CN117384920A - 一种栽培茄来源的马铃薯晚疫病主效抗性基因Rpi-mel1及其编码蛋白与应用 - Google Patents
一种栽培茄来源的马铃薯晚疫病主效抗性基因Rpi-mel1及其编码蛋白与应用 Download PDFInfo
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Abstract
本发明属于植物基因工程技术领域，具体涉及一种栽培茄来源的马铃薯晚疫病主效抗性基因Rpi‑mel1及其编码蛋白与应用，该发明将栽培茄来源的主效抗性基因Rpi‑mel1克隆到马铃薯中，通过异源过表达SEQ ID NO:1序列提高马铃薯中Rpi‑mel1编码蛋白的水平，从而使马铃薯叶片具有更强的晚疫病抗性。本发明SEQ ID NO:1所示的抗性基因序列来源于栽培茄(Solanummelongena)；对上述序列的异源过表达提高马铃薯中Rpi‑mel1编码蛋白的水平；遗传改造后马铃薯叶片具有更强的晚疫病抗性。
Description
技术领域
本发明属于植物基因工程技术领域，更具体地，一种栽培茄来源的马铃薯晚疫病主效抗性基因Rpi-mel1及其编码蛋白与应用。
背景技术
马铃薯晚疫病是马铃薯生产过程中容易遭受的毁灭性农业病害，严重影响马铃薯产量及品质，每年造成近百亿美元的经济损失(Guha Roy,S.,Dey,T.,Cooke,D.E.L.,Cooke,L.R.(2021).The dynamics of Phytophthora infestans populations in themajor potato-growing regions of Asia—A review.Plant Pathology,70:1015–1031)。我国晚疫病年均发生面积约占种植总面积的40％左右，导致马铃薯年平均产量损失约占所有病害产量损失的63.5％，其危害程度远高于其他农业病害(李洁，闫硕，张芳，李小波，任彬元，胡同乐，国立耘，窦道龙，王晓丹.2021.近年来我国马铃薯晚疫病的时空演变特征及防控情况分析.植物保护学报，48(4):703-711)。前期科研工作者从野生马铃薯等近缘抗病资源中不断挖掘新的R基因，将其引入栽培品种以实现晚疫病有效控制，但由于近缘抗病物种的R基因识别的效应蛋白有限，其介导的抗性容易因病原菌突变而丧失(Hein,I.,Birch,P.R.J.,Danan,S.,Lefebvre,V.,Achieng Odeny,D.,Gebhardt,C.,Trognitz,F.,Bryan,G.J.(2009).Progress in Mapping and Cloning Qualitative and QuantitativeResistance Against Phytophthora infestans in Potato and Its WildRelatives.Potato Research,52:215–227)。因此挖掘马铃薯远缘物种的R基因，并验证其功能成为了新的研究方向。
目前，栽培马铃薯的R基因主要来源于野生抗病马铃薯，其介导的寄主抗性具有小种专化性且效应蛋白识别谱较窄，其抗性容易因病原菌的突变而丧失。同时，新的R基因的筛选和鉴定仍需依赖传统的正向遗传学，时间成本和经济成本较高。
发明内容
本发明的目的针对现有技术存在的问题，本发明提供一种栽培茄来源的马铃薯晚疫病主效抗性基因Rpi-mel1及其编码蛋白与应用。
为实现上述发明目的，经研究，本发明提供如下技术方案：
一种栽培茄来源的马铃薯晚疫病主效抗性基因Rpi-mel1，包括以下核苷酸序列：SEQ ID NO:1中所示的基因组核苷酸序列；或SEQ ID NO:2中所示的SEQ ID NO:1编码区的核苷酸序列；或SEQ ID NO:3所示的氨基酸序列经人为工程改造或自然核酸多态性所致的替换、缺失、或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列编码所对应的核苷酸序列。
优选的，所述SEQ ID NO:1所示的抗性基因序列来源于栽培茄(Solanummelongena)。
一种栽培茄来源的马铃薯晚疫病主效抗性基因Rpi-mel1编码的蛋白，包括以下氨基酸序列：SEQ ID NO:3中所示的氨基酸序列；或SEQ ID NO:3所示的氨基酸序列经人为工程改造或自然核酸多态性所致的替换、缺失、或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
