專利名稱：絲狀病原真菌中一類控制侵染釘形成效率和致病力的新基因ppf1及其用途的制作方法
技術領域：
本發(fā)明涉及微生物基因工程和植物保護領域中真菌致病性基因及其編碼蛋白與用途。本發(fā)明提供了一類僅在絲狀真菌中高度保守、但功能未知的新基因，該基因在梨孢菌中控制侵染釘?shù)男纬尚?、其缺失導致該菌的致病力顯著下降。本發(fā)明所提供的基因及其編碼蛋白質(zhì)的表達以及參與的信號途徑可以作為靶位點用于抗真菌藥劑的篩選和設計中。
背景技術：
梨孢菌(Magnaporthe grisea)是子囊菌亞門的真菌，能侵染水稻、小麥、大麥、粟以及其它多種禾本科植物，導致瘟病。尤其是該菌侵染水稻引起的稻瘟病在世界各個栽培稻區(qū)每年均有發(fā)生，危害廣泛、嚴重。一般情況下，稻瘟病的危害可使水稻減產(chǎn)5-10％，重病田可導致水稻絕收。稻瘟病在我國曾多次流行，也是我國水稻的主要病害之一。1993年，稻瘟病在我國發(fā)生大流行，在全面防治的情況下仍然減產(chǎn)五十億公斤。梨孢菌還可侵染鈍葉草、狗牙草、假儉草等屬的草坪草，造成大面積枯死，是草坪草常見病害。因此，急需研發(fā)新的途徑和方法控制梨孢菌引起的植物病害。
梨孢菌還是研究植物病原真菌致病性和致病型變異的模式系統(tǒng)之一。這首先是因為該菌具有許多重要作物的病原真菌所共有的侵染過程，包括分生孢子產(chǎn)生與萌發(fā)、附著胞形成、侵入、侵染菌絲的擴展。其次，梨孢菌可以進行遺傳雜交和轉(zhuǎn)化，尤其是最近完成的梨孢菌全基因組序列草圖測定為科學家研究有關基因提供了極大的方便，是一個極好的分子遺傳和功能基因組研究材料。
鑒于梨孢菌在上述理論和應用上的重要性，許多發(fā)達國家的研究者已經(jīng)全面、深入地開展了該菌致病性和致病型變異的分子機理研究。已有研究表明在濕潤條件下，梨孢菌的分生孢子尖端釋放出孢子尖端粘液(STM)，該物質(zhì)能以非特異的形式將分生孢子附著在寄主臘質(zhì)表面。在適宜的溫度和水的存在下，分生孢子30-90分鐘即產(chǎn)生芽管，通常情況下只從分生孢子頂端或基部細胞產(chǎn)生單個芽管。隨后，芽管生長和分化成附著胞的形成，繼而產(chǎn)生侵染釘，侵染釘借助胞內(nèi)產(chǎn)生的高達8.0Mpa的機械膨壓穿透寄主的角質(zhì)層和細胞壁后，頂端膨大形成泡狀的初級侵染菌絲。一般情況下，梨孢菌在接種后9小時內(nèi)完成附著胞的形成與成熟，在12至15小時開始侵入植物細胞內(nèi)。隨后，初級侵染菌絲分化成次級侵染菌絲在細胞內(nèi)或細胞間蔓延，進一步分化為分生孢子梗，突破表皮，產(chǎn)生大量的分生孢子。分生孢子在風、雨的沖刷下釋放、附著，引起植物的再侵染。梨孢菌從分生孢子附著到分生孢子再產(chǎn)生的周期一般所需時間為3-5天；在植物的生長季節(jié)，如果條件適宜，能夠多次侵染，造成危害。
如上所述，在梨孢菌的侵染過程中，分生孢子、附著胞、侵染釘和侵染菌絲的形成在致病性中起著關鍵的作用。因而，阻斷其任何一個環(huán)節(jié)，即可破壞整個侵染循環(huán)，就可以達到控制病害的目的。為此，多年來，世界許多國家的科學家紛紛針對這些侵染器官的形成開展了較為廣泛的分子水平研究。但從目前的研究報道來看，已有的研究大都集中在侵染之前附著胞的發(fā)育階段，已克隆到的一些致病相關基因大都參與調(diào)控附著胞的形成，如PMK1、MPG1、MST7、MST11等，而對附著胞形成后侵染釘?shù)漠a(chǎn)生和侵染性菌絲的擴展研究相對較少。
2001年，Pierre等從梨孢菌中克隆了侵染釘產(chǎn)生所必需的基因PLS1(Proc.Natl.Acad.Sci.USA 2001，98(12)6963～6968)。該基因被破壞后產(chǎn)生的突變體puntchless在水稻葉片表面、洋蔥表皮及疏水性表面上能形成正常的附著胞，但是不能形成侵染釘，因而該突變體完全喪失了對水稻葉片的致病性。PLS1在侵染釘產(chǎn)生之前的附著胞里特異性表達，這也說明了它是一個控制侵染釘形成的基因。之后，Park等又克隆了侵入所必需的基因Mst12(Mol.Plant-Microbe Interact.2002，(15)183～192)。mst12-與puntchless類似，能形成正常的、黑色素化的附著胞，但不能分化成侵染釘，同時完全喪失了對水稻葉片的致病性。mst12-也不能侵染傷害處理后的水稻葉片，說明喪失侵染性生長的能力。進一步的研究表明，Mst12編碼的轉(zhuǎn)錄因子在PMK1調(diào)控下控制稻瘟菌的侵染性菌絲生長，可能由PMK1和CPKA共同調(diào)控侵染釘?shù)男纬?。此外，CHM1在梨孢菌侵染過程中發(fā)揮重要的作用(Li L.et al.Mol.Plant-Microbe Interact.2004，17547-556)。chm1-附著胞的形成率比野生型明顯的減少，而且少數(shù)形成的附著胞也不能產(chǎn)生侵染釘，完全喪失對水稻葉片的致病性。但是，chm1-與puntchless和mst12-不同，它能夠在傷害處理的水稻葉片上形成侵染菌絲，病斑與野生型一致。因此，表明CHM1只是在附著胞和侵染釘?shù)男纬呻A段發(fā)揮關鍵的作用，它并不控制侵染菌絲在寄主細胞內(nèi)的擴展。
盡管如此，侵染釘形成是一個由很多個基因控制的復雜過程，其機理遠未解明。本發(fā)明提供的MgPPF1(MagnaporthegriseaPenetrationPegFormation)是一類僅在絲狀真菌中高度保守、但功能未知的新基因。該基因可能編碼與幾丁質(zhì)合成有關的蛋白，在梨孢菌中控制侵染釘?shù)男纬尚?，其缺失導致該菌的致病力顯著下降。因此，該基因及其編碼蛋白質(zhì)的表達以及參與的信號途徑可以作為重要候選靶位點用于抗真菌藥劑的篩選和設計中。

發(fā)明內(nèi)容
本發(fā)明旨在提供一個控制梨孢菌侵染釘形成、并影響其對植物致病性的新基因及其編碼蛋白。本發(fā)明所提供的控制梨孢菌侵染釘形成的蛋白，名稱為MgPPF1，是具有下述氨基酸殘基序列之一的蛋白質(zhì)1)序列表中的SEQ ID №.3；2)將序列表中SEQ ID №.3的氨基酸殘基序列經(jīng)過一個或幾個氨基酸殘基的取代和/或缺失和/或添加且控制真菌侵染釘形成的的蛋白質(zhì)。
本發(fā)明還發(fā)現(xiàn)MgPPF1的同源蛋白僅在絲狀真菌中廣泛存在，且高度保守，屬絲狀真菌特異蛋白。尤其是病原真菌中存在的與MgPPF1在氨基酸序列一致性高于40％的同源蛋白可能在致病性其著相同的作用，也屬于本發(fā)明的保護范圍。這些同源蛋白的特征在于具有序列表中的SEQ ID №..氨基酸序列。
MGPPF1的編碼基因MgPPF1也屬于本發(fā)明的保護范圍，其具有下述核苷酸序列之一1)序列表中SEQ ID №.1的DNA序列；2)編碼序列表中SEQ ID №.3蛋白質(zhì)序列的多核苷酸；3)與序列表中SEQ ID №.1限定的DNA序列具有80％以上同源性，且編碼相同功能蛋白質(zhì)的DNA序列。
序列表中的序列1由4119個堿基組成，包括六個外顯子和五個內(nèi)含子，自序列1的5′端第1117位至1139位堿基為第一外顯子，自序列1的5′端第1140位至1207位堿基為第一內(nèi)含子，自序列1的5′端第1208位至1334位堿基為第二外顯子，自序列1的5′端第1335位至1411位堿基為第二內(nèi)含子，自序列1的5′端第1412位至1542位堿基為第三外顯子，自序列1的5′端第1543位至1621位堿基為第三內(nèi)含子，自序列1的5′端第1622位至1902位堿基為第四外顯子，自序列1的5′端第1903位至1972位堿基為第四內(nèi)含子，自序列1的5′端第1973位至2955位堿基為第五外顯子，自序列1的5′端第2956位至3029位堿基為第五內(nèi)含子，自序列1的5′端第3030位至3572位堿基為第六外顯子；其編碼區(qū)位于序列1的5′端第1208位至3572位堿基之間。自序列1的5′端第1位至1207位堿基為啟動子序列。
本發(fā)明還提供了MgPPF1基因的cDNA，其具有下述核苷酸序列之一1)序列表中SEQ ID №.2的DNA序列；2)編碼序列表中SEQ ID №.3蛋白質(zhì)序列的多核苷酸；3)與序列表中SEQ ID №.2限定的DNA序列具有80％以上同源性，且編碼相同功能蛋白質(zhì)的DNA序列。
序列表中的序列2由2207個堿基組成，其編碼框的序列為自SEQ ID №.2的5′端第70位至2157位堿基。
利用MgPPF1基因或其部分序列構建的表達載體，細胞系和宿主菌均屬于本發(fā)明的保護范圍。
本發(fā)明證明MgPPF1基因的缺失、表達受阻或變異導致梨孢菌侵染釘?shù)男纬陕拭黠@下降，且對水稻的致病力顯著減弱。因此，本發(fā)明的重要用途可以是應用上述成果，設計和篩選能夠破壞MgPPF1基因表達、剪切及其編碼蛋白的表達的化合物；或設計和篩選能對MgPPF1蛋白的氨基酸序列進行修飾或阻斷其細胞定位并影響其功能的化合物；或者以上述所克隆基因MgPPF1及其編碼蛋白MgPPF1的表達為報告基因或蛋白，設計、篩選相關化合物，阻斷該基因及其蛋白參與的信號途徑；繼而從上述化合物中開發(fā)新型的抗真菌的藥物；或者利用與MgPPF1蛋白在氨基酸序列一致性高于40％的同源蛋白的表達、修飾和參與的信號途徑為靶標，設計和篩選對MgPPF1同源蛋白的功能有影響的化合物；繼而從上述化合物中開發(fā)新型的抗真菌的藥物，用于病害的控制。此外，本發(fā)明的用途還包括以該基因的DNA或cDNA的保守區(qū)段作為探針、或根據(jù)該保守區(qū)段的序列設計引物通過PCR在梨孢菌中或者在其它真菌中分離的、與該基因具有一定序列同源性的序列。


圖1野生型菌株P131與其突變體S1104對水稻品種麗江新團黑谷上的致病性比較。

上為野生型菌株P131接種的水稻葉片，下為突變體S1104接種的水稻葉片。接種濃度為每毫升1×104個孢子。
圖2，野生型菌株P131與其突變體S1104在洋蔥表皮上侵入釘形成率的比較。
圖3，MgPPF1互補載體示意圖與S1104的MgPPF1互補轉(zhuǎn)化體的基因組Southern雜交確認。
圖4，野生型菌株P131、突變體S1104與S1104的MgPPF1互補轉(zhuǎn)化體C3對水稻品種麗江新團黑谷的致病性比較。

接種濃度5×104個孢子/毫升。
圖5，野生型菌株P131、突變體S1104與S1104的MgPPF1互補轉(zhuǎn)化體C3在洋蔥表皮上不同時間段的侵入釘形成率的比較。
圖6，MgPPF1的同源置換敲除原理(A圖)、敲除載體示意圖(B圖)與敲除體的基因組Southern雜交確認(C圖)。
圖7，野生型菌株P131(上葉片)與MgPPF1敲除體K68(下葉片)對水稻致病力的比較。

接種濃度為每毫升1×104個孢子。
圖8，野生型菌株P131與MgPPF1敲除體K68在洋蔥表皮上不同時間段的侵入釘形成率的比較。
圖9，MGPPF1與其他真菌中同源蛋白的親緣關系比較。

MGPPF1、GZPPF1、GMPPF1、SSPPF1、BCPPF1、PNPPF1、UMPPF1、NUPPF1、ANPPF1、CIPPF1、CNPPF1、SCCHS6P和CCPPF1分別為梨孢菌(Magnaporthe grisea)、小麥赤霉病菌(Gibberella zeae)、玉米莖腐病菌(Gibberella moniliformis)、油菜菌核病菌(Sclerotiniasclerotiorum)、灰葡萄孢菌(Botrytis cinerea)、小麥穎枯病菌(Phaeosphaerianodorum)、玉米瘤黑粉菌(Ustilago maydis)、粗糙脈孢霉菌(Neurospora crassa)、構巢曲霉菌(Aspergillus nidulans)、粗球孢子菌(Coccidioides immitis)和新生隱球菌(Cryptococcus neoformans)、啤酒酵母(Saccharomyces cervisiae)和球毛殼菌(Chaetomium globosum)中的同源蛋白質(zhì)。
具體實施例方式
為了更好的理解本發(fā)明，以下通過實施例予以進一步地說明，但并非對本發(fā)明的限制。
下述實施例中的實驗方法，如無特別說明，均為常規(guī)方法。
下述實施例中的百分含量，如無特別說明，均為質(zhì)量百分含量。
實施例1、MgPPF1及其編碼基因的獲得。
1、突變體的篩選與鑒定1)梨孢菌菌株P131的REMI轉(zhuǎn)化體庫的構建通過堿裂解法大量提取pUCATPH(Lu，S.，Lyngholm，L.，Yang，G.，Bronson，C.，Yoder，O.C.1994.Tagged mutants at the Toxl locus of Cochliobolusheterostrophus by restriction enzyme-mediated integration.Proc.Natl.Acad.Sci.USA 911264-12653)的質(zhì)粒DNA，分別用限制性內(nèi)切酶HindIII、KpnI、SacI和SmaI酶切將其線性化，按REMI轉(zhuǎn)化的方法(Shi Z.，Christian D.，&Leung H.，Enhanced transformation in Magnaporthe grisea by restriction enzyme mediatedintegration of plasmid DNA.Phytopathology，1995，85329-333.)轉(zhuǎn)化野生型的梨孢菌菌株P131。每個轉(zhuǎn)化體系中加入2μg線性化的質(zhì)粒和相應的限制性內(nèi)切酶，其中KpnI、HindIII、SacI和SmaI分別采用30U、45U、30U和15U。具體的步驟見實施例二的第二部分梨孢菌的轉(zhuǎn)化。
2)致病性檢測將活化的梨孢菌的菌絲充分打斷，均勻地涂布到西紅柿汁燕麥片培養(yǎng)基(每升含150ml西紅柿汁、30-50克燕麥片煮沸30分鐘過濾取濾液、16克瓊脂)平板上，26℃-28℃培養(yǎng)，當肉眼可見新生菌絲長出培養(yǎng)基表面時，用棉簽輕輕將菌絲洗下，并用水沖洗干凈，蓋上單層紗布，于26℃-28℃光照培養(yǎng)48小時，在培養(yǎng)基表面即可見大量的梨孢菌孢子。用水將分生孢子洗下，并用單層擦鏡紙過濾，然后用0.25‰(體積比)的吐溫20將孢子濃度調(diào)整為每毫升1-5×104個孢子。均勻的噴霧接種于五葉一心期的親和性水稻麗江新團黑谷幼苗的葉片正面，72小時后開始觀察葉片的發(fā)病情況。
3)突變體的篩選通過對REMI轉(zhuǎn)化體進行致病性檢測，獲得了一個對水稻麗江新團黑谷致病性顯著減弱的突變體S1104。該突變體與野生型相比，在水稻上形成的病斑數(shù)明顯減少(圖1)。
4)突變體與野生型在洋蔥表皮上侵入過程的比較用0.25‰(體積比)的吐溫20將孢子濃度調(diào)整為每毫升4×105個孢子，均勻的噴霧接種于新鮮的洋蔥內(nèi)表皮的正面。分別在接種后2小時、6小時、12小時、24小時、36小時、48小時和72小時于顯微鏡下觀察分生孢子的萌發(fā)，附著胞、侵染釘及侵染性菌絲的形成等過程，突變體與野生型每時間點各觀察100個附著胞，同時計算分生孢子的萌發(fā)率和附著胞、侵染釘及侵染性菌絲的形成率。如圖3所示結(jié)果在洋蔥表皮上突變體S1104的侵染釘形成率僅僅達到了野生型菌株的45％，表明該突變體致病性減弱是由于侵染釘?shù)男纬陕式档投鸬摹?br> 2、突變體的表型變化與插入標記的共分離分析用遺傳雜交的方法對突變體中插入標記潮霉素抗性基因與突變表型的共分離進行分析。具體方法是將突變體S1104與一個不具有潮霉素抗性、并且對水稻品種麗江新團黑谷的致病性同野生型一致的、并且交配型與野生型P131相反的菌株S1528在番茄燕麥片培養(yǎng)基上進行對峙培養(yǎng)。首先將菌株置于26℃下培養(yǎng)，待相鄰的菌落邊緣即將接觸時，將它們轉(zhuǎn)移至20℃的光照條件下培養(yǎng)。經(jīng)過20天左右，在菌落的交界處形成黑色突起的子囊殼。挑取成熟的子囊殼至無菌水中，輕輕將其擠破，釋放出殼內(nèi)的子囊，然后將去除雜質(zhì)的子囊懸浮液均勻的鋪到水瓊脂平板上，24-36小時后，挑取子囊內(nèi)的子囊孢子萌發(fā)的單根菌絲至番茄燕麥片平板上培養(yǎng)。檢測所有子囊孢子后代的潮霉素抗性以及對親和性水稻麗江新團黑谷的致病性。如表1所示，所有不抗潮霉素的子囊孢子后代均表現(xiàn)為野生型，表明突變體S1104致病性減弱的突變表型與插入標記是共分離的，即突變表型是由于外源質(zhì)粒的插入而引起的；在抗潮霉素的子囊孢子后代中，有一半表現(xiàn)為突變的表型，一半表現(xiàn)為野生的表型，說明在突變體中，外源質(zhì)粒有兩個插入位點，其中在一個位點處破壞了目標基因(本發(fā)明中的MgPPF1)。
表1、S1528與突變體S1104有性雜交后代的潮霉素抗性及致病性分析

