CN111235164A - 疫霉菌抗性的负调控因子AtPPR1基因及其同源基因 - Google Patents
疫霉菌抗性的负调控因子AtPPR1基因及其同源基因 Download PDFInfo
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Abstract
本发明公开了疫霉菌抗性的负调控因子AtPPR1基因及其同源基因，包括AtPPR1基因和AtPPR1基因编码的蛋白，AtPPR1基因的核苷酸序列如Tair Gene Locus：At4G02820.1所示，AtPPR1基因编码的蛋白的氨基酸序列如TAIR Accession AASequence NO.：1009127979所示；本发明的负调控因子AtPPR1基因作为负调控因子在提高植物疫霉菌抗性上的用途，可用于抗疫霉菌品种的选育；AtPPR1作为一个新型的植物免疫负调控因子，通过干扰植物内源茉莉酸和水杨酸的下游信号传递以及ROS信号，负调控植物对疫霉菌的抗性。
Description
技术领域
本发明涉及生物技术领域，尤其涉及疫霉菌抗性的负调控因子AtPPR1基因及其同源基因。
背景技术
卵菌(Oomycetes)是一类包括疫霉属在内的、类似真菌菌丝生长的真核微生物，在进化上与藻类同界，但却是独立的、具有独特分布地位的群体，包括疫霉属(Phytophthora)、水霉目(Saprolegniales)和霜霉目(Peronosporales)等。疫霉属中的许多成员都能引起毁灭性的植物病害。例如，致病疫霉菌(Phytophthora infestans)引起马铃薯晚疫病曾经引发了19世纪的爱尔兰大饥荒，造成爱尔兰人口锐减近四分之一并有约一百万人因饥饿移居海外，该事件对爱尔兰的政治、经济和文化产生了深远的影响。寄生疫霉菌(P.parasitica)导致的烟草黑胫病是世界性的烟草主要病害之一，每年在中国的各大主要产烟区都有不同程度的发生，是全球烟草产业的严重威胁；
目前对于卵菌如疫霉菌的防控主要依赖于利用已有的抗病品种以及化学防治的方法，但由于疫霉菌进化速度快，易通过变异使抗病品种抗性丧失并且易对化学药剂产生抗药性，因此开发新型抗病途径挖掘新的抗病资源势在必行；
在植物与病原菌的亲和互作过程中，病原菌会依赖于一系列由植物调控因子参与的营养传递、分子交换以及激素信号转导过程来实现成功的侵染和定植，当这些感病因子缺失时，植物就会表现出对于病原菌的抗性，这为植物抗病育种打开了新的思路。通过寻找植物感病因子的天然突变体或定点突变使其丧失功能使植物的抗性增强的策略已经在植物抗真菌的育种中得到了应用；
目前已经鉴定到多个植物对卵菌抗性的免疫负调控因子，但多集中于抗霜霉目卵菌，对于疫霉菌的免疫负调控因子研究较少，并且许多免疫负调控因子的具体作用机制还不明晰，虽然近几年在马铃薯中对于卵菌免疫负调控因子的应用有所探索，但到目前还没有应用于作物抗病育种中，因此，本发明提出疫霉菌抗性的负调控因子AtPPR1基因及其同源基因，以解决现有技术中的不足之处。
发明内容
针对上述问题，本发明提出疫霉菌抗性的负调控因子AtPPR1基因及其同源基因，本发明的负调控因子AtPPR1基因作为负调控因子在提高植物疫霉菌抗性上的用途，可用于抗疫霉菌品种的选育；AtPPR1作为一个新型的植物免疫负调控因子，通过干扰植物内源茉莉酸和水杨酸的下游信号传递以及ROS信号，能负调控植物对疫霉菌的抗性。
本发明提出一种疫霉菌抗性的负调控因子AtPPR1基因，包括AtPPR1基因和AtPPR1基因编码的蛋白，所述AtPPR1基因的核苷酸序列如Tair Gene Locus：At4G02820.1所示，所述AtPPR1基因编码的蛋白的氨基酸序列如TAIR Accession AASequence NO.：1009127979所示。
所述AtPPR1基因在本氏烟草中的1个同源基因为NbPPR1；
所述AtPPR1基因在马铃薯测序基因组中的2个同源基因的氨基酸序列如GenBankAccession Number：XP_015167864.1和XP_006366127.1所示。
进一步改进在于：所述NbPPR1的氨基酸序列如SequenceIDNiben101Scf00317g06017.1所示。
进一步改进在于：所述NbPPR1与AtPPR1蛋白序列相似度大于50％，所述NbPPR1与AtPPR1基因有相似的负调控植物对疫霉菌抗性的功能。
进一步改进在于：所述AtPPR1基因在马铃薯测序基因组中的2个同源基因与AtPPR1基因具有＞50％的氨基酸序列相似度。
所述AtPPR1基因和AtPPR1基因编码的蛋白在马铃薯晚疫病和烟草黑胫病防治中的应用。
