CN117286153A - OsRLK40基因及其在水稻抗病方面的应用 - Google Patents
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Abstract
本发明公开了OsRLK40基因及其在水稻抗稻瘟病方面的应用，它是通过敲除水稻OsRLK40基因实现的；所述的OsRLK40基因，它的核苷酸序列如SEQ ID NO.1所示；所述的敲除是利用CRISPR/Cas9基因编辑技术敲除；本发明通过CRISPR/Cas9技术敲除所述基因OsRLK40，获得敲除突变体osrlk40；通过稻瘟病抗性鉴定发现，与野生型水稻相比，突变体osrlk40更抗病，病斑面积小，病叶携带稻瘟菌量少，表明OsRLK40负向调控水稻对稻瘟病的抗性。通过白叶枯病抗性鉴定发现，与野生型水稻相比，突变体osrlk40更抗病，病斑更短，表明OsRLK40负向调控水稻对白叶枯病的抗性。本发明有利于培育抗稻瘟病和白叶病的水稻品种，为后期筛选高抗和广谱抗性的水稻品种提供依据。
Description
技术领域
本发明属于分子生物技术领域，具体涉及OsRLK40基因及编码蛋白在提高水稻抗病性的应用。
背景技术
稻瘟病是由稻梨孢属真菌(Pyricularia oryzae)所引发的一种易流行的水稻重要真菌病害，对水稻产量造成严重的影响，是东北稻区水稻高产、稳产的重要限制因素之一。在稻瘟病害盛行的时候，产量一般减少10%～20%，较严重的地区可达40%～50%，局部区域甚至颗粒无收。
水稻白叶枯病是由革兰氏阴性细菌稻黄单胞菌（Xanthomonas oryzae pv.oryzae）所引起的一种全国各地均有发生，且南方地区更为严重的水稻细菌病害。水稻在整个生育期均可受到水稻白叶枯病危害,在孕穗期受害最为严重。受害的水稻地区一般减产约10%～30%，较严重的地区可达50%以上。
预计2030年,水稻年产量需在现有基础上再增产40%才能满足世界人口的需求。因此，由稻瘟病和白叶枯病引起的粮食安全问题一直受到广泛关注。目前的防治措施主要以化学防治和抗病育种为主，以生物防治为辅。虽然化学防治成本低见效快，但由于病原菌生理小种复杂易变，化学农药施用数年后常常出现药效下降甚至丧失，且长期施用易造成环境污染，难以持续施用。生物防治虽然对稻瘟病和白叶枯病的防治效果较为显著，但存在着见效慢，生防菌活性受环境影响等问题，很难大规模应用。
培育抗病品种有着安全、有效且可持续的优势。而传统抗病育种主要通过田间杂交和回交，再结合田间抗性鉴定和农艺性状的综合选择才能完成，需要经过多年多代的筛选才能选育出抗性品种。CRISPR/Cas9技术是近年来被广泛应用的基因编辑技术，有着效率高、操作简便、成本低等特点。该技术原理是借助gRNA与目标序列的特异性识别，引导核酸酶Cas9在靶位点定向切割DNA使之双链断裂，进而激发胞内DNA修复系统，引起基因突变，包括碱基插入、缺失和替换。近年来，利用该系统已经对水稻等植物进行了相关性状的遗传改良。在育种过程中，利用CRISPR/Cas9技术能够迅速得到安全、稳定遗传的纯合突变体，可应用于改良和培育抗病水稻品种，推进优异种质资源创制。
发明内容
本发明目的是为解决传统抗病育种的技术难题，而提供了OsRLK40基因及其在提高水稻抗性的应用。
水稻OsRLK40基因，它的核苷酸序列如SEQ ID NO.1所示。
如SEQ ID NO.1所示的水稻OsRLK40基因在水稻抗稻瘟病方面的应用。
一种水稻抗稻瘟病育种方法，它是通过敲除如SEQ ID NO.1所示的水稻OsRLK40基因实现的；
所述的敲除是利用CRISPR/Cas9基因编辑技术敲除；所述的OsRLK40基因的靶点核苷酸序列为：CACCATGGATGACGTGGCGCagg。
一种基因敲除载体，它是将OsRLK40基因的靶点核苷酸序列CACCATGGATGACGTGGCGCagg插入了载体Pylcrispr/Cas9P35S-H中。
