leu Leu Lys Glu
275 280 285
Gly Ser Gly Tyr Cys Pro Pro Ser Pro Ala His Ser Ala Gly Ala Asp
290 295 300
Glu Glu Glu Ala Cys Asn Ile Cys Phe Glu Gln Ala Cys Ser Met Glu
305 310 315 320
Val Lys Glu Cys Gly His Gln Met Cys Ala Ala Cys Thr Leu Ala Leu
325 330 335
Cys Cys His Ser Lys Pro Asn Pro Lys Thr Leu Leu Leu His Pro Pro
340 345 350
Ala Cys Pro Phe Cys Arg Thr Thr Ile Ser Arg Leu Val Val Ala Thr
355 360 365
Lys Ala Ala Ala Asp Arg Pro Ala Ser Pro Arg Leu Ser Arg Arg Arg
370 375 380
Ser Arg Arg Ser Arg Asp Gly Ser Ser Ser Phe Lys Gly Leu Ser Ser
385 390 395 400
Ala Met Gly Ser Phe Ser Lys Ile Gly Arg Gly Ser Gly Arg Leu Val
405 410 415
Asp Gly Gly Glu Leu Ala Asp Lys Pro Asp His Asp Leu Ser Ser Val
420 425 430
Val Val Gly Val Met
435
SEQUENCE LISTING 3
<110> 云南省农业科学院生物技术与种质资源研究所
<120> 云南疣粒野生稻MeXB3基因及其应用
<130> 云南省农业科学院生物技术与种质资源研究所
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Oryza meyeriana Baill
<220>
<221> 1-20
<222> (1)..(20)
<400> 1
ggccatgggg tcattttcca 20
SEQUENCE LISTING 4
<110> 云南省农业科学院生物技术与种质资源研究所
<120> 云南疣粒野生稻MeXB3基因及其应用
<130> 云南省农业科学院生物技术与种质资源研究所
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Oryza meyeriana Baill
<220>
<221> 1-20
<222> (1)..(20)
<400> 1
gacaccgacg acgacagaag 20
SEQUENCE LISTING 5
<110> 云南省农业科学院生物技术与种质资源研究所
<120> 云南疣粒野生稻MeXB3基因及其应用
<130> 云南省农业科学院生物技术与种质资源研究所
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> Oryza meyeriana Baill
<220>
<221> 1-25
<222> (1)..(25)
<400> 1
gcgcgacacc gtacgcgatc ctgcc 25
SEQUENCE LISTING 6
<110> 云南省农业科学院生物技术与种质资源研究所
<120> 云南疣粒野生稻MeXB3基因及其应用
<130> 云南省农业科学院生物技术与种质资源研究所
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> Oryza meyeriana Baill
<220>
<221> 1-25
<222> (1)..(25)
<400> 1
cgcgcttcgt caacgtcagg gacca 25
SEQUENCE LISTING 7
<110> 云南省农业科学院生物技术与种质资源研究所
<120> 云南疣粒野生稻MeXB3基因及其应用
<130> 云南省农业科学院生物技术与种质资源研究所
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1321
<212> DNA
<213> Oryza meyeriana Baill
<220>
<221> 1-480
<222> (1)..(480)
<400> 1
tggctatggg gcacggcctc agctgcagcc gcgacaccga cgagcaccac ttgttccgcg 60
cggcgcaggt gggcgacatc tacgcactgg gcgacctcct cgccgccgac cccgcgctcg 120
ctcgccgcgc aaccatatac ggccgcttca ccgctctgca catcgccgcc ggcaatggca 180
gcctccaggt ggtgtccatg ttgctggatc gcggtgttat gccggacgtg ttgaaccgga 240
agaggcagac gccgctgatg ctggcggcca tgcatggcaa caccgactgc gtgctcgtcc 300
tcctccacgc cggcgccaac atcctcacct tcgactcgcc gcgcgccagg acgtgcctcc 360
accacgccgc ctactgcggc cacgccgact gcctgcaggc catcctcgcc gcctcctggg 420
gcttcgcgcg cttcgtcaac gtcagggacg agcagggcgc cacgccgctg catctcgccg 480