所述AtPPR1基因、AtPPR1基因编码的蛋白及其同源基因在植物疫霉菌抗性上的应用。
本发明的有益效果为：本发明的负调控因子AtPPR1基因作为负调控因子在提高植物疫霉菌抗性上的用途，可用于抗疫霉菌品种的选育；AtPPR1作为一个新型的植物免疫负调控因子，通过干扰植物内源茉莉酸和水杨酸的下游信号传递以及ROS信号，能负调控植物对疫霉菌的抗性。
附图说明
图1为本发明实施例二中AtPPR1与NbPPR1、StPPR1-1和StPPR1-2蛋白序列比对示意图；
图2为本发明实施例三中AtPPR1拟南芥敲除突变体表现出对寄生疫霉菌的抗性结果示意图；
图3为本发明实施例三中AtPPR1拟南芥敲除突变体表现出对辣椒疫霉菌的抗性结果示意图；
图4为本发明实施例三中AtPPR1拟南芥敲除突变体表现出对植物病原细菌丁香假单胞菌的抗性结果示意图；
图5为本发明实施例四中在本氏烟中沉默NbPPR1增强对寄生疫霉菌的抗性结果示意图；
图6为本发明实施例四中在本氏烟中沉默NbPPR1增强对致病疫霉菌的抗性结果示意图；
图7为本发明实施例四中NbPPR1沉默植株生长情况示意图。
具体实施方式
为了加深对本发明的理解，下面将结合实施例对本发明做进一步详述，本实施例仅用于解释本发明，并不构成对本发明保护范围的限定。
实施例一
本实施例提出拟南芥负调控因子AtPPR1基因的克隆，包括以下步骤：
步骤一：准备植物材料，野生型拟南芥Col-0(可以通过公开渠道获得)，RNA的抽提，用RNA提取试剂盒(OMGA，Lot#:R6827-01)抽提RNA，用琼脂糖凝胶电泳鉴定RNA的完整性，然后在分光光度计上测定RNA的纯度和浓度；
步骤二：基因克隆，用反转录试剂盒(TaKaRa，Lot#:AHE3187A)获得Col-0的cDNA，根据拟南芥网站Tair中提供的AtPPR1(At4G02820)的全长编码序列设计上下游引物AtPPR1-F/R，以cDNA为模板进行扩增；PCR产物经酶切、连接、菌液PCR验证后构建成载体pKannibal-AtPPR1，然后送去测序；将测序结果与已公布序列进行比对，正确的质粒用于后续实验；
引物序列：AtPPR1-F：CCGCTCGAGATGAATAAAAACATGTTGGTTCGCT
AtPPR1-R：GCTCTAGACTAAGAAATGGTGGACGAGATTTCA
实施例二
本实施例提出拟南芥AtPPR1基因的序列信息与同源性分析，拟南芥AtPPR1基因全长CDS序列为1599bp，详细序列见Tair Gene Locus：At4G02820.1，氨基酸序列共532个氨基酸，详细序列见TAIR Accession AASequence NO.：1009127979；
将拟南芥AtPPR1基因编码蛋白的氨基酸序列用BLAST程序进行同源性检索，结果表明与本氏烟草中的1个基因NbPPR1同源；同时在马铃薯中基因组的检索中也找到2个AtPPR1的同源基因，它们的氨基酸序列的相似度都在50％以上，推断3个同源基因具有和AtPPR1相同的功能；
拟南芥AtPPR1基因与本氏烟草和马铃薯中3个同源基因的氨基酸序列的比对结果如图1所示。
实施例三
本实施例提出AtPPR1拟南芥T-DNA插入突变体表现出对寄生疫霉菌、辣椒疫霉菌和丁香假单胞菌的抗性，具体步骤为：
步骤一：首先设计特异引物，检测突变体中AtPPR1的表达情况；
步骤二：收取约0.1g拟南芥样品提取RNA、反转录为cDNA后，设计特异性引物利用实时荧光定量PCR技术检测AtPPR1的表达情况；
步骤三：采集苗龄为4-5周的atppr1突变体拟南芥叶片以野生型拟南芥Col-0为对照，划伤叶片后接种GFP标记的寄生疫霉菌游动孢子约2000个，于23℃培养箱黑暗培养约48个小时后进行荧光观察并统计病级；
步骤四：接种辣椒疫霉时调整游动孢子数为约800个，其余培养条件与寄生疫霉菌接种类似，于约40小时后观察并测量病斑直径；
步骤五：于含有相应抗生素的LB平板上划线活化保存的Pst DC3000甘油菌，于28℃，黑暗，过夜培养；
步骤六：挑取单菌落与3mL含有抗生素的LB液体培养基中，28℃，220rpm培养至OD600＝1左右，收集细菌，利用dH2O重悬，调整OD600至0.1，稀释1000倍后注射拟南芥叶片，注射后以及接种三天后分别收取等量样品研磨后涂板分析丁香假单胞的繁殖量。
结果如图2、3、4所示，图2中左图为接种寄生疫霉菌游动孢子后约2天后病斑观察结果；左图为病级统计结果；图3中左图为接种寄生疫霉菌游动孢子后约46小时后病斑观察结果；左图为病斑直径统计结果；图4中左图为在接菌三天后，atppr1-1突变体丁香假单胞菌cfu数显著小于野生型拟南芥；右图为定量PCR技术检测AtPPR1在两个突变体中的表达量。