一种权利要求1所述基因或权利要求2所述蛋白在增强马铃薯晚疫病抗性中的应用，在马铃薯栽培种中对SEQ ID NO:1所示基因进行异源过表达，从而提高马铃薯中Rpi-mel1编码蛋白的水平。
优选的，所述对SEQ ID NO:1所示基因异源过表达通过马铃薯稳定遗传转化技术实现。
一种通过异源过表达Rpi-mel1培育晚疫病抗性增强的马铃薯的方法，包括以下步骤：
S1、以栽培茄‘桂茄1号’cDNA为模板，将SEQ ID NO:2所示的Rpi-mel1蛋白编码区的核苷酸序列克隆至双元植物表达载体pCHF3-YFP中；
S2、将S1中得到的pCHF3-YFP-Rpi-mel1转染到大肠杆菌菌株中；
S3、对S2中的菌株进行阳性筛选，并提取其质粒DNA
S4、将S3得到的质粒DNA转染至农杆菌菌株；
S5、对S4中的菌株进行阳性筛选；
S6、将S5得到的阳性植株与马铃薯茎段共培养；
S7、将S6中共培养分化芽进行抗性筛选并转移至生根培养基，认根系发育定植后移栽至日光大棚；
S8、将S7得到的阳性植株进行Rpi-mel1蛋白编码基因表达水平检测；
S9、将S8中得到的过表达倍数较高的株系进行晚疫病抗性鉴定。
优选的，所述S2中所用的大肠杆菌菌株为DH5α。
优选的，所述S4中所用的农杆菌菌株为EHA105，GV3101。
本发明的有益效果至少包括：
1、SEQ ID NO:1所示的抗性基因序列来源于栽培茄(Solanummelongena)；
2、对上述序列的异源过表达提高马铃薯中Rpi-mel1编码蛋白的水平；
3、遗传改造后马铃薯叶片具有更强的晚疫病抗性。
本发明的特征和优点将在随后的具体实施方式部分予以详细说明。
附图说明
图1为本发明实施例1的Rpi-mel1过表达载体示意图；
其中，抗性筛选标记为卡那霉素(kanamycin),壮观霉素(Spectaculinresistan)。
图2为不同Rpi-mel1过表达株系马铃薯和野生型马铃薯的Rpi-mel1基因表达量示意图(****代表Pvalue<0.0001)。
图3为转Rpi-mel1马铃薯离体叶片接种致病疫霉后的晚疫病叶片坏死症状示意图(a)及叶片病变区域直径示意图(b)。
图4为全植株喷洒接种致病疫霉导致晚疫病后转Rpi-mel1马铃薯和野生型马铃薯的叶片症状对比图(a)及植株地上部生物量对比图(b)。
具体实施方式
为了使本发明的目的、技术方案和有益效果更加清楚，下面结合附图和具体的实施方式对本发明作进一步详细的说明。所述实施例的示例在附图中示出。应理解，在下述本发明的实施方式中描述的具体的实施例仅作为本发明的具体实施方式的示例性说明，旨在用于解释本发明，而不构成对本发明的限制。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。在本申请的描述中，除非另有说明，“一个/一种”、“多个/多种”等类似用词的含义是两个/种或两个/种以上。
在本发明的描述中，需要说明的是，实施例中未注明具体条件者，按照常规条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1 Rpi-mel1过表达载体构建
使用南京诺唯赞生物科技股份有限公司生产的RNA提取试剂(RNA-easyIsolation Reagent)提取栽培茄叶片的RNA，并利用该公司的反转录试剂盒(HiScript III1st Strand cDNA Synthesis Kit)获得相应的cDNA。以栽培茄‘桂茄1号’cDNA为模板，利用南京诺唯赞生物科技股份有限公司生产的无缝克隆试剂盒(ClonExpress Ultra One StepCloning Kit)，将SEQ ID NO:2所示的Rpi-mel1蛋白编码区的核苷酸序列克隆至双元植物表达载体pCHF3-YFP，酶切位点选择Kpn I和Sal I，上游引物序列为：AGAACACGGGGGACGAGCTCGGTACCATGACAACCCCAAAATCCTC ACA；下游引物序列为：TCCTCGCCCTTGCTCACCATGTCGACTGGGAAGAATCTTTTGAGGCCA。并转染至大肠杆菌菌株DH5α中。在含壮观霉素Spe(浓度50ug/mL)的LB固体培养基上培养，挑选单克隆并转至含壮观霉素的液体LB培养基中培养扩繁。利用PCR鉴定阳性克隆菌液并送至上海生工生物工程有限公司进行Sanger测序。序列确认无误后，使用天根生化科技(北京)有限公司生产的质粒小提试剂盒(TIANGEN:#DP103)提取质粒DNA，取5μL重组质粒，加入100μL农杆菌EHA105感受态，冰上放置30分钟后，液氮速冻3分钟，37℃热激5分钟，加入600μL LB液体培养基，置于28℃摇床，200转/分钟，复苏4小时后涂在含有利福平Rif和壮观霉素Spe(工作浓度均为50ug/mL)的固体LB培养基上，28℃培养2-3天。待长出菌斑后，挑取单克隆扩繁培养，使用扩增引物筛选阳性克隆，用于后续农杆菌浸染外植体。
