CN108291235B - 苹果中的白叉丝单囊壳抗性赋予基因 - Google Patents
苹果中的白叉丝单囊壳抗性赋予基因 Download PDFInfo
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Abstract
本发明涉及白粉菌抗性赋予基因，包含该抗性赋予基因的植物、植物部分和种子以及其在选择抗白叉丝单囊壳植物的用途。具体地，本发明涉及白叉丝单囊壳抗性赋予基因，其中由所述抗性赋予基因编码的氨基酸序列是由SEQ ID No.1所代表的一级氨基酸序列，或与SEQ ID No.1具有超过70％的同一性，优选超过80％的同一性，更优选超过90％的同一性，并且最优选超过95％的同一性的一级氨基酸序列；且其中所述抗性赋予基因是受损的。
Description
技术领域
描述
本发明涉及白叉丝单囊壳(Podosphaera leucotricha)抗性赋予基因、包含本发明的抗性提供基因的植物、植物部分和种子以及其在选择白叉丝单囊壳抗性植物的用途。
背景技术
白粉病(Powdery mildew，PM)是成千上万种植物的主要真菌病害，包括苹果(Malus domestica)、杏(Prunus armeniaca)、桃(Prunus persica)和草莓(Fragaria xananassa)等栽培蔷薇科。所有主要的蔷薇科作物生长地区均发生白粉病，导致严重损失。
由真菌白叉丝单囊壳引起的苹果白粉病是世界上与苹果最经济相关的病害之一。其症状是年幼绿色组织上的白色斑点，尤其是打开后第一天的叶子，而成熟的叶子表现出一定的抗性。被感染的叶子起皱、卷曲，并过早掉落。花和果实不是PM真菌的主要目标，但这些组织是有可能感染的。白粉病孢子在芽中越冬，然后在春季，随着植物生长的复制，它们开始新的感染。苹果PM在世界所有苹果产区都有发生。该病通过降低树木活力、花芽产量和果实质量而造成经济损害。苹果PM在幼芽、叶子、花朵和果实中产生症状。一般来说，叶子和果实的症状最为明显。
白叉丝单囊壳是白粉菌科家族的子囊菌真菌。在生长季节期间，这种专性活体营养不断在专门的短茎上产生无性孢子(分生孢子)，这种孢子被称为分生孢子，并且在子实体(子囊果)包裹的囊状子囊中产生有性孢子(子囊孢子)。分生孢子是透明的(透明，没有颜色)并且含有不同的纤维蛋白体，而每个子囊果(黑色)含有单个子囊和八个密集组合在一起的椭圆形子囊孢子。透明纤维蛋白体是折射包涵体，所述折射包涵体呈现出包括棒和圆锥在内的各种形状。子囊果在启动新的流行病中不起任何已知作用，因为子囊孢子不容易发芽。分生孢子是风分散性的，其发芽不需要游离水分。如果它们落在易感染组织上，它们会启动感染并产生菌丝体。
在商业果园中，杀真菌剂几乎总是用于控制霉变以及其他苹果病害。然而，白叉丝单囊壳在杀真菌剂重复使用时表现出对这些杀真菌剂产生抗性的能力。苯并咪唑对白叉丝单囊壳有活性，但由于苹果黑星病(黑星病)的广泛抗性发展，它们在苹果病管理中的应用受到限制。杀真菌剂通常在密集丛生阶段以7到10天的间隔施用，直到末端新梢生长结束，以确保施用与快速叶发育和开花后期相一致，并且确保新的生长不会长时间不受保护。进一步地，频繁使用杀真菌剂有几个缺点。首先，已有文件很好地记载了对环境的影响。第二，在部分地区，化学品及其应用的成本可以高达苹果生产总支出的20％。第三，Baudoin等(2008)和Dufour等(2011)已经记载了病原体的抗性种群的发展，其大大降低了化学处理的功效。因此，开发新的控制白粉病的替代方法引起了越来越多的兴趣。
抗PM品种的产生是使可持续的苹果种植成为现实可能并同时维持种植者的收入的最佳选择之一。使用较不易感染的苹果品种可能是预防PM最有效的手段。包括“Jonafree”、“Prima”和“Enterprise”在内的苹果品种表现出对PM的自然抗性，但它们没有得到广泛的培育。品种选择受商业吸引力、果实品质、适销性和传粉特性的影响大于疾病抗性。“Golden Delicious”、“Idared”和“Granny Smith”等苹果品种得到广泛种植，但其非常容易受到PM感染，需要化学病害管理。
开发抗性植物最常见的策略是关注抗性基因(R基因)的渗入。R基因编码识别病原体效应物并触发防御反应的蛋白质，由植物激素在其中起主要作用的信号传导网络介导(Pavan et al.，2010)。抗性表现为感染部位的局部过敏反应(Robert-Seilaniantz etal.,2007；Bruce and Pickett,2007；Bari and Jones,2009)。R基因几乎不能持久，因为病原体效应物突变使其能够克服抗性(Parlevliet et al.，1993)。此外，R基因通常来源于栽培种的野生近缘种，因此种间杂交障碍可阻止其渗入。此外，在成功杂交的情况下，R基因会引入一些不需要的特性，从而需要进行大量的回交，这在木质物种如苹果中是非常耗时的。