本发明提供了一种水稻抗病育种方法，它是通过敲除水稻OsRLK40基因实现的；所述的OsRLK40基因，它的核苷酸序列如SEQ ID NO.1所示；所述的敲除是利用CRISPR/Cas9基因编辑技术敲除；所述的OsRLK40基因的靶点核苷酸序列为：CACCATGGATGACGTGGCGCagg。本发明通过CRISPR/Cas9技术敲除所述基因OsRLK40，获得敲除突变体osrlk40。通过稻瘟病抗性鉴定发现，与野生型水稻相比，敲除突变体osrlk40水稻更抗病，病斑面积小，病叶携带稻瘟菌量少，表明OsRLK40负向调控水稻对稻瘟病的抗性。敲除突变体osrlk40缺失了OsRLK40基因，对稻瘟病表现为抗性增强。通过水稻白叶枯病抗性鉴定发现，与野生型水稻相比，敲除突变体osrlk40水稻更抗病，病斑长度显著小于野生型，表明OsRLK40负向调控水稻对水稻白叶枯病的抗性。敲除突变体osrlk40缺失了OsRLK40基因，对水稻白叶枯病表现为抗性增强，可推广实际应用。本发明有利于培育抗稻瘟病和白叶枯病的水稻品种，为后期筛选高抗和广谱抗性的水稻品种提供依据。
附图说明
图1 利用CRISPR/Cas9基因编辑技术在水稻中花11（ZH11）背景中敲除所述基因OsRLK40示意图；
图2 osrlk40突变体对稻瘟病的抗性分析；A表示野生型中花11（ZH11）和osrlk40突变体接种稻瘟病菌后的表型；B表示野生型中花11（ZH11）和osrlk40突变体接种稻瘟病菌后，叶片中稻瘟病菌的菌量检测结果；
图3 osrlk40突变体对白叶枯病的抗性分析；A表示野生型中花11（ZH11）和osrlk40突变体接种白叶枯病菌后的表型；B表示野生型中花11（ZH11）和osrlk40突变体接种白叶枯病菌后，叶片病斑长度的统计结果。
具体实施方式
下面结合附图来进一步说明发明的具体实施方式，但本发明不仅仅限于以下实施例。在本发明的范围内或者在不脱离本发明的内容、精神和范围内，对本发明进行的变更、组合或替换，对于本领域的技术人员来说是显而易见的，且包含在本发明的范围之内。
实施例1 构建OsRLK40基因突变体
一、利用CRISPR/Cas9基因编辑技术构建OsRLK40基因突变体
利用http://skl.scau.edu.cn/网站，对目的基因OsRLK40（碱基序列如序列表SEQID NO.1所示）进行编辑靶点的设计，靶点位置如图1所示，其中靶点核苷酸序列为：CACCATGGATGACGTGGCGCagg（最后三个碱基为PAM位点）。将候选靶点核苷酸序列插入中间载体pYLsgRNA-OsU6a中，随后通过Golden Gate克隆方式，利用BsaI酶切位点将OsU6a-target-sgRNA片段插入到最终载体BGK03中（具体构建步骤参考文献：Lu Yuming, YeXiao, Guo Renming, Huang Jing, Wang Wei, Tang Jiuyou, Tan Longtao et al.(2017) Genome-wide targeted mutagenesis in rice using the CRISPR/Cas9 system.Molecular Plant, 10, 1242–1245.）。将构建成功的CRISPR/Cas9载体，通过农杆菌介导的方法，转化至野生型中花11的幼胚诱导的愈伤组织中，经潮霉素筛选获得阳性转基因植株。水稻品种中花11是由中国农科院作物研究所水稻花培组用京丰2号特特普的杂种后代与福锦杂交后，娶妻F2代花药经花培接种，从试管苗后代中选育而成。1988年3月经天津市农作物品种审定委员会审定命名。吉林农业大学吉林省作物病虫害绿色防控重点实验室保存。
二、阳性转基因植株编辑形式分子鉴定
以水稻叶片的基因组DNA为模板，用如下鉴定引物进行PCR扩增，对扩增产物进行Sanger测序，解析突变体编辑形式。
OsRLK40基因突变体鉴定引物为：
GP13932-10194-F：GGGGTGCTGCTAATGGAG；
GP13932-10194-R：CGATTTACAGTAGCCGTGT。
PCR扩增体系根据2×Es Taq MasterMix（Dye）使用说明书配制，具体为：2×EsTaq MasterMix（Dye）10 μL，上游引物（10 μM）0.8 μL，下游引物（10 μM）0.8 μL，DNA模板1μg，ddH2O补至20 μL。
PCR反应体系为：94℃预变性2 min；94℃变性30 s，58℃退火30 s，72℃延伸30 s，循环35次；72℃延伸2 min。