实施例2 Rpi-mel1过表达马铃薯品系的转染与筛选
将实施例一中所获含双元植物表达载体(如图1)的转基因农杆菌菌株与马铃薯茎段在无菌条件下置于Murashige-Skoog培养基上共培养2天。将共培养后分化出的再生芽转移到新的含有卡那霉素(浓度4mg/L)的MS培养基上进行筛选培养。将所得抗性阳性分化芽转移至生根培养基上，培养3周后移栽至日光大棚。
根据SEQ ID NO:2所示的Rpi-mel1蛋白编码区的核苷酸序列，设计并合成对应的上下游引物，上游引物是GCTTCACAAACCAAGGCA，下游引物是TCGTCGTCTCGGAAAGTTC，使用北京艾德莱生物科技有限公司生产的植物基因组DNA快速提取试剂盒提取转基因马铃薯叶片DNA，利用PCR技术，PCR扩增反应程序是95℃预变性3分钟，95℃变性15秒，60℃退火10秒，72℃延伸1分钟，循环数为35，最后72℃延伸10分钟。利用2％琼脂糖凝胶电泳初步确认条带，将扩增片段送至上海生工生物工程有限公司进行Sanger测序确认，筛选T0代转基因马铃薯阳性植株。在此基础上，使用南京诺唯赞生物科技股份有限公司生产的RNA提取试剂(RNA-easy Isolation Reagent)提取阳性植株的RNA，并利用该公司的反转录试剂盒(HiScriptIII 1st Strand cDNA Synthesis Kit)获得相应的cDNA。以野生型马铃薯为对照，通过实时荧光定量PCR技术，实时荧光定量PCR的反应程序为：95℃预变性10分钟，95℃变性15秒，60℃退火30秒，并收集荧光信号，循环数为40，以此验证Rpi-mel1蛋白编码基因的表达。实时荧光定量PCR结果显示转基因马铃薯品系Rpi-mel1-23/36/39的基因表达水平显著提高(如图2)。选择Rpi-mel1蛋白编码基因表达水平较高的转基因马铃薯株系用于后续晚疫病抗性鉴定。
实施例3转基因马铃薯的晚疫病抗性鉴定
基于抗病基因Rpi-mel1在转基因马铃薯中的表达结果，选择过表达倍数较高的株系(Rpi-mel1-23)进行晚疫病抗性鉴定。
致病疫霉孢子悬浮液的制备：黑麦固体培养基中接种致病疫霉T30-4，培养15-20天后，加入15毫升无菌水，4℃下放置3-5小时后，刮取平板内的孢子悬浮液，3000rpm离心10分钟，利用血球计数板，将孢子数量调整至1×108个/mL，用于离体叶片的晚疫病抗性鉴定。用于活体植物晚疫病抗性鉴定的孢子悬浮液，孢子数量控制在5×104个/mL。
离体叶片晚疫病抗性鉴定：以野生型植株为对照，吸取10μL孢子悬浮液滴于马铃薯离体叶片中央，对照组使用无菌水替代，每组3-6片叶。将叶片放在铺有湿纸巾的方形培养皿中，18℃，暗培养3-5天后，观察叶片发病情况，并进行病斑大小测量和台盼蓝染色。抗性鉴定试验需重复至少三次。通过叶片病斑大小和和病变区域直径看出转Rpi-mel1马铃薯离体叶片的晚疫病发病症状减弱(如图3)
活体植物晚疫病抗性鉴定：以野生型植株为对照，对马铃薯植株进行喷施致病疫霉孢子悬浮液，对照组使用无菌水替代，保证喷施均匀，23℃，16/8小时(光/暗)条件下培养，观察叶片发病情况，统计并比较发病后植株地上部的生物量。抗性鉴定试验需重复至少三次。通过接种致病疫霉前后转Rpi-mel1马铃薯与野生型叶片症状对照和喷洒致病疫霉后植株地上部生物量对照可以看出，转Rpi-mel1马铃薯植株对晚疫病抗性提升(如图4)。
SEQ ID NO:1Rpi-mel1基因组核苷酸序列
CCTCGACATTTTGTTCCTCCACGCGGGCCTTTCCATTGACTTAGAGTCCTCAAACTTGTCGCTCTGAATTTTCCTTTTTCCTTCTCTATTAAAAATCTCAATTGGATCATTATCCTCTTCGTTTCTGCACAAAATTATCACTTTCGATAACATCTCTCACTTGGTCTATCATTTTCTTGGACTATTTCCGATGACAACCCCAAAATCCTCACAAACCCGTTATTCTTTCCACGTTTTCTTGAGTTTCAGAGGCGAAGACACCCGGAAAAACTTCACTGATCATCTCTACACAGCTCTCATCAACGCCGGAATTCGAACTTTCCGAGACGACGACGAAATCCGCAGGGGAGAAAACATAGAATCTGAACTGCAGAAGGGTATTCGGGAATCCAGGATTTCCCTAATTGTCTTCTCCAAAGACTACGCTTCTTCAAGATGGTGTCTTGATGAGCTTGTCAACATTCTGGATCGAAGGAAGAAAGAGGGTCATACTGTTTTGCCTGTGTTTTACACTGTGAGCCCTGAGGATGTCCAGAACCAAACTGGGAGTTTTGCTGAAGCTTTTGTGAATCATGAAAAGAGAGGAAATGCAGAGACTGGAGAGAAAAGAAAGGAATGGATGGAGAAGATGGACAAGTGGAGAGTAGCTCTCAAACAAGTTGCTGAATTGGAAGGAATGTGCTTAGCCAAAGAAGTAGACGGGTCAGTTTCCTAAATTTCTATTTTCTTCTTCTTATTCAACATTTGTAGCTATTCCCCCTCCACCCTCCTTTTATAGTTAGTCATCAGGTTTATGGTAGTTCTAATTGTTTTTGGTGAAAGAAAAAAGTGTACTTCATAAGAAATTGCAAAATCGGCAGCACAATAATTGCTTGAATCGTTACTTGAAAGGGAATTTGGCTGATGATGTTAGTGAAGTACCATAGATGATAGTCTAGAACTCTGTTTGAAGGAGTAAGCAGTCAAAGAATTGCTTTGAGCCGAGGGTCTATCTGACACAACCTCTTTACCTCCCAAGGTATGGGGTGAGGTCTGGTTACACACTGTACTCTCCGAACCCCACTTGTGGAATTACACTGGTTATGTTGTTGTTGAAGTAGTCTACCAATTGATTTCATCTTTCTAACTCTGAAAGTTTCTCTTTTGGAGATCAACGGTATTCCAACTTAGTCGACACGTGATTCTCCCCAACAAGCAAGTCTATTCATCAGTTGAAAGTTCTAATATAAATTTACCATTCACTTAGTACTAGTTGTCCTATATTCTTGGGCTGTGACAATTGCAATGATGTGGTGCTAGTTTGGAAATTCACGAGTAAACAAGTAAGTAGTAAATGATATTTTCTTAGAATAGAGAGTGAAAAGCATGATTGTTAGTCTATCTATATATCTGACCGTCATATATATCGATGTGCTTGCCAAGTCCAACGACTAATATGCGCTGCAAAATTATCTATTGTAAAAGGTTACTTTAGGAAAAGATACTCTGGGAATGCTAGAGTGGGCAAAATTTTATATGGAGGATTGAGGAAATTACCTCCACCATGGACATGCAAAGTTAAATGTGAACAGCCCAAACTTGAAAGAGATTGAATATATCAAAGACCCTAAATAGTATGTTGAGCTCTAAAGGAATTTGAGCAAAAAGTGTAACAGCGTTGGCATAAATCTAATTTGGAAGACTAATCAGATGAAATACACAAAGTTCAAAGCTTAAGATGATTAAATGCTATGTCAGGTTAGATTCTAGTCAAACGTGAAAACTTACGTGTGAGTACTCTAAAAGGTAAATACTACGCATACTCTCAATGACTGAACATCCTGGGGTTTCCCTAAAAGATAGCGATTCCTAGCTTTCATATCTAGAGACAAATGAGGGTTGTCTTTGGGCTTCTTTTGTTGCTAAATGTATCGTGTGGATCCCATTAGAAAAAGCCTCATTACTGATCCTGTTTTAATATTTACAACCTCTAAAGGCTCAAACCACTGAAAATAGAGGTCATTTCCCTATTAGATCTATGCTGATCTTGAAAGGAGTCCTCATAGAGAAGATAGTGACTTGTACATTTATCCATTACAATCACCTATATACTTAGGTAGCAATGACATGAAACACATTCTAATGATCAATATTTCTGGCGACTCTTCCATTAGATTTTGTGTCTCCTTTTCCATAGATGTGTGCTGCCTTCTACAAACTTGGTCAACTAGGGACTCTCAGATTTAAAGATTGCCCTCAAATTATTCTGATTAAGATAGATGGGAGCAATAATTTCTTGCTTTCGACGTAATCTTCCTTCATGTCTATGAATGCTCTTAACTTGTATATGCTTTTAGCTTTTCCAATCAGCAGTTCTTCAAGCCATTGTGCCCTCAATTATTTGTTATCAGAGTCAAAATCATCTATTTAGACTATCTCTAACTAGGGCATGCAAAAATTGAACCGAAAAAATATTATTGGTTTATGGCTATTGGGTTGACAGGTTTTTAATGGTTTTGTAAAAAAAATTATCGGGTTATTGGTTTTTTAATATTGGGTTATGGGCAGCATGGTCAAAGCTGCAGTTTAAGCACGCTTAAGCCTTGAAGCGAGGCTCAAAACGTGGTGAGCACTTTGTCTCGCTTTATGTGCGCTTCAATGTTGTCATCAAGGTTCTAAGGCAACTTTTCCTTGCCAATGAACCTCTTCTGAAGAAGTGACACTAGACAATTGATATTGCACTTTATCATATTTTTTTTTCAATTTCTTCTATCATATAATTTTTTTATTCATATTTATAATTATTACTCTTGGACTAAACATATGTTTTTATATTTTTTTCTCCATTTACACCTTTTTTTAATGAAAGCTCATGCCTTATTTGCGTCTTCCGCTTAAAGCCCCAAAGGACCTTAGAGCTTTTTTGCATTTTTTTCCTTACTATAGTAATTTATTACTTTTGCCCTTATATGAATATTAAATATTAATATCACTACCTTACTAGTTACTGTTGCTCCCCTTTAACTTTCAATTCACACTTCACAATGACTTTGAGCTTAAAAAAACAAACTTCTTTATTTGTGTTGCACTTGCATAGTTTTTTTCATGTTAATTGCTAATCCCTCCGTCCCATTTTTATGTGGTACCGTTTGACTTGGCATGGCTTTTGAAACTTGTGGTCTAAAACAACTCTTAGATTTTTGTGTGGTTTAGAATCATTTCATTAAGAGTAAACGAGGAATTTTAAAGTTAAATTATTTCCAATTATAGCAATGTGACATTCTTCTTGGAATGGACTAAAAAGGAAAGGGTGTTACATGAAATGAGACAGAGGGAGTAAAGGTTATTTATAGTAAAATACAATTTTATATTAGATAAAAGATTTGAAAAGAATAAAGTTTTATATTACATGAAGATTATATGGAGTGATTATTTGGGAAAAAAAAGAAAGGATTTGTATTATGAATATGAAAGTATAAATATAATAAAAATAATAATATAATATTGAGGAGAGAGGAAAGAGTAAAATAGAGAGTTGGATTGGAGTTGGTTGTCTTGAAATGCTCTCTATTCTCTATATTTGGAAAGTGAAATAGAGTGGGTTGGAGATGGCCTTAAAGAGTGAAGGATATATAAAGGGAGGACACCAAATAGCTGTATAGACGCTTCAGAAGGTACAAAAGAACGAAATTTTGTCATGATACACTCCATTTTCCAAGTGTGGGTGCATTTTGTATCAGCATCTTTTGGAAATTTTGGTATAATATAACCTCAGTTATGATGAGGCTAAACGTGCCTTGTCTTGATCATTTTCCTAACTGAAAATGGTTCCCTGTAATATTTCATGGTTCTCTATTTACCACACTCTTTATTTTGCTGTCCATTTCCCGCTTTGTTGCTCCCACAGTATTGTCAAATTCTTCACAGCACACAACATTCTTTCTATTCTTCACAGTACATAAGTAAATACCGCTCATTCTTTCTCCTGTAGTACCAGTAAGCCACATATATTTCATCTTTGTTGCTTTTCTGGGAAATGTAAAAGCTTTATTAATTTAGCATTAAGAAATTGCTTTAAAGGTATACTTTCACGTTTGTTCTTAATAGTGCCTAGGCACCTAACCAGATTGTTTCATTTGTTGATGCTTAATATGACTGATCATCCCAGTGTGTTATGTTTGCTCTAAGAAGTATTATTATATCAGTGAACTCTTTTTATTTGTTCTCCAGTACTTGGCAAGTTGCAGCGCTTTACTGATTTTTCATCTTGTTTCGTCTCTAACATTTGCTCTGCATCTCAGGCGCGAGGCAAAATTCATCCAAAAGATCATCAAGGAGATACTGAAAAGATTGAATCGCACAGTACTTAGCATACCTTCATACACAATTGGATTAGAGTCTCGAGTGAAGGACATTAACTCATGGTTAGAGGATGAATCCAGTGAAGTTGGCATTGGGGTGATCTGTGGACTTGGTGGCGTAGGAAAGACTACTGTTGCTAAAGTGGCTTACAACTCTAACTATGACAGATTTGATGGCAGTTGCTTTCTTGCAAATGTCAGAGAAATCTCAGAGAAACATTCAAATGGTCAGGTTTATTTGCAAAAACAAATTTTTGAAAGTATTTTGAAGGGCAGAAAGGAAAAAATATACAATGCTGATGAAGGAATTGTCAAAATGAAAGATGCTATAGGCAACAAAAAGGTTTTTATTGTATTTGATGATGTGGATCAGCTGGATGTCTTAGATTCACTTATTGGGACAAGGGATTGGTTTTATCCAGGGAGTAAGATTCTCATAACGACAAGGTGCGAGAAATTGTTGAAGGCTCATGAGAGACACATGCTATTCAAGATTAAGGAACTTGGGGATGATGAGTCCCTAAAGCTCTTCAGTTGGTATGCTTTTGCACAAGACCACCCATTAGAAGAATTCAAGGTGCTTTCAACAGAGGCAATACAGCATTGTGGTGGGCTTCCATTAGCTCTTTGTGATTTGGGTTCGTTTCTTTCAGGGAGAGGTATGGAAATATGGAGAAGTAAATTGCAGAAATTGGAAGCAATCCCTCATAGCAGAGTTCAAAAGAATCTTGAGATAAGCTACAAGTCTCTGGATGATCATGACCAGAGGTTGTTCCTCGTTATTGCTTCATATTTTGTTGGGAAGGACAAAGATTATGTTATTAAAATTTTAGAAGACACTGATTTGCACCCAACAGTTGGAATTCAGAACCTCACCGACAGATCCCTCATAAGTATCGACGACGAAAATAGATTGATGATGCCTCCACTTATTCAGGACATGGGGAAAGAAATTATTCGTCGAGAATCACCAGACGATCCCGAGAGATTATGTAAATTGTTGAACCAATGGGGTCTATCAAATTACTTGACAGAAAACAACGTAAGAATCTTGAGTTTTCTATTCATTTTAAGCTTGTCTTGGTTGATTCTGAAAGTGTTCTTGTTTCTGACACAAATCTTTCTTCAGCAGGAGATTGATGTAGATGGAGGAGACCTCCCAAGTGCACATCTGCCTGAAGATGGTCTTGTCCCAGCAGTTGGTCCTAATGGTGCGAAAAGACCATATTATCAAGATTTTCCTGAAATAGCTATTCTGCCATACCTAGGTAATTCGTTGAAAAGGCGCTTTATAGGACTTCTTTCAGCATTCCCAATATCTGGTGGCCTCAAAAGATTCTTCCCATGAAGATGCTTTCTTTCTTTAGGGAAAAAACCAACATCGTACTGCTTAATCATATGGAACTAGAAGATTTACAGGAGTGTTTTGAGAGTCAGGGCTTCAATTAAGGATCACTGTCGGATAATATCTGAGCATTTTTTGTGCTTGCCATACCATCTTAGTTGCACAAGTTTGTTAGTTGTAGAGGTAGATTCAGTCCTGTTTGTTTTTTTATCAGCTTCGGGTGGGCGATCATGTAAGATATGTTTCATTAACTCATTGTACAGTCATGAGGTCATTTCTTGCTAGCTGCATAGTTTGTTTTAGCAAACAGTTCACTGGAAGAAACTAAAAATGTTACCTTTAACTATTCCTTTCTAGTCTCTGCAAAATGTCTTGAAGTTTTGCCTCTTTCATTCTGATGGAACTGCAGGGCTCAAATAAGAAATATTCTAATTTTGGTGTATATATGTAACTGAATTCAATTACTGGTGATACCAATATCTAGAAAACATCATCCAAC
