CN106676114B - 水稻基因OsUEP3及抗病调控功能的应用 - Google Patents
水稻基因OsUEP3及抗病调控功能的应用 Download PDFInfo
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- CN106676114B CN106676114B CN201710050804.6A CN201710050804A CN106676114B CN 106676114 B CN106676114 B CN 106676114B CN 201710050804 A CN201710050804 A CN 201710050804A CN 106676114 B CN106676114 B CN 106676114B
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Abstract
本发明提供一个水稻抗病调控基因,该基因的开放阅读框长390bp,编码一种泛素延伸蛋白,由129个氨基酸组成。该蛋白N端为76个氨基酸的泛素分子,C端则是53个氨基酸的60S核糖体蛋白L40e。本发明通过构建该基因的超表达转基因水稻及其抗病分析,首次阐明了该基因对水稻细菌性病害抗性的调控作用。超表达转基因水稻植株显著提高了对水稻白叶枯病菌菌株PXO99和PXO112的抗性,从而揭示了该基因对水稻白叶枯病抗性的正调控功能,而且调控作用具有针对不同菌株的广谱性。本发明提供的OsUEP3基因是一种适用于创制和选育广谱抗白叶枯病和其它细菌病害的水稻新材料和新品种的新基因资源。
Description
技术领域
本发明属生物技术领域,涉及一个水稻基因(OsUEP3)的抗病调控功能分析及其应用。
背景技术
1、植物基因克隆技术
植物基因克隆方法有很多种,随着基因组序列测定完成的植物物种的增多,基于数据库序列的植物基因克隆方法得到越来越广泛的应用。该方法操作步骤主要包括基于保守序列的引物设计,目的植物组织RNA提取,反转录酶-聚合酶链式反应(RT-PCR),PCR产物与载体的连接,连接产物的细菌转化,质粒提取,酶切检验,测序分析等。
2、植物基因表达控制和基因功能分析技术
植物基因的功能通过基因表达和蛋白积累来执行。因此,基因功能通常通过比较分析基因的超常规表达与正常表达情况下的表型(Phenotype)或功能发挥情况来判断和明确。基因的超常规表达包括高于正常水平的表达和低于正常水平的表达两大类。高于正常水平的表达主要为超表达/过表达(Over-expression),主要通过连接一个强启动子来驱动目的基因表达的方式来达到。低于正常水平的表达主要通过RNA干扰(RNA interfering,超表达)、基因敲除(Knock-out)等方式来达到。超表达通过构建和转化一个含有同一序列片段相反方向***一个内含子或其它不表达的序列形成的发卡结构来实现,结果是目的基因表达的降低。基因敲除(Knock-out)则通过在植物基因组中的目的基因中***一个长的非植物序列或切除目的基因等方式来达到,结果是目的基因表达的完全或近乎完全的抑制。构建基因超表达植株和/或超表达植株、基因敲除突变体,比较这些植株的表型和性状与野生型/正常植株的差异,就能明确目的基因对该性状的调节功能。
3、植物转基因技术
用于将外源基因导入目的植物,从而获取携带外源基因的转基因植物。包括基因枪法,农杆菌介导法等。农杆菌介导法包括将目的基因克隆入植物表达载体,表达载体对农杆菌的转化,携带表达载体的农杆菌对目的植物组织的感染,转基因植株的再生以及植株中基因转化和表达情况的检测分析等步骤。
4、植物抗病性检测分析技术
植物病原物包括真菌、卵菌、细菌、菌原体、病毒和线虫等多种类型。不同类型病原物的接种方法各不相同。对于真菌,能产生分生孢子的,通常以分生孢子悬浮液喷雾接种植物。不产生分生孢子的,则以菌丝块等接种植物。对于卵菌,一般先培养产生大量孢子囊,而后低温培养使之释放游动孢子,最后以游动孢子悬浮液接种植物。对于细菌,常以菌体悬浮液剪叶、针刺、注射、喷雾等法接种植物。对于病毒,常以提纯的病毒粒子或人工体外转录产物摩擦、注射、或通过昆虫等传播媒介接种植物。对于菌原体,常通过传播介体接种或者嫁接接种。对于线虫,常以一定数量的二龄幼虫接触植物根部茎基部或根围土壤中接种植物。多数情况下,接种后需要保持较高的相对湿度,合适的温度和光照条件。接种后按不同时间点连续观察病害发生症状,统计发病率和发病严重度,计算病情指数,采用RT-PCR检测病原物生物量。通过与感病对照植物发病情况的对比分析,明确目标植物的抗病性。