另一种替代方法是以易感基因(S基因)的失活为基础，易感基因的定义为其功能丧失导致隐性遗传抗性的基因(Pavan et al.，2010)。一些病原体能够通过激活植物蛋白来抑制植物防御，这些功能是植物免疫系统的负调控。编码这些植物蛋白的基因被称为易感基因(S基因)，它们的敲除释放了对植物防御的抑制并导致抗性(Pavan et al.，2010)。S基因的缺点是有时与敲除有关的多效表型(Pavan et al.，2011)。霉病基因座O(MLO)基因是PM S基因的典型例子。
由于敲除MLO基因而产生的抗性(mlo抗性)是1992年在大麦中发现的(
1992)，长期以来被认为是一种独特的抗性形式。然而，进一步的研究表明，MLO基因在整个植物界中大部分是保守的，它们的功能缺失导致在几个物种如拟南芥(Consonni et al.，2006)、豌豆(Pavan et al.，2011)、番茄(Bai et al.，2008)和辣椒(Zheng et al.，2013)中的抗性。并非所有的MLO基因都是S基因，MLO家族成员分为七个进化枝(Acevedo-Garciaet al.，2014；Pessina et al.，2014)。只有两个进化枝含有S基因：进化枝IV包含所有单子叶植物S基因(Panstruga et al.，2005；
et al.，2010)；进化枝V含有所有双子叶植物S基因(Consonni et al.，2006；Bai et al.，2008；Feechan et al.，2008；Winterhagen et al.，2008)。并非所有的进化枝IV和V的成员都是S基因。
考虑到白叉丝单囊壳感染对苹果生产的经济影响，本领域一直需要白叉丝单囊壳抗性提供基因。
发明内容
除其它目的之外，本发明的目的是满足本领域的这种需要。
根据本发明，除其它目的之外，通过提供所附权利要求书中概述的受损的白叉丝单囊壳抗性提供基因来满足上述目的。
具体而言，根据本发明的第一方面，除其它目的之外，上述目的是通过提本发明的供白叉丝单囊壳抗性赋予基因来实现的，其中由所述抗性赋予基因编码的氨基酸序列是SEQ ID No.1所示的一级氨基酸序列，或在本发明抗性赋予基因受损的条件下与SEQ IDNo.1具有超过70％同一性，优选超过80％同一性，更优选超过90％同一性，最优选超过95％同一性的一级氨基酸序列。
本文使用的序列同一性定义为，在本发明序列的全长上相同的连续一致的核苷酸或氨基酸的数目除以本发明序列全长的核苷酸或氨基酸的数目，并乘以100％。例如，与SEQID No.1具有80％同一性的序列，包含在SEQ ID No.1的589个氨基酸的总长度上有471或472个相同的一致氨基酸，即471或472/589*100％＝80％。
根据本发明的受损的抗性赋予基因，意在表示对白叉丝单囊壳提供降低的或甚至不存在的易感性的基因，所述白叉丝单囊壳如叶和茎上的粉状斑点所示。
根据本发明的受损的抗性赋予基因是突变的基因。目前的基因中的一个或多个突变可以通过不同的机制导致受损。例如，编码DNA序列的蛋白质中的一个或多个突变可导致突变的、截短的或非功能性的蛋白质。非编码DNA序列中的一个或多个突变可导致选择性剪接、翻译或蛋白质运输。或者，导致基因转录活性改变的一个或多个突变决定了可用于翻译成蛋白质的mRNA的量，由于编码蛋白质水平低或完全不存在，可导致抗性。此外，本发明的基因的受损可能是在翻译后引起的，即在蛋白质水平上。
在本文中，受损也表示为编码非功能性基因或蛋白质。虽然本发明基因的功能尚未确定，但通过建立植物中的白叉丝单囊壳抗性(非功能性)或白叉丝单囊壳易感性(功能性)，可以容易地确定非功能性基因或蛋白质。白叉丝单囊壳抗性(非功能性)植物通过包含发生突变的基因来表示，相对于SEQ ID No.1，该突变发生在蛋白质水平上，或无，或在观察到的包含SEQ ID No.2的mRNA的降低水平上。
功能性和非功能性基因或蛋白质也可以使用互补实验来确定。例如，在组成型启动子的控制下用本发明基因的复制将抗白叉丝单囊壳的苹果植物进行转化，这将导致白叉丝单囊壳易感苹果植物。
根据本发明，本发明的白叉丝单囊壳抗性赋予基因在受损时提供白叉丝单囊壳抗性。根据本发明，受损可以通过在本文中由SEQ ID No.1确定的蛋白质的缺失或减少来指示。在本领域中，已知许多机制导致基因在转录、翻译或蛋白质水平的受损。
例如，在转录水平的受损可以是转录调控序列(例如启动子、增强子、起始、终止或内含子剪接序列)中一个或多个突变的结果。这些序列通常位于由SEQ ID No.2所表示的编码序列的5'、3'或其内部。受损也可以由本基因中的删除、重排或插入来提供。
翻译水平的受损可以通过提前终止密码子或其他RNA至蛋白质的控制机制或翻译后修饰影响(例如，蛋白质折叠或细胞运输)来提供。