3、控制梨孢菌侵染釘形成的基因MgPPF1的克隆1)粒拯救及基因組TAC文庫的篩選由于突變體S1104有兩個位點的插入，所以從子囊孢子后代中找到一個只有MgPPF1被破壞，即單位點插入、表現(xiàn)為突變表型的后代S9，進行質(zhì)粒拯救。獲得了插入位點旁側(cè)的基因組DNA的序列，并且確定了突變體中質(zhì)粒的插入位點。具體方法如下選取插入質(zhì)粒pUCATPH(Lu，S.，Lyngholm，L.，Yang，G.，Bronson，C.，Yoder，O.C.1994.Tagged mutants at the Tox1 locus of Cochliobolus heterostrophusby restriction enzyme-mediated integration.Proc.Natl.Acad.Sci.USA911264-12653)中只含有一個酶切位點但不影響質(zhì)粒復制功能的酶HindIII完全酶切子囊后代S9的基因組DNA后，用25∶24∶1的酚、氯仿、異戊醇抽提后，加入1/10體積的醋酸鈉和兩倍體積的無水乙醇沉淀后，用T4DNA連接酶在16℃水浴中進行自連接，16小時后電激轉(zhuǎn)化感受態(tài)細胞JM109。提取轉(zhuǎn)化體的質(zhì)粒進行酶切鑒定，其中正確的拯救到的質(zhì)粒應該含有pUCATPH中pUC18部分的序列外，還含有部分插入位點側(cè)端的基因組的序列。并且對質(zhì)粒帶有的側(cè)端基因組序列進行測序，根據(jù)測序的結(jié)果在梨孢菌的全基因組數(shù)據(jù)庫中檢索，分析插入位點附近的基因的范圍和可能的外顯子。結(jié)果表明，在突變體中，外源質(zhì)粒插入在限制性內(nèi)切酶SacI的位點處，SEQ ID NO.1中的第854位。將獲得的側(cè)端序列與國際稻瘟菌基因組協(xié)會的官方網(wǎng)站(http://www.riceblast.org/)已公布的梨孢菌的基因組數(shù)據(jù)庫比較，插入位點在梨孢菌的第六號染色體的contig2.26上。以拯救得到的質(zhì)粒上所帶有的基因組DNA7.3Kb為探針，篩選基因組TAC文庫，該基因組文庫平均克隆片段大小為50kb，共覆蓋全基因組的16-18倍(魏士平等，植物病理學報，2003，3357-62)。獲得兩個陽性克隆，并且都包含完整的MgPPF1(SEQ ID NO.1所示的第1位到第4119位的核苷酸序列)。
質(zhì)粒拯救過程中涉及到的限制性內(nèi)切酶和T4DNA連接酶由寶生物工程(大連)有限公司生產(chǎn)，參照使用說明書使用。
2)RT-PCR獲得MgPPF1基因的cDNA將野生型菌株P131接種親和性水稻麗江新團黑谷，分別在接種后1小時、3小時、6小時、9小時、12小時、24小時、36小時、48小時、72小時、96小時剪取接種葉片，分別用異硫氰酸胍(GT)法抽取總RNA(包括水稻和梨孢菌)，消解其中的DNA，然后以poly(T)為引物用SuperscriptIITM的反轉(zhuǎn)錄酶進行反轉(zhuǎn)錄，得到總cDNA的第一條鏈，然后將所有的反轉(zhuǎn)錄產(chǎn)物混合到一起，獲得了梨孢菌P131侵染的水稻麗江新團黑谷的過程中所有表達的基因的cDNA。將所有的cDNA片段兩端分別加上限制性內(nèi)切酶EcoRI和XhoI的酶切位點，酶切后克隆到λ噬菌體中，構建了梨孢菌P131侵染親和性水稻麗江新團黑谷的感病cDNA文庫。從λ噬菌體中提取擴繁后的總cDNA，用于擴增MgPPF1的全長cDNA。
利用英國Exeter大學的建立的植物病原真菌的EST數(shù)據(jù)庫(http://cogeme.ex.ac.uk/)，分別從5’和3’的非翻譯區(qū)以及不同的外顯子中設計了三對引物5’non5’-TTTCGCGAAGCAACAAACC-3’和R35’-TAAACGTCCTTGGGCTCCG-3’；F25’-ACGAGCTATGGCTGGAGACG-3’和R25’-ATGTTGCACAAAGGGCGG-3’；F35’-ACTTTACGAGGACCTTCGGC-3’和3’non5’-AAGCTGACTGTGGACCAGT-3’擴增全長的cDNA。將擴增得到的片段分別連接到pMD18-T載體上測序，并且將這三個cDNA片段拼接后，得到MgPPF1的全長cDNA。結(jié)果表明MgPPF1的cDNA具有SEQ ID NO.2所示的第1位到第2207位的堿基對，其編碼序列為自SEQ ID NO.2的第70位到第2157位堿基，編碼具有序列表中SEQ ID NO.3所示的氨基酸殘基序列的蛋白質(zhì)MGPPF1。將MGPPF1與NCBI的蛋白質(zhì)數(shù)據(jù)庫進行比對，發(fā)現(xiàn)它與釀酒酵母的一個幾丁質(zhì)合成酶CHS6(登錄號為CAA89393.1)有28％的一致性。
將SEQ ID NO.1的基因組基因的核苷酸序列與SEQ ID NO.2的cDNA序列進行比較，結(jié)果表明MgPPF1的基因組基因中包含六個外顯子和五個內(nèi)含子，這五個內(nèi)含子的剪切都符合“GT-AG”原則，自序列1的5′端第1117位至1139位堿基為第一外顯子，自序列1的5′端第1140位至1207位堿基為第一內(nèi)含子，自序列1的5′端第1208位至1334位堿基為第二外顯子，自序列1的5′端第1335位至1411位堿基為第二內(nèi)含子，自序列1的5′端第1412位至1542位堿基為第三外顯子，自序列1的5′端第1543位至1621位堿基為第三內(nèi)含子，自序列1的5′端第1622位至1902位堿基為第四外顯子，自序列1的5′端第1903位至1972位堿基為第四內(nèi)含子，自序列1的5′端第1973位至2955位堿基為第五外顯子，自序列1的5′端第2956位至3029位堿基為第五內(nèi)含子，自序列1的5′端第3030位至3572位堿基為第六外顯子；其開放閱讀框從序列1的5′端第1208位至3572位堿基之間。
實施例2、MGPPF1及其編碼基因功能的確認該部分通過互補實驗和基因同源置換實驗來證明該基因的功能，包括互補載體和基因置換載體的構建，以及將上述兩種載體導入梨孢菌中，得到梨孢菌的重組轉(zhuǎn)化體。互補載體的構建是指將包含預測的MgPPF1全長的基因組基因(梨孢菌基因組數(shù)據(jù)庫中contig2.26的第32031位的核苷酸至第36150位的核苷酸)的DNA片斷與一個帶有新霉素抗性基因的載體相連，在這里新霉素抗性基因并不是對本發(fā)明的限制，其他能導致抗生素抗性的基因，即潮霉素抗性基因之外的導致真菌抗性的基因都可以達到同樣的效果?；パa載體所導入的梨孢菌受體菌株是突變體S1104，看導入一個完整MgPPF1后所得的轉(zhuǎn)化體是否能夠恢復野生的表型。基因置換載體的構建是指將包括MgPPF1全長基因的基因組序列(梨孢菌基因組數(shù)據(jù)庫中contig2.26的第32031位的核苷酸至第36150位的核苷酸)連入一個載體中，然后用潮霉素抗性基因替換掉MgPPF1的大部分編碼區(qū)序列。基因置換載體所導入的梨孢菌菌株是野生型梨孢菌P131，基因置換載體通過基因兩側(cè)的側(cè)翼序列與野生型菌株基因組的相應序列發(fā)生同源重組，從而將基因組中相應位點MgPPF1的基因組基因部分序列與潮霉素基因置換，看野生菌株缺失MgPPF1后是否具有突變表型。
1、互補載體和基因置換載體的構建1)互補載體的構建首先合成帶有限制性內(nèi)切酶XbaI識別序列的引物從克隆質(zhì)粒載體pS65T-C1(gi1019893)中擴增出新霉素磷酸轉(zhuǎn)移酶基因連入pBlueScriptKS(+)的限制性內(nèi)切酶切位點XbaI間，得到的質(zhì)粒載體命名為KN。然后從實施例1中的步驟三的1中獲得的陽性克隆中用限制性內(nèi)切酶SphI切下包含有預測的MgPPF1的基因組基因(梨孢菌基因組數(shù)據(jù)庫中contig2.26的第23322位的核苷酸至第36150位核苷酸)的DNA片斷，末端補平后與限制性內(nèi)切酶EcoRV酶切的KN連接，但是該片段還包括有MgPPF1之外另一個基因，因而再將上面載體用內(nèi)切酶BamHI切開后回收9.1Kb的片段自連接，得到只包含有預測的MgPPF1的基因組基因(梨孢菌基因組數(shù)據(jù)庫中contig2.26的第32031位的核苷酸至第36150位的核苷酸)和選擇標記新霉素磷酸轉(zhuǎn)移酶基因的互補載體pS1104-C(圖3)。
構建過程中涉及到的限制性內(nèi)切酶和連接酶，末端補平酶由寶生物工程(大連)有限公司生產(chǎn)，參照使用說明書使用。
2)基因置換載體的構建考慮在基因置換過程中不能破壞相鄰的其它基因，來構建基因置換的載體。因此，首先將克隆的MgPPF1基因序列在梨孢菌基因組數(shù)據(jù)庫中進行比隊和基因預測，發(fā)現(xiàn)contig2.26的第33442位核苷酸至35262位核苷酸包括了MgPPF1基因的大部分基因組序列，而沒有包含任何別的基因。從實施例1中的步驟三獲得的陽性克隆中用內(nèi)切酶SacI和XbaI切下一個片段(從contig第32886位核苷酸至第33441位核苷酸)作為同源置換的左臂，連接到pBlueScriptKS(+)上，命名為KS-L，再從質(zhì)粒pUCATPH上用XbaI切下潮霉素磷酸轉(zhuǎn)移酶基因與KS-L連接，命名為KSH-L；然后從陽性克隆中用一對引物Up5’-AGCGAATTCACCACACAGATGAGGC-3’和Down5’-AAGCTCGAGACTGGTGATGCTGA-TTCC-3’擴增出一個片段(從contig2.26的第33263位核苷酸至第36118位核苷酸)作為同源置換的右臂，用內(nèi)切酶EcoRI和XhoI連接到pGEM7zf(+)上，命名為7zf-R。最后用內(nèi)切酶SacI和KpnI從KSH-L上切下左臂和潮霉素磷酸轉(zhuǎn)移酶基因連接到7zf-R上，得到基因置換載體pS1104-K(圖6A、B)。
2、梨孢菌的轉(zhuǎn)化梨孢菌的轉(zhuǎn)化首選REMI的方法。REMI(Restriction Enzyme MediatedIntegration)為限制性內(nèi)切酶介導的整合，采用不破壞載體內(nèi)基因的限制性內(nèi)切酶將載體線性化，在限制性內(nèi)切酶的介導下轉(zhuǎn)化梨孢菌的原生質(zhì)體，被轉(zhuǎn)化的質(zhì)粒隨機的整合到基因組中相應的限制性內(nèi)切酶的切點處。但是在互補實驗和基因置換實驗中，為了能夠盡量減少基因組DNA其它位置的變化，使載體更為隨機的導入梨孢菌的基因組中，在轉(zhuǎn)化的過程中省略了限制性內(nèi)切酶的介導。雖然轉(zhuǎn)化效率會比添加限制性內(nèi)切酶的方法低，但不影響被轉(zhuǎn)化質(zhì)粒整合到基因組中。原生質(zhì)體的制備和轉(zhuǎn)化方法如下1)原生質(zhì)體的制備500毫升三角瓶裝入150毫升液體CM培養(yǎng)基(酵母提取物0.1％，酶水解干酪素0.05％，葡萄糖1％，硝酸鈣0.1％，磷酸二氫鉀0.02％，硫酸鎂0.025％，氯化鈉0.015％)，分別接入梨孢菌突變體和野生型菌株的1×106個分生孢子，在26-28℃、100轉(zhuǎn)/分條件下?lián)u培30-32小時，三層滅菌擦鏡紙過濾收集菌絲體，菌絲體用0.7M氯化鈉溶液洗滌后轉(zhuǎn)移至滅菌的50毫升離心管中，每1克菌絲加入1毫升的酶滲透液(含20毫克/毫升崩潰酶，用0.7M氯化鈉配制)，26-28℃、100轉(zhuǎn)/分條件下酶解3-4小時后，用0.7M氯化鈉洗滌菌絲體，經(jīng)三層滅菌擦鏡紙過濾，收集原生質(zhì)體，4,000轉(zhuǎn)/分離心15分鐘，先用25毫升STC(1.2M山梨醇，10mM Tris-Cl，pH7.5，50mM氯化鈣)洗滌原生質(zhì)體一次，然后分別用10毫升STC洗2次，離心沉淀后用STC將原生質(zhì)體濃度調(diào)至0.5-1×108個/毫升。
2)梨孢菌的轉(zhuǎn)化分別將梨孢菌突變體和野生型菌株的原生質(zhì)體分裝于滅菌的50毫升離心管中，每管150微升，分別加入等體積的線性化的載體(約2微克，梨孢菌突變體S1104的離心管中分別加用限制性內(nèi)切酶BamHI線性化的互補載體pS1104-C；梨孢菌野生型菌株的離心管中加入用限制性內(nèi)切酶SacI線性化的基因置換載體pS1104-K)和STC混合液，冰上放置20分鐘，然后逐滴緩慢加入2毫升/管PTC溶液(60％聚己二醇3350，10mM Tris-pH 7.5，50mM氯化鈣)，冰上靜止20分鐘，加入25毫升/管冰冷的STC，緩慢混勻，4,000轉(zhuǎn)/分、4℃離心15分鐘，去上清，然后每管加入3毫升的LR培養(yǎng)基(0.1％酵母提取物，0.1％酶水解干酪素，1M蔗糖)，室溫靜置培養(yǎng)12-13小時后，轉(zhuǎn)入培養(yǎng)皿，加入12毫升冷卻至50℃的SR(LR+1.6％瓊脂)，混勻，待其凝固吹干后，上面鋪一層12毫升的0.7％上層瓊脂(含400微克/毫升的G418，互補實驗；含300微克/毫升美國Roche公司生產(chǎn)的潮霉素，基因置換試驗)。28℃培養(yǎng)4-6天后，將長出的轉(zhuǎn)化體(互補轉(zhuǎn)化體為S1104-C3，基因置換轉(zhuǎn)化體為S1104-K68)轉(zhuǎn)至固體CM培養(yǎng)基(0.6％酵母提取物，0.3％酶水解干酪素，0.3％酸水解干酪素，1％的蔗糖，16％瓊脂)(含400微克/毫升的G418，互補實驗；含250微克/毫升的潮霉素，基因置換試驗)上進行二次篩選，然后將確認抗性的單個菌落轉(zhuǎn)入燕麥片番茄培養(yǎng)基上培養(yǎng)，并進行單孢分離后，檢測對水稻麗江新團黑谷的致病性。
互補實驗證明互補載體在突變體S1104的基因組DNA中異位整合后，能夠使突變體對水稻麗江新團黑谷的致病性恢復到與野生表型，如圖4所示。接種洋蔥表皮后，侵染釘?shù)男纬梢不謴偷搅艘吧硇?，突變體S1104和相應的互補轉(zhuǎn)化體S1104/C3以及野生型菌株在接種后12小時、24小時、36小時和48小時在洋蔥表皮上形成侵染釘?shù)谋嚷实谋容^如圖5所示。
基因置換實驗證明MgPPF1的部分基因組基因序列與潮霉素基因序列的置換導致轉(zhuǎn)化體S1104-K68與突變體S1104非常相似，對水稻品種麗江新團黑谷的致病性顯著減弱。野生型菌株P131、突變體S1104和基因置換轉(zhuǎn)化體S1104-K68對水稻品種麗江新團黑谷的致病性比較如圖7。在洋蔥表皮上接種后，基因置換轉(zhuǎn)化體S1104-K68等與突變體S1104一樣，其侵染釘形成率只有野生型菌株P131的45％。野生型P131、突變體S1104和基因置換轉(zhuǎn)化體S1104-K68接種洋蔥表皮后12小時、24小時、36小時和48小時侵染釘形成率的比較如圖8所示。
互補實驗能夠使突變體S1104恢復到野生表型，基因置換實驗能夠使野生型菌株P131變?yōu)橥蛔兊谋硇停浞肿C明了MgPPF1在梨孢菌控制侵染釘形成中其極其重要作用。MgPPF1的破壞導致梨孢菌對水稻品種麗江新團黑谷的致病性顯著減弱，說明該基因是病原真菌的一個重要致病力基因。
實施例3，其它真菌中MgPPF1同源蛋白的生物信息學分析。
對梨孢菌MGPPF1的氨基酸序列對NCBI蛋白質(zhì)和基因組數(shù)據(jù)庫進行檢索，發(fā)現(xiàn)該蛋白在植物其它病原真菌如小麥赤霉病菌(Gibberella zeae)、玉米莖腐病菌(Gibberellamoniliformis)、油菜菌核病菌(Sclerotinia sclerotiorum)、灰葡萄孢菌(Botrytiscinerea)、小麥穎枯病菌(Phaeosphaeria nodorum)、玉米瘤黑粉菌(Ustilago maydis)等中均存在一同源蛋白。本發(fā)明將這些真菌中的同源蛋白分別命名為GZPPF1、GMPPF1、SSPPF1、BCPPF1、PNPPF1和UMPPF1，它們與MGPPF1在氨基酸序列上的一致性分別為74％、74％、72％、68％、60％和37％，并分別具有如序列表中SEQ.ID.NO4、5、6、7、8和9所示的氨基酸序列。同時，本發(fā)明還發(fā)現(xiàn)MGPPF1與GenBank登錄的啤酒酵母(Saccharomyces cervisiae)中的一種幾丁質(zhì)合成相關蛋白CHS6P(本發(fā)明中為SCCHS6P)在氨基酸序列上的一致性為20％。此外，在人類、動物病原真菌和腐生真菌如粗糙脈孢霉菌(Neurospora crassa)、構巢曲霉菌(Aspergillus nidulans)、粗球孢子菌(Coccidioides immitis)新生隱球菌(Cryptococcus neoformans)和球毛殼菌(Chaetomium globosum)也存在與MGPPF1同源的蛋白質(zhì)，本發(fā)明將這些同源蛋白分別命名為NUPPF1、ANPPF1、CIPPF1、CNPPF1和CGPPF1，ANPPF1、CIPPF1、CNPPF1和CGPPF1序列分別如SEQ.ID.NO10、11、12和13所示。不同真菌中MGPPF1蛋白的同源關系如圖9所示。