5、植物抗病育种技术
主要可以分为传统抗病育种和通过基因工程抗病育种两大类。传统抗病育种因为有天然遗传隔离现象使抗病资源可用范围受到显著限制,只能应用遗传关系较近的抗病资源,而且需要多次杂交和回交等,因此选育周期长,且需要大量人力物力。而基因工程育种方法是通过将外源的抗病调控基因通过农杆菌介导的方法等技术导入植物,使其获得原来不具有的抗病性。因此,基因工程育种方法打破了天然遗传隔离现象的限制,拓宽了抗病资源可用范围,而且具有操作相对简单方便、培育周期短、无需大量人工物力的特点。另外,可通过导入一个广谱抗病调控基因,或者多个不同抗病谱的基因,创制具有广谱抗病的品种。因此具有特别适宜培育广谱、持久抗病品种等优点。
发明内容
本发明的目的是提供一个水稻(Oryza sativa)基因OsUEP3,对水稻细菌性病害抗性具有调控作用。本发明克隆的水稻基因OsUEP3的核苷酸序列如SEQ ID NO:1所示,该基因的开放阅读框(ORF)长390bp,编码一种泛素延伸蛋白(Ubiquitin extension protein),由129个氨基酸组成,其序列如SEQ ID NO:2所示。该蛋白N端为76个氨基酸的泛素分子,C端则是53个氨基酸的60S核糖体蛋白L40e。本发明克隆的序列与水稻品种日本晴的基因组注释的LOC_Os03g13170.1有很高的相似性,仅有2个碱基的区别:LOC_Os03g13170.1序列的A6替换为G;G15替换为C。这些点突变没有导致氨基酸的变化。在本发明之前,该基因功能没有任何公开报告。本发明首次通过构建该基因的超表达转基因水稻及其抗病分析,阐明了该基因对水稻细菌性病害抗性的调控作用。结果显示,超表达转基因水稻植株显著降低了对水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)菌株PXO99和PXO112的抗性,从而揭示了该基因对水稻白叶枯病抗性的正调控功能(具体见实施例中的说明)。
本发明的另一个目的是提供所述的水稻OsUEP3基因通过创制基因超表达水稻来获取抗细菌性病害水稻材料的应用,是在通过创制超表达OsUEP3的转基因水稻纯合系来获得针对水稻白叶枯病菌不同菌株的广谱抗病材料中的应用,通过以下步骤实现:
(1)OsUEP3基因超表达结构的构建和获取
将OsUEP3基因ORF克隆入一个植物表达载体,使其受强启动子的驱使表达。
(2)转化OsUEP3基因超表达结构的农杆菌的获取
将构建好的OsUEP3基因超表达结构通过电击等方法转化对水稻具有强侵染能力的农杆菌菌株。
(3)超表达OsUEP3的转基因水稻创制和获取
通过农杆菌介导法将OsUEP3基因超表达结构导入水稻,获取转OsUEP3基因超表达结构的水稻。
(4)超表达OsUEP3的转基因水稻纯合系的获取
分别以抗生素抗性和OsUEP3基因表达为检测指标,检测转基因植株后代的性状分离情况,获取后代性状不再分离的、并能稳定遗传的超表达OsUEP3的转基因水稻纯合系。
(5)抗细菌病害的超表达OsUEP3的转基因水稻纯合系筛选鉴定和获取
以超表达OsUEP3的转基因水稻纯合系为材料,检测分析对各类水稻细菌病害的抗性,获取抗细菌病害的转OsUEP3基因水稻。
本发明的优点:(1)本发明提供的OsUEP3基因是优质抗细菌病害调控基因资源,利用该基因获取的抗病材料具有抗性强烈、抗病谱广等优点。水稻白叶枯病菌(Xoo)有明显的致病性分化,可以分为很多个小种。水稻对不同Xoo小种的抗性机制有显著差异,因此,获取同时兼抗不同Xoo小种的水稻基因资源对于水稻白叶枯病的防控具有重要价值。本发明提供的OsUEP3基因此前功能完全未知,本发明研究发现该基因兼具对Xoo菌株PXO99和PXO112的正调控作用,可通过构建OsUEP3-超表达水稻创制和获取兼具对Xoo不同菌株广谱抗性的新材料。因此,OsUEP3基因是一种适用于创制和选育广谱抗白叶枯病和其它细菌病害的水稻新材料和新品种的全新基因资源。(2)获取抗病材料周期短。获取抗病植物材料和品种的方法主要有常规的传统育种方法和利用抗病调控基因的基因工程育种方法。传统育种方法具有可用抗病资源范围受天然遗传隔离限制,选育周期长,需要大量人工物力等缺点。而基因工程育种方法则具有可用抗病资源范围广、操作相对简单方便、培育周期短、无需大量人工物力、特别适宜培育广谱、持久、高抗病性品种等优点。本发明利用抗病调控基因OsUEP3,采用基因工程方法,创制培育强烈抗细菌病害的水稻材料,具有周期短,选育快速等特点。