蛋白质水平的受损可以通过截短的、错误折叠的或受干扰的蛋白质-蛋白质相互作用来提供。
除潜在机制之外，通过减少或缺失根据SEQ ID No.1的功能性蛋白来指示根据本发明的受损。
鉴于此，根据本发明第一方面的一个实施例，根据本发明的受损包括在本发明基因中的一个或多个突变，其分别导致具有SEQ ID No.1所示的一级氨基酸序列或包含SEQID No.2的mRNA的蛋白质表达产物的缺失。
根据本发明的第一方面的另一个实施例，本发明的受损包括在本发明基因中的一个或多个突变，其导致非功能性的蛋白质表达产物。
根据本发明第一方面的另一个实施例，本发明的受损包括转录水平降低，其导致包含SEQ ID No.2的mRNA水平降低。
根据本发明第一方面的另一个实施例，本发明的受损包括包含SEQ ID No.2的mRNA的转录水平降低。
根据本发明的一个特别优选的实施例，本发明的白叉丝单囊壳抗性赋予基因来源于苹果(Malus domestica)。
根据第二方面，本发明涉及苹果植物，其在基因组中包含如上所述的受损的白叉丝单囊壳抗性赋予基因，其中该受损提供白叉丝单囊壳抗性。
根据本发明第二方面的一个优选实施例，与易感染白叉丝单囊壳的苹果植物相比，本发明的苹果植物显示，本发明的白叉丝单囊壳抗性赋予基因的表达或转录降低了至少10％，优选降低了至少20％，优选至少30％，更优选至少50％，甚至更优选至少70％，最优选至少80％，例如25％、35％、40％、45％、55％、60％、65％或75％。
根据本发明第二方面的另一个优选实施例，本发明的苹果植物显示本发明的白叉丝单囊壳抗性赋予基因的缺失表达或转录。
根据本发明第二方面的一个特别优选的实施例，本发明的苹果植物在其基因组中包含受损的白叉丝单囊壳抗性赋予基因，所述基因编码具有SEQ ID No.1的一级氨基酸序列或与SEQ ID No.1具有超过70％的同一性，优选超过80％的同一性，更优选超过90％的同一性，并且最优选超过95％的同一性的一级氨基酸序列的蛋白质；不同地表述，根据特别优选的实施例，本发明涉及包含受损的Mdmlo19基因或受损的MdMLO19基因的苹果植物。
根据第三方面，本发明涉及本发明的白叉丝单囊壳抗性植物的种子、植物部分或繁殖材料，其在其基因组中包含提供白叉丝单囊壳抗性的本发明的受损的白叉丝单囊壳抗性赋予基因。
根据第四方面，本发明涉及由SEQ ID No.2表示的分离的核苷酸序列，或与其具有超过70％同一性，优选超过80％同一性，更优选超过90％同一性，并且最优选超过95％同一性的核苷酸序列。
根据第五方面，本发明涉及由SEQ ID No.1表示的分离的氨基酸序列，或与其具有超过70％同一性，优选超过80％同一性，更优选超过90％同一性，并且最优选超过95％同一性的氨基酸序列。
根据第六方面，本发明涉及本发明的白叉丝单囊壳抗性赋予基因的应用，用于选择抗白叉丝单囊壳苹果植物应用的本发明的分离的核苷酸序列或本发明的分离的氨基酸序列，例如用于开发分子标记的序列。
附图说明
在下面的实施例中将进一步详细描述本发明。在该实施例中，参考了附图，其中：
图1：显示四个苹果mlo株系和对照“Gala”的病害进展曲线下的面积(AUDPC)，接种白叉丝单囊壳。在四次实验中考虑的平均AUDPC计算从15到24个生物学重复。误差线显示平均值的标准误差。根据Tukey和Games-Howell事后检验(P＝0.05)，与“Gala”比较的统计学显著性差异用星号表示。
图2：显示“Gala”和在21dpi下接种白叉丝单囊壳的mlo株系TG0、TG11、TG19和TG11+19的每cm2叶表面的分生孢子数目。柱表示在两个实验中测量的分生孢子的平均数量。误差线显示平均值的标准误差。根据Tukey事后检验，星号表示与'Gala'相比具有统计学显著差异(P＝0.01)。
图3：显示受感染的'Gala'，以及以3、10和21dpi下获得的线TG11+19和TG19叶片的明场显微镜图像。对于3dpi下的Gala，在更高的放大倍数下，显示出白叉丝单囊壳孢子的萌发。
图4：显示“Gala”、易感染株系TG0和抗性株系TG11+19的感染叶子的SEM显微图像。照片在21dpi下拍摄。
图5：在“Gala”(A，B)和抗性株系TG11+19(C，D)和TG19(E)的感染叶中显示在3dpi下形成的乳突。左侧的图像用明场显微镜拍摄，右侧用荧光显微镜拍摄。对于TG19株系，只显示用荧光显微镜拍摄的图像。
图6：显示在不存在感染白叉丝单囊壳的情况下在五个mlo株系中的两个苹果MLO基因的表达。每条柱代表从三到五个植物评估的线平均相对表达。误差线显示平均值的标准误差。星号表明，根据Tukey或Games-Howell事后检验，mlo株系与“Gala”的比较有显著性差异(P＝0.05)。
实施例
苹果属是由许多广泛分散和多样化的分类群组成的一个属，但几乎所有的世界商业苹果生产都集中在单一物种，即亚洲原产的苹果(Malus domestica)。苹果高度易受PM影响，品种和选择间差异较小。