实施例四
本实施例提出在本氏烟中沉默NbPPR1可以提高对寄生疫霉和致病疫霉的抗性，包括以下步骤：
步骤一：选取NbPPR1特异的区段约300bp，构建pTRV2-NbPPR1沉默载体；
步骤二：将pTVR1、pTRV2-GFP和已连入目的片段的pTRV2载体电转入农杆菌，利用农杆菌渗透法将pTRV1与pTRV-NbPPR1两种转化的农杆菌在本氏烟上共同瞬时表达，一般注射时终浓度为OD600＝0.25；
步骤三：利用一起沉默的PDS基因作为阳性对照，GFP为阴性对照，待阳性对照出现较明显的沉默效果时(2-3周后)选取实验组的叶片接菌分析，同时设计特异引物，检测沉默植株中PPR1的表达情况；
步骤四：收取约0.1g本氏烟叶片样品提取RNA、反转录为cDNA后，设计特异性引物利用实时荧光定量PCR技术检测PPR1的表达情况；
步骤五：采集沉默NbPPR1的本氏烟叶片以GFP沉默植株为对照，划伤叶片后接种GFP标记的寄生疫霉菌游动孢子约2000个，于23℃培养箱黑暗培养约40个小时后观察并统计病斑直径；
步骤六：将新鲜培养的致病疫霉菌于培养后10天左右刺激游动孢子，在培养物中加入约5mL无菌水，4℃刺激约1-2h，待大量游动孢子释放后进行接菌，每个接种点接种约1500个游动孢子，于16℃培养5天后观察并统计病斑直径，在接种10天后，收取叶片计数并统计孢子囊产生情况；
步骤七：在注射后3-6周持续观察沉默植株的生长及开花情况。
结果如图5、6、7所示，图5中左图为接种寄生疫霉菌游动孢子后约46小时后病斑观察结果；左图为病斑直径统计结果；图6中左图为接种致病疫霉菌游动孢子后约5天后病斑观察结果；中图为病斑直径统计结果；右图为致病疫霉接种10天后的产孢情况。
本发明负调控因子AtPPR1基因，首次作为影响植物ROS以及激素信号传递的免疫负调控因子被克隆，通过基因工程构建AtPPR1基因的过表达转基因拟南芥材料，离体叶片接菌实验证明AtPPR1过表达拟南芥更感寄生疫霉菌侵染，通过基因工程构建AtPPR1基因缺失突变体，发现该突变体能够显著增加对疫霉菌、丁香假单胞等病原菌的抗性；通过病毒介导的基因沉默技术(VIGS)获得NbPPR1缺陷型本氏烟草植物材料，通过离体叶片接菌证明此材料增强对寄生疫霉、致病疫霉菌的抗性，实验结果证明了AtPPR1基因作为负调控因子在提高植物疫霉菌抗性上的用途，可用于抗疫霉菌品种的选育；
另外，还实时荧光定量PCR技术，分析发现AtPPR1基因干扰植物内源水杨酸和茉莉酸信号通路，并且通过活性氧测定发现AtPPR1缺失后植物表现出更强的ROS，得出结果表明，AtPPR1作为一个新型的植物免疫负调控因子，通过干扰植物内源茉莉酸和水杨酸的下游信号传递以及ROS信号，负调控植物对疫霉菌的抗性。
以上显示和描述了本发明的基本原理、主要特征和优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (5)
1.一种疫霉菌抗性的负调控因子AtPPR1基因，包括AtPPR1基因和AtPPR1基因编码的蛋白，其特征在于：所述AtPPR1基因的核苷酸序列如Tair Gene Locus：At4G02820.1所示，所述AtPPR1基因编码的蛋白的氨基酸序列如TAIR Accession AASequence NO.：1009127979所示。
2.根据权利要求1所述的一种疫霉菌抗性的负调控因子AtPPR1基因的同源基因，其特征在于：所述AtPPR1基因在本氏烟草中的1个同源基因为NbPPR1；
所述AtPPR1基因在马铃薯测序基因组中的2个同源基因的氨基酸序列如GenBankAccession Number：XP_015167864.1和XP_006366127.1所示。
3.根据权利要求2所述的一种疫霉菌抗性的负调控因子AtPPR1基因的同源基因，其特征在于：所述NbPPR1的氨基酸序列如Sequence ID Niben101Scf00317g06017.1所示。
4.根据权利要求2所述的一种疫霉菌抗性的负调控因子AtPPR1基因的同源基因，其特征在于：所述NbPPR1与AtPPR1蛋白序列相似度大于50％，所述NbPPR1与AtPPR1基因有相似的负调控植物对疫霉菌抗性的功能。
5.根据权利要求2所述的一种疫霉菌抗性的负调控因子AtPPR1基因的同源基因，其特征在于：所述AtPPR1基因在马铃薯测序基因组中的2个同源蛋白与AtPPR1蛋白具有＞50％的氨基酸序列相似度。
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