附图说明
图1为OsUEP3超表达水稻纯合系及其亲本水稻叶片接种水稻白叶枯病菌不同菌株后的症状和菌量检测分析结果比较图。OsUEP3超表达水稻纯合系169-6及其亲本水稻叶片采用剪叶法接种水稻白叶枯病菌(Xoo)菌株PXO99和PXO112。上:接种Xoo菌株14天后的症状;下:接种后叶片内的Xoo菌量检测结果。结果表明,与亲本相比,OsUEP3超表达水稻纯合系169-6形成的枯死病斑明显更短,菌量也显著降低。表明OsUEP3基因的超表达显著增强了对水稻白叶枯病不同菌株的抗性,揭示OsUEP3对水稻白叶枯病抗性起广谱正调控作用。
具体实施方式
本发明结合附图和实施例作进一步的说明。
实施例1
本发明克隆了一个水稻基因OsUEP3,首次通过构建超表达转基因水稻,阐明了该基因的广谱抗白叶枯病不同菌株的调控功能。同时,本发明构建的超表达转基因水稻具有增强的抗病性,因此,本发明提供了一套通过构建该基因的超表达转基因水稻、采用基因工程技术创制和获取广谱抗水稻细菌病害水稻新材料的技术体系。主要步骤包括:
1)水稻OsUEP3基因的克隆和保存
本发明提供的水稻OsUEP3基因通过以下步骤克隆获得。先根据水稻基因组数据库中OsUEP3序列设计了引物OsUEP3-F3(5’-caggcgcgcc atg cag atc ttc gtc aag acc-3’,斜体部分为Asc I酶切位点)(序列如SEQ ID NO:3所示),以及OsUEP3-R2(5’-caggtacc gttctt gat ctt ctt ctt ggg-3’,斜体部分为Kpn I酶切位点)(序列如SEQ ID NO:4所示)。采用TRIZOL试剂提取水稻叶片总RNA,采用RT-PCR方法扩增获取OsUEP3cDNA,通过1%琼脂糖凝胶电泳后割胶纯化回收PCR产物,连接pMD19-mT载体,热击转化大肠杆菌DH5α,在LB培养基中摇菌培养过夜,提取质粒,采用AscI/KpnI酶切的方法和以OsUEP3-F3/OsUEP3-R2为引物对的PCR方法分别检验所提取的质粒是否包含OsUEP3基因,最后送公司测序验证,从而成功克隆和获取OsUEP3基因cDNA全长序列。
克隆获得的OsUEP3核苷酸序列如SEQ ID NO:1所示,该基因的开放阅读框(ORF)长390bp,编码一种泛素延伸蛋白(Ubiquitin extension protein),由129个氨基酸组成,其序列如SEQ ID NO:2所示。该蛋白N端为76个氨基酸的泛素分子,C端则是53个氨基酸的60S核糖体蛋白L40e。本发明克隆的序列与水稻品种日本晴的基因组注释的LOC_Os03g13170.1有很高的相似性,仅有2个碱基的区别:LOC_Os03g13170.1序列的A6替换为G;G15替换为C。这些点突变没有导致氨基酸的变化。在本发明之前,该基因功能没有任何公开报告。
转化了携带有OsUEP3基因序列的载体的大肠杆菌,保存于-80℃冰箱。因此,可随时通过活化菌株,提取质粒,通过PCR扩增和酶切,将OsUEP3基因亚克隆至目的载体中,用于转基因等研究。
2)OsUEP3基因超表达结构的构建和获取
对上述1)中获取的pMD19-OsUEP3质粒以及植物超表达载体pCZD分别先进行Kpn I酶切、随后进行Asc I酶切(因为两种酶的buffer差异大,因此不能同时进行双酶切),将酶切后的目的基因与同样酶切后的pCZD载体进行连接、连接产物进行大肠杆菌转化、氨苄抗生素培养基平板筛选、酶切和PCR鉴定等步骤获取OsUEP3基因超表达结构pCZD-OsUEP3。超表达载体pCZD以玉米Ubq启动子驱动目的基因的表达,同时携带HA标签,便于分子鉴定和基因功能研究。
3)转化OsUEP3基因超表达结构pCZD-OsUEP3的农杆菌的获取