在商业果园中控制PM通常需要大量使用杀真菌剂，这对苹果生产者具有经济影响。许多有机和无机杀真菌剂被用来控制白叉丝单囊壳。然而，白叉丝单囊壳在反复使用时表现出对这些材料的耐受性，而且这些合成产品不具有环境友好性。
为了提高苹果对这种病害的抗性，育种者开始向苹果(M.domestica)引入野生苹果属植物的遗传抗性，导致许多苹果属杂种(例如White Angel)。
开发了三个二元RNAi载体来沉默2个MLO基因。对MLO基因突变的转基因“Gala”的接种显示MdMLO19基因的沉默增加了苹果对白叉丝单囊壳的抗性。在突变的“Gala”株系中观察到对这种病害的易感性降低高达75％，MLO沉默的水平在90％左右。
筛选63个苹果品种的重测序数据，我们发现MdMLO19中存在一个天然的功能缺失突变。在其他四种考虑的MLO基因中未发现类似的突变(MdMLO5、7、11和18)。当这种突变存在于两个等位基因时，苹果栽培品种就具有抗性。
材料和方法
构建苹果中的MdMLO11和MdMLO19敲除
使用表2中列出的引物从MdMLO11和MdMLO19(表1中的编号)扩增RNAi的基因片段，并在关口pENTR/SD-TOPO((hermo Fisher Scientific，沃尔瑟姆，美国)克隆。此外，还开发了一种嵌合构建体，用于连接同时沉默MdMLO11和MdMLO19的RNAi片段(Abbott et al.,2002)。为此目的，在MdMLO11RNAi片段的3'末端和MdMLO19RNAi片段的5'末端添加EcoRI的限制性位点。两个片段都用EcoR1限制并且与T4DNA连接酶(New England Biolabs，Ipswich，美国)连接。将所得到的构建体克隆到pENTR载体中。测序后，将所有片段克隆到目的载体pHELLSGATE 12(Thermo Fisher Scientific，沃尔瑟姆，美国)中通过测序验证最终构建体，并通过电穿孔将其插入根癌农杆菌(A.tumefaciens)菌株AGL0中。使用设计用于在载体和MLO序列上退火的特异性引物，通过PCR测试根癌农杆菌转化的细胞中存在的构建体。
表1qPCR引物
| 名称 | 编号 | 正(‘5–‘3) | 逆(‘5–‘3) |
| EF1 | MD09G014760<sup>a</sup> | TACTGGAACATCACAGGCTGAC | TGGACCTCTCAATCATGTTGTC |
| 泛激素 | MD05G001920<sup>a</sup> | CATCCCCCCAGACCAGCAGA | ACCACGGAGACGAAGCACCAA |
| Md8283 | MDP0000375455<sup>b</sup> | CTCGTCGTCTTGTTCCCTGA | GCCTAAGGACAGGTGGTCTATG |
| MdMLO11 | MDP0000239643<sup>b</sup> | ATCGAAGGCTGTTGGAGCAA | AAGCACGTGAAAGACGGCTA |
| MdMLO19 | MDP0000168714<sup>b</sup> | CAGAGTGGCGACTGCACTTA | GGGACATGGAGTGCAAAGGA |
a可在http://bioinformatics.psb.ugent.be/plaza查阅
b可在http://www.rosaceae.org/gb/gbrowse/malus_x_domestica/查阅
表2RNAi引物
a可在http://www.rosaceae.org/gb/gbrowse/malus_x_domestica/.查阅
RNAi苹果苗木的研制
如Joshi等(2011)所述将RNAi构建体转移到苹果中。将来自栽培品种Gala的4周龄体外繁殖芽的顶部四片叶子的外植体保持在含有卡那霉素(Joshi et al.,2011)的培养基中，并在24℃下在具有16小时光照/8小时黑暗循环的生长室中生长。为了用PCR证实存在构建体，用Illustra Nucleon Phytopure试剂盒(GE Healthcare)提取来自再生苗木的基因组DNA。在CaMV 35S启动子(5’-CGCACAATCCCACTATCCTT-3')上退火的正向引物和反向引物对于RNAi片段是特异性的(表2)。使用
Green Master Mix(Promega，Fitchburg，美国)进行PCR。将构建体阳性的植物移至嫩枝繁殖培养基(SPM)：含有维生素的4.4g/LMurashige和Skoog培养基、30g/L蔗糖、0.7mg/L BAP、96mg/L FeEDDHA，pH 5.8。为了促进生根，将植物转移到含有IBA的培养基中以促进生根。一旦形成根，植物在125ml的覆盖有塑料袋且含有湿热压处理的草皮的盆中(“Terriccio Vegetal Radic”-Tercomposti Spa,Brescia,意大利)逐渐适应温室条件(25℃，16小时/8小时黑暗循环，相对湿度70±5％)。在三周中，每隔5-7天降低空气湿度以促进叶角质层的形成。然后取出塑料袋并将植物转移到1L盆中。如上所述使对照(未转化的体外生长的“Gala”)适应。