序列表<110>中國農(nóng)業(yè)大學<120>絲狀病原真菌中一類控制侵染釘形成效率和致病力的新基因PPF1及其用途<160>13<210>1<211>4119<212>DNA<213>梨孢菌(Magnaporthe grisea)<400>1ggatccgatc gagtcgaaag tgaccatggt tcttgtatgc taaatagtag acttgaggca 60ggcttgtggc ggaagggtcg ccatcgggat ttgcaacgtc gagagctgca gagaatgcgg 120tttcttggga caagaagggc gtttgtgggc gctgtgcacg ctcatactcg gtgatcgctt 180cgggtagctt gaatattgcg gattcaaaaa gctggtcata cgccttggag gcatcgtcgt 240aggactttgg gccttgtgca tgtagcttca atgcattctg gtatagtttc aaggcttcgt 300cgacctgtat ttgtctagta tcttcgatat cctcatctaa gttttcctcc ggttcgacat 360tgatggcagc gaacgagggc tgcaagtgga gcgagggtta gtactgggct agccgtctgt 420ttccaatgcg gaggcaaatt gagggaagat tgttgttacc attgtggcga cttggcggca 480tcagcctagc cattgccctt taggaattct gctttccaat tgggattata tgattgtgtg 540tattcaagct caaaggtacc tctgatgcag tttgcaggtt gtgaataatt acaacaaatg 600aacaagtcgc gatcgacgcg ccgcacgcta ccaagggtac cacactacgt aaagtgcttg 660gcgaccgttt ctccaacctg aacttctttt actgtagata acactgtgct agagtggtcg 720tatacgagga tcaccagcca aactgctgcg gggactacct agacaggtac cgggccaagg 780atcaagcaac gtgggaactg ggaaggtacg cgcagattga tggtctgtgc acatgctaag 840ctggtctttg agctcgattg attgatggac cggcgggcgc ttccaacttg tgctaatctt 900tcaagcccgt gaattaaatc ccaccaacat cagtcgactg ccagcagctg cgggactgca 960tcgggcgcca atcgcagcta gacaccttgt ccttctccaa gcttgaaatc aaaacaacaa 1020ggcgctatcc gcccttccaa ccacgatttc gcgaagcaac aaaccctctc ccttttcgca 1080agctcgagtc ttgcgacttg ataactgaat ttcgtgatgg ttgcgccggc tgtacccgag 1140ttcgtatccg cccgtatttc cttgcagctc aaccacggaa ggatttctaa ccggcccata 1200acatcagaat caccgaggag atcctccacg aggccattga tgcccgcaca gatcagttga 1260tcgccttgcg cgagctgggc cctccagacc ttgtccacct cacaaaacaa cagacacgga 1320gccctggaaa gcaggtacgg aagaagggtt tcttgctctc tctctctctg gctctgtcga 1380aaatgtctga cactcgtcta caatactcta gattggcgtc taccaccatg tcacaggtgt 1440cgacgcgtca tcctcggcga gcttggcggc atatatcaac acactcacct acaaagacca 1500tggccaggct gcaacaacca agatagtcca gggagtgtac tggtacgggc gccgtcactc 1560tcgtcatgcc aacgactgtt tgaagaagag aaaggggctg actggtatat tcgttaaaca 1620gttgctacaa tgcgctatct cgcattgata tgcgagtaca cgtcactatc ccgggttcgg 1680tcgaaagcta tggtgttgat gagaggggag agaagcggaa ggctaccgac gagctatggc 1740tggagacgta cctctgcagc gttctgcgcg cctactcata cgcggacgac ggcagtggag 1800agaccatcag gaagataatg ggcgttcgca gattcaatcc cgtcaccagc acggaaactg 1860
agcatcgatt cctcagcgct gccgagcaac tattcttcag aggtgagaaa gaccagacct 1920cagggtgcgt tctggtgtgc aatatgcaat acgttactga cgcaaccttc aggttggcaa 1980cttgggtcgg actcgactgt ccaagttccc aacgttgtct ctaatcactt ggcaagtggt 2040ctcctgagat atttccagac gactggaagg ttcgtctcag gtatcaacct tttcgagaag 2100ttgaggtcac aaaatgttga ggttgcgtca ttgctctcca aggtgctttt cttgggcaac 2160gaggaggtga agggtgtgcg cgtgctgcat caagccttgc aggaaattcc tatggactat 2220gttatgcttg acgctcaggc agaattcttg ctgaagaagg ccaagaccgc cactacccct 2280gagcagaagg aggagaggtt acgaatggca ctggcttgtg ccgataggag tacgattgcg 2340gcaccgagcg agtttggcac gtgggcccgg ctggccgagg tatacgttgc gatggaggac 2400tgggaaaacg ccctgacaat cctcaactca tgcccaatgt tcacctacca ggacaaggac 2460gcacctctga tgccggagcc caaggacgtt tatttgccca ccttggtcga gacacgattg 2520gacgagatcg acagcgagcc agagggtagg tacaatgagc aggtcgctga gccacttctt 2580aacttgcggg cagccggtta caggggtact ttcaagcacg cctacaagat tctgaccgag 2640atgacttcaa agattggatg ggatcagttg ctcaagacca ggagtattgt gttcgtaatg 2700gaagatgaat accatatgga gaggacagaa gtgagggact cgactcagga acgtagcgcg 2760agcaccgacg gtcttcgggg atcttctccg aacaacacca ataccgctgc tgctcaggac 2820ggtgagtctg acacagactc tcacattgag acagggcccg cggaaaaggc cgactcggcg 2880gcgccgaacg ggcagggcct ggaaaagccg ctgaccactg ctgatccttc cgaggtgaaa 2940gaggatcagg agaatgtaag tctatgtcca tttttcgggt ctgttccttg gtcgtgcaac 3000ataaattcgt gtgctgacta ctagaaaagg gcatcgtacc gagcgatcat tccaaactga 3060acaacaagcg tctttgtgaa cgctggctag acagcttgtt catggtactt tacgaggacc 3120ttcggcaata tacaatttgg cgcacacaaa tggcgcaact caagctgcaa tctttgcagt 3180acaaaaagtc agccgaggaa tgggagatac tgggagcgct ggccgagcgc cttcaccaca 3240cagatgaggc tgtcgaggcg taccgggcct gtctgagcca acgatttagc cccaaggctc 3300tcacaggcat actacgagcc tatgaacgga agccaaacag cacccgcgac acagtggccg 3360cggtgattag gcttgtcacc tggcaatacc gttggtactc ggagttctct cccgagctac 3420tcaacactat ccggtcactc atcgaggatg agggcgccgt caaggtccgc agcattatcc 3480aggctacgag cctgccgcag aacgtgcttg acctaacgca ccactacgcc gccctttgtg 3540caacattccg cagcagcgga acggaaggct agccgtaggg ttatcttgtg tatttatgac 3600tttactggtc cacagtcagc tttctcagag gtgtgaaaaa aaaagaaaat gcgcatttac 3660gttttgtggg cggctttttc ccgcattaaa taaacatttc gggttctttt cacagggcta 3720gcggtcacgg gaggcttttt gacagatgaa ggcaatgatt tatactacag attcggcaga 3780actcggtgaa ctccttggtc ttgctctaga cggataacca atacatttta cgcaacgggc 3840tccattgtta tatgctctat cttatacaca ttattgacac ctgccatggc ttgcttcaaa 3900tagggccgaa cccattgtct gcaacatccg caaatttgaa ccagtgagcc caggcgaata 3960ctacgctcat cacaccgcct aggaccagaa gccctccttg gcctgtccgg ctccctcgcg 4020aatccctcga ccccgacaag caacgtctac cactcccatc tgcggcggaa tcagcatcac 4080cagtcgggtt cttcgacctt ggcatgtggc gctgcatgc4119<210>2<211>2207
<212>DNA<213>梨孢菌(Magnaporthe grisea)<400>2ttcgcgaagc aacaaaccct ctcccttttc gcaagctcga gtcttgcgac ttgataactg 60aatttcgtga tggttgcgcc ggctgtaccc gaaatcaccg aggagatcct ccacgaggcc 120attgatgccc gcacagatca gttgatcgcc ttgcgcgagc tgtgccctcc agaccttgtc 180cacctcacaa aacaacagac acggagccct ggaaagcaga ttggcgtcta ccaccatgtc 240acaggtgtcg acgcgtcatc ctcggcgagc ttggcggcat atatcaacac actcacctac 300aaagaccatg gccaggctgc aacaaccaag atagtccagg gagtgtactg ttgctacaat 360gcgctatctc gcattgatat gcgagtacac gtcactatcc cgggttcggt cgaaagctat 420ggtgttgatg agaggggaga gaagcggaag gctaccgacg agctatggct ggagacgtac 480ctctgcagcg ttctgcgcgc ctactcatac gcggacgacg gcagtggaga gaccatcagg 540aagataatgg gcgttcgcag attcaatccc gtcaccagca cggaaactga gcatcgattc 600ctcagcgctg ccgagcaact attcttcaga ggttggcaac ttgggtcgga ctcgactgtc 660caagttccca acgttgtctc taatcacttg gcaagtggtc tcctgagata tttccagacg 720actggaaggt tcgtctcagg tatcaacctt ttcgagaagt tgaggtcaca aaatgttgag 780gttgcgtcat tgctctccaa ggtgcttttc ttgggcaacg aggaggtgaa gggtgtgcgc 840gtgctgcatc aagccttgca ggaaattcct atggactatg ttatgcttga cgctcaggca 900gaattcttgc tgaagaaggc caagaccgcc actacccctg agcagaagga ggagaggtta 960cgaatggcac tggcttgtgc cgataggagt acgattgcgg caccgagcga gtttggcacg 1020tgggcccggc tggccgaggt atacgttgcg atggaggact gggaaaacgc cctgacaatc 1080ctcaactcat gcccaatgtt cacctaccag gacaaggacg cacctctgat gccggagccc 1140aaggacgttt atttgcccac cttggtcgag acacgattgg acgagatcga cagcgagcca 1200gagggtaggt acaatgagca ggtcgctgag ccacttctta acttgcgggc agccggttac 1260aggggtactt tcaagcacgc ctacaagatt ctgaccgaga tgacttcaaa gattggatgg 1320gatcagttgc tcaagaccag gagtattgtg ttcgtaatgg aagatgaata ccatatggag 1380aggacagaag tgagggactc gactcaggaa cgtagcgcga gcaccgacgg tcttcgggga 1440tcttctccga acaacaccaa taccgctgct gctcaggacg gtgagtctga cacagactct 1500cacattgaga cagggcccgc ggaaaaggcc gactcggcgg cgccgaacgg gcagggcctg 1560gaaaagccgc tgaccactgc tgatccttcc gaggtgaaag aggatcagga gaatggcatc 1620gtaccgagcg atcattccaa actgaacaac aagcgtcttt gtgaacgctg gctagacagc 1680ttgttcatgg tactttacga ggaccttcgg caatatacaa tttggcgcac acaaatggcg 1740caactcaagc tgcaatcttt gcagtacaaa aagtcagccg aggaatggga gatactggga 1800gcgctggccg agcgccttca ccacacagat gaggctgtcg aggcgtaccg ggcctgtctg 1860agccaacgat ttagccccaa ggctctcaca ggcatactac gagcctatga acggaagcca 1920aacagcaccc gcgacacagt ggccgcggtg attaggcttg tcacctggca ataccgttgg 1980tactcggagt tctctcccga gctactcaac actatccggt cactcatcga ggatgagggc 2040gccgtcaagg tccgcagcat tatccaggct acgagcctgc cgcagaacgt gcttgaccta 2100acgcaccact acgccgccct ttgtgcaaca ttccgcagca gcggaacgga aggctagccg 2160tagggttatc ttgtgtattt atgactttac tggtccacag tcagctt 2207
