CRN类效应蛋白基因在诱导植物抗性中的应用

技术领域

本发明书属于基因工程技术领域，具体涉及一种CRN类效应蛋白基因及其在诱导植物抗性中的应用。

背景技术

在植物与病原菌的长期协同进化过程中，病原菌为了更好地侵染植物，进化出大量的攻击寄主的武器，例如效应蛋白(effector)。病原菌分泌这些效应蛋白到植物体内为了促进其更好地侵染寄主植物，然而，伴随着植物的共同进化，有些效应蛋白会被植物自身进化的调控抗病相关的基因识别。对病原体的识别通常会触发局部抗性反应，称为超敏反应(HR)，其特征在于感染部位的快速细胞死亡。对于研究植物与病原菌互作而言，这一明显的HR反应也是作为激发植物免疫的重要信号。活性氧积累也是一个触发植物免疫反应的关键信号，也可以作为植物防御反应触发的信号标识。利用农杆菌介导的瞬时表达系统接种病原菌是最直接评估基因是否能够诱导植物抗性的方法。

寡雄腐霉(Pythium oligandrum)

发明内容

本发明的目的在于提供一种CRN类效应蛋白基因及其在诱导植物抗性中的应用。本发明鉴定到的五个CRN类效应蛋白基因可以作为诱导植物产生抗性和开发植物免疫诱抗剂的理论知识补充，为防控作物病虫害提供理论指导。

本发明的目的可以通过以下技术方案实现：

本发明鉴定到来自寡雄腐霉(Pythium oligandrum)和缠器腐霉(Pythiumperiplocum)的五个CRN类效应蛋白基因CRN12、CRN13、CRN20、CRN23和CRN19，所述CRN12、CRN13、CRN20、CRN23和CRN19基因依次分别具有如SEQ ID NO.1～5所示的核苷酸序列。五个CRN类效应蛋白基因编码的蛋白质为五个CRN类效应蛋白，其依次分别具有如SEQ ID NO 6～10所示的氨基酸序列。

含有上述CRN类效应蛋白基因的表达盒、重组表达载体、转基因细胞系或转基因重组菌。

含有上述CRN类效应蛋白基因的重组表达载体PBIN-PLUS:CRN12、PBIN-PLUS:CRN13、PBIN-PLUS:CRN20、PBIN-PLUS:CRN23和PBIN-PLUS:CRN19。

构建所述CRN类效应蛋白基因CRN12、CRN13、CRN20、CRN23和CRN19的重组表达载体PBIN-PLUS:CRN12、PBIN-PLUS:CRN13、PBIN-PLUS:CRN20、PBIN-PLUS:CRN23和PBIN-PLUS:CRN19。以植物表达载体PBIN-PLUS为出发载体，将CRN12、CRN13、CRN20、CRN23和CRN19基因分别插入PBIN-PLUS的KpnⅠ和XbaⅠ酶切位点获得。

所述CRN类效应蛋白基因及其表达载体可以激发植物免疫反应，为开发免疫诱抗剂提供知识基础，为防控作物病虫害提供理论指导。

一种植物免疫诱抗剂，该诱抗剂含有上述的寡雄腐霉CRN类效应蛋白或上述寡雄腐霉CRN类效应蛋白的发酵液。

上述的CRN类效应蛋白基因、上述的CRN类效应蛋白或上述的表达盒、重组表达载体、转基因细胞系或转基因重组菌在触发HR ROS诱导植物产生抗性中的应用。

上述的CRN类效应蛋白基因、上述的CRN类效应蛋白或上述的表达盒、重组表达载体、转基因细胞系或转基因重组菌在开发植物免疫诱抗剂中的应用。

上述的CRN类效应蛋白基因在培育抗病作物新品种中的应用。

一种诱导植物抗性的方法，将上述的CRN类效应蛋白基因或上述的表达盒、重组表达载体、转基因细胞系或转基因重组菌导入植株中，触发HR ROS诱导植物产生抗性。

一种培育抗病作物品种的方法，将上述的CRN类效应蛋白基因或上述的表达盒、重组表达载体、转基因细胞系或转基因重组菌导入植株中，经过抗性筛选得到阳性转化植株，获得抗病作物品种。

本发明的有益效果：

本发明从寡雄腐霉(Pythium oligandrum)和缠器腐霉(Pythium periplocum)中鉴定到五个CRN类效应蛋白基因，将其插入表达载体PBIN-PLUS。将所述载体导入植物中，能够产生显著的HR ROS反应，激发植物免疫。所述五个CRN类效应蛋白基因可以作为开发植物免疫诱抗剂的潜在候选者，为防控植物病害理论支撑。同时也可以直接导入作物中制备转基因植株，提供新型转基因抗病策略。

附图说明

图1为CRN19触发HR ROS诱导植物抗性。

其中，A为农杆菌瞬时表达注射本式烟叶片会诱导HR表型；B为农杆菌瞬时表达注射本式烟叶片会诱导活性氧积累；C为农杆菌瞬时表达CRN19基因在烟草上能够提高植物对辣椒疫霉的抗性；D为农杆菌诱导抗病表型的病斑面积统计。

图2为CRN12/13触发HR ROS诱导植物抗性。

其中，A为农杆菌瞬时表达注射本式烟叶片会诱导HR表型；B为农杆菌瞬时表达注射本式烟叶片会诱导活性氧积累；C为农杆菌瞬时表达CRN12基因在烟草上能够提高植物对辣椒疫霉的抗性和病斑面积统计；D为农杆菌瞬时表达CRN13基因在烟草上能够提高植物对辣椒疫霉的抗性和病斑面积统计。

图3为CRN20触发HR ROS诱导植物抗性。

其中，A为农杆菌瞬时表达注射本式烟叶片会诱导HR表型；B为农杆菌瞬时表达注射本式烟叶片会诱导活性氧积累；C为农杆菌瞬时表达CRN20基因在烟草上能够提高植物对辣椒疫霉的抗性；D为农杆菌诱导抗病表型的病斑面积统计。

图4为CRN23触发HR ROS诱导植物抗性。

其中，A为农杆菌瞬时表达注射本式烟叶片会诱导HR表型；B为农杆菌瞬时表达注射本式烟叶片会诱导活性氧积累；C为农杆菌瞬时表达CRN23基因在烟草上能够提高植物对辣椒疫霉的抗性；D为农杆菌诱导抗病表型的病斑面积统计。

具体实施方式

以下结合具体实施例对本发明做出详细的描述。根据以下的描述和这些实施例，本领域技术人员可以确定本发明的基本特征，并且在不偏离本发明精神和范围的情况下，可以对本发明做出各种改变和修改，以使其适用各种用途和条件。

实施例1五个CRN类基因的扩增和测序

1.实验菌株

供试菌株寡雄腐霉(Pythium oligandrum)和缠器腐霉(Pythium periplocum)由南京农业大学植病系植物与疫霉互作实验室保存菌株保存于10％V8固体培养基，保存温度为10℃。

2.供试本氏烟草幼苗的准备

本氏烟草(Nicotiana benthamiana)播种于装有蛭石(2-4mm)的塑料花盆(d＝10cm)里，放到14h光照(强光照射)/10h黑暗的温室中生长。使其生长7天，生长出两片真叶后，将其移栽至蛭石黑土提及体积比例为5：1的培养土里生长30天，取相同3，4，5叶位的叶片用作表达实验基因，然后持续观察7天叶片表型是否有明显HR。

3.提取寡雄腐霉(Pythium oligandrum)和缠器腐霉(Pythium periplocum)RNA，并获得目的基因的cDNA

收集寡雄腐霉(Pythium oligandrum)和缠器腐霉(Pythium periplocum)菌丝，然后液氮速冻后放入研钵冷却研磨。参照天根公司总RNA抽提纯化试剂盒推荐方法进行终极腐霉总RNA提取和纯化。参照诺唯赞公司RNA反转录试剂盒提供的方法，以Oligo(dT)为引物反转录合成c D N A。以上述c D N A为模板，用相应上下游引物进行PCR扩增(引物序列见表1)。

表1引物序列

PCR反应体系为：2.5μL 10×PCR反应缓冲液；1.5μL 1.5mM MgCl2；0.5μL 2.5mMdNTPs；0.25μL Taq DNA聚合酶(5.0U/μL)；0.5μL引物；0.5μL模板；无菌水补足至25μL。

反应程序为：94℃5min预变性；94℃变性15s，58℃退火15s，72℃延伸1:30min，35个循环；72℃延伸10min；4℃保存。

上述PCR扩增产物经琼脂糖凝胶电泳、回收，经测序确认无误后，获得的序列为所述5个CRN类基因的核苷酸序列。该CRN类基因包括分别来自寡雄腐霉(Pythiumoligandrum)和缠器腐霉(Pythium periplocum)的CRN12、CRN13、CRN19、CRN20、CRN23，所述CRN12、CRN13、CRN19、CRN20、CRN23基因的核苷酸序列依次分别如SEQ ID NO.1—5所示。五个CRN类基因编码的蛋白质的氨基酸序列依次分别如SEQ ID NO.6—10所示。后续研究发现其具有诱导植物产生坏死和活性氧积累的强大功能。