白叉丝单囊壳接种和苹果病害严重程度评估
为了产生PM接种物，从位于特伦蒂诺省(意大利)的果园的感染叶中分离出白叉丝单囊壳的局部菌株。通过在温室条件下在苹果幼苗上连续接种来维持真菌。通过用感染幼苗的叶子刷新近轴表皮来对植物进行干接种。为了促进真菌渗透，将植物在温度为25℃，相对湿度为100％的温室中温育6小时。然后将植物保持在25℃和80±10％相对湿度下直至评估结束。
四个接种实验在一年的不同时期进行。在每个测试中，考虑每个转基因株系的三至八个生物学重复。在四次实验中至少进行了三次品株系测试，总重复次数在15次和24次之间变化。在所有接种的叶7、14和21dpi下用肉眼评估病害严重程度。病害严重程度表示为PM菌丝体覆盖的近轴叶面积的百分比(间隔5％)，并计算单个植物平均值。转化植物中病害严重程度的降低表示为[(对照的严重程度-转基因的严重程度)/对照的严重程度]×100％。为了考虑所有的时间点，计算病害进展曲线下面积(AUDPC)，计算一段时间内的病害强度的总和(Campbell和Madden，1990；Madden et al.，2007)。如Angeli等稍作修改(2012)，评估存在于感染叶片上的白叉丝单囊壳分生孢子的数量：在21dpi下从每个重复中收集三片叶子，每片叶片切割四个直径为0.8cm的圆盘，每个重复总共12个圆盘。将叶片圆盘转移至含有5mL含0.01％吐温20(Sigma-Aldrich，圣路易斯，美国)的蒸馏水的50mL管中。将管涡旋1分钟，并在光学显微镜下用血球计数器测定每毫升分生孢子浓度。分生孢子量表示为每平方厘米(cm2)叶片的量。
接种苹果叶的组织学分析
在接种后3、10和21天收集每个重复的两片接种叶用于明场显微镜观察。为了显现真菌菌丝，将叶子在乙醇：乙酸(3:1，v/v)中清洗，直到叶绿素去除(大约48小时)。用250μg/ml的溶于乳酸、甘油和水(1:1:1)中的台盼蓝染色样品15分钟。在Vogel和Somerville(2000)中漂洗和固定后，在Leica LMD7000显微镜(韦茨拉尔，德国)的明场照明下观察菌丝。
将考虑用于扫描电子显微镜(Hitachi S-2300，东京，日本)的叶子固定在0.1M、pH7的Sorensen磷酸盐缓冲液和3％戊二醛中。24小时后，叶片在温和搅拌(80-100rpm)下在没有戊二醛的Sorensen缓冲液中洗涤两小时。之后，逐渐用四次乙醇洗涤脱水样品，乙醇浓度从40至100％，干燥并保存在离心管(falcon tube)中直至观察。在观察之前，用金金属化叶片的片段。用ImageJ软件(http://imagej.nih.gov/ij/)处理图像。
为了检测乳突，在乙醇：冰醋酸(3:1，v/v)中清除叶片直到叶绿素被除去，并在乳酸、甘油和水(1:1:1)的溶液中平衡过夜。使用Leica LMD6500显微镜(Leica Microsystem,韦茨拉尔德国)的LMD滤波器(BP滤波器380-420nm激发，415分色镜和BP 445-485nm发射)使乳突显现。
基因表达分析
为了鉴定显示沉默效应的株系，使用体外生长的转基因植物的第一个基因表达研究重复三次。在关于适应的转基因植物的第二项研究中，在PM接种之前立即在24hpi和10dpi下收集叶样品。对于每个时间点的每个株系，从五个不同的植物收集叶片样品。将样品冷冻在液氮中，并在80℃下保存。用Spectrumtmtm植物总RNA试剂盒(Sigma-Aldrich)提取总RNA，用DNAse I(Sigma-Aldrich)处理并使用SuperScript III逆转录酶(Invitrogen，Life Technologies，韦茨拉尔，美国)进行逆转录。根据Advanced Universal SYBR GreenSupermix(Bio-Rad，赫拉克勒斯，美国)以15μL反应体积进行qPCR分析，使用CFX96TouchReal-Time PCR检测系统(Bio-Rad，赫拉克勒斯，美国)和CFX Manager软件。样品在两个技术重复中运行，根据以下热循环参数：95℃3分钟，95℃10秒，然后以55℃30秒(40个循环)和95℃10秒。为了分析MdMLO19，使用了以前工作中考虑的引物对(表1；Pessina et al.,2014)。对于MdMLO11和涉及苹果与白叉丝单囊壳之间相互作用的17个基因的表达，使用NCBI引物设计工具设计新的引物对(http://www.ncbi.nlm.nih.gov/tools/primer-blast/)(表1)。