<210>3<211>695<212>RTP<213>梨孢菌(Magnaporthe grisea)<400>3Met Val Ala Pro Ala Val Pro Glu Ile Thr Glu Glu Ile Leu His Glu1 5 10 15Ala Ile Asp Ala Arg Thr Asp Gln Leu Ile Ala Leu Arg Glu Leu Cys20 25 30Pro Pro Asp Leu Val His Leu Thr Lys Gln Gln Thr Arg Ser Pro Gly35 40 45Lys Gln Ile Gly Val Tyr His His Val Thr Gly Val Asp Ala Ser Ser50 55 60Ser Ala Ser Leu Ala Ala Tyr Ile Asn Thr Leu Thr Tyr Lys Asp His65 70 75 80Gly Gln Ala Ala Thr Thr Lys Ile Val Gln Gly Val Tyr Cys Cys Tyr85 90 95Asn Ala Leu Ser Arg Ile Asp Met Arg Val His Val Thr Ile Pro Gly100105 110Ser Val Glu Ser Tyr Gly Val Asp Glu Arg Gly Glu Lys Arg Lys Ala115 120 125Thr Asp Glu Leu Trp Leu Glu Thr Tyr Leu Cys Ser Val Leu Arg Ala130 135 140Tyr Ser Tyr Ala Asp Asp Gly Ser Gly Glu Thr Ile Arg Lys Ile Met145 150 155 160Gly Val Arg Arg Phe Asn Pro Val Thr Ser Thr Glu Thr Glu His Arg165 170 175Phe Leu Ser Ala Ala Glu Gln Leu Phe Phe Arg Gly Trp Gln Leu Gly180 185 190Ser Asp Ser Thr Val Gln Val Pro Asn Val Val Ser Asn His Leu Ala195 200 205Ser Gly Leu Leu Arg Tyr Phe Gln Thr Thr Gly Arg Phe Val Ser Gly210 215 220Ile Asn Leu Phe Glu Lys Leu Arg Ser Gln Asn Val Glu Val Ala Ser225 230 235 240Leu Leu Ser Lys Val Leu Phe Leu Gly Asn Glu Glu Val Lys Gly Val
245 250 255Arg Val Leu His Gln Ala Leu Gln Glu Ile Pro Met Asp Tyr Val Met260 265 270Leu Asp Ala Gln Ala Glu Phe Leu Leu Lys Lys Ala Lys Thr Ala Thr275 280 285Thr Pro Glu Gln Lys Glu Glu Arg Leu Arg Met Ala Leu Ala Cys Ala290 295 300Asp Arg Ser Thr Ile Ala Ala Pro Ser Glu Phe Gly Thr Trp Ala Arg305 310 315 320Leu Ala Glu Val Tyr Val Ala Met Glu Asp Trp Glu Asn Ala Leu Thr325 330 335Ile Leu Asn Ser Cys Pro Met Phe Thr Tyr Gln Asp Lys Asp Ala Pro340 345 350Leu Met Pro Glu Pro Lys Asp Val Tyr Leu Pro Thr Leu Val Glu Thr355 360 365Arg Leu Asp Glu Ile Asp Ser Glu Pro Glu Gly Arg Tyr Asn Glu Gln370 375 380Val Ala Glu Pro Leu Leu Asn Leu Arg Ala Ala Gly Tyr Arg Gly Thr385 390 395 400Phe Lys His Ala Tyr Lys Ile Leu Thr Glu Met Thr Ser Lys Ile Gly405 410 415Trp Asp Gln Leu Leu Lys Thr Arg Ser Ile Val Phe Val Met Glu Asp420 425 430Glu Tyr His Met Glu Arg Thr Glu Val Arg Asp Ser Thr Gln Glu Arg435 440 445Ser Ala Ser Thr Asp Gly Leu Arg Gly Ser Ser Pro Asn Asn Thr Asn450 455 460Thr Ala Ala Ala Gln Asp Gly Glu Ser Asp Thr Asp Ser His Ile Glu465 470 475 480Thr Gly Pro Ala Glu Lys Ala Asp Ser Ala Ala Pro Asn Gly Gln Gly485 490 495Leu Glu Lys Pro Leu Thr Thr Ala Asp Pro Ser Glu Val Lys Glu Asp500 505 510Gln Glu Asn Gly Ile Val Pro Ser Asp His Ser Lys Leu Asn Asn Lys515 520 525
Arg Leu Cys Glu Arg Trp Leu Asp Ser Leu Phe Met Val Leu Tyr Glu530 535 540Asp Leu Arg Gln Tyr Thr Ile Trp Arg Thr Gln Met Ala Gln Leu Lys545 550 555 560Leu Gln Ser Leu Gln Tyr Lys Lys Ser Ala Glu Glu Trp Glu Ile Leu565 570 575Gly Ala Leu Ala Glu Arg Leu His His Thr Asp Glu Ala Val Glu Ala580 585 590Tyr Arg Ala Cys Leu Ser Gln Arg Phe Ser Pro Lys Ala Leu Thr Gly595 600 605Ile Leu Arg Ala Tyr Glu Arg Lys Pro Asn Ser Thr Arg Asp Thr Val610 615 620Ala Ala Val Ile Arg Leu Val Thr Trp Gln Tyr Arg Trp Tyr Ser Glu625 630 635 640Phe Ser Pro Glu Leu Leu Asn Thr Ile Arg Ser Leu Ile Glu Asp Glu645 650 655Gly Ala Val Lys Val Arg Ser Ile Ile Gln Ala Thr Ser Leu Pro Gln660 665 670Asn Val Leu Asp Leu Thr His His Tyr Ala Ala Leu Cys Ala Thr Phe675 680 685Arg Ser Ser Gly Thr Glu Gly690 695<210>4<211>664<212>RTP<213>小麥赤霉病菌(Gibberella zeae)<400>4Met Val Ala Pro Ala Val Pro Glu Ile Thr Glu Glu Ile Leu His Glu1 5 10 15Ser Ile Asp Ala Arg Thr Glu Ser Leu Ala Gly Leu Arg Glu Leu Gly20 25 30Pro Pro Asp Leu Val His Leu Leu Lys His Gly Val Arg Asn Pro Ala35 40 45Lys Gln Thr Gly Val Tyr His His Val Thr Gly Val Asp Ala Ser Ser50 55 60
Ser Ala Ser Leu Ala Ala Tyr Ile Asn Thr Leu Thr Tyr Lys Glu Ser65 70 75 80Gly Pro Asn Ala Thr Asn Lys Ile Val Glu Gly Val Phe Cys Cys Tyr85 90 95Asn Ala Phe Ser Arg Leu Asp Met Arg Val His Val Ser Ile Pro Gly100 105 110Thr Val Glu Ser Tyr Cys Val Asp Glu Arg Gly Glu Lys Arg Lys Ala115 120 125Ser Glu Asp Leu Trp Leu Glu Thr Tyr Leu Cys Ser Val Leu Arg Ala130 135 140Tyr Ser Tyr Ala Asp Asp Gly Ser Gly Asp Thr Ile Arg Lys Ile Met145 150 155 160Gly Val Arg Arg Phe Asn Pro Val Thr Asn Thr Glu Thr Glu His Arg165 170 175Phe Leu His Ala Ala Glu Gln Leu Phe Phe Arg Gly Trp Gln Leu Gly180 185 190Ser Asp Ser Val Val Gln Val Pro Thr Asn Val Ser Asn His Leu Thr195 200 205Thr Gly Leu Leu Lys Tyr Leu Glu Thr Thr Gly Arg Tyr Ala Ser Gly210 215 220Ile Asn Leu Phe Glu Lys Leu Arg Thr Gln Ser Val Glu Val Ser Ser225 230 235 240Leu Leu Ala Lys Val Met Phe Met Gly His Glu Glu Val Ala Gly Val245 250 255Arg Thr Leu His Gln Ser Leu Lys Glu Asn Pro Met Asp Tyr Val Met260 265 270Leu Asp Thr Gln Ala Glu Phe Leu Leu Gly Lys Ala Lys Asn Ser Ala275 280 285Thr Pro Glu Leu Lys Glu Glu Arg Leu Lys Leu Ser Leu Gly Cys Ala290 295 300Asp Arg Ala Thr Val Ala Ala Pro Thr Glu Phe Ser Thr Trp Ala Arg305 310 315 320Leu Ala Gln Val Tyr Val Ala Met Glu Asp Trp Asp Asn Ala Leu Thr325 330 335Ile Leu Asn Ser Cys Pro Met Phe Thr Tyr Gln Asp Lys Asp Thr Pro
340 345 350Leu Met Pro Glu Pro Lys Glu Val Ile Leu Pro Thr Leu Pro Glu Thr355 360 365Arg Leu Asp Glu Ile Asp Ser Glu Pro Glu Ser Arg Tyr Ser Glu Gln370 375 380Val Asp Pro Ser Leu Leu Asn Leu Arg Ala Ala Ser Tyr Arg Gly Thr385 390 395 400Phe Lys Lys Ala Tyr Glu Ile Leu Thr Glu Met Thr Ala Lys Ile Gly405 410 415Trp Asp Gln Leu Leu Lys Ile Arg Ser Asn Val Phe Val Met Glu Asp420 425 430Glu Tyr Arg Thr Glu Lys Gln Glu Ser Thr His Pro Gly Ala Ser Lys435 440 445Arg Thr Pro Ser Thr Asp Gly Leu Arg Gly Thr Pro Asp Gln Thr Val450 455 460Asn Arg Asp Asn Glu Ala Glu Gly Glu Asn Ala Glu Lys Pro Ala Glu465 470 475 480Glu Arg Gln Arg Gln Ile Leu Ser Asp Glu Asn Leu Ser Lys Leu Asn485 490 495Thr Lys Arg Leu Cys Glu Arg Trp Leu Asp Ser Leu Phe Met Val Leu500 505 510Tyr Glu Asp Leu Arg Val Tyr Thr Ile Trp Arg Thr Gln Met Ala Gln515 520 525Tyr Arg Ala Gln Gln Met Gln Tyr Lys Lys Ser Ala Glu Glu Trp Glu530 535 540Ile Leu Gly Ser Leu Ala Glu Arg Leu Gln His Met Asp Glu Ala Val545 550 555 560Glu Ala Tyr Arg Ala Cys Leu SerIle Arg Phe Ser Pro Lys Ala Leu565 570 575Ala Gly Ile Leu Arg Val Phe Glu Lys Thr Lys Ser Thr Arg Glu Thr580 585 590Val Ala Ser Val Ile Arg Leu Val Thr Trp Gln Tyr Arg Trp Tyr Ser595 600 605Glu Phe Ser Pro Glu Leu Leu His Thr Ile Arg Thr Leu Ile Glu Asp610 615 620
Glu Gly Ala Val Lys Val Arg Ser Ile Ile Gln Ala Thr Ser Leu Pro625 630 635 640Gln Asn Val Leu Asp Leu Thr His His Tyr Ala Ala Leu Cys Ala Thr645 650 655Phe Arg Ser Ser Gly Thr Asp Gly660<210>5<211>692<212>RTP<213>玉米莖腐病菌(Gibberella moniliformis)<400>5Met Pro Cys Glu Thr Gln Leu Asn Arg Tyr Ala Ser Ser Ser Ala Gln1 5 10 15Gly Arg Pro Gly Thr Asp Phe Leu Lys Thr Gly Val Tyr His His Val20 25 30Thr Gly Val Asp Ala Ser Ser Ser Ala Ser Leu Ala Ala Tyr Ile Asn35 40 45Thr Leu Thr Tyr Lys Glu Ser Gly Pro Asn Ala Thr Asn Lys Ile Val50 55 60Glu Gly Val Tyr Cys Cys Tyr Asn Ala Phe Ser Arg Leu Asp Met Arg65 70 75 80Val His Val Ser Ile Pro Gly Thr Val Glu Ser Tyr Cys Val Asp Glu85 90 95Arg Gly Glu Lys Arg Lys Ala Ser Glu Glu Leu Trp Leu Glu Thr Tyr100 105 110Leu Cys Ser Val Leu Arg Ala Tyr Ser Tyr Ala Asp Asp Gly Ser Gly115 120 125Asp Thr Ile Arg Lys Ile Met Gly Val Arg Arg Phe Asn Pro Val Thr130 135 140Asn Thr Glu Thr Glu His Arg Phe Leu His Ala Ala Glu Gln Leu Phe145 150 155 160Phe Arg Gly Trp Gln Leu Gly Ser Asp Ser Val Val Gln Val Pro Thr165 170 175Asn Val Ser Asn His Leu Thr Thr Gly Leu Leu Lys Tyr Leu Glu Thr180 185 190