结果如图1所示，获得1个纯合敲除突变体株系osrlk40。在osrlk40中，OsRLK40基因起始密码子ATG下游2896 bp处插入一个碱基A，产生移码突变，导致了2953-2955 bp处提前形成了终止密码子。
实施例2 喷雾接种法进行稻瘟病抗性鉴定
将野生型中花11（ZH11）和纯合敲除突变体株系osrlk40种植于苗盘中，待其生长至四叶期时接种稻瘟病菌。
将保存在滤纸上的稻瘟病菌RB22菌株接种于番茄燕麦固体培养基平板上活化。刮取菌丝转至新的番茄燕麦培养基平板上扩大培养。将菌丝进行涂断，放置26℃黑光灯培养箱中诱导产孢。用0.1%吐温涂洗平板，过滤后，得到稻瘟菌孢子洗脱液，将孢子浓度调至5.0×105个/mL，待用。
接种时，用0.1%吐温均匀喷洒水稻叶片，然后将稻瘟菌孢子洗脱液均匀喷洒至水稻叶片上，接种后黑暗保湿培养24小时，撤去遮光布后，继续正常光照培养，接种7天后进行拍照记录发病情况。同时利用实时荧光定量PCR检测发病叶片的带菌量，取等量的野生型中花11（ZH11）和纯合敲除突变体osrlk40的叶片，提取基因组DNA，用如下引物进行荧光定量PCR，比较野生型ZH11与纯合敲除突变体osrlk40叶片所携带的稻瘟菌量，从而完成敲除突变体对稻瘟病的抗性评价。
实时荧光定量PCR引物为：
MoPot2-F：ACGACCCGTCTTTACTTATTTGG；
MoPot2-R：AAGTAGCGTTGGTTTTGTTGGAT；
OsUbi-F：GCCCAAGAAGAAGATCAAGAAC；
OsUbi-R：AGATAACAACGGAAGCATAAAAGTC。
荧光定量PCR扩增体系根据2×SYBR qPCR Mixture使用说明书配制，具体为：2×SYBR qPCR Mixture 5 μL，上游引物（10 μM）1 μL，下游引物（10 μM）1 μL，DNA模板0.6 μL，ddH2O补至10 μL。每个样3次重复。
荧光定量PCR反应体系为：95℃预变性10 min；95℃变性30 s，60℃退火1 min，循环40次；72℃延伸2 min。
结果如图2所示，相较于野生型，纯合敲除突变体株系osrlk40对稻瘟病的抗性显著增强，表现为纯合敲除突变体株系osrlk40叶片中的稻瘟菌量显著低于野生型。
实施例3 剪叶接种法进行白叶枯病抗性鉴定
将野生型中花11（ZH11）和纯合敲除突变体株系osrlk40种植于大棚温室中，对生长6周的水稻接种白叶枯病菌。
先将冻存于-80℃的白叶枯病菌PXO99A菌株划线活化于含有头孢氨苄抗生素的牛肉膏蛋白胨固体培养基平板上，于28℃培养一天。挑取单菌落至含有头孢氨苄抗生素的牛肉膏蛋白胨液体培养基，28℃摇培48 h后，9000 rpm离心3 min收集菌体。用0.1 M的MgCl2溶液重悬菌体，9000 rpm离心3 min，弃上清，重复一次，最后0.1 M的MgCl2溶液重悬菌体，利用分光光度计将菌悬液的OD值调至0.8，待用。
接种时，将生长6周的水稻移至光照适宜没有雨水的地方，用灭过菌的剪刀蘸取菌悬液，于每株水稻倒数第二叶的尖部向下约10 cm处剪下，放置2周，期间定期用蒸馏水喷洒叶面保持湿润。于两周后测量发病的长度，比较野生型ZH11与纯合敲除突变体株系osrlk40叶片上的病斑长度，从而完成敲除突变体对白叶枯病的抗性评价。
结果如图3所示，相较于野生型，纯合敲除突变体株系osrlk40对白叶枯病的抗性显著提高，表现为纯合敲除突变体株系osrlk40叶片上的病斑长度显著低于野生型。
Claims (5)
1.水稻OsRLK40基因，它的核苷酸序列如SEQ ID NO.1所示。
2.如SEQ ID NO.1所示的水稻OsRLK40基因在水稻抗稻瘟病方面的应用。
3.一种水稻抗稻瘟病育种方法，它是通过敲除如SEQ ID NO.1所示的水稻OsRLK40基因实现的。
4.根据权利要求3所述的一种抗病水稻育种方法，其特征在于：所述的敲除是利用CRISPR/Cas9基因编辑技术敲除；所述的OsRLK40基因，其靶点核苷酸序列为：CACCATGGATGACGTGGCGCagg。
5.一种基因敲除载体，它是将OsRLK40基因的靶点核苷酸序列CACCATGGATGACGTGGCGCagg插入了载体Pylcrispr/Cas9P35S-H中。
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