使用cDNA的连续稀释(1/10-1/100-1/1000和1/10000)来计算引物对的效率，通过琼脂糖凝胶电泳来确认产物的期望大小。在每次qPCR运行后，确定特定最终解离曲线的存在，其中温度从65℃到95℃递增(每步0.5℃，5秒)。考虑的参考基因是延伸因子1、泛素蛋白和8283(表1)。已知它们都是苹果稳定的参考基因(Botton et al.,2011；Pessinaet al.,2014)。使用geNorm软件(medgen.ugent.be/～jvdesomp/genorm)进行的分析导致所有三个参考基因的M值低于1，在M值低于1.5的条件下被认为是足够的(Ling andSalvaterra,2011)。将阈值周期(Ct)转换为Hellemans等(2007)的相对表达水平，使用两个技术重复的平均Ct作为输入。作为参考Ct，采用野生型“Gala”在0hpi时的平均Ct。
统计
病害严重程度
严重程度数据通过SPSS统计软件包(IBM，Armonk，美国)进行分析。对于苹果和拟南芥(A.thaliana)，在进一步分析之前，来自相同植物的叶子的严重程度数据被平均。考虑了植物八片幼叶的苹果严重程度数据，而拟南芥数据来自所有叶片。在进行任何分析之前，数据显示为正态分布(Kolmogorov-Smirnov和Shapiro-Wilk检验P>0.05)并且具有齐次方差(Levene检验，P>0.05)。采用Tukey's事后检验的单因素方差分析检测各时间点的显著性差异(P<0.05)。为了满足方差分析的先决条件，根据方程y＝arcsin(x)将数据进行变换。在非齐次方差的情况下，应用Games-Howell的事后检验。在汇集来自独立实验的数据之前，测试单个实验的效果：实验没有显著效果出现。独立分析时间点14和21dpi下汇集的数据。如上所述处理了AUDPC数据用于严重程度数据。使用单因素方差分析，应用Tukey事后检验分析分生孢子数据的数量(P<0.05)。
qPCR数据分析
为了评估基因表达，根据式Y＝ln(x)(Pessina et al.,2014)将相对表达值按对数尺度转换，以获得方差的正态分布和同质性，并用Shapiro-Wilk(P≤0.05)和Levene(P≤0.05)检验分别评估。用Tukey's检验(P<0.05)对同方差数据进行成对比较，而采用统计软件包SPSS(IBM)用Games-Howell检验(P<0.05)对非方差数据进行分析。为了检测表达差异，将Tukey事后检验用单因素方差分析(P<0.05)应用于在0hpi收集的样品数据。防御基因表达分析用Fisher事后检验进行检验。
相关性
采用双尾Pearson相关性检验来研究AUDPC与10dpi下MLO基因的相对表达以及21dpi下严重度与分生孢子数量之间的相关性。所有数据都已经按照y＝arcsin(x)进行了变换，以实现正态分布。
代谢物
来自酚类代谢物的数据用Fisher事后检验进行单因素方差分析。在非同方差的情况下，应用Games-Howell事后检验，数据的Kruskall-Wallis非参数检验未正态分布。
结果
RNAi苹果苗木的研制
生成三种RNAi构建体，两种分别针对MdMLO11和MdMLO19单独敲除(i＝KD-MdMLO11，ii＝KD-MdMLO19)，第三种旨在同时敲除MdMLO11和MdMLO19(iii＝KD-MdMLO11+19)。如材料和方法(表3)中所述，获得80个再生株系，其中48个携带RNAi插入物。通过qPCR检测48个转基因株系以评估MLO基因表达的水平，但仅在其中三个中观察到显著的敲除(表3)。在这三个株系中，苹果的其他两个进化枝V基因(MdMLO5和7)未检测到脱靶敲除。将三个敲除株系(命名为TG11(转基因Gala)MdMLO11)，TG19和TG11+19适应温室条件，以及对照野生型“Gala”和TG0(携带MdMLO19的RNAi构建体的株系)没有显示显著的MLO基因敲除。将TG0、TG11、TG19和TG11+19表示为转基因株系，但只有TG11、TG19和TG11+19作为mlo株系。
进入适应程序的植物的存活率在90％以上。在温室条件下，与温室条件下的“Gala”相比，mlo株系显示正常生长。
表3.基因转移结果总结
| 基因转移 | 敲除基因 | 再生株系 | 确认转基因 | 选择的株系 |
| i | MdMLO11 | 39 | 23 | TG11 |
| ii | MdMLO19 | 33 | 19 | TG0,TG19 |
| iii | MdMLO11+MdMLO19 | 8 | 5 | TG11+19 |
RNAi苹果植物的降低的白叉丝单囊壳易感性