Thr Gly Arg Tyr Ala Ser Gly Ile Asn Leu Phe Glu Lys Leu Arg Thr195 200 205Gln Ser Val Glu Val Ser Ser Leu Leu Ala Lys Val Met Phe Met Gly210 215 220His Glu Glu Val Ala Gly Val Arg Thr Leu His Gln Ser Leu Gln Glu225 230 235 240Thr Pro Met Asp Tyr Val Met Leu Asp Thr Gln Ala Glu Phe Leu Leu245 250 255His Lys Ala Lys Ser Ala Pro Thr Pro Glu Leu Arg Asp Glu Arg Leu260 265 270Lys Met Ser Leu Gly Cys Ala Asp Arg Ala Thr Val Ala Ala Pro Thr275 280 285Glu Phe Ser Thr Trp Ala Arg Leu Ala Gln Val Tyr Val Ala Met Glu290 295 300Asp Trp Asp Asn Ala Leu Thr Ile Leu Asn Ser Cys Pro Met Phe Thr305 310 315 320Tyr Gln Asp Lys Asp Thr Pro Leu Met Pro Glu Pro Lys Glu Val His325 330 335Leu Pro Thr Leu Pro Glu Thr Arg Leu Asp Glu Ile Asp Ser Glu Pro340 345 350Glu Ser Arg Tyr Ser Glu Gln Val Asp Pro Ser Leu Leu Asn Leu Arg355 360 365Ala Ala Ser Tyr Arg Gly Thr Phe Lys Lys Ala Tyr Asp Ile Leu Thr370 375 380Glu Met Thr Ala Lys Ile Gly Trp Asp Gln Leu Leu Lys Ile Arg Ser385 390 395 400Asn Val Phe Val Met Glu Asp Glu Tyr Arg Ser Glu Lys Gln Glu Ala405 410 415Thr Gln Pro Ala Ser Ser Ala Lys Arg Thr Pro Ser Thr Asp Gly Leu420 425 430Arg Gly Thr Pro Asp Gln Thr Val Asn Gln Asp Glu Glu Thr Glu Asp435 440 445Glu Glu Val Glu Lys Leu Asp Glu Ala Ala Ser Lys Ala Glu Glu Ala450 455 460Gly Glu Val Pro Ala Thr Asn Gly Thr Ser Glu Gln Asp Lys Ile Glu
465 470 475 480Lys Pro Ser Asn Ile Ile Asp Pro Gly Glu Ala Lys Ala Asp Ser Glu485 490 495Val Gly Phe Ile Ser Thr Pro Cys Lys Thr Glu Ala Asn Asn Pro Gln500 505 510Thr Thr Lys Asn Asp Glu His Leu Ser Lys Leu Asn Thr Lys Arg Leu515 520 525Cys Glu Arg Trp Leu Asp Ser Leu Phe Met Val Leu Tyr Glu Asp Leu530 535 540Arg Val Tyr Thr Ile Trp Arg Thr Gln Met Ala Gln Tyr Arg Ala Gln545 550 555 560Gln Met Gln Tyr Lys Lys Ser Ala Glu Glu Trp Glu Ile Leu Gly Ser565 570 575Leu Ala Glu Arg Leu Gln His Val Asp Glu Ala Val Glu Ala Tyr Arg580 585 590Ala Cys Leu Ser Ile Arg Phe Ser Pro Lys Ala Leu Ala Gly Ile Leu595 600 605Arg Val Phe Glu Lys Thr Lys Ser Thr Arg Glu Thr Val Ala Ser Val610 615 620Ile Arg Leu Val Thr Trp Gln Tyr Arg Trp Tyr Ser Glu Phe Ser Pro625 630 635 640Glu Leu Leu His Thr Ile Arg Thr Leu Ile Glu Asp Gln Gly Ala Val645 650 655Lys Val Arg Ser Ile Ile Gln Ala Thr Ser Leu Pro Gln Asn Val Leu660 665 670Asp Leu Thr His His Tyr Ala Ala Leu Cys Ala Thr Phe Arg Ser Ser675 680 685Gly Thr Asp Gly690<210>6<211>711<212>RTP<213>油菜菌核病菌(Sclerotinia sclerotiorum)<400>6Met Val Ala Pro Ala Val Pro Glu Leu Thr Glu Glu Leu Val His Glu
1 5 10 15Ser Ile Asp Ala Arg Ser Glu Val Leu Ala Ala Leu Arg Glu Leu Gly20 25 30Pro Pro Asp Leu Val Gln Leu Ile Lys Gln Ala Pro Arg Asn Pro Val35 40 45Lys Gln Ile Gly Val Tyr His His Val Thr Gly Val Asp Ala Ser Ser50 55 60Ser Ala Ser Leu Ala Ala Tyr Ile Asn Thr Leu Thr Tyr Lys Glu Asn65 70 75 80His Gln Ser Thr Thr Lys Ile Val Glu Gly Val Tyr Cys Cys Tyr Asn85 90 95Ala Phe Ser Arg Leu Asp Met Arg Val His Val Thr Ile Pro Gly Ser100 105 110Val Glu Ser Tyr Cys Val Asp Glu Arg Gly Asp Lys Arg Thr Thr Thr115 120 125Asp Glu Leu Trp Leu Glu Thr Tyr Leu Cys Ser Val Leu Arg Ala Tyr130 135 140Ser Tyr Ala Asp Asp Gly Ser Gly Asp Thr Ile Arg Lys Ile Met Gly145 150 155 160Val Arg Arg Phe Asn Pro Val Thr Thr Thr Glu Thr Glu His Arg Phe165 170 175Leu Gln Ala Ala Glu Gln Leu Phe Phe Arg Gly Ile Pro Ser Leu Val180 185 190Lys Gln Lys Thr Met Leu Thr Ala Ile Gly Trp Gln Leu Gly Ser Asp195 200 205Ser Ile Val Gln Val Pro Asn Asn Val Ser Asn His Leu Thr Thr Gly210 215 220Leu Leu Lys Tyr Phe Gln Thr Thr Gly Arg Tyr Ala Ser Gly Ile Asn225 230 235 240Leu Phe Glu Lys Leu Arg Thr Arg Asn Ile Glu Val Ser Ser Leu Leu245 250 255Ala Lys Val Leu Phe Met Gly Asn Glu Glu Val Gln Ala Val Arg Val260 265 270Leu His Glu Ser Val Lys Gln Ser Pro Met Asp Tyr Val Met Leu Asp275 280 285
Thr Gln Ala Glu Phe Leu Lys Lys Lys Ala Met Asp Ile Asn Lys Pro290 295 300Glu Phe Lys Thr Glu Arg Leu Gln Met Ala Leu Gly Cys Ala Asp Arg305 310 315 320Ser Thr Ile Ala Ala Pro Ser Glu Phe Gly Thr Trp Ala Arg Leu Ala325 330 335Glu Val Tyr Val Ala Met Glu Asp Trp Glu Asn Ala Leu Thr Thr Leu340 345 350Asn Ser Cys Pro Met Phe Thr Tyr Gln Asp Lys Asp Ala Pro Asn Met355 360 365Pro Glu Pro Lys Glu Ile His Leu Pro Thr Leu Pro Glu Thr Arg Leu370 375 380Asp Glu Ile Asp Ser Glu Pro Glu Ser Arg Leu Ser Glu Gln Val His385 390 395 400Pro Ser Leu Gln Asn Leu Arg Ala Ala Met Tyr Lys Gly Thr Phe Lys405 410 415His Ala Tyr Ser Ile Leu Thr Glu Met Thr Ala Lys Ile Gly Trp Asp420 425 430Gln Leu Leu Lys Ile Arg Ser Asn Val Phe Val Met Glu Glu Glu Tyr435 440 445Arg Ser Glu Lys Gln Pro Pro Val Gly Gln Gln Gly Glu Pro Thr Lys450 455 460Arg Ser Ala Ser Thr Asp Val Leu Arg Gly Ser Pro Gly Pro Arg Val465 470 475 480Asn Gly Asp Asp His Ser Asp Asp Glu Lys Ser Thr Asn Gly Asp Ser485 490 495Ala Ala Leu Lys Pro Asn Ala Glu Asp Gly Ser Gly Ala Asp Asp Ser500 505 510Val Glu Lys Pro Ser His Thr Val Ala Ala Glu Glu Val Lys Ser Gly515 520 525Asn Glu Asp Asn Glu Thr Thr Glu Glu Gln Leu Ser Arg Phe Asn Asn530 535 540Lys Arg Leu Cys Glu Arg Trp Leu Asp Ser Leu Phe Met Val Leu Tyr545 550 555 560Glu Asp Leu Arg Ile Tyr Thr Ile Trp Arg Thr Glu Met Ala Gln Tyr
565 570 575Arg Ala Gln Gln Met Gln Tyr Lys Lys Ser Ala Glu Glu Trp Glu Ile580 585 590Leu Gly Ser Leu Ala Asp Arg Leu His His Gln Pro Glu Ala Val Glu595 600 605Ala Tyr Arg Asn Cys Leu Gln Ile Arg Phe Ser Pro Lys Ala Leu Ala610 615 620Gly Ile Leu Ala Glu Leu Glu Lys Asn Lys Asn Thr Arg Asp Thr Val625 630 635 640Ala Ser Val Ile Arg Leu Val Thr Trp Gln Tyr Arg Trp Tyr Ser Glu645 650 655Phe Ser Pro Glu Leu Leu Tyr Thr Ile Arg Thr Leu Ile Glu Asp Glu660 665 670Gly Ala Val Lys Val Arg Ser Ile Ile Gln Ala Thr Ser Leu Pro Gln675 680 685Asn Val Leu Asp Leu Thr His His Tyr Ala Ala Leu Cys Ala Ala Phe690 695 700Arg Ser Ser Gly Thr Glu Gly705 710<210>7<211>699<212>RTP<213>灰葡萄孢菌(Botrytis cinerea)<400>7Met Val Ala Pro Ala Val Pro Glu Ile Thr Glu Glu Ile Val His Glu1 5 10 15Ser Ile Asp Ala Arg Ser Glu Val Leu Ala Ala Leu Arg Glu Leu Gly20 25 30Arg Pro Asp Leu Val Gln Leu Ile Lys His Ala Pro Arg Asn Pro Arg35 40 45Lys Gln Ile Gly Val Tyr His His Val Thr Asp Ala Ser Ser Ser Ala50 55 60Ser Leu Ala Ala Tyr Ile Asn Thr Leu Thr Tyr Lys Glu Thr His Gln65 70 75 80Ser Thr Pro Lys Ile Leu Arg Gly Tyr Tyr Cys Cys Phe Asn Ala Phe
85 90 95Pro Arg Leu Ile Trp Arg Val Lys Val Pro Ile Gln Gly Ser Val Ala100 105 110Ala Tyr Gly Thr Asp Asp Glu Ala Ile Lys Arg Arg Gln Thr Asp Lys115 120 125Trp Leu Glu Thr Tyr Trp Cys Ser Val Phe Gly Ala Tyr Ser Tyr Pro130 135 140Asp Asp Gly Ser Gly Asp Thr Phe Asp Lys Ile Met Gly Val Arg Arg145 150 155 160Phe Asn Pro Val Thr Thr Thr Glu Thr Glu His Arg Phe Leu Gln Ala165 170 175Ala Glu Gln Leu Phe Phe Arg Gly Ile Leu Ser Ser Cys Ser Gln Asn180 185 190Pro Gly Leu Thr Ser Val Gly Trp Gln Leu Gly Ser Asp Ser Ile Val195 200 205Gln Val Pro Asn Asn Val Ser Asn His Leu Thr Thr Gly Leu Leu Lys210 215 220Tyr Phe Gln Thr Thr Gly Arg Tyr Ala Ser Gly Ile Asn Leu Phe Glu225 230 235 240Lys Leu Arg Thr Arg Asn Ile Glu Val Ser Ser Leu Leu Ala Lys Ile245 250 255Leu Phe Met Gly Asn Glu Glu Val Gln Ala Val Arg Val Leu His Glu260 265 270Ser Val Lys Gln Ser Pro Met Asp Tyr Val Met Leu Asp Thr Gln Ala275 280 285Glu Phe Leu Lys Lys Lys Ala Met Asp Pro Asn Lys Pro Glu Phe Lys290 295 300Thr Glu Arg Leu Gln Met Ala Leu Gly Cys Ala Asp Arg Ser Thr Ile305 310 315 320Ala Ala Pro Ser Glu Phe Gly Thr Trp Ala Arg Leu Ala Glu Val Tyr325 330 335Val Ala Met Glu Asp Trp Glu Asn Ala Leu Thr Thr Leu Asn Ser Cys340 345 350Pro Met Phe Thr Tyr Gln Asp Lys Asp Ala Pro Asn Met Pro Glu Pro355 360 365