SEQ ID NO:2Rpi-mel1编码区核苷酸序列
ATGACAACCCCAAAATCCTCACAAACCCGTTATTCTTTCCACGTTTTCTTGAGTTTCAGAGGCGAAGACACCCGGAAAAACTTCACTGATCATCTCTACACAGCTCTCATCAACGCCGGAATTCGAACTTTCCGAGACGACGACGAAATCCGCAGGGGAGAAAACATAGAATCTGAACTGCAGAAGGGTATTCGGGAATCCAGGATTTCCCTAATTGTCTTCTCCAAAGACTACGCTTCTTCAAGATGGTGTCTTGATGAGCTTGTCAACATTCTGGATCGAAGGAAGAAAGAGGGTCATACTGTTTTGCCTGTGTTTTACACTGTGAGCCCTGAGGATGTCCAGAACCAAACTGGGAGTTTTGCTGAAGCTTTTGTGAATCATGAAAAGAGAGGAAATGCAGAGACTGGAGAGAAAAGAAAGGAATGGATGGAGAAGATGGACAAGTGGAGAGTAGCTCTCAAACAAGTTGCTGAATTGGAAGGAATGTGCTTAGCCAAAGAAGTAGACGGGCGCGAGGCAAAATTCATCCAAAAGATCATCAAGGAGATACTGAAAAGATTGAATCGCACAGTACTTAGCATACCTTCATACACAATTGGATTAGAGTCTCGAGTGAAGGACATTAACTCATGGTTAGAGGATGAATCCAGTGAAGTTGGCATTGGGGTGATCTGTGGACTTGGTGGCGTAGGAAAGACTACTGTTGCTAAAGTGGCTTACAACTCTAACTATGACAGATTTGATGGCAGTTGCTTTCTTGCAAATGTCAGAGAAATCTCAGAGAAACATTCAAATGGTCAGGTTTATTTGCAAAAACAAATTTTTGAAAGTATTTTGAAGGGCAGAAAGGAAAAAATATACAATGCTGATGAAGGAATTGTCAAAATGAAAGATGCTATAGGCAACAAAAAGGTTTTTATTGTATTTGATGATGTGGATCAGCTGGATGTCTTAGATTCACTTATTGGGACAAGGGATTGGTTTTATCCAGGGAGTAAGATTCTCATAACGACAAGGTGCGAGAAATTGTTGAAGGCTCATGAGAGACACATGCTATTCAAGATTAAGGAACTTGGGGATGATGAGTCCCTAAAGCTCTTCAGTTGGTATGCTTTTGCACAAGACCACCCATTAGAAGAATTCAAGGTGCTTTCAACAGAGGCAATACAGCATTGTGGTGGGCTTCCATTAGCTCTTTGTGATTTGGGTTCGTTTCTTTCAGGGAGAGGTATGGAAATATGGAGAAGTAAATTGCAGAAATTGGAAGCAATCCCTCATAGCAGAGTTCAAAAGAATCTTGAGATAAGCTACAAGTCTCTGGATGATCATGACCAGAGGTTGTTCCTCGTTATTGCTTCATATTTTGTTGGGAAGGACAAAGATTATGTTATTAAAATTTTAGAAGACACTGATTTGCACCCAACAGTTGGAATTCAGAACCTCACCGACAGATCCCTCATAAGTATCGACGACGAAAATAGATTGATGATGCCTCCACTTATTCAGGACATGGGGAAAGAAATTATTCGTCGAGAATCACCAGACGATCCCGAGAGATTATGTAAATTGTTGAACCAATGGGGTCTATCAAATTACTTGACAGAAAACAACCAGGAGATTGATGTAGATGGAGGAGACCTCCCAAGTGCACATCTGCCTGAAGATGGTCTTGTCCCAGCAGTTGGTCCTAATGGTGCGAAAAGACCATATTATCAAGATTTTCCTGAAATAGCTATTCTGCCATACCTAGGTAATTCGTTGAAAAGGCGCTTTATAGGACTTCTTTCAGCATTCCCAATATCTGGTGGCCTCAAAAGATTCTTCCCATGA
SEQ ID NO:3Rpi-mel1编码蛋白氨基酸序列