在四个独立实验中测试四种转基因株系和对照对PM的易感性。TG0，不显示任何MLO基因敲除的株系，显示出对白叉丝单囊壳的易感性水平与对照相当；TG11也有同样的结果，而TG11+19和TG19的病害严重程度明显下降(图1)。虽然TG11+19和TG19植物的叶子部分感染(图1)，但与对照相比，孢子覆盖的近轴叶面积的延伸明显减少(图1)。表4总结了病害严重程度降低的结果。
所有的转基因株系叶片上存在的分生孢子数量减少(图2)，但仅TG11+19和TG19的降低有统计学意义(P<0.05)。这与图1所示的病害严重程度评估相比较：与“Gala”相比，TG11+19显示分生孢子数减少63.3％，TG19为64.8％。在21dpi下的病害严重程度和在21dpi下的分生孢子计数之间发现显著(P＝0.01)但中度正相关(Pearson系数为0.525)。
通过明场显微镜和扫描电子显微镜(SEM)进一步分析TG11+19和TG19连同“Gala”，以跟踪白叉丝单囊壳感染的发展。在“Gala”中，在3dpi下已经观察到发育良好的叶子感染(图3A)，此时在TG11+19和TG19中真菌发育仍然受限(图3B和3C)。在10dpi下，在所有被考虑的株系的叶子上观察到分生孢子，但是在“Gala”中它们的数目更高(图3)。在21dpi下，“Gala”叶完全被孢子覆盖，并且可见大量分生孢子(图3A)。TG11+19和TG19的叶表面被孢子化的菌丝体部分定殖，但也观察到不能发育的分离的孢子，以及与“Gala”所指出的情况比较少的分生孢子梗(图3B和3C)。与TG0和“Gala”相比，SEM图像显示TG11+19上菌丝体的生长减少(图4)。
在3dpi下的所有株系(抗性的和易感的)中都观察到乳突的形成(图6)。与TG11+19和TG19相比，“Gala”(图6A，B)的乳突更小，形状更明确，发射的荧光更强烈(图6C，D，E)。
表4：用MLO RNAi构建体转化的株系中的白粉病病害严重程度降低(％)。
*根据Tukey事后检验(P＝0.05)，与对照相比统计学显著差异。
#Gala被用作对照(19个植物)，并假定有0％的病害减少。
°与21dpi下的Gala相比，TG11株系感染的水平更高。
MLO基因在mlo苹果株系中的表达
先前选择的mlo系的基因表达分析在温室适应的植物中重复。在TG11+19(P＝0.01)和TG11(P＝0.05)(图6A)中MdMLO11的表达显著减少，而TG11+19(P＝0.01)和TG19(P＝0.01)中MdMLO19的表达减少(图6B)。进化枝V的另外两个苹果成员MdMLO5和MdMLO7也进行了测试，但没有观察到任何转基因株系的显著降低，这一发现支持不存在脱靶沉默(数据未显示)。
尽管中等(皮尔逊系数＝0.515)，但MdMLO19和AUDPC-病害严重程度的量度-表达之间的相关性具有统计学显著性(P＝0.05)。相反，AUDPC与MdMLO11的表达之间没有显著相关性。
讨论
MLO S基因的天然和人工功能缺失突变降低了大麦(Büschges et al.，1997)、拟南芥(Consonni et al.，2006)、豌豆(Pavan et al.，2011)、番茄(Bai et al.，2008)和辣椒(Zheng et al.，2013)中对PM病原体的易感性。在双子叶植物中，所有PM易感基因都属于进化枝V(Consonni et al.,2006；Baiet al.,2008；Feechan et al.,2008；Winterhagenet al.,2008)。在之前的一篇文章中，我们确定了3个PM感染早期阶段上调的苹果MLO基因(Pessina et al.,2014)。其中两个，MdMLO11和MdMLO19属于双子叶植物进化枝V和MdMLO18进化枝VII。因为作为S基因的进化枝V外的MLO基因尚不清楚，所以只有MdMLO11和MdMLO19被认为是在苹果中被敲除的合理候选者。
MdMLO11和MdMLO19被敲除以评估它们在支持苹果对PM的易感性中的作用。RNAi被用来降低两种MLO基因的表达，并且尽管产生了大量的转基因“Gala”株系(48)，但只有其中3种检测到靶基因表达的显著减少。部分原因是由于短RNAi片段小于150bp，与我们实验中使用的片段相似，因其有限的敲除效率而闻名。另一方面，它们具有更具体的优势，从而避免了在我们的实验中检测到的其他进化枝V MLO基因的脱靶沉默的产生。
在一些物种中，MLO基因的剔除或敲除诱导引起多效表型，如叶片上的坏死斑点的形成和大麦的减产(
1992)、拟南芥的缓慢生长(Consonni et al.，2006)、辣椒的植株减小(Zheng et al.，2013)。在材料和方法中规定的温室条件下未观察到这种或其他意外的多效表型。