Lys Glu Ile His Leu Pro Thr Leu Pro Glu Thr Arg Leu Asp Glu Ile370 375 380Asp Ser Glu Pro Glu Ser Arg Leu Ser Glu Gln Val His His Ser Leu385 390 395 400Leu Asn Val Arg Ala Ala Met Tyr Lys Gly Thr Phe Lys His Ala Tyr405 410 415Ser Ile Leu Thr Glu Met Thr Ala Lys Ile Gly Trp Asp Gln Leu Leu420 425 430Lys Ile Arg Ser Asn Val Phe Val Met Glu Glu Glu Tyr Arg Ser Glu435 440 445Lys Gln Pro Pro Val Gly Gln Gln Gly Glu Ser Thr Lys Arg Ser Ala450 455 460Ser Thr Asp Val Leu Arg Gly Ser Pro Gly Pro Arg Ala Asn Gly Asp465 470 475 480Asp His Ser Asp Asp Gly Lys Ser Thr Asn Gly Asp Ser Ala Gly Pro485 490 495Lys Pro Asn Ala Glu Asp Gly Ser Gly Gly Asp Asp Ser Val Glu Lys500 505 510Pro Ser His Thr Val Ala Ala Glu Glu Val Lys Ser Gly Asn Glu Gln515 520 525Ser Glu Thr Thr Glu Glu Gln Leu Ser Arg Phe Asn Asn Lys Arg Leu530 535 540Cys Glu Arg Trp Leu Asp Ser Leu Phe Met Val Leu Tyr Glu Asp Leu545 550 555 560Arg Ile Tyr Thr Ile Trp Arg Thr Glu Met Ala Gln Tyr Arg Ala Gln565 570 575Gln Met Gln Tyr Lys Lys Ser Ala Glu Glu Trp Glu Ile Leu Gly Ser580 585 590Leu Ala Asp Arg Leu His His Gln Pro Glu Ala Val Glu Ala Tyr Arg595 600 605Asn Cys Leu Gln Ile Arg Phe Ser Pro Lys Ala Leu Ala Gly Ile Leu610 615 620Ala Glu Leu Glu Lys Asn Lys Asn Thr Arg Asp Thr Val Ala Ser Val625 630 635 640Ile Arg Leu Ser Glu Phe Ser Pro Glu Leu Leu Tyr Ile Arg Thr Leu
645 650 655Ile Glu Asp Glu Gly Ala Val Lys Val Arg Ser Ile Ile Gln Ala Thr660 665 670Ser Leu Pro Gln Asn Val Leu Asp Leu Thr His His Tyr Ala Ala Leu675 680 685Cys Ala Ala Phe Arg Ser Ser Gly Thr Glu Gly690 695<210>8<211>681<212>RTP<213>小麥穎枯病菌(Phaeosphaeria nodorum)<400>8Met Ser Pro His Ser Asp Arg Ala Thr Glu Ser Met Arg Met Asn Tyr1 5 10 15Thr Pro Gln Ser Lys Leu Ala Gln Ser Leu Tyr Lys Arg Ser Ala Asn20 25 30Leu Ala Arg Gln Thr Ser Ser His Leu Ile Lys Pro Ala Pro Val Lys35 40 45Val His Glu Arg Lys Pro Glu Ser Ser Val Tyr Pro Pro Ser Arg Gln50 55 60Thr Thr Gln Glu Leu Cys Arg Glu Phe Thr Val Ala Tyr Asn Ala Leu65 70 75 80Phe Ala Ser Trp Thr Cys Val Phe Lys Ser Glu Ile Pro Gly Thr Val85 90 95Glu Ser Tyr Cys Ile Asp Glu Arg Gly Asp Lys Arg Val Ala Thr Glu100 105 110Glu Leu Trp Leu Glu Thr Tyr Leu Cys Ser Val Leu Arg Ala Tyr Ser115 120 125Tyr Ala Asp Asp Gly Ser Gly Asp Thr Ile Lys Lys Ile Val Gly Val130 135 140Arg Arg Phe Asn Pro Ile Thr Ser Thr Glu Ser Glu His Lys Phe Leu145 150 155 160Asp Ala Ala Glu Arg Leu Phe Phe Ser Gly Trp Gln Leu Gly Ser Asp165 170 175Pro Glu Ile Gln Val Pro Asn Leu Val Ser Asn His Leu Thr Thr Gly
180 185 190Leu Leu Ser Tyr Ile Gln Thr Thr Gly Arg Tyr Ala Ser Gly Ile Asn195 200 205Leu Phe Glu Lys Leu Arg Thr Arg Asp Pro Glu Ile Ser Ser Leu Leu210 215 220Ala Arg Val Tyr Ile Glu Ala Asp Glu Glu Val Lys Ala Val Gln Leu225 230 235 240Leu His Glu Ala Ile Gln Asp Leu Pro Met Asp Tyr Ser Leu Leu Asp245 250 255Cys Gln Ala Glu Phe Cys Asn Lys Lys Asn Arg Gly Asp Leu Ala Leu260 265 270Glu Ile Ala Lys Arg Ser Val Val Ser Ala Pro Ser Glu Phe Gly Thr275 280 285Trp Ala Arg Leu Ala Glu Ile Tyr Val Ser Leu Glu Gln Trp Asp Leu290 295 300Ala Leu Leu Thr Leu Asn Ser Cys Pro Met Phe Thr Tyr Gln Asp Lys305 310 315 320Asp Ala Pro Arg Met Pro Glu Pro Gln Lys Val Phe Leu Pro Ile Met325 330 335Pro Glu Ala Met Cys Asp Glu Ile Glu Asp Ser Asn Leu Asp Asp Met340 345 350Glu Leu Val His Pro Thr Leu Arg Lys Leu His Ala Ala Gly Phe Lys355 360 365Gly Thr Phe Leu Lys Ala Tyr Val Leu Leu Thr Glu Ile Thr Lys Lys370 375 380Ile Gly Trp Asp Gln Leu Leu Lys Ile Arg Ser Gln Val Phe Val Met385 390 395 400Glu Glu Glu Tyr Arg His Glu Lys Thr Gly Gln Gln Pro His Ser Arg405 410 415Ser Ala Ser Thr Thr Ala Leu His Ser Pro Ser Pro Gly Ala Gly Ala420 425 430Gln Pro Asn Gly Thr Ser His Gly Glu Ala Glu Glu Arg Pro Ser Thr435 440 445Glu Gln Pro Ser Thr Asp Gly Glu Ser Ser Thr Pro Thr Thr Val Ala450 455 460
Ala Ala Glu Glu Ser Ile Glu Lys Pro Ser His Thr Val Ala Ala Glu465 470 475 480Val Val Lys Ala Gly Ser Glu Asp Pro His Pro Leu Pro Ser Thr Ser485 490 495Asn Thr Thr Asp Pro Ser Gln Pro Asn Tyr Thr His Phe Gln Asn Lys500 505 510Arg Leu Cys Glu Arg Trp Leu Asp Asn Leu Phe Met Val Leu Tyr Glu515 520 525Asp Leu Arg Val Tyr Thr Ile Trp Arg Thr Glu Met Ala Gln Tyr Arg530 535 540Gln Gln Gln Leu Leu Tyr Lys Lys Ser Ala Glu Glu Trp Glu Ile Leu545 550 555 560Gly Glu Leu Ala Glu Arg Leu His His Glu Asp Asp Ala Leu Glu Ala565 570 575Tyr Glu Ser Cys Leu Gln Ile Arg Phe Ser Pro Lys Ala Met Lys Gly580 585 590Val Leu Lys Tyr Gly Phe Val Gly Glu Asp Arg Gly Lys Thr Arg Asp595 600 605Ala Cys Ala Ala Leu Ile Arg Leu Val Thr Trp Gln Tyr Arg Trp Tyr610 615 620Ser Glu Phe Ser Pro Ser Leu Leu Tyr Thr Ile Arg Lys Leu Ile Glu625 630 635 640Glu Glu Gly Ala Val Lys Ile Arg Ser Ile Ile Gln Gly Thr Ser Leu645 650 655Pro Gln His Val Leu Asp Leu Thr His Gln Tyr Ala Ala Leu Cys Ala660 665 670Ala Phe Arg Ser Ser Gly Ser Asp Gly675 680<210>9<211>813<212>RTP<213>玉米瘤黑粉菌(Ustilago maydis)<400>9Met Gly Asp Thr Phe Leu Ser Gln Pro Glu Phe Phe Glu Val Glu Leu1 5 10 15
Gly Glu Ser Leu Leu Ser Arg Thr Glu Thr Leu Ala Ser Phe Arg Glu20 25 30Leu Gly Pro Pro Asp Leu Val His Val Ile Lys Phe Thr Gly Thr Ser35 40 45Ala Arg Ser Arg Asp Ile Gly Ser Tyr His Tyr Val Ser Gly Val Asp50 55 60Ala Ser Ser Ser Ala Ala Leu Ala Ala Tyr Ile Asn Ser Leu Thr Tyr65 70 75 80Glu Leu Asp Gln Asn Pro Gly Phe Phe Ser Ser Lys Ala Ala Tyr Lys85 90 95Leu Lys Ser Gly Ala Tyr Cys Cys Phe Asn Ala Phe Ser Arg Val Asp100 105 110Val Arg Val Glu Val Arg Ile Pro Gly Ser Val Asp Ala Tyr Val Val115 120 125Asp Leu Arg Gly Glu Arg His Glu Thr Thr Pro Glu Ile Trp Gln Glu130 135 140Val Tyr Leu Ser Ala Leu Leu Arg Ser Ile Leu Tyr Ala Asp Asp Ala145 150 155 160Asn Tyr Arg Leu Ala Gly Tyr Arg Lys Leu Asp Pro Ile Ser Ser Pro165 170 175Asp Ala Glu His Arg Phe Leu Gln Ala Ala Glu Asn Leu Phe Phe Lys180 185 190Gly Trp Gln Leu Gly Ser Asp Pro Glu Val Gln Val Ala Thr Val Val195 200 205Ser Asn His Leu Thr Ala Gly Ile Leu Lys Tyr Phe Gly Glu Ala Ser210 215 220Arg Tyr Glu Gln Ala Val Asn Leu Phe Glu Lys Leu Trp Ala Arg Glu225 230 235 240Pro Glu Val Ala Ala Leu Val Ala Gln Ser Tyr Ile Gly Met Asn Glu245 250 255Glu Ile Lys Ala Val Gln Val Met His Ser Ala Ile Arg Glu Thr Pro260 265 270Gln Ser Tyr Ala Leu Leu His Ala Gln Val Asp Phe Leu Arg Ser Lys275 280 285Gly Lys Tyr Glu Trp Ala Leu Lys Val Ala Lys Gln Ala Val Asn Cys
290 295 300Ala Pro Ser Glu Phe Val Thr Trp Ala Lys Leu Thr Glu Cys Tyr Ile305 310 315 320Asp Leu Glu Gln Trp Glu Ser Ala Leu Tyr Thr Leu Asn Ser Cys Pro325 330 335Met Phe Thr Tyr Asn Glu Arg Asp Leu His Arg Met Pro Thr Pro Ala340 345 350Arg Ser His Leu Pro Ile Lys Thr Phe Ile Ala Glu Ser Gly Leu Val355 360 365Asp Glu Glu Ser Ala Arg Asp Asn Glu Ala Asp Val Ala Leu Leu Arg370 375 380Leu Pro Ala Pro Ala Leu Arg Gly Thr Phe Ala Lys Ala Tyr Ala Leu385 390 395 400Leu Gly Arg Leu Val Asn Ser Ile Gly Trp Asp Glu Leu Leu Arg Tyr405 410 415Arg Ser Asn Val Phe Val Met Glu Glu Glu Tyr Arg Ala Gln Lys Ala420 425 430Ala Ala Thr Gln Ala Glu Gln Ala Glu Glu Asp Ala Ser Ile Arg Gly435 440 445Val Ala Pro Ser Asp Ser Pro Ala Ile Ala Ser Ser Thr Pro Pro Gln450 455 460Ala Ser Asn Ala Ala Ala Gly Thr Glu Gly Val Glu Thr Glu Ala Asn465 470 475 480Asp Ser Ser Glu Gln Ser Asp Asp Glu Gln Asp Ser Ser Glu Ile Arg485 490 495Pro Ser Asn Gln Ala Leu Thr Ser Gln Leu Gly Ile Gln Ser Thr Ala500 505 510Asn Ala Ser Ser Gln Ser Ile Pro Met Ile Lys Ile Ser Thr Glu Ser515 520 525Asp His Glu Arg Glu Lys Gln Glu Leu Glu Asp Tyr Met Lys Thr Met530 535 540Asn Val Ser Ala Ser Asp Gln Pro Asp Asp Glu Asp Ala Ser Ala His545 550 555 560Ala Ser Ile Gln Val Glu Pro Pro Pro Leu Glu Lys Pro Ala Gln Ala565 570 575