MTTPKSSQTRYSFHVFLSFRGEDTRKNFTDHLYTALINAGIRTFRDDDEIRRGENIESELQKGIRESRISLIVFSKDYASSRWCLDELVNILDRRKKEGHTVLPVFYTVSPEDVQNQTGSFAEAFVNHEKRGNAETGEKRKEWMEKMDKWRVALKQVAELEGMCLAKEVDGREAKFIQKIIKEILKRLNRTVLSIPSYTIGLESRVKDINSWLEDESSEVGIGVICGLGGVGKTTVAKVAYNSNYDRFDGSCFLANVREISEKHSNGQVYLQKQIFESILKGRKEKIYNADEGIVKMKDAIGNKKVFIVFDDVDQLDVLDSLIGTRDWFYPGSKILITTRCEKLLKAHERHMLFKIKELGDDESLKLFSWYAFAQDHPLEEFKVLSTEAIQHCGGLPLALCDLGSFLSGRGMEIWRSKLQKLEAIPHSRVQKNLEISYKSLDDHDQRLFLVIASYFVGKDKDYVIKILEDTDLHPTVGIQNLTDRSLISIDDENRLMMPPLIQDMGKEIIRRESPDDPERLCKLLNQWGLSNYLTENNQEIDVDGGDLPSAHLPEDGLVPAVGPNGAKRPYYQDFPEIAILPYLGNSLKRRFIGLLSAFPISGGLKRFFP
以上序列将会通过WIPOSequence软件提交Xml格式的序列表。
最后说明的是，以上的实施例仅用于说明本发明的技术方案，并不构成对本发明内容的限制。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。
Claims (8)
1.一种栽培茄来源的马铃薯晚疫病主效抗性基因Rpi-mel1，其特征在于，包括以下核苷酸序列：SEQ ID NO:1中所示的基因组核苷酸序列；或SEQ ID NO:2中所示的SEQ ID NO:1编码区的核苷酸序列；或SEQ ID NO:3所示的氨基酸序列经人为工程改造或自然核酸多态性所致的替换、缺失、或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列编码所对应的核苷酸序列。
2.根据权利要求1所述一种栽培茄来源的马铃薯晚疫病主效抗性基因Rpi-mel1，其特征在于，所述SEQ ID NO:1所示的抗性基因序列来源于栽培茄(Solanum melongena)。
3.一种栽培茄来源的马铃薯晚疫病主效抗性基因Rpi-mel1编码的蛋白，其特征在于，包括以下氨基酸序列：SEQ ID NO:3中所示的氨基酸序列；或SEQ ID NO:3所示的氨基酸序列经人为工程改造或自然核酸多态性所致的替换、缺失、或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
4.一种权利要求1所述基因或权利要求3所述蛋白在增强马铃薯晚疫病抗性中的应用，其特征在于，在马铃薯栽培种中对SEQ ID NO:1所示基因进行异源过表达，从而提高马铃薯中Rpi-mel1编码蛋白的水平。
5.根据权利要求4所述的应用，其特征在于，所述对SEQ ID NO:1所示基因异源过表达通过马铃薯稳定遗传转化技术实现。
6.一种通过异源过表达Rpi-mel1培育晚疫病抗性增强的马铃薯的方法，其特征在于，包括以下步骤：
S1、以栽培茄‘桂茄1号’cDNA为模板，将SEQ ID NO:2所示的Rpi-mel1蛋白编码区的核苷酸序列克隆至双元植物表达载体pCHF3-YFP中；
S2、将S1中得到的pCHF3-YFP-Rpi-mel1转染到大肠杆菌菌株中；
S3、对S2中的菌株进行阳性筛选，并提取其质粒DNA
S4、将S3得到的质粒DNA转染至农杆菌菌株；
S5、对S4中的菌株进行阳性筛选；
S6、将S5得到的阳性植株与马铃薯茎段共培养；
S7、将S6中共培养分化芽进行抗性筛选并转移至生根培养基，认根系发育定植后移栽至日光大棚；
S8、将S7得到的阳性植株进行Rpi-mel1蛋白编码基因表达水平检测；
S9、将S8中得到的过表达倍数较高的株系进行晚疫病抗性鉴定。
7.根据权利要求6所述的一种通过异源过表达Rpi-mel1培育晚疫病抗性增强的马铃薯的方法，其特征在于，所述S2中所用的大肠杆菌菌株为DH5α。
8.根据权利要求6所述的一种通过异源过表达Rpi-mel1培育晚疫病抗性增强的马铃薯的方法，其特征在于，所述S4中所用的农杆菌菌株为EHA105，GV3101。
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