苹果转基因株系的温室接种导致TG11+19和TG19株系的病害严重程度统计学显著降低。由于MdMLO19在两个抗性系中都被敲除，因此认为这是导致PM易感性降低的最有效基因。MdMLO11的敲除并未导致敏感性的显著降低，甚至与MdMLO19组合的敲除导致敏感性的任何额外降低。结论是，苹果中由PM诱导的两个进化枝V基因中，仅MdMLO19是功能性S基因。MdMLO18，可以通过白叉丝单囊壳接种诱导的进化枝VII基因，不应该被认为是PM S基因。为了评估插入“目标无效”RNAi构建体对PM易感性的影响，考虑了TG0株系。TG0是从敲除MdMLO19的转移中获得的。在该株系中，记录了MdMLO19表达的降低(尽管不显著)以及PM敏感性的中度非显著降低。结论是插入“无效”RNAi构建体可能具有功能相关性，但是这不能被统计学证明。
MLO S基因功能缺失降低对PM病原菌的易感性的确切机制尚不完全清楚。然而，已知mlo抗性与分泌囊泡的运输(Miklis et al.，2007；Feechan et al.，2011)以及称为乳突的细胞壁附着物的形成有关(Consonni et al.，2006)。乳突由富含蛋白质和自发荧光的酚类化合物的胼胝质基质组成(Vanacker et al.，2000)，其形成取决于肌动蛋白依赖性的内膜运输(Hückelhoven，2014)。“Gala”、TG11+19和TG19株系的特征是在3dpi下存在乳突，但其形状和尺寸在抗性和易感性株系中不同。快速乳突形成(
et al.,2000)，在试图穿透部位(Stolzenburg et al。，1984)和不同生化组成(Chowdhury等，2014)增加乳突大小，可以解释有效和无效乳突之间的显著差异。在mlo株系中，特别是在TG19中，乳突的大小大于对照，这支持了较大尺寸增加乳突功效的假设。Chowdhury等(2014)表示，有效和无效乳突之间的差异在于有效乳突具有较高浓度的胼胝质、纤维素和阿拉伯糖基本聚糖。这可能反映了观察到的抗性和易感染株系乳突之间的荧光差异。事实上，MLO蛋白被认为是囊泡相关和肌动蛋白依赖的防御途径的负调节因子(Panstruga，2005)，一旦在真菌的控制下，它就会诱导肌动蛋白丝为生长中的菌丝提供营养(Miklis et al.,2007)。本文提供的数据支持这样的观点，即在苹果野生型中，病原体在穿透后控制物质向细胞壁的运输，改变乳突的组成并将其变为无效。在葡萄树中进行的类似工作(Pessina等人，未发表)支持这种解释：与对照相比，mlo葡萄株系显示较大和较少定义的乳头，类似于在mlo苹果中观察到的那些。
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序列表
<110> 埃德蒙马赫基金会
<120> 苹果中的白叉丝单囊壳抗性赋予基因
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Claims (3)
1.一种分离的白叉丝单囊壳抗性赋予基因，其特征在于，由所述抗性赋予基因编码的氨基酸序列是由SEQ ID No. 1所代表的一级氨基酸序列，并且其中所述分离的白叉丝单囊壳抗性赋予基因的表达、转录减少至少70%。
2.提供白叉丝单囊壳抗性苹果植物的方法，所述方法包括在其基因组中引入权利要求1所述的分离的白叉丝单囊壳抗性赋予基因。
3.根据权利要求1所述的分离的白叉丝单囊壳抗性赋予基因在选择、识别白叉丝单囊壳抗性苹果植物上的用途。
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| CN104561027A (zh) * | 2013-10-28 | 2015-04-29 | 常熟市董浜东盾蔬菜专业合作社 | 梨抗白粉病基因快速鉴定 |
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Also Published As
| Publication number | Publication date |
|---|---|
| US20180298403A1 (en) | 2018-10-18 |
| CN108291235A (zh) | 2018-07-17 |
| CL2018000874A1 (es) | 2018-10-05 |
| WO2017060294A1 (en) | 2017-04-13 |
| BR112018007149A2 (pt) | 2018-10-30 |
| EP3359673B1 (en) | 2021-05-19 |
| US11028406B2 (en) | 2021-06-08 |
| RU2018115275A (ru) | 2019-11-07 |
| RU2742492C2 (ru) | 2021-02-08 |
| RU2018115275A3 (zh) | 2020-04-02 |
| EP3359673A1 (en) | 2018-08-15 |
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