Ser Ala Gly His Glu Arg Ser Glu Ser Thr Ser Thr Ala Val Ala Ala580 585 590Thr Met Ala Gly Asp Leu Ser Ala Ala Leu His Ala Asn Ala Asn Ala595 600 605Asn Gly Lys Ala Gly Pro Ile Val Asn Gly Glu His Asp Ala Ser Ile610 615 620Ala Met Ala Glu Ala Arg Gln Lys Glu Lys Asn Thr Gly Phe Ser Phe625 630 635 640Arg Asp Lys Arg Leu Cys Glu Arg Trp Leu Asp Asn Leu Phe Met Val645 650 655Leu Tyr Glu Asp Leu Arg Val Tyr Thr Ile Trp Arg Ala Glu Ile Ala660 665 670His Phe Arg Ser Gln Asn Leu Ala Tyr Arg Lys Thr Gly Thr Glu Trp675 680 685Glu Ile Leu Gly Glu Leu Ala Leu Arg Leu Leu His Lys Glu Glu Ala690 695 700Arg Asp Ala Phe Gln Arg Cys Val Asp Val Lys Phe Ser Ala Lys Ala705 710 715 720Tyr Leu Arg Leu Leu Glu Ile Tyr Thr Glu Ala Arg Asp Val Gln Arg725 730 735Ser Leu Trp Thr Ala Ile Arg Leu Thr Ala Tyr His His Arg Trp Tyr740 745 750Met Glu Gly Ser Phe Pro Gly Ala Val Ala Thr Ser Leu Phe Lys Leu755 760 765Ile Lys Thr Glu Gly Leu Ala Lys Val Ser Tyr Thr Leu Val Ser Met770 775 780Ser Pro Pro Pro Ala Ile Leu Arg Leu Met Gln Asn Tyr Phe Ala Tyr785 790 795 800Ala Thr Ala Phe Lys Val Pro Gly Tyr Asp Phe805 810<210>10<211>689<212>RTP<213>粗球孢子菌(Coccidioides immitis)<400>10
Met Ile Ala Pro Ala Val Pro Glu Leu Ile Glu Asp Glu Asp Ile His1 5 10 15Ala Ser Ile Glu Ala Arg Thr Glu Ser Leu Gln Ser Leu Arg Glu Leu20 25 30Gly Pro Pro Asp Leu Val His Val Val Lys Gln Ser Lys Ala Asn Ala35 40 45Asn His Gln Val Thr Gly Val Tyr His His Val Thr Gly Ile Asp Ala50 55 60Ser Ser Ser Ala Ser Leu Ala Ala Tyr Val Thr Thr Leu Thr Tyr Ser65 70 75 80Pro Leu Asp Lys His Asn Lys Val Ile Ser Gly Ile Tyr Trp Ser Cys85 90 95Tyr Asn Ala Phe Ser His Leu Asp Met Arg Val Glu Val Lys Ile Pro100 105 110Gly Thr Leu Glu Ser Tyr Cys Ile Asp Glu Arg Gly Asp Lys Arg Val115 120 125Pro Thr Glu Gln Leu Trp Leu Glu Thr Phe Val Cys Gly Val Leu Arg130 135 140Ala Tyr Ala Tyr Ala Asp Asp Gly Asn Gly Asp Ala Ile Lys Lys Ile145 150 155 160Val Ala Val Arg Arg Phe Asn Pro Val Thr Asn Thr Glu Met Glu His165 170 175Lys Phe Leu Asp Ala Ala Glu Lys Leu Phe Phe Asn Gly Gly Arg Gln180 185 190Leu Ser Ser Asp Pro Glu Thr Gln Val Pro Asn Thr Val Ser Asn His195 200 205Leu Ala Ser Gly Leu Leu Lys Tyr Ile His Thr Thr Gly Arg Tyr Ala210 215 220Ser Gly Ile Asn Leu Phe Glu Lys Leu Arg Thr Lys Asp Val Glu Val225 230 235 240Ser Ser Leu Leu Ala Arg Val Leu Ile Ser Ala Ser Glu Glu Val Gln245 250 255Ala Val Arg Leu Leu His Asp Ala Leu Gln Asp Val Pro Met Asp Tyr260 265 270Ala Leu Leu Asp Cys Gln Ala Val Phe Cys Gln Ser Lys Gly Glu Tyr
275 280 285Glu Met Ala Leu Glu Cys Ala Lys Arg Ser Val Thr Ala Ala Pro Ser290 295 300Glu Phe Ser Ser Trp Ile Arg Leu Ala Glu Val Tyr Val His Leu Glu305 310 315 320Gln Trp Asp Leu Ala Leu Leu Thr Leu Asn Ser Cys Pro Met Phe Thr325 330 335Tyr Gln Asp Lys Asp Ser Pro Arg Leu Pro Gln Pro Ser Arg Ile Leu340 345 350Leu Pro Ile Leu Pro Glu Met Leu Asp Glu Leu Asp Glu Gly Gln Pro355 360 365Lys Pro Gly Asp Pro Val Asp Met Val Asn Pro Ile Leu Lys Lys Leu370 375 380His Ala Ala Thr Tyr Gln Gly Thr Phe Leu Lys Ala Tyr Ser Leu Leu385 390 395 400Thr Lys Ile Ala Ala Ala Ile Gly Trp Asp Gln Leu Leu Arg Ile Arg405 410 415Ser Glu Val Phe Val Met Glu Glu Glu Tyr Arg Val Glu Arg Gln Ala420 425 430Ser Ser Ala Arg Pro Val Thr Ser Asn Asn Ala Ser Thr Val Ala Leu435 440 445His Glu Ala Ser Ser Asn Gly Asn Gly Val His Asp Ser Asp Asp Val450 455 460Glu Glu Asn Gly Asp Arg Asn Ser Thr His Gly Glu Thr Pro Lys Glu465 470 475 480Asn Gly Asp Leu Asp Gln Ser Glu Ser His Ser Ile Glu Ser Arg Ile485 490 495Glu Lys Pro Glu Gln Ser Met Ala Ser Glu Val Val Lys Ser Gly Glu500 505 510Ser Pro Asn Ala Tyr Thr His Phe Asn Asn Lys Arg Leu Cys Glu Arg515 520 525Trp Leu Asp Asn Leu Phe Met Val Leu Tyr Glu Asp Leu Arg Val Tyr530 535 540Thr Ile Trp Arg Ser Glu Thr Ala Gln Ser Arg Ala Gln Ser Ile Glu545 550 555 560
Tyr Lys Lys Ser Ala Thr Glu Trp Glu Ile Leu Gly Glu Leu Ala Glu565 570 575Arg Leu His His Phe Asn Glu Ala Ile Glu Ala Tyr Gln His Cys Leu580 585 590Gln Ile Arg Phe Ser Pro Lys Ala Met Lys Gly Ile Leu Lys Met Tyr595 600 605Glu Arg Gln Asn Asp Thr Arg Gly Met Leu Asn Ala Leu Ile Arg Leu610 615 620Ile Ala Trp Gln Tyr Arg Trp Tyr Ser Glu Phe Ser Pro Glu Leu Leu625 630 635 640Phe Thr Ile Arg Arg Leu Ile Glu Glu Glu Gly Ala Val Lys Val Arg645 650 655Ser Ile Val Gln Ala Thr Asn Leu Pro Gln Pro Val Leu Asp Leu Thr660 665 670His His Tyr Cys Gln Leu Cys Ala Thr Phe Arg Ser Thr Gly Ser Asp675 680 685Gly<210>11<211>576<212>RTP<213>構巢曲霉菌(Aspergillus nidulans)<400>11Met Arg Val Glu Val Lys Ile Pro Gly Ser Leu Glu Ser Tyr Cys Ile1 5 10 15Asp Glu Arg Gly Asp Lys Arg Val Ala Thr Asp Ala Leu Trp Leu Glu20 25 30Thr Phe Leu Cys Gly Val Leu Arg Ala Tyr Ser Tyr Ala Asp Asp Gly35 40 45Ser Gly Asp Ala Ile Arg Lys Ile Val Gly Val Arg Arg Phe Asn Pro50 55 60Val Thr Asn Thr Glu Met Glu His Arg Phe Leu Asp Ala Ala Glu Lys65 70 75 80Leu Phe Phe Leu Gly Arg Gln Leu Ser Ser Asp Pro Glu Thr Gln Val85 90 95Pro Asn Thr Val Ser Asn His Leu Thr Ala Gly Leu Leu Lys Tyr Ile
100 105 110Gln Thr Thr Gly Arg Tyr Ala Ser Gly Ile Asn Leu Phe Glu Lys Leu115 120 125Arg Thr Arg Asp Val Glu Val Ser Ser Leu Leu Ala Gln Val Phe Arg130 135 140Met Ala Asp Glu Glu Val Gln Ala Val Arg Leu Met Tyr Asp Ala Leu145 150 155 160Gln Asp Val Pro Met Asp Tyr Ala Leu Leu Asp Cys Gln Ser Ala Phe165 170 175Cys Ala Ser Lys Gly Glu Gly Glu Met Ala Leu Glu Cys Ala Lys Arg180 185 190Ala Val Thr Ala Ala Pro Ser Glu Phe Ser Thr Trp Ala Arg Leu Ala195 200 205Glu Val Tyr Val Cys Leu Glu Lys Trp Asp Leu Ala Leu Leu Thr Leu210 215 220Asn Ser Cys Pro Met Phe Thr Tyr Gln Asp Lys Asp Thr Pro Arg Met225 230 235 240Pro Gln Pro Ser Arg Ile Met Leu Pro Ile Leu Ala Glu Ser Met Leu245 250 255Asp Glu Ile Asp Glu Gly Gln Pro Lys Gln Gly Asp Pro His Asp Tyr260 265 270Val His Pro Ser Leu Arg Arg Leu His Ala Ala Thr Tyr Gln Gly Thr275 280 285Phe Leu Lys Ala Tyr Asn Leu Leu Thr Lys Ile Ala Ala Ala Ile Gly290 295 300Trp Asp Gln Leu Leu Lys Val Arg Ser Glu Val Phe Val Met Glu Glu305 310 315 320Glu Tyr Arg Ser Glu Arg Gln His Ser Thr Lys Pro Ser Ile His Ser325 330 335His Gly Asp Ser Pro Ala Glu Ser Ala Glu Ser Thr Glu Pro Ala Glu340 345 350Thr Ala Glu Thr Asn Gly Ser Arg Glu Gln Glu Asn Gly Asp Gly Arg355 360 365Pro Val Glu Ser Val Thr Glu Ile Val Asn Gly Asp Asp His Thr Gln370 375 380
Gly Glu Asn Ser Ile Glu Arg Pro Glu Gln Thr Met Ala Ser Glu Val385 390 395 400Val Lys Ser Gly Asn Asp Asp Pro Asp Pro Ser His Ala Ser Tyr Thr405 410 415Gln Phe Lys Asn Lys Arg Leu Cys Glu Arg Trp Leu Asp Asn Leu Phe420 425 430Met Val Leu Tyr Glu Asp Leu Arg Ile Tyr Thr Ile Trp Arg Thr Glu435 440 445Val Ala Gln Tyr Arg Gln Gln Ala Met Glu Tyr Lys Lys Ser Ala Thr450 455 460Glu Trp Glu Ile Leu Gly Glu Leu Ala Glu Arg Leu His His Phe Asp465 470 475 480Glu Ala Ile Glu Ala Tyr Gln Ala Cys Leu Ser Ile Arg Phe Ser Pro485 490 495Lys Ala Met Arg Gly Ile Leu Lys Leu His Glu Lys Gln Asn Asp Thr500 505 510Arg Gly Met Leu Ser Ala Leu Ile Arg Leu Ile Ala Trp Gln Tyr Arg515 520 525Trp Tyr Ser Glu Leu Ile Glu Asp Glu Gly Ala Val Lys Val Arg Ser530 535 540Ile Val Gln Ala Thr Asn Leu Pro Gln His Val Leu Asp Leu Thr His545 550 555 560Gln Tyr Cys Gln Leu Cys Ala Thr Phe Arg Ser Ser Gly Ser Asp Gly565 570 575<210>12<211>697<212>RTP<213>新生隱球菌(Cryptococcus neoformans)<400>12Met Ser Glu Leu Phe Lys Asp Ile Pro Glu Phe Val Glu Thr Asp Ile1 5 10 15Gly Glu Ser Leu Ala Ala Arg Thr Glu Thr Leu Gly Ser Phe Arg Glu20 25 30Leu Gly Pro Pro Asp Leu Cys His Val Met Lys Val Tyr Gly Lys Pro35 40 45