实施例2构建PBIN-PLUS:GFP、PBIN-PLUS:INF1、PBIN-PLUS:CRN12、PBIN-PLUS:CRN13、PBIN-PLUS:CRN19、PBIN-PLUS:CRN20和PBIN-PLUS:CRN23表达载体

将PBIN-PLUS(BIOVECTOR中国质粒载体菌株细胞株基因保藏中心)空载体质粒用KpnⅠ和XbaⅠ进行酶切。利用诺唯赞公司的同源重组酶将目的片段(CRN12、CRN13、CRN20、CRN23和CRN19)和载体连接。PBIN-PLUS:GFP、PBIN-PLUS:INF1载体是用SmaⅠ单酶切PBIN-PLUS空载体质粒后与相应的基因片段进行连接构建的，构建方法为现有技术公开的常规方法。

反应体系如下：2μL 10×CEⅡ反应缓冲液；2μL上述PCR回收产物；2μL上述酶切后的空载体；1μL同源重组酶；无菌水补足至10μL。

反应程序为：37℃条件下反应30min。将连接产物转化大肠杆菌DH5α，在Kan抗性培养基上筛选转化子，挑取阳性克隆提取质粒进行菌落PCR鉴定。将鉴定到的阳性克隆测序，正确即获得了PBIN-PLUS:GFP、PBIN-PLUS:INF1

实施例3表达载体转化农杆菌及功能验证

1、获得农杆菌感受态细胞

在LB固体培养基平板上划线培养农杆菌GV3101菌株，28℃条件下倒置培养18-20h；将单菌落置于LB液体培养基(含有100mg/L Rif)，28℃条件下振荡培养18-20h；100倍体积的不含抗生素的菌液加入菌液中，28℃条件下振荡培养OD600至0.3-0.5；冰上冷却后，4℃条件下4000r/min离心5min，弃上清；加入20mmol/L的CaCl

2、PBIN-PLUS:GFP、PBIN-PLUS:INF1、PBIN-PLUS:CRN12、PBIN-PLUS:CRN13、PBIN-PLUS:CRN19、PBIN-PLUS:CRN20和PBIN-PLUS:CRN23表达载体转化农杆菌

利用电转法将PBIN-PLUS:GFP、PBIN-PLUS:INF1、PBIN-PLUS:CRN12、PBIN-PLUS:CRN13、PBIN-PLUS:CRN19、PBIN-PLUS:CRN20和PBIN-PLUS:CRN23表达载体转化农杆菌细胞；利用70％酒精冲洗电击杯三次，然后完全风干酒精。将100ng表达载体加入农杆菌感受态，冰上放置30min。将农杆菌感受态细胞转移至电击杯中，利用2.5kV的电压进行电击转化。电击完成后将农杆菌感受态细胞加入到400μL LB液体培养基中，28℃振荡培养2h。吸取20μL菌液，均匀涂抹在含有50mm卡那霉素和25mm的利福平固体LB培养基平板上，28℃倒置培养48h。挑取单菌落进行菌落PCR，获取阳性克隆。

3、在本氏烟草中瞬时表达PBIN-PLUS:GFP、PBIN-PLUS:INF1、PBIN-PLUS:CRN12、PBIN-PLUS:CRN13、PBIN-PLUS:CRN19、PBIN-PLUS:CRN20和PBIN-PLUS:CRN23蛋白

将上述阳性克隆放置于LB液体培养基中，28℃震荡培养30h。将菌液收集，用8000rpm离心2min，利用10mM MgCl2清洗三次。最后用10mM的MgCl2稀释菌液至OD600值0.3。将菌液与含有沉默抑制子P19的菌液1：1混合。用1mL注射器在本式烟叶片上分别注射所有基因，每个最少5个重复，实验重复三次，结果如图1～4所示。

4、瞬时表达PBIN-PLUS:CRN12、PBIN-PLUS:CRN13、PBIN-PLUS:CRN19、PBIN-PLUS:CRN20和PBIN-PLUS:CRN23蛋白的本氏烟抗病性检测

将确认表达蛋白的叶片剪下，放置于滤纸保湿的托盘内。用7mm打孔器在辣椒疫霉的平板生长边缘打孔，将打下的菌碟菌面向下得左右对称放置在本氏烟叶片的左右两侧。将托盘放置于25℃培养箱，黑暗，36h。取出叶片，然后用手持紫外荧光仪照射叶片。颜色变深的区域即为辣椒疫霉的侵染区域。用量尺测量侵染区域的面积，表达PBIN-PLUS:GFGP蛋白的叶面为对照，比较两种蛋白对本氏烟抗性的影响。发现来自寡雄腐霉和缠器腐霉的5个CRN蛋白显著提高了本氏烟草对辣椒疫霉的抗性(如图1～4所示)。

7、活性氧(ROS)积累检测

注射所有处理基因36h后，用1mg/ml DAB溶液于黑暗中染色8h，然后用95％乙醇脱色处理，最后用相机拍照记录表型，实验结果如图1～4所示。

参考文献

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2.Paul,B.&Masih,I.ITS1 region of the nuclear ribosomal DNA of themycoparasite Pythium periplocum,its taxonomy,and its comparison with relatedspecies.FEMS Microbiology Letters(2000).

3.Jambunathan,N.Determination and detection of reactive oxygenspecies(ROS),lipid peroxidation,and electrolyte leakage in plants.Methods MolBiol 639,292-298,doi:10.1007/978-1-60761-702-0_18(2010).

4.Qi Li1,Gan Ai1,2,Danyu Shen1,Fen Zou1,Ji Wang1,Tian Bai1,YanyuChen1,Shutian Li1,Meixiang Zhang1,Maofeng Jing1 and Daolong Dou1,APhytophthora capsici Effector Targets ACD11 Binding Partners that RegulateROS-Mediated Defense Response in Arabidopsis.Mol Plant12,565-581(2019).

序列表

CRN12基因

ATGGTGTTCTCCGTCGAGGTTGAGAGAAACGCAGACGTGGAAGCGCTGCAGGAGGCTATCTTTGTCAAGAAGCGATACAAAGAGCGCTATACATTCGATGCAAGCGCATTGACGCTGTACTTGGCGAAGAAGGACGGCGCATGGCTCAAGCATGATCACACTGTGAAGGCGTTCTTGCAAGGTGACATCGACACTGAGTACGAGGAGATGCTTTCGACGTGGAAGCTCAATAAGGAGGAGTTGTTTGGGGACTTCCAGCTCGGAGGAGAAGAGATTCACGTGCTGGTGGAGCTCCCAGAGCGCCAGTCTGCTGATCCTCAGCTGATCGAGCTTCAGGAATCACTTTTGCAGCATATCCTGATGGATGCTCCAACTTCGACGTCCGTGCGAAGCAATCATTTCAAGAAGAAGCTGTGTGCAACGTACAATTGCGACATGGGAAGTGGCCAGTTGCGCTGTATGCTACTGGACACAGCTCTTCCGTCGGAGTTGGTGATTGCGTCTCATTTGTTTCGCCGAAAAAACGAATTTATCTCTGAGAAGTTTATGGGATTCTCGGATATCGACGACGTGAGAAACGGTTTGCTGCTTTTCAAGCCGCTGGAGCATGCGCTCGACCATTTCCAGATCAGCTTTATTTACGACCCAAGCAGCAACGAGTTTCGTTTGAAGATTTTCGACCGATCCATGAGAAAACAGCGATTGCTTGGAAAATTAGACAAAACGCAGCGTGCGATACTTCTTCAAGGTCAGGTGTTGCCGAAAAACTGGAGATCGCGAGGTCGAAGACTTGCACCCGGGACAAACTATGACCTCCAGACGACTTTTGGTGATCTTGAAGGTCGGACACTTTGCTTTCGGGGTATCGAGCGTCCGTACAAGCGTTGTTTGAACTTGCAGGCCCGATTAGCCCGAAAACAAGCAATCAAGAAACAGTGGATCGAGCCAGAGGAAGATGACTTTCAAGACTTTTGGTCCGAAGGCATGTCGTTGGCAGAGAAGATGGAGTTTTACTACGCGAACGAAACCTGA