将OsUEP3基因超表达结构pCZD-OsUEP3通过电击等方法转化对水稻具有强侵染力的农杆菌菌株,如EHAl05,在含链霉素和卡那霉素的YEP培养基上筛选转化子,再通过Asc I+Kpn I酶切和PCR鉴定,获取携带OsUEP3基因超表达结构pCZD-OsUEP3的农杆菌。用于下一步水稻的遗传转化。
4)转OsUEP3基因超表达结构pCZD-OsUEP3水稻的创制和获取
通过农杆菌介导法将OsUEP3基因超表达结构pCZD-OsUEP3导入水稻。分为水稻愈伤组织的诱导、携带pANDA-OsUEP3的农杆菌感染水稻愈伤组织、潮霉素抗性植株的再生和鉴定等几大步骤,最后获取再生的转pCZD-OsUEP3的水稻T0代。具体操作步骤如下:
(i)水稻愈伤组织的诱导
种胚表面的消毒和清洗:取成熟完好的日本晴水稻种子,剥去种皮,留下完整的种胚。用70%乙醇溶液浸泡种胚1min,倒掉乙醇后用50%次氯酸钠溶液浸泡种胚,放在摇床上28℃,180rpm消毒40min。倒掉次氯酸钠溶液,用灭菌的ddH2O洗涤种胚至少10次,直至液体干净透明为止。用灭菌的滤纸风干种胚后,用镊子小心把种胚放入NB1固体培养基上,每个培养皿约30粒种子。之后用保鲜膜密封,置于培养箱中28℃暗培养。
愈伤组织的诱导:将种胚放在NB1上培养15天后,将分化出的愈伤组织转移到新鲜的NB2培养基上,在28℃黑暗条件下再培养10天。将健康亮黄的愈伤组织转移到NB3上培养大约10天。注意,为了提供充足营养,一个NB2板转成2个NB3板进行培养。
愈伤组织的继代培养:将健康的愈伤组织从NB3换至NB4上,在28℃黑暗条件下培养9天,此时的愈伤组织最适合农杆菌感染。
(ii)水稻愈伤组织的农杆菌感染
农杆菌的培养:将携带有OsUEP3基因超表达结构pANDA-OsUEP3的根癌农杆菌EHA105菌株从-80℃中取出,在含链霉素和卡那霉素的LB平板上划线,置于28℃恒温箱内培养(约2d),长出菌落后挑取单菌落,分别接种于含相应抗生素的50mL YEP培养液中,27℃恒温摇床(180rpm)培养约48h至OD600为0.5-0.8,转入无菌离心管中,室温条件下5000×g离心10min,去掉上清液,菌体用25mL AAM-AS培养液重新悬浮清洗后,再在相同的条件下离心一次,最后用约50mL AAM-AS培养液重新悬浮菌体到OD600为0.05-0.1(约109个细胞/mL),置于冰上即可用于水稻的转化。
农杆菌侵染:选择生长健康带亮黄色的愈伤组织,用勺子小心地刮进无菌的的小培养皿中,然后将重悬好的农杆菌液倒入培养皿中,尽量使菌液浸没所有愈伤组织,保持浸泡约30min,期间不时地轻轻摇晃。之后用灭菌的胶头滴管将菌液尽可能多地吸出。
共培养:将侵染过的愈伤组织放入NB-AS培养基上22℃黑暗培养3d。
带菌愈伤组织在抗性筛选前的清洗:共培养3d后,将带菌的愈伤组织集中放到500mL无菌瓶中,用灭菌的蒸馏水清洗愈伤组织8~10次直到液体透明,然后加入200mL含Amp(200mg/mL)和Cef(300mg/mL)的ddH2O,将其放到摇床,180rpm、27℃恒温下摇动30min,然后用灭菌的胶头滴管将洗出液吸出,最后将清洗干净的愈伤转入NB-CHA培养基,在28℃黑暗下培养14d后用于抗性筛选。
(iii)潮霉素抗性植株的再生
抗性筛选培养:将愈伤组织分系转入含抗生素(Cef,300mg/mL;Hyg,50mg/mL)的NB-CH1筛选培养基上,在28℃黑暗下培养,进行抗生素抗性筛选;
继代培养:将NB-CH1上经抗性筛选再生的潮霉素抗性愈伤组织,以系的方式转入含抗生素(Cef,300mg/mL;Hyg,50mg/mL)的NB-CH2筛选培养基上,在28℃黑暗下继代培养一次,培养时间大约1周。
抗性愈伤组织的预分化培养:将具有抗潮霉素的愈伤组织以系的方式转入含抗生素(Hyg,50mg/mL)预分化培养基Pre-H上,在28℃黑暗下培养15d。
抗性愈伤组织的再分化培养:将预分化充分的抗性愈伤以系的方式转入含抗生素(Hyg,50mg/mL)再分化培养基R-50上,在27℃光条件下培养大约3周。
生根培养:待潮霉素抗性愈伤组织分化出小根和绿色小芽,且小根长到1cm以上进行生根培养。用镊子将变绿的抗性芽从分化培养基上挑出,转入到含有抗生素(Hyg,50mg/mL)的1/2MS生根培养基上进行生根培养。