ccaggcaggg ccgcccggcc tgcgtgcgcc tgctgctcga caagggcgcc atcgtctccg 540
ccgccaccgg cgcctacggc ttccccggga gcacgccact gcacctggcc gcccgcgcgg 600
gcaacctgga ctgcatccgg gagctgctcg cctggggggc ggaccgcctc cagagggact 660
cggccggcag gatcgcgtac ggtgtcgcgc tcaggcgggg ccatcgcgcg tgcgccgcgc 720
tgctcaaccc tgcggcggcg gagcccattg tgtggccgtc cccgctcaag ttcatcgccg 780
agctcgaggc ggacgccaag gctctgctgg aggcggcgct catggaggcc aacagggaga 840
gggagaagag gatcctccta ctgaaggagg gaagcgggta ctgtccgccg tcaccggctc 900
actccgccgg cgcggacgag gaggaagctt gcaacatctg cttcgagcag gcgtgcagca 960
tggaggtgaa ggagtgcggg caccagatgt gcgcggcgtg cacgctggcg ctctgctgcc 1020
acagcaagcc caaccccaag acactcctcc tgcacccgcc ggcctgcccc ttctgccgca 1080
ccaccatctc ccgcctcgtc gtcgccacca aggccgccgc cgacaggccg gcgtcaccgc 1140
gcctaagccg gcggcggtcg aggcgatctc gcgatgggag cagcagcttc aaggggctct 1200
cgtcggccat ggggtcattt tccaagatag ggcgcggctc cggccgattg gttgacggag 1260
gcgagctcgc ggacaagcct gatcacgacc tctcttctgt cgtcgtcggt gtcatgtgat 1320
t 1321
SEQUENCE LISTING 8
<110> 云南省农业科学院生物技术与种质资源研究所
<120> 云南疣粒野生稻MeXB3基因及其应用
<130> 云南省农业科学院生物技术与种质资源研究所
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Oryza meyeriana Baill
<220>
<221> 1-20
<222> (1)..(20)
<400> 1
ttgctggatc gcggtgttat 20
SEQUENCE LISTING 9
<110> 云南省农业科学院生物技术与种质资源研究所
<120> 云南疣粒野生稻MeXB3基因及其应用
<130> 云南省农业科学院生物技术与种质资源研究所
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Oryza meyeriana Baill
<220>
<221> 1-20
<222> (1)..(20)
<400> 1
ccgacgacga cagaagagag 20
Claims (3)
1.一种云南疣粒野生稻MeXB3基因，其特征在于其cDNA由SEQ ID NO.1所示的DNA序列组成。
2.编码云南疣粒野生稻MeXB3基因，其特征在于由EQ ID NO.2所示的氨基酸序列组成。
3.根据权利要求1所述的云南疣粒野生稻MeXB3基因在抗水稻白叶枯病中的应用。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111116722A (zh) * | 2020-01-20 | 2020-05-08 | 华中农业大学 | 野生稻抗菌肽OrR935及其应用 |
| CN111116723A (zh) * | 2020-01-20 | 2020-05-08 | 华中农业大学 | 野生稻抗菌肽OrR214及其应用 |
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| CN103194431A (zh) * | 2013-04-17 | 2013-07-10 | 北京大学 | Ring1蛋白在提高植物对水稻矮缩病毒抗性中的应用 |
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| 付坚等: "云南疣粒野生稻抗白叶枯病相关基因分析", 《2015年中国作物学会学术年会论文摘要集》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111116722A (zh) * | 2020-01-20 | 2020-05-08 | 华中农业大学 | 野生稻抗菌肽OrR935及其应用 |
| CN111116723A (zh) * | 2020-01-20 | 2020-05-08 | 华中农业大学 | 野生稻抗菌肽OrR214及其应用 |
| CN111116722B (zh) * | 2020-01-20 | 2022-03-08 | 华中农业大学 | 野生稻抗菌肽OrR935及其应用 |
| CN111116723B (zh) * | 2020-01-20 | 2022-03-08 | 华中农业大学 | 野生稻抗菌肽OrR214及其应用 |
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