CRN13基因

ATGGTGTTCTCCGTCGAGGTTGAGAGAAACGCAGACGTGGAAGCGCTGCAGGAGGCTATCTTTGTCAAGAAGCGATACAAAGAGCGCTATACATTCGACGCAAGCGCATTGACGCTGTACTTGGCGAAGAAGGACGGCGCATGGCTCAAGCATGATCACACTGTGAAGGCGTTCTTGCAAGGTGACATCGACACTGAGTACGAGGAGATGCTTTCGACGTGGAAGCTCAATAAGGAGGAGTTGTTTGGGGACTTCCAGCTCGGAGGAGAAGAGATTCACGTGCTGGTGGAGCTCCCAGAGCGCCAGTCTGCTGATCCTCAGCTGATCGAGCTTCAGGAATCACTTTTGCATCATATCTTGATGGATGCTCCAACTTCGACGTCCGTGCGAAGCAATCATTTCAAGAAGAAGCTGTGTGCAACGTACAATTGCGACATGGGAAATGGCCAGTTGCGCTGTATGCTACTGGACACAGCTCTTCCGTCGGAGTTGGTGATTGCGTCTCATTTGTTTCGCCGAAAAAACGAATTTATCTCTGAGAAGTTTATGGGATTCTCGGACATCGACGATGTGAGAAACGGTTTGCTGCTTTTCAAGCCGCTGGAGCATGCGTTCGACCATTTCCAGATCAGCTTTATTTACGACCCAAGCAGCAACGAGTTTCGTTTGAAGATTTTCGACAGATCCATGAGAAAACAGCGATTGTTTGGAAAATTAGACAAAACGCAGCGTGCGATACTTCTTCAAGGTCAGGTGTTGCCGAGAAACTGGAGATCGCGAGGTCGAAGACTTGCACCCGGGACAAACTATGACCTCCAGACGACTTTTGGTGATCTTGAAGGTCGGACGCTTTGCTTTCGGGGTATCGAGCGTCCGTACAAGCGTTGTTTGAACTTGCAGGCCCGACTAGCCCGAAAACAAGCAATCAAGAAACTGTGGATCGAGCCAGAGAAAGATGACTTTCAAGACTTTTGGTCCGAAGGCATGTCGTTGGCAGAGAAGATGGAGTTTTACTACGCGAACGAAACCTAA

CRN20基因

ATGTTTCCTGTGGACATTGATGAGAGTCTGACGGTTGGGGACTTGAAGGAAGTGATCCGGGGGAAGAAGCCGCGCAAGATCACGTGCGATGCCGATGAGTTGGAGTTGTACTTGGCCAAGAGGGGCGACGCGTGGCTGACAGAAAACGAGGTCAGAGGGATAAGTGACACCAATGGCCTCAAGTATCTGGGTGCTGCGCGAGCGGAACTCGACGTAGTTGGCCTTTCGGAGGAAGATCTGCGATTCGAAATCGACAAACACCGAGTTGCAGCAGGATATGGCCCTGTAAATGTGTTGGTGGTGGTCCCGTCAAATGGAACGAGCATGTCTGTGGTGGGGACGAACATTGAAAAGTTCAGGACTCCACTTCAGTTCTCCACTGCGGAACACGAAGGTGGCGTTCCTTCAACATTCAGGACCTATCGATCAGCCACTGTTCACGCATTTTTCAGGCTGCTGTTTCCTCCGGGCGGGAACTTTCTCCCATTAATATTCGTCCGTGCACCACCGTTATCCGGAAAGTCTGCCATGTTCGACCTACTCTACAATCACATTGTGCTCTCCCAGCCGGATGCGCTTGTTGCGCGGGTGTCTGCGAATTTTATGCCTGAGTCGACCACATTCTGTCAATACTTCAGCTCAACCTATGGCTGCGATTTTAAAGCATTCTGCAAGCTCGACTACGAGAAAGTGGTATTGATTGATGAGGCTCAAGTGACGTACGATGATGATCTACTTTGGCTGGGTTACCTGAAGAATACGTTGGAGGGGAAAGTCCACGGCCTACGATTTGTTCTCTTCTCGTCATACGGCAGTTTTGACATTTACCGGGAGGATGTACGTCCTGGGACTCCTATTCTCGTCCCGCCGGGAAATACTTTTGGATTGAATGCGGCGCCATCGAAGCCTGGCTTGCAACTCACGCGCGTAGAACTGGACGAAATGATTCACGGAAGCATCGGAGCAGCGGTGGGTGATCTCATTTGGATTCTGTGCTCTGGACATATCGGGATTGCTCGAGCAATACTCAGGTTTTTGCATGGCAAGTTTGGATCAAAGAAAGCATCCATTGCTCCTGAAGATCTGGAAGCAGAACTTCGTTCAGTCCGCTTGCTACGATACATCCGCGACAGTTATCGTGGCATACCGACGCTTGATGCTTTTCAGCGAGTGATCCAGGGTCGGCAATTGGCTGACGAGTCGAAGTTGAAGATGAGTGAAGTGATGAGTAGTGTTGCTTCGGGCAAGATTGTGCTTGCATCTGATGGGGAGCGATCTCCTAGAAGTCGTCTTGCAGTGGAATTACTTACCCGATTCGGTTTCCTCTACGAGGATTACAACAAGCAACTCCAGTTCGCATCGAATATGCATCTGAAGATTTGGCTGCATTCAAATCGATCGGATCCAATCGGACATATGGTCTCCAACATCTCGCATGACGAATTCTTGGTTGCTTGTATAACGAGAATGCGTAGTTCGCAACTTCAGCATTTTGCGACTGGAAACACCAGCAACACGTTGGTTGCTCGAGAGCGCCAAATCCAAATGGAGCTGTACAACGCCACCACTTCATGTTTACCGCGGGACGTGCTAGTTACTCCAGAATGGCGCACGAGTGATGAGATAGGCTTCGTTGACCTGGTTATTCAAGGCGGAGATATCCTCTGGTTCTGGGAGCTGCTTGTGAATGGAGACGATGCTGTTAGCCATTCGAAGCGATTCAAAACAGGTGGCAAGTACTATGGAAGCCTTACGAGCCATTCTCGATACGTACTCATTGACTTCCGTCAGAACAAAGGCGTTCGAGAGCAGAAGCTTGGTTTTTTGTACGTGTCCTTTGTGGATTCTTACTCTAAGGCGAAGATCTTTGGGCTTCATAGGGACGAGATCACTGTGGAGTTATCGTGA

CRN23基因

ATGGTGTTCCCCGTGAGGATCGCACGTGATGCTAGAGTGAGCGCACTGCAGAAGGCTATCTTTGACGAGAAACGATACAGAGAGCGCTACACCTTTGACGCAAGCGCATTGACGCTGTACTTGGCGCGGAAGAAGGAAGGTGACGAAAGCAAGTGGCTGACGGATGACGACAATTTGGATGCTATCTTGGGAGGAGATGTCGACAAGCAGTTCGTGAAAATGCGATCGTTGTGGATTCTCAGCGAAGATTACCTTGGCGCGAATTTTCAGCCTGGGCGCAAAGAGATCCACGTGCTGGTGGAACTCCCAGAAGGTGCGGCCGGTGAGAAATCGGAAATGGCACAAATGATGAAGGAAATGTACGAGCATATTGTGCATCCTGTGCAGACTAAGCGCAAATACGTCCATTCCGAGATGAACTCAAGCAAGGGAATCGCGCTTCTGAAAGACCTGAATATTCGAGTGAAGGACGCGGACTCGGTTCCATTCTCTACTGAAGATCCAACTCCGGTTGAACCATTCACATGGGAAAGTGTTTGTAATGAACATGGCCAACTCATTGTGCTTACCGAGGAGCAGCAACGAGAACGATATCGTAGATACGTGGAAGACAACATTGGCGATGTGCTCGCGGAGAAGAGGCTTTGCGTGCTCGGTGTGGAAAAAGGCAAGAATATTCTCACTGCTGACGTCCCTGGTCACGACATTGAACTTGCTGGACGGACGGATATGCTCATACTGAGGGATGTCACAAAGCAGTTCCCGGACCATTTGGAGATGTTGGATGAAGTGAAGATGCTTATCGAAGTGAAGAGGAACGTAAAAGGAGGCTCTGTTTTCCAGGCGTTGTCGGAACTGATTGCGCTAGATGTTCTTGTCGATGACCCAGTGATGGCACTGCTGACCGACTTGACTGGCCGTTGGCAGTTCTTCTGGGTCTCCGAGAAGAGCGACAACCATGTCATCATCCGAACAGTGATTATATCTGATCCAAGCGCGGCGTTTGCCGTGATCAGGACGCTACTTGCACAGTCGCCAAATGGTGATGCAGATATCACACTTCCATGTTTTGGAGAACCCGTGAAACGACGCAAGTTGGCTCAATTGCTGCCGTCTATTGGTGAAGGAGGTGAAAGCGGCGGCATACGTGAGGCTATCGAACGGTACTATGATATCGCAAGCGTGCTGGGTCCCGACATTGACATGGCACGTTCTGTGGCTCACCAACTTGTCCGAACGATTCCCACGTTCAGCTATTACACGTAA