土壤种植:等潮霉素抗性苗长到根系完全后,将T0代无菌小苗在27℃下开盖培养锻炼2d,然后移栽到水田取回的熟土中,置于温室中进行常规管理,最后收获得到T0种子。
5)转OsUEP3基因超表达结构pCZD-OsUEP3的水稻纯合系的筛选鉴定和获取
分别以潮霉素抗性和OsUEP3基因表达为检测指标,检测转基因植株后代的性状分离情况。潮霉素抗性筛选在含潮霉素的平板上针对水稻种子开展进行,观察能否在潮霉素抗性平板上正常生长成健康小苗。基因表达采用实时荧光定量PCR等方法进行检测。获取后代性状不再分离的、并能稳定遗传的转OsUEP3基因超表达结构pCZD-OsUEP3的水稻纯合系。这些水稻纯合系在潮霉素抗性平板上全部能正常生长成健康小苗、且OsUEP3基因表达水平显著高于转空载体的对照。
6)OsUEP3基因超表达水稻纯合系的抗病性检测分析
以5)中获取的OsUEP3基因超表达水稻纯合系为材料,检测分析其对水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)菌株PXO99和PXO112的抗性。采用剪叶法接种。先摇菌制成浓度为OD600=1.0的Xoo细菌悬浮液,再以无菌剪刀蘸取菌液,在离叶尖3cm处剪叶,置于大塑料筐中覆膜保湿48h。
接种结果显示,在剪叶接种Xoo菌株PXO99后14天,亲本对照水稻叶片形成长度达16.4cm的枯死病斑,而OsUEP3基因超表达水稻纯合系169-6叶片则只形成平均长为9.3cm的枯死病斑,长度仅为对照的56.7%(图1左上)。菌量检测分析结果显示,亲本对照水稻叶片的菌量达1.25×109cfu/叶,而OsUEP3超表达水稻纯合系169-6叶片的菌量平均仅有6.50×108cfu/叶,仅为亲本对照的52.0%(图1左下)。剪叶接种Xoo另一个菌株PXO112后14天,亲本对照水稻叶片形成长度达15.4cm的枯死病斑,而OsUEP3基因超表达水稻纯合系169-6叶片则只形成平均长为8.4cm的枯死病斑,长度仅为对照的54.5%(图1右上)。菌量检测分析结果显示,亲本对照水稻叶片的菌量达3.10×108cfu/叶,而OsUEP3超表达水稻纯合系169-6叶片的菌量平均仅有9.96×107cfu/叶,仅为亲本对照的32.1%(图1右下)。
这些结果表明,OsUEP3超表达水稻纯合系169-6对两个Xoo菌株PXO99和PXO112的抗性均显著高于亲本对照。OsUEP3基因的超表达导致水稻对这些菌株抗性的显著增高,因此,OsUEP3对水稻白叶枯病菌不同菌株均起正调控作用。可以通过构建OsUEP3-超表达水稻纯合系来创建获取广谱抗水稻白叶枯病菌不同菌株的水稻新材料和新品种。
7)广谱抗细菌病害的超表达OsUEP3基因水稻纯合系的筛选鉴定和获取
以超表达OsUEP3基因的水稻纯合系为材料,检测分析对各类细菌病原物所致病害的抗性,比如水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola,Xoc)不同菌株、水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)的其它菌株等。通过接种分析和抗病评价,筛选和获取对Xoc和Xoo多个菌株均表现出抗性的、广谱抗细菌病害的OsUEP3基因超表达水稻。
<110> 浙江大学
<120> 水稻基因OsUEP3及抗病调控功能的应用
<160> 4
<210> 1
<211> 390
<212> DNA
<213> 水稻(Oryza sativa)
<220>
<221> CDS
<222> (1)…(390)
<400> 1
atgcagatct tcgtcaagac cctgactggg aagaccatca ccctcgaggt ggagagcagc 60
gacaccatcg acaatgtcaa ggctaagatc caggacaagg agggaatccc gccggaccag 120
cagcggctga tcttcgccgg gaagcagctg gaggacggac gcaccctggc tgactacaac 180
atccagaagg agtccaccct ccacctcgtc ctcaggctcc gtggcggtat catcgagccg 240