Pro Thr Gln Arg Glu Ile Gly Ser Tyr His Tyr Cys Ser Gly Ile Glu50 55 60Ala Ser Ser Ser Ala Ser Leu Ala Ala Tyr Leu Asn Ser Leu Gln Phe65 70 75 80Ser Val Glu Asp Ser Ser Ala Trp Phe Gly Lys Gly Ser Ala Trp Lys85 90 95Val Arg Ser Gly Thr Tyr Cys Cys Phe Asn Ala Phe Ser Arg Val Asp100 105 110Met Arg Val Glu Ala Asn Ile Pro Gly Gly Val Asp Ala Phe Val Val115 120 125Asp Leu His Gly Gln Arg His Pro Ala Thr Pro Glu Leu Trp Gln Glu130 135 140Thr Tyr Leu Ser Ala Ile Leu Arg Ala Ile Arg Tyr Ala Asp Asp Ala145 150 155 160Ser Tyr Arg Leu Ala Gly Tyr Arg Lys Leu Asp Pro Ile Thr Thr Pro165 170 175Glu Ala Glu Glu Arg Phe Leu Lys Ala Ala Glu Ala Leu Phe Phe Lys180 185 190Gly Trp Gln Leu Gly Ser Asp Pro Glu Ile Gln Val Ala Thr Val Val195 200 205Thr Asn His Leu Thr Ser Ala Ile Leu Lys Tyr Phe Ser Asp Ser Phe210 215 220Arg Leu His Arg Ala Ala Asn Leu Phe Glu Arg Met Met Asp Lys Glu225 230 235 240Pro Glu Val Ala Ala Leu Val Ala Lys Ser Tyr Ile Gly Met Asn Glu245 250 255Glu Ile Lys Ala Val Lys Ile Met Asn Ala Ala Leu Ala Ala Asn Pro260 265 270Gln Ser Tyr Pro Ile Leu His Ala Gln Val Asp Phe Leu Leu Ser Lys275 280 285His Lys Tyr Glu Trp Ala Gln Gln Val Ala Gln Gln Ala Val Asn Ser290 295 300Ala Pro Ser Glu Phe Thr Thr Trp Ala Lys Leu Thr Glu Thr Tyr Ile305 310 315 320Glu Leu Gly Gln Leu Asp Gln Ala Leu Leu Thr Leu Asn Ser Cys Pro
325 330 335Met Phe Thr Tyr Asn Glu Arg Asp Leu His Arg Met Pro Thr Pro Ala340 345 350Lys Ser Asn Met Pro Val Lys Lys Phe Ile Ala Asp Ser Asn Leu Val355 360 365Asp Glu Asp Ser Ser Arg Glu Asn Glu Ala Asp Ile Ala Leu Leu Arg370 375 380Leu Pro Ala Pro Asn Leu Arg Gly Thr Phe Ala Lys Ala Tyr Ser Leu385 390 395 400Leu Thr Leu Leu Val Ser Lys Ile Gly Trp Asp Glu Leu Leu Lys Ile405 410 415Arg Ser Ser Val Phe Val Met Glu Glu Glu Tyr Arg Leu His Lys Thr420 425 430Asn Val Ser Val Asp Met Asn Gly Glu Ala Gly Asp Gly Ala Ser Ile435 440 445Ala Gly Leu Lys Arg Thr Ser Ser Glu Glu Val Asn Thr Pro Ser Asp450 455 460Ile Pro Thr Ile Arg Ile Ser Ser Glu Ser Met Arg Thr Pro Asn Thr465 470 475 480Ala Pro Gly Pro Gly Phe Ser Glu Lys Ala Ser Thr His Lys Pro Ala485 490 495Leu Glu Lys Pro Glu Thr Ala Gln Ala Asn Glu Asp Pro Asn Ser Pro500 505 510Leu Gly Met Lys Ser Glu Gly Glu Gln Pro Val Ser Ala Phe Ser His515 520 525Lys Arg Leu Cys Glu Arg Trp Leu Asp Asn Leu Phe Leu Val Leu Tyr530 535 540Glu Asp Leu Arg Val Tyr Thr Ile Trp Arg Ala Glu Ile Ser His Phe545 550 555 560Lys Thr Gln His Met Ser Tyr Arg Lys Thr Gly Thr Glu Trp Glu Ile565 570 575Leu Gly Glu Leu Ala Thr Arg Leu His His Lys Glu Glu Ala Lys Asp580 585 590Ala Tyr Gln Arg Cys Leu Asp Ser Lys Phe Ser Ala Lys Ala Leu Met595 600 605
Lys Leu Leu Glu Thr Tyr Ala Asn Glu Gly Asp Leu Gln Lys Thr Leu610 615 620Thr Ala Ala Val Arg Leu Thr Thr Tyr His His Arg Trp Tyr Met Asp625 630 635 640Ala Ser Tyr Pro Ser Met Val Ala His Tyr Leu Tyr Lys Val Gly Leu645 650 655Ile His Gly His Ala Lys Leu Gln Tyr Thr Met Leu Ser Met Asn Leu660 665 670Pro Val Gly Ile Phe Glu Ile Met Gln Gly Tyr Met Lys Tyr Gly Ala675 680 685Thr Phe Asn Val Glu Gly Ser Glu Phe690 695<210>13<211>727<212>RTP<213>球毛殼菌(Chaetomium globosum)<400>13Met Val Ala Pro Ala Val Pro Glu Ser Val Pro Pro Ala Ile Ala Leu1 5 10 15Pro Arg Leu Thr Glu Glu Val Leu His Glu Ala Ile Asp Ala Arg Thr20 25 30Asp Ser Leu Ala Ser Leu Arg Glu Leu Gly Pro Pro Asp Leu Val His35 40 45Leu Ile Lys Gln Pro Leu Arg Ser Gly Gly Lys His Thr Gly Val Tyr50 55 60His His Val Thr Gly Val Asp Ala Ser Ser Ser Ala Ser Leu Ala Ala65 70 75 80Tyr Ile Asn Thr Leu Ala Tyr Lys Glu Phe Gly Pro Ser Ala Thr Thr85 90 95Lys Thr Leu Glu Gly Thr Tyr Cys Cys Tyr Asn Ala Phe Ser Arg Val100 105 110Asp Met Arg Val His Ala Pro Phe Pro Gly Ser Val Glu Ser Phe Cys115 120 125Ile Asp Glu Arg Gly Glu Lys Arg Lys Ala Thr Asp Glu Leu Trp Leu130 135 140
Glu Thr Tyr Leu Cys Ser Val Leu Arg Ala Tyr Ser Tyr Ala Asp Asp145 150 155 160Gly Thr Gly Asp Thr Ile Arg Lys Ile Met Gly Val Arg Arg Phe Asn165 170 175Pro Val Thr Asn Thr Glu Thr Glu His Arg Phe Leu Ser Ala Ala Glu180 185 190His Leu Phe Phe Arg Gly Trp Gln Leu Gly Ser Asp Ser Ile Val Gln195 200 205Val Pro Asn Val Val Ser Asn His Leu Thr Ala Gly Leu Leu Arg Tyr210 215 220Phe His Thr Thr Gly Arg Tyr Thr Ser Gly Ile Asn Leu Phe Glu Lys225 230 235 240Leu Arg Thr Glu Asn Val Glu Val Ala Ser Leu Leu Ser Lys Val Leu245 250 255Phe Met Gly Asn Glu Glu Val Gln Gly Ile Arg Ile Leu His Asp Ala260 265 270Leu Lys Gln Ser Pro Met Asp Tyr Val Met Leu Asp Thr Gln Ala Glu275 280 285Phe Leu Leu Lys Lys Ala Lys Thr Ala Ala Thr Pro Glu Gln Lys Asp290 295 300Glu Arg Leu Arg Met Ser Leu Gly Cys Ala Asp Arg Ser Thr Ile Ala305 310 315 320Ala Pro Ser Glu Phe Gly Thr Trp Ala Arg Leu Ala Glu Val Tyr Val325 330 335Thr Met Glu Asp Trp Glu Asn Ala Leu Thr Thr Leu Asn Ser Cys Pro340 345 350Met Phe Thr Tyr Gln Asp Lys Asp Ala Pro Ile Met Pro Glu Pro Arg355 360 365Asp Ile His Leu Pro Thr Leu Gln Glu Thr Arg Leu Asp Glu Ile Asp370 375 380Ser Glu Pro Glu Ser Arg Tyr Ser Glu Gln Val Asp Pro Ser Leu Leu385 390 395 400Asn Leu Arg Ala Ala Ala Tyr Arg Gly Thr Phe Lys His Ala Tyr Ser405 410 415Ile Leu Thr Glu Met Thr Ala Lys Ile Gly Trp Asp Gln Leu Leu Lys
420 425 430Ile Arg Ser Asn Val Phe Val Met Glu Asp Glu Tyr Arg Glu Lys Gln435 440 445Glu Ser Thr Ser Tyr Pro Lys Asn Arg Asn Ala Ser Thr Asp Ala Leu450 455 460Arg Gly Thr Pro Asp Pro Thr Ile Asn Gly Ala Ala Ser Glu Ala Gly465 470 475 480Gly Asp Glu Glu Lys Glu Gly Ala Glu Gly Val Asp Lys Gly Lys Lys485 490 495Lys Glu Lys Asn Glu Gly Glu Gly Ala Glu Gly Glu Gln Glu Ala Asp500 505 510Ser Glu Ser Ala Ala Gln Thr Leu Ala Pro Gly Asn Arg Ala Glu Asp515 520 525Ile Glu Arg Pro Ser Ser Ala Val Asp Pro Asp Glu Val Lys Lys Gly530 535 540Asp Asp Ser Gly Ser Lys Glu Asp His Phe Ser Arg Leu Asn Asn Lys545 550 555 560Arg Leu Cys Glu Arg Trp Leu Asp Ser Leu Phe Met Val Leu Tyr Glu565 570 575Asp Leu Arg Val Tyr Thr Ile Trp Arg Thr Gln Met Ala Gln Tyr Arg580 585 590Ala Gln Ser Met Gln Tyr Lys Lys Ser Ala Glu Glu Trp Glu Ile Leu595 600 605Gly Ser Leu Ala Glu Arg Leu Gln His Val Asp Glu Ala Val Glu Ala610 615 620Tyr Arg Ala Cys Leu Ala Gln Arg Phe Ser Pro Lys Ala Leu Ala Gly625 630 635 640Ile Leu Lys Val Phe Glu Lys Thr Lys Gly Ser Thr Arg Asp Thr Val645 650 655Ala Ala Thr Ile Arg Leu Val Thr Trp Gln Tyr Arg Trp Tyr Ser Glu660 665 670Phe Ser Pro Glu Leu Leu His Thr Val Arg Met Leu Ile Glu Asp Glu675 680 685Gly Ala Val Lys Ile Arg Ser Ile Ile Gln Ala Thr Asn Leu Pro Gln690 695 700
Asn Val Leu Asp Leu Thr His His Tyr Ala Ala Leu Cys Ala Thr Phe705 710 715 720Arg Ser Ser Gly Thr Asp Gly72權利要求
1.控制病原真菌的侵染釘形成并影響致病力的蛋白PPF1，其特征在于是具有下述氨基酸殘基序列之一的蛋白質(zhì)1)序列表中的SEQ ID №3、4、5、6、7、8、9、10、11、12、13；2)將序列表中SEQ ID №3、4、5、6、7、8、9、10、11、12、13的氨基酸殘基序列經(jīng)過一個或幾個氨基酸殘基的取代和/或缺失和/或添加且控制梨孢菌的侵染釘形成并影響致病力的蛋白質(zhì)。3)具有與SEQ ID №3所示氨基酸序列一致性高于40％、且控制真菌的侵染釘形成并影響致病力的的同源蛋白。
2.權利要求1所述的控制真侵染釘?shù)男纬刹⒂绊懼虏×Φ牡鞍譖PF1的編碼基因PPF1。該基因的特征在于具有下述核苷酸序列之一1)序列表中SEQ ID №1的DNA序列；2)編碼序列表中SEQ ID №3、4、5、6、7、8、9、10、11、12、13蛋白質(zhì)序列的多核苷酸；3)在高嚴謹條件下可與序列表中SEQ ID №1限定的DNA序列雜交的核苷酸序列；4)與序列表中SEQ ID №1限定的DNA序列具有80％以上同源性，且編碼相同功能蛋白質(zhì)的DNA序列。
3.根據(jù)權利要求3所述的基因，其特征在于所述控制真菌侵染釘?shù)男纬刹⒂绊懼虏×Φ牡鞍椎幕虻木幋a區(qū)位于自SEQ ID №1的5′端第1208位至3572位堿基之間。
4.根據(jù)權利要求2所述的基因，其特征在于所述控制真菌侵染釘?shù)男纬刹⒂绊懼虏×Φ牡鞍椎腸DNA，可具有下述核苷酸序列之一1)序列表中SEQ ID №2的DNA序列；2)編碼序列表中SEQ ID №3、4、5、6、7、8、9、10、11、12、13蛋白質(zhì)序列的多核苷酸；；3)在高嚴謹條件下可與序列表中SEQ ID №2限定的DNA序列雜交的核苷酸序列；4)與序列表中SEQ ID №2限定的DNA序列具有80％以上同源性，且編碼相同功能蛋白質(zhì)的DNA序列。
5.根據(jù)權利要求4所述基因的cDNA，其特征在于所述控制真菌侵染釘?shù)男纬刹⒂绊懼虏×Φ牡鞍椎腸DNA的編碼序列為自SEQ ID №2的5′端第70位至2157位堿基。
6.利用權利要求2所述控制真菌侵染釘?shù)男纬刹⒂绊懼虏×Φ牡鞍椎木幋a基因或其cDNA或其任一方面的部分核苷酸序列構建的表達載體，細胞系和宿主菌。
7.控制真菌侵染釘?shù)男纬刹⒂绊懼虏×Φ牡鞍椎木幋a基因的啟動子，具有自SEQID №1的5′端第1位至1207位堿基的核苷酸序列。
8.權利要求1或2所述的控制真菌侵染釘?shù)男纬刹⒂绊懼虏×Φ牡鞍准捌渚幋a基因的表達、修飾以及參與的信號途徑在設計和篩選抗真菌藥物中的應用。
9.根據(jù)權利要求9所述的抗真菌藥物，包括抗梨孢菌(Magnaporthe grisea)、小麥赤霉病菌(Gibberella zeae)、玉米莖腐病菌(Gibberella moniliformis)