CRN19基因

ATGGGTGTGTACGGCGAAGGGAGTGTCTTCTCGGTCGAGATCCAGCGCAATGCAGATGTGGAGGCGCTGCAGAAGAAGATCGCAAGTACATACAGAGTCGTGAGCAACCGCGTTGAGGTTTACCCTGCCACCCTGACGCTGTACTTGGCGCGGAAAGAGGGTGGCCCGTGGTTGGCGGATGACCACCATGTGAAGAATTTTTTGCAAGGTGGCATCAACACTGAGTACGAGGAGATGCGTCCGTCGTGGCATCTCGACGAAGACTACTTTGGAGCGGACTTTCAGCCTGGACGCAAAGAGATTCACGTGCTGGTGCAAGTGCCGGAAGTGCTGGAAACTGCGGTAGCCAATAAGAAGCAGCGCCTGGAGTGGCGGTCGACCCGATGGGGATCACACGCTTATGACCCGAACTCACAATACTTCCTGCTGGAGAAAGAGGATGTGGACGAGTCCGGACTTCCACCCGTTCGCTTGATGCTCTACTGCAGACCCGCATTCCACCGCCAATTCGAGTTCTTGCGCGATGAAGTGTTGAACGAAGGTCATTTGGGCTGGATTCTCGGCCCACCTGGAACGGGAAAGTCAACCACAGCCATGGCTTTCGCGTCGACTCTGGACAGAAGTGAATGGATCGTGACGTGGATCCACGTGGGGAAATTCATGTCTTGGCGATGCGTGCGCCTCATCGGCGAAGACCGGAGAAGTCGAATTCTTGATATCTCGGACGTGGATGAAGTGTTGATGGTTGACGATACGAGGCATCACGTAGTGCTCGTTGACGGGTGGACTGCTGCAGACGAGTTTATTGAGCTGACACGCAAGTGCGTCGAATGGTTCCTTGTCGGTACAAAGGTGAAGCGGCGGCTCGCATTTGTTTGTTCAGTTGCTGCTCGTGGGAAGTTCCAAGAAGACATGGAGATTCTGACTCGTGCAAGAGAGTTCCGCGTGTGGTCATGGACTCTGGATGAGTACCTCAATGCGATCAAGGATGACGAGGTGTTCCAGAGTGTCTCGCAATGTCTGGATGCCCCTGCAGTGTTGCCGGACGAAGAGATGTCTCGCGCGGCAATGGTTGAGTCCAAGTATTACTACGCAGGTGGTTCCTGTCGTTATATGTTTCACTTCCGCACTGTGACCGTGGCGACGAAGTTGAGTGATGCCGTGGATTCATTAAACGATGCCGCAATTGTTGCCACGAGTGGTCAGCGTTCATCTTCAAGCATCAACCGTCTGTTTGGGATGTTCCAGCGACCTCACGACGTGGGTGCGGTGTCGCCTGTGATCAGTCAGTATGCTGCGACGCTGATTGCCATCAGATGCGGTCCAGAGGCTATCAAGAAGTTCATATCGACGCATCGAGACAATTCAAACCCAGCATTGGATGGATGGATGTTGGAGATGGTGTTCTTCGCAAGTCTTCGAAACGGCGGTCTCGCTCTCGTCGATGCGGCAGGGAACACAGTTGACACGTGGGGTCAATCAGTTGTTGTGATCGCGGATGGAATACCTGCGCTTTCTTCTGCTCACCCCGTTTGGATCAAGCCAGAAAAATGGAACCAAGGCGGATATGACGCTATTATGGTGTGCAAGCGTACTCAGCACGTGCGTTTGGTGCAAGTGACGAGTGCCCACAAGCATTCTTTCCGCATCGATTATTTTTACGGATGGCTGAAGATGCTGTCGGAGTCGCCCGAAAGTTTTGAGGTGAAGACAATGGAGATTGTCTTCGTTGTCGAGCAAGACAAGCTGAGCACGTTCGACTTCTCGACTGTGAAGGGAGCCGGACTTCTCCGACCGTTTGGTTGGTCGAAGGGCAAGGAGACGGACTTAGTACGGCTGGTAGGGATTCGTGGGTTATATAATCTATCACCATAA

CRN12蛋白

MVFSVEVERNADVEALQEAIFVKKRYKERYTFDASALTLYLAKKDGAWLKHDHTVKAFLQGDIDTEYEEMLSTWKLNKEELFGDFQLGGEEIHVLVELPERQSADPQLIELQESLLQHILMDAPTSTSVRSNHFKKKLCATYNCDMGSGQLRCMLLDTALPSELVIASHLFRRKNEFISEKFMGFSDIDDVRNGLLLFKPLEHALDHFQISFIYDPSSNEFRLKIFDRSMRKQRLLGKLDKTQRAILLQGQVLPKNWRSRGRRLAPGTNYDLQTTFGDLEGRTLCFRGIERPYKRCLNLQARLARKQAIKKQWIEPEEDDFQDFWSEGMSLAEKMEFYYANET*

CRN13蛋白

MVFSVEVERNADVEALQEAIFVKKRYKERYTFDASALTLYLAKKDGAWLKHDHTVKAFLQGDIDTEYEEMLSTWKLNKEELFGDFQLGGEEIHVLVELPERQSADPQLIELQESLLHHILMDAPTSTSVRSNHFKKKLCATYNCDMGNGQLRCMLLDTALPSELVIASHLFRRKNEFISEKFMGFSDIDDVRNGLLLFKPLEHAFDHFQISFIYDPSSNEFRLKIFDRSMRKQRLFGKLDKTQRAILLQGQVLPRNWRSRGRRLAPGTNYDLQTTFGDLEGRTLCFRGIERPYKRCLNLQARLARKQAIKKLWIEPEKDDFQDFWSEGMSLAEKMEFYYANET*

CRN20蛋白

MFPVDIDESLTVGDLKEVIRGKKPRKITCDADELELYLAKRGDAWLTENEVRGISDTNGLKYLGAARAELDVVGLSEEDLRFEIDKHRVAAGYGPVNVLVVVPSNGTSMSVVGTNIEKFRTPLQFSTAEHEGGVPSTFRTYRSATVHAFFRLLFPPGGNFLPLIFVRAPPLSGKSAMFDLLYNHIVLSQPDALVARVSANFMPESTTFCQYFSSTYGCDFKAFCKLDYEKVVLIDEAQVTYDDDLLWLGYLKNTLEGKVHGLRFVLFSSYGSFDIYREDVRPGTPILVPPGNTFGLNAAPSKPGLQLTRVELDEMIHGSIGAAVGDLIWILCSGHIGIARAILRFLHGKFGSKKASIAPEDLEAELRSVRLLRYIRDSYRGIPTLDAFQRVIQGRQLADESKLKMSEVMSSVASGKIVLASDGERSPRSRLAVELLTRFGFLYEDYNKQLQFASNMHLKIWLHSNRSDPIGHMVSNISHDEFLVACITRMRSSQLQHFATGNTSNTLVARERQIQMELYNATTSCLPRDVLVTPEWRTSDEIGFVDLVIQGGDILWFWELLVNGDDAVSHSKRFKTGGKYYGSLTSHSRYVLIDFRQNKGVREQKLGFLYVSFVDSYSKAKIFGLHRDEITVELS*

CRN23蛋白

MVFPVRIARDARVSALQKAIFDEKRYRERYTFDASALTLYLARKKEGDESKWLTDDDNLDAILGGDVDKQFVKMRSLWILSEDYLGANFQPGRKEIHVLVELPEGAAGEKSEMAQMMKEMYEHIVHPVQTKRKYVHSEMNSSKGIALLKDLNIRVKDADSVPFSTEDPTPVEPFTWESVCNEHGQLIVLTEEQQRERYRRYVEDNIGDVLAEKRLCVLGVEKGKNILTADVPGHDIELAGRTDMLILRDVTKQFPDHLEMLDEVKMLIEVKRNVKGGSVFQALSELIALDVLVDDPVMALLTDLTGRWQFFWVSEKSDNHVIIRTVIISDPSAAFAVIRTLLAQSPNGDADITLPCFGEPVKRRKLAQLLPSIGEGGESGGIREAIERYYDIASVLGPDIDMARSVAHQLVRTIPTFSYYT*

CRN19蛋白

MGVYGEGSVFSVEIQRNADVEALQKKIASTYRVVSNRVEVYPATLTLYLARKEGGPWLADDHHVKNFLQGGINTEYEEMRPSWHLDEDYFGADFQPGRKEIHVLVQVPEVLETAVANKKQRLEWRSTRWGSHAYDPNSQYFLLEKEDVDESGLPPVRLMLYCRPAFHRQFEFLRDEVLNEGHLGWILGPPGTGKSTTAMAFASTLDRSEWIVTWIHVGKFMSWRCVRLIGEDRRSRILDISDVDEVLMVDDTRHHVVLVDGWTAADEFIELTRKCVEWFLVGTKVKRRLAFVCSVAARGKFQEDMEILTRAREFRVWSWTLDEYLNAIKDDEVFQSVSQCLDAPAVLPDEEMSRAAMVESKYYYAGGSCRYMFHFRTVTVATKLSDAVDSLNDAAIVATSGQRSSSSINRLFGMFQRPHDVGAVSPVISQYAATLIAIRCGPEAIKKFISTHRDNSNPALDGWMLEMVFFASLRNGGLALVDAAGNTVDTWGQSVVVIADGIPALSSAHPVWIKPEKWNQGGYDAIMVCKRTQHVRLVQVTSAHKHSFRIDYFYGWLKMLSESPESFEVKTMEIVFVVEQDKLSTFDFSTVKGAGLLRPFGWSKGKETDLVRLVGIRGLYNLSP*

序列表

<110> 南京农业大学

<120> CRN类效应蛋白基因在诱导植物抗性中的应用

<160> 10

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1032

<212> DNA

<213> 寡雄腐霉(Pythium oligandrum)

<400> 1

atggtgttct ccgtcgaggt tgagagaaac gcagacgtgg aagcgctgca ggaggctatc 60

tttgtcaaga agcgatacaa agagcgctat acattcgatg caagcgcatt gacgctgtac 120

ttggcgaaga aggacggcgc atggctcaag catgatcaca ctgtgaaggc gttcttgcaa 180

ggtgacatcg acactgagta cgaggagatg ctttcgacgt ggaagctcaa taaggaggag 240

ttgtttgggg acttccagct cggaggagaa gagattcacg tgctggtgga gctcccagag 300

cgccagtctg ctgatcctca gctgatcgag cttcaggaat cacttttgca gcatatcctg 360

atggatgctc caacttcgac gtccgtgcga agcaatcatt tcaagaagaa gctgtgtgca 420

acgtacaatt gcgacatggg aagtggccag ttgcgctgta tgctactgga cacagctctt 480

ccgtcggagt tggtgattgc gtctcatttg tttcgccgaa aaaacgaatt tatctctgag 540

aagtttatgg gattctcgga tatcgacgac gtgagaaacg gtttgctgct tttcaagccg 600

ctggagcatg cgctcgacca tttccagatc agctttattt acgacccaag cagcaacgag 660

tttcgtttga agattttcga ccgatccatg agaaaacagc gattgcttgg aaaattagac 720

aaaacgcagc gtgcgatact tcttcaaggt caggtgttgc cgaaaaactg gagatcgcga 780

ggtcgaagac ttgcacccgg gacaaactat gacctccaga cgacttttgg tgatcttgaa 840

ggtcggacac tttgctttcg gggtatcgag cgtccgtaca agcgttgttt gaacttgcag 900

gcccgattag cccgaaaaca agcaatcaag aaacagtgga tcgagccaga ggaagatgac 960

tttcaagact tttggtccga aggcatgtcg ttggcagaga agatggagtt ttactacgcg 1020

aacgaaacct ga 1032

<210> 2

<211> 1032

<212> DNA

<213> 缠器腐霉(Pythium periplocum )

<400> 2

atggtgttct ccgtcgaggt tgagagaaac gcagacgtgg aagcgctgca ggaggctatc 60

tttgtcaaga agcgatacaa agagcgctat acattcgacg caagcgcatt gacgctgtac 120

ttggcgaaga aggacggcgc atggctcaag catgatcaca ctgtgaaggc gttcttgcaa 180

ggtgacatcg acactgagta cgaggagatg ctttcgacgt ggaagctcaa taaggaggag 240

ttgtttgggg acttccagct cggaggagaa gagattcacg tgctggtgga gctcccagag 300

cgccagtctg ctgatcctca gctgatcgag cttcaggaat cacttttgca tcatatcttg 360

atggatgctc caacttcgac gtccgtgcga agcaatcatt tcaagaagaa gctgtgtgca 420

acgtacaatt gcgacatggg aaatggccag ttgcgctgta tgctactgga cacagctctt 480

ccgtcggagt tggtgattgc gtctcatttg tttcgccgaa aaaacgaatt tatctctgag 540

aagtttatgg gattctcgga catcgacgat gtgagaaacg gtttgctgct tttcaagccg 600

ctggagcatg cgttcgacca tttccagatc agctttattt acgacccaag cagcaacgag 660

tttcgtttga agattttcga cagatccatg agaaaacagc gattgtttgg aaaattagac 720

aaaacgcagc gtgcgatact tcttcaaggt caggtgttgc cgagaaactg gagatcgcga 780

ggtcgaagac ttgcacccgg gacaaactat gacctccaga cgacttttgg tgatcttgaa 840

ggtcggacgc tttgctttcg gggtatcgag cgtccgtaca agcgttgttt gaacttgcag 900

gcccgactag cccgaaaaca agcaatcaag aaactgtgga tcgagccaga gaaagatgac 960

tttcaagact tttggtccga aggcatgtcg ttggcagaga agatggagtt ttactacgcg 1020

aacgaaacct aa 1032

<210> 3

<211> 1908

<212> DNA

<213> 寡雄腐霉(Pythium oligandrum)

<400> 3

atgtttcctg tggacattga tgagagtctg acggttgggg acttgaagga agtgatccgg 60

gggaagaagc cgcgcaagat cacgtgcgat gccgatgagt tggagttgta cttggccaag 120

aggggcgacg cgtggctgac agaaaacgag gtcagaggga taagtgacac caatggcctc 180

aagtatctgg gtgctgcgcg agcggaactc gacgtagttg gcctttcgga ggaagatctg 240

cgattcgaaa tcgacaaaca ccgagttgca gcaggatatg gccctgtaaa tgtgttggtg 300

gtggtcccgt caaatggaac gagcatgtct gtggtgggga cgaacattga aaagttcagg 360

actccacttc agttctccac tgcggaacac gaaggtggcg ttccttcaac attcaggacc 420

tatcgatcag ccactgttca cgcatttttc aggctgctgt ttcctccggg cgggaacttt 480

ctcccattaa tattcgtccg tgcaccaccg ttatccggaa agtctgccat gttcgaccta 540

ctctacaatc acattgtgct ctcccagccg gatgcgcttg ttgcgcgggt gtctgcgaat 600

tttatgcctg agtcgaccac attctgtcaa tacttcagct caacctatgg ctgcgatttt 660

aaagcattct gcaagctcga ctacgagaaa gtggtattga ttgatgaggc tcaagtgacg 720

tacgatgatg atctactttg gctgggttac ctgaagaata cgttggaggg gaaagtccac 780

ggcctacgat ttgttctctt ctcgtcatac ggcagttttg acatttaccg ggaggatgta 840

cgtcctggga ctcctattct cgtcccgccg ggaaatactt ttggattgaa tgcggcgcca 900

tcgaagcctg gcttgcaact cacgcgcgta gaactggacg aaatgattca cggaagcatc 960

ggagcagcgg tgggtgatct catttggatt ctgtgctctg gacatatcgg gattgctcga 1020

gcaatactca ggtttttgca tggcaagttt ggatcaaaga aagcatccat tgctcctgaa 1080

gatctggaag cagaacttcg ttcagtccgc ttgctacgat acatccgcga cagttatcgt 1140

ggcataccga cgcttgatgc ttttcagcga gtgatccagg gtcggcaatt ggctgacgag 1200

tcgaagttga agatgagtga agtgatgagt agtgttgctt cgggcaagat tgtgcttgca 1260

tctgatgggg agcgatctcc tagaagtcgt cttgcagtgg aattacttac ccgattcggt 1320

ttcctctacg aggattacaa caagcaactc cagttcgcat cgaatatgca tctgaagatt 1380

tggctgcatt caaatcgatc ggatccaatc ggacatatgg tctccaacat ctcgcatgac 1440

gaattcttgg ttgcttgtat aacgagaatg cgtagttcgc aacttcagca ttttgcgact 1500

ggaaacacca gcaacacgtt ggttgctcga gagcgccaaa tccaaatgga gctgtacaac 1560

gccaccactt catgtttacc gcgggacgtg ctagttactc cagaatggcg cacgagtgat 1620

gagataggct tcgttgacct ggttattcaa ggcggagata tcctctggtt ctgggagctg 1680

cttgtgaatg gagacgatgc tgttagccat tcgaagcgat tcaaaacagg tggcaagtac 1740

tatggaagcc ttacgagcca ttctcgatac gtactcattg acttccgtca gaacaaaggc 1800

gttcgagagc agaagcttgg ttttttgtac gtgtcctttg tggattctta ctctaaggcg 1860

aagatctttg ggcttcatag ggacgagatc actgtggagt tatcgtga 1908

<210> 4

<211> 1266

<212> DNA

<213> 缠器腐霉(Pythium periplocum )

<400> 4

atggtgttcc ccgtgaggat cgcacgtgat gctagagtga gcgcactgca gaaggctatc 60

tttgacgaga aacgatacag agagcgctac acctttgacg caagcgcatt gacgctgtac 120

ttggcgcgga agaaggaagg tgacgaaagc aagtggctga cggatgacga caatttggat 180

gctatcttgg gaggagatgt cgacaagcag ttcgtgaaaa tgcgatcgtt gtggattctc 240

agcgaagatt accttggcgc gaattttcag cctgggcgca aagagatcca cgtgctggtg 300

gaactcccag aaggtgcggc cggtgagaaa tcggaaatgg cacaaatgat gaaggaaatg 360

tacgagcata ttgtgcatcc tgtgcagact aagcgcaaat acgtccattc cgagatgaac 420

tcaagcaagg gaatcgcgct tctgaaagac ctgaatattc gagtgaagga cgcggactcg 480

gttccattct ctactgaaga tccaactccg gttgaaccat tcacatggga aagtgtttgt 540

aatgaacatg gccaactcat tgtgcttacc gaggagcagc aacgagaacg atatcgtaga 600

tacgtggaag acaacattgg cgatgtgctc gcggagaaga ggctttgcgt gctcggtgtg 660

gaaaaaggca agaatattct cactgctgac gtccctggtc acgacattga acttgctgga 720

cggacggata tgctcatact gagggatgtc acaaagcagt tcccggacca tttggagatg 780

ttggatgaag tgaagatgct tatcgaagtg aagaggaacg taaaaggagg ctctgttttc 840

caggcgttgt cggaactgat tgcgctagat gttcttgtcg atgacccagt gatggcactg 900

ctgaccgact tgactggccg ttggcagttc ttctgggtct ccgagaagag cgacaaccat 960

gtcatcatcc gaacagtgat tatatctgat ccaagcgcgg cgtttgccgt gatcaggacg 1020

ctacttgcac agtcgccaaa tggtgatgca gatatcacac ttccatgttt tggagaaccc 1080

gtgaaacgac gcaagttggc tcaattgctg ccgtctattg gtgaaggagg tgaaagcggc 1140

ggcatacgtg aggctatcga acggtactat gatatcgcaa gcgtgctggg tcccgacatt 1200

gacatggcac gttctgtggc tcaccaactt gtccgaacga ttcccacgtt cagctattac 1260

acgtaa 1266

<210> 5

<211> 1875

<212> DNA

<213> 缠器腐霉(Pythium periplocum )

<400> 5

atgggtgtgt acggcgaagg gagtgtcttc tcggtcgaga tccagcgcaa tgcagatgtg 60

gaggcgctgc agaagaagat cgcaagtaca tacagagtcg tgagcaaccg cgttgaggtt 120

taccctgcca ccctgacgct gtacttggcg cggaaagagg gtggcccgtg gttggcggat 180

gaccaccatg tgaagaattt tttgcaaggt ggcatcaaca ctgagtacga ggagatgcgt 240

ccgtcgtggc atctcgacga agactacttt ggagcggact ttcagcctgg acgcaaagag 300

attcacgtgc tggtgcaagt gccggaagtg ctggaaactg cggtagccaa taagaagcag 360

cgcctggagt ggcggtcgac ccgatgggga tcacacgctt atgacccgaa ctcacaatac 420

ttcctgctgg agaaagagga tgtggacgag tccggacttc cacccgttcg cttgatgctc 480

tactgcagac ccgcattcca ccgccaattc gagttcttgc gcgatgaagt gttgaacgaa 540

ggtcatttgg gctggattct cggcccacct ggaacgggaa agtcaaccac agccatggct 600

ttcgcgtcga ctctggacag aagtgaatgg atcgtgacgt ggatccacgt ggggaaattc 660

atgtcttggc gatgcgtgcg cctcatcggc gaagaccgga gaagtcgaat tcttgatatc 720

tcggacgtgg atgaagtgtt gatggttgac gatacgaggc atcacgtagt gctcgttgac 780

gggtggactg ctgcagacga gtttattgag ctgacacgca agtgcgtcga atggttcctt 840

gtcggtacaa aggtgaagcg gcggctcgca tttgtttgtt cagttgctgc tcgtgggaag 900

ttccaagaag acatggagat tctgactcgt gcaagagagt tccgcgtgtg gtcatggact 960

ctggatgagt acctcaatgc gatcaaggat gacgaggtgt tccagagtgt ctcgcaatgt 1020

ctggatgccc ctgcagtgtt gccggacgaa gagatgtctc gcgcggcaat ggttgagtcc 1080

aagtattact acgcaggtgg ttcctgtcgt tatatgtttc acttccgcac tgtgaccgtg 1140

gcgacgaagt tgagtgatgc cgtggattca ttaaacgatg ccgcaattgt tgccacgagt 1200

ggtcagcgtt catcttcaag catcaaccgt ctgtttggga tgttccagcg acctcacgac 1260

gtgggtgcgg tgtcgcctgt gatcagtcag tatgctgcga cgctgattgc catcagatgc 1320

ggtccagagg ctatcaagaa gttcatatcg acgcatcgag acaattcaaa cccagcattg 1380

gatggatgga tgttggagat ggtgttcttc gcaagtcttc gaaacggcgg tctcgctctc 1440

gtcgatgcgg cagggaacac agttgacacg tggggtcaat cagttgttgt gatcgcggat 1500

ggaatacctg cgctttcttc tgctcacccc gtttggatca agccagaaaa atggaaccaa 1560

ggcggatatg acgctattat ggtgtgcaag cgtactcagc acgtgcgttt ggtgcaagtg 1620

acgagtgccc acaagcattc tttccgcatc gattattttt acggatggct gaagatgctg 1680

tcggagtcgc ccgaaagttt tgaggtgaag acaatggaga ttgtcttcgt tgtcgagcaa 1740

gacaagctga gcacgttcga cttctcgact gtgaagggag ccggacttct ccgaccgttt 1800

ggttggtcga agggcaagga gacggactta gtacggctgg tagggattcg tgggttatat 1860

aatctatcac cataa 1875

<210> 6

<211> 343

<212> PRT

<213> 寡雄腐霉(Pythium oligandrum)

<400> 6

Met Val Phe Ser Val Glu Val Glu Arg Asn Ala Asp Val Glu Ala Leu

1 5 10 15

Gln Glu Ala Ile Phe Val Lys Lys Arg Tyr Lys Glu Arg Tyr Thr Phe

20 25 30

Asp Ala Ser Ala Leu Thr Leu Tyr Leu Ala Lys Lys Asp Gly Ala Trp

35 40 45

Leu Lys His Asp His Thr Val Lys Ala Phe Leu Gln Gly Asp Ile Asp

50 55 60

Thr Glu Tyr Glu Glu Met Leu Ser Thr Trp Lys Leu Asn Lys Glu Glu

65 70 75 80

Leu Phe Gly Asp Phe Gln Leu Gly Gly Glu Glu Ile His Val Leu Val

85 90 95

Glu Leu Pro Glu Arg Gln Ser Ala Asp Pro Gln Leu Ile Glu Leu Gln

100 105 110

Glu Ser Leu Leu Gln His Ile Leu Met Asp Ala Pro Thr Ser Thr Ser

115 120 125

Val Arg Ser Asn His Phe Lys Lys Lys Leu Cys Ala Thr Tyr Asn Cys

130 135 140

Asp Met Gly Ser Gly Gln Leu Arg Cys Met Leu Leu Asp Thr Ala Leu

145 150 155 160

Pro Ser Glu Leu Val Ile Ala Ser His Leu Phe Arg Arg Lys Asn Glu

165 170 175

Phe Ile Ser Glu Lys Phe Met Gly Phe Ser Asp Ile Asp Asp Val Arg

180 185 190

Asn Gly Leu Leu Leu Phe Lys Pro Leu Glu His Ala Leu Asp His Phe

195 200 205

Gln Ile Ser Phe Ile Tyr Asp Pro Ser Ser Asn Glu Phe Arg Leu Lys

210 215 220

Ile Phe Asp Arg Ser Met Arg Lys Gln Arg Leu Leu Gly Lys Leu Asp

225 230 235 240

Lys Thr Gln Arg Ala Ile Leu Leu Gln Gly Gln Val Leu Pro Lys Asn

245 250 255

Trp Arg Ser Arg Gly Arg Arg Leu Ala Pro Gly Thr Asn Tyr Asp Leu

260 265 270

Gln Thr Thr Phe Gly Asp Leu Glu Gly Arg Thr Leu Cys Phe Arg Gly

275 280 285

Ile Glu Arg Pro Tyr Lys Arg Cys Leu Asn Leu Gln Ala Arg Leu Ala

290 295 300

Arg Lys Gln Ala Ile Lys Lys Gln Trp Ile Glu Pro Glu Glu Asp Asp

305 310 315 320

Phe Gln Asp Phe Trp Ser Glu Gly Met Ser Leu Ala Glu Lys Met Glu

325 330 335

Phe Tyr Tyr Ala Asn Glu Thr

340

<210> 7

<211> 343

<212> PRT

<213> 缠器腐霉(Pythium periplocum)

<400> 7

Met Val Phe Ser Val Glu Val Glu Arg Asn Ala Asp Val Glu Ala Leu

1 5 10 15

Gln Glu Ala Ile Phe Val Lys Lys Arg Tyr Lys Glu Arg Tyr Thr Phe

20 25 30

Asp Ala Ser Ala Leu Thr Leu Tyr Leu Ala Lys Lys Asp Gly Ala Trp

35 40 45

Leu Lys His Asp His Thr Val Lys Ala Phe Leu Gln Gly Asp Ile Asp

50 55 60

Thr Glu Tyr Glu Glu Met Leu Ser Thr Trp Lys Leu Asn Lys Glu Glu

65 70 75 80

Leu Phe Gly Asp Phe Gln Leu Gly Gly Glu Glu Ile His Val Leu Val

85 90 95

Glu Leu Pro Glu Arg Gln Ser Ala Asp Pro Gln Leu Ile Glu Leu Gln

100 105 110

Glu Ser Leu Leu His His Ile Leu Met Asp Ala Pro Thr Ser Thr Ser

115 120 125

Val Arg Ser Asn His Phe Lys Lys Lys Leu Cys Ala Thr Tyr Asn Cys

130 135 140

Asp Met Gly Asn Gly Gln Leu Arg Cys Met Leu Leu Asp Thr Ala Leu

145 150 155 160

Pro Ser Glu Leu Val Ile Ala Ser His Leu Phe Arg Arg Lys Asn Glu

165 170 175

Phe Ile Ser Glu Lys Phe Met Gly Phe Ser Asp Ile Asp Asp Val Arg

180 185 190

Asn Gly Leu Leu Leu Phe Lys Pro Leu Glu His Ala Phe Asp His Phe

195 200 205

Gln Ile Ser Phe Ile Tyr Asp Pro Ser Ser Asn Glu Phe Arg Leu Lys

210 215 220

Ile Phe Asp Arg Ser Met Arg Lys Gln Arg Leu Phe Gly Lys Leu Asp

225 230 235 240

Lys Thr Gln Arg Ala Ile Leu Leu Gln Gly Gln Val Leu Pro Arg Asn

245 250 255

Trp Arg Ser Arg Gly Arg Arg Leu Ala Pro Gly Thr Asn Tyr Asp Leu

260 265 270

Gln Thr Thr Phe Gly Asp Leu Glu Gly Arg Thr Leu Cys Phe Arg Gly

275 280 285

Ile Glu Arg Pro Tyr Lys Arg Cys Leu Asn Leu Gln Ala Arg Leu Ala

290 295 300

Arg Lys Gln Ala Ile Lys Lys Leu Trp Ile Glu Pro Glu Lys Asp Asp

305 310 315 320

Phe Gln Asp Phe Trp Ser Glu Gly Met Ser Leu Ala Glu Lys Met Glu

325 330 335

Phe Tyr Tyr Ala Asn Glu Thr

340

<210> 8

<211> 635

<212> PRT

<213> 寡雄腐霉(Pythium oligandrum)

<400> 8

Met Phe Pro Val Asp Ile Asp Glu Ser Leu Thr Val Gly Asp Leu Lys

1 5 10 15

Glu Val Ile Arg Gly Lys Lys Pro Arg Lys Ile Thr Cys Asp Ala Asp

20 25 30

Glu Leu Glu Leu Tyr Leu Ala Lys Arg Gly Asp Ala Trp Leu Thr Glu

35 40 45

Asn Glu Val Arg Gly Ile Ser Asp Thr Asn Gly Leu Lys Tyr Leu Gly

50 55 60

Ala Ala Arg Ala Glu Leu Asp Val Val Gly Leu Ser Glu Glu Asp Leu

65 70 75 80

Arg Phe Glu Ile Asp Lys His Arg Val Ala Ala Gly Tyr Gly Pro Val

85 90 95

Asn Val Leu Val Val Val Pro Ser Asn Gly Thr Ser Met Ser Val Val

100 105 110

Gly Thr Asn Ile Glu Lys Phe Arg Thr Pro Leu Gln Phe Ser Thr Ala

115 120 125

Glu His Glu Gly Gly Val Pro Ser Thr Phe Arg Thr Tyr Arg Ser Ala

130 135 140

Thr Val His Ala Phe Phe Arg Leu Leu Phe Pro Pro Gly Gly Asn Phe

145 150 155 160

Leu Pro Leu Ile Phe Val Arg Ala Pro Pro Leu Ser Gly Lys Ser Ala

165 170 175

Met Phe Asp Leu Leu Tyr Asn His Ile Val Leu Ser Gln Pro Asp Ala

180 185 190

Leu Val Ala Arg Val Ser Ala Asn Phe Met Pro Glu Ser Thr Thr Phe

195 200 205

Cys Gln Tyr Phe Ser Ser Thr Tyr Gly Cys Asp Phe Lys Ala Phe Cys

210 215 220

Lys Leu Asp Tyr Glu Lys Val Val Leu Ile Asp Glu Ala Gln Val Thr

225 230 235 240

Tyr Asp Asp Asp Leu Leu Trp Leu Gly Tyr Leu Lys Asn Thr Leu Glu

245 250 255

Gly Lys Val His Gly Leu Arg Phe Val Leu Phe Ser Ser Tyr Gly Ser

260 265 270

Phe Asp Ile Tyr Arg Glu Asp Val Arg Pro Gly Thr Pro Ile Leu Val

275 280 285

Pro Pro Gly Asn Thr Phe Gly Leu Asn Ala Ala Pro Ser Lys Pro Gly

290 295 300

Leu Gln Leu Thr Arg Val Glu Leu Asp Glu Met Ile His Gly Ser Ile

305 310 315 320

Gly Ala Ala Val Gly Asp Leu Ile Trp Ile Leu Cys Ser Gly His Ile

325 330 335

Gly Ile Ala Arg Ala Ile Leu Arg Phe Leu His Gly Lys Phe Gly Ser

340 345 350

Lys Lys Ala Ser Ile Ala Pro Glu Asp Leu Glu Ala Glu Leu Arg Ser

355 360 365

Val Arg Leu Leu Arg Tyr Ile Arg Asp Ser Tyr Arg Gly Ile Pro Thr

370 375 380

Leu Asp Ala Phe Gln Arg Val Ile Gln Gly Arg Gln Leu Ala Asp Glu

385 390 395 400

Ser Lys Leu Lys Met Ser Glu Val Met Ser Ser Val Ala Ser Gly Lys

405 410 415

Ile Val Leu Ala Ser Asp Gly Glu Arg Ser Pro Arg Ser Arg Leu Ala

420 425 430

Val Glu Leu Leu Thr Arg Phe Gly Phe Leu Tyr Glu Asp Tyr Asn Lys

435 440 445

Gln Leu Gln Phe Ala Ser Asn Met His Leu Lys Ile Trp Leu His Ser

450 455 460

Asn Arg Ser Asp Pro Ile Gly His Met Val Ser Asn Ile Ser His Asp

465 470 475 480

Glu Phe Leu Val Ala Cys Ile Thr Arg Met Arg Ser Ser Gln Leu Gln

485 490 495

His Phe Ala Thr Gly Asn Thr Ser Asn Thr Leu Val Ala Arg Glu Arg

500 505 510

Gln Ile Gln Met Glu Leu Tyr Asn Ala Thr Thr Ser Cys Leu Pro Arg

515 520 525

Asp Val Leu Val Thr Pro Glu Trp Arg Thr Ser Asp Glu Ile Gly Phe

530 535 540

Val Asp Leu Val Ile Gln Gly Gly Asp Ile Leu Trp Phe Trp Glu Leu

545 550 555 560

Leu Val Asn Gly Asp Asp Ala Val Ser His Ser Lys Arg Phe Lys Thr

565 570 575

Gly Gly Lys Tyr Tyr Gly Ser Leu Thr Ser His Ser Arg Tyr Val Leu

580 585 590

Ile Asp Phe Arg Gln Asn Lys Gly Val Arg Glu Gln Lys Leu Gly Phe

595 600 605

Leu Tyr Val Ser Phe Val Asp Ser Tyr Ser Lys Ala Lys Ile Phe Gly

610 615 620

Leu His Arg Asp Glu Ile Thr Val Glu Leu Ser

625 630 635

<210> 9

<211> 421

<212> PRT

<213> 缠器腐霉(Pythium periplocum)

<400> 9

Met Val Phe Pro Val Arg Ile Ala Arg Asp Ala Arg Val Ser Ala Leu

1 5 10 15

Gln Lys Ala Ile Phe Asp Glu Lys Arg Tyr Arg Glu Arg Tyr Thr Phe

20 25 30

Asp Ala Ser Ala Leu Thr Leu Tyr Leu Ala Arg Lys Lys Glu Gly Asp

35 40 45

Glu Ser Lys Trp Leu Thr Asp Asp Asp Asn Leu Asp Ala Ile Leu Gly

50 55 60

Gly Asp Val Asp Lys Gln Phe Val Lys Met Arg Ser Leu Trp Ile Leu

65 70 75 80

Ser Glu Asp Tyr Leu Gly Ala Asn Phe Gln Pro Gly Arg Lys Glu Ile

85 90 95

His Val Leu Val Glu Leu Pro Glu Gly Ala Ala Gly Glu Lys Ser Glu

100 105 110

Met Ala Gln Met Met Lys Glu Met Tyr Glu His Ile Val His Pro Val

115 120 125

Gln Thr Lys Arg Lys Tyr Val His Ser Glu Met Asn Ser Ser Lys Gly

130 135 140

Ile Ala Leu Leu Lys Asp Leu Asn Ile Arg Val Lys Asp Ala Asp Ser

145 150 155 160

Val Pro Phe Ser Thr Glu Asp Pro Thr Pro Val Glu Pro Phe Thr Trp

165 170 175

Glu Ser Val Cys Asn Glu His Gly Gln Leu Ile Val Leu Thr Glu Glu

180 185 190

Gln Gln Arg Glu Arg Tyr Arg Arg Tyr Val Glu Asp Asn Ile Gly Asp

195 200 205

Val Leu Ala Glu Lys Arg Leu Cys Val Leu Gly Val Glu Lys Gly Lys

210 215 220

Asn Ile Leu Thr Ala Asp Val Pro Gly His Asp Ile Glu Leu Ala Gly

225 230 235 240

Arg Thr Asp Met Leu Ile Leu Arg Asp Val Thr Lys Gln Phe Pro Asp

245 250 255

His Leu Glu Met Leu Asp Glu Val Lys Met Leu Ile Glu Val Lys Arg

260 265 270

Asn Val Lys Gly Gly Ser Val Phe Gln Ala Leu Ser Glu Leu Ile Ala

275 280 285

Leu Asp Val Leu Val Asp Asp Pro Val Met Ala Leu Leu Thr Asp Leu

290 295 300

Thr Gly Arg Trp Gln Phe Phe Trp Val Ser Glu Lys Ser Asp Asn His

305 310 315 320

Val Ile Ile Arg Thr Val Ile Ile Ser Asp Pro Ser Ala Ala Phe Ala

325 330 335

Val Ile Arg Thr Leu Leu Ala Gln Ser Pro Asn Gly Asp Ala Asp Ile

340 345 350

Thr Leu Pro Cys Phe Gly Glu Pro Val Lys Arg Arg Lys Leu Ala Gln

355 360 365

Leu Leu Pro Ser Ile Gly Glu Gly Gly Glu Ser Gly Gly Ile Arg Glu

370 375 380

Ala Ile Glu Arg Tyr Tyr Asp Ile Ala Ser Val Leu Gly Pro Asp Ile

385 390 395 400

Asp Met Ala Arg Ser Val Ala His Gln Leu Val Arg Thr Ile Pro Thr

405 410 415

Phe Ser Tyr Tyr Thr

420

<210> 10

<211> 624

<212> PRT

<213> 缠器腐霉(Pythium periplocum)

<400> 10

Met Gly Val Tyr Gly Glu Gly Ser Val Phe Ser Val Glu Ile Gln Arg

1 5 10 15

Asn Ala Asp Val Glu Ala Leu Gln Lys Lys Ile Ala Ser Thr Tyr Arg

20 25 30

Val Val Ser Asn Arg Val Glu Val Tyr Pro Ala Thr Leu Thr Leu Tyr

35 40 45

Leu Ala Arg Lys Glu Gly Gly Pro Trp Leu Ala Asp Asp His His Val

50 55 60

Lys Asn Phe Leu Gln Gly Gly Ile Asn Thr Glu Tyr Glu Glu Met Arg

65 70 75 80

Pro Ser Trp His Leu Asp Glu Asp Tyr Phe Gly Ala Asp Phe Gln Pro

85 90 95

Gly Arg Lys Glu Ile His Val Leu Val Gln Val Pro Glu Val Leu Glu

100 105 110

Thr Ala Val Ala Asn Lys Lys Gln Arg Leu Glu Trp Arg Ser Thr Arg

115 120 125

Trp Gly Ser His Ala Tyr Asp Pro Asn Ser Gln Tyr Phe Leu Leu Glu

130 135 140

Lys Glu Asp Val Asp Glu Ser Gly Leu Pro Pro Val Arg Leu Met Leu

145 150 155 160

Tyr Cys Arg Pro Ala Phe His Arg Gln Phe Glu Phe Leu Arg Asp Glu

165 170 175

Val Leu Asn Glu Gly His Leu Gly Trp Ile Leu Gly Pro Pro Gly Thr

180 185 190

Gly Lys Ser Thr Thr Ala Met Ala Phe Ala Ser Thr Leu Asp Arg Ser

195 200 205

Glu Trp Ile Val Thr Trp Ile His Val Gly Lys Phe Met Ser Trp Arg

210 215 220

Cys Val Arg Leu Ile Gly Glu Asp Arg Arg Ser Arg Ile Leu Asp Ile

225 230 235 240

Ser Asp Val Asp Glu Val Leu Met Val Asp Asp Thr Arg His His Val

245 250 255

Val Leu Val Asp Gly Trp Thr Ala Ala Asp Glu Phe Ile Glu Leu Thr

260 265 270

Arg Lys Cys Val Glu Trp Phe Leu Val Gly Thr Lys Val Lys Arg Arg

275 280 285

Leu Ala Phe Val Cys Ser Val Ala Ala Arg Gly Lys Phe Gln Glu Asp

290 295 300

Met Glu Ile Leu Thr Arg Ala Arg Glu Phe Arg Val Trp Ser Trp Thr

305 310 315 320

Leu Asp Glu Tyr Leu Asn Ala Ile Lys Asp Asp Glu Val Phe Gln Ser

325 330 335

Val Ser Gln Cys Leu Asp Ala Pro Ala Val Leu Pro Asp Glu Glu Met

340 345 350

Ser Arg Ala Ala Met Val Glu Ser Lys Tyr Tyr Tyr Ala Gly Gly Ser

355 360 365

Cys Arg Tyr Met Phe His Phe Arg Thr Val Thr Val Ala Thr Lys Leu

370 375 380

Ser Asp Ala Val Asp Ser Leu Asn Asp Ala Ala Ile Val Ala Thr Ser

385 390 395 400

Gly Gln Arg Ser Ser Ser Ser Ile Asn Arg Leu Phe Gly Met Phe Gln

405 410 415

Arg Pro His Asp Val Gly Ala Val Ser Pro Val Ile Ser Gln Tyr Ala

420 425 430

Ala Thr Leu Ile Ala Ile Arg Cys Gly Pro Glu Ala Ile Lys Lys Phe

435 440 445

Ile Ser Thr His Arg Asp Asn Ser Asn Pro Ala Leu Asp Gly Trp Met

450 455 460

Leu Glu Met Val Phe Phe Ala Ser Leu Arg Asn Gly Gly Leu Ala Leu

465 470 475 480

Val Asp Ala Ala Gly Asn Thr Val Asp Thr Trp Gly Gln Ser Val Val

485 490 495

Val Ile Ala Asp Gly Ile Pro Ala Leu Ser Ser Ala His Pro Val Trp

500 505 510

Ile Lys Pro Glu Lys Trp Asn Gln Gly Gly Tyr Asp Ala Ile Met Val

515 520 525

Cys Lys Arg Thr Gln His Val Arg Leu Val Gln Val Thr Ser Ala His

530 535 540

Lys His Ser Phe Arg Ile Asp Tyr Phe Tyr Gly Trp Leu Lys Met Leu

545 550 555 560

Ser Glu Ser Pro Glu Ser Phe Glu Val Lys Thr Met Glu Ile Val Phe

565 570 575

Val Val Glu Gln Asp Lys Leu Ser Thr Phe Asp Phe Ser Thr Val Lys

580 585 590

Gly Ala Gly Leu Leu Arg Pro Phe Gly Trp Ser Lys Gly Lys Glu Thr

595 600 605

Asp Leu Val Arg Leu Val Gly Ile Arg Gly Leu Tyr Asn Leu Ser Pro

610 615 620