tcgcttcagg cgcttgcccg caagtacaac caggacaaga tgatctgccg caaatgctat 300
gcgcgcctgc accctagggc tgtcaactgc cgcaagaaga agtgtggtca cagcaaccag 360
ctgaggccca agaagaagat caagaactag 390
<210> 2
<211> 129
<212> PRT
<213> 水稻(Oryza sativa)
<400> 2
Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu
1 5 10 15
Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ala Lys Ile Gln Asp
20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys
35 40 45
Gln Leu Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu
50 55 60
Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Ile Ile Glu Pro
65 70 75 80
Ser Leu Gln Ala Leu Ala Arg Lys Tyr Asn Gln Asp Lys Met Ile Cys
85 90 95
Arg Lys Cys Tyr Ala Arg Leu His Pro Arg Ala Val Asn Cys Arg Lys
100 105 110
Lys Lys Cys Gly His Ser Asn Gln Leu Arg Pro Lys Lys Lys Ile Lys
115 120 125
Asn
<210> 3
<211> 31
<212> DNA
<213> 人工序列
<220>
<223> 根据水稻基因组数据库提供的水稻基因序列设计和人工合成,用作引物
<400> 3
caggcgcgcc atg cag atc ttc gtc aag acc
<210> 4
<211> 29
<212> DNA
<213> 人工序列
<220>
<223> 根据水稻基因组数据库提供的水稻基因序列设计和人工合成,用作引物
<400> 4
caggtacc gtt ctt gat ctt ctt ctt ggg
Claims (2)
1.一种水稻抗病调控基因OsUEP3在通过创制超表达OsUEP3的转基因水稻来获取广谱抗病材料中的应用,其特征在于,在通过创制超表达OsUEP3的转基因水稻纯合系来获得针对水稻白叶枯病菌不同菌株的广谱抗病材料中的应用,所述水稻白叶枯病菌株为PXO99和PXO112,所述抗病调控基因OsUEP3的核苷酸序列如SEQ ID NO:1所示,该基因的开放阅读框长390 bp,编码一种泛素延伸蛋白,由129个氨基酸组成,其序列如SEQ ID NO:2所示。
2.根据权利要求1所述的应用,其特征在于,通过以下步骤实现:
(1)OsUEP3基因超表达结构的构建和获取
将OsUEP3基因ORF克隆入一个植物表达载体,使其受强启动子的驱使表达;
(2)转化OsUEP3基因超表达结构的农杆菌的获取
将构建好的OsUEP3基因超表达结构通过电击等方法转化对水稻具有强侵染能力的农杆菌菌株;
(3)超表达OsUEP3的转基因水稻创制和获取
通过农杆菌介导法将OsUEP3基因超表达结构导入水稻,获取转OsUEP3基因超表达结构的水稻;
(4)超表达OsUEP3的转基因水稻纯合系的获取
分别以抗生素抗性和OsUEP3基因表达为检测指标,检测转基因植株后代的性状分离情况,获取后代性状不再分离的、并能稳定遗传的超表达OsUEP3的转基因水稻纯合系;
(5)抗细菌病害的超表达OsUEP3的转基因水稻纯合系筛选鉴定和获取
以超表达OsUEP3的转基因水稻纯合系为材料,检测分析对水稻白叶枯病菌PXO99和PXO112病害的抗性,获取抗细菌病害的转OsUEP3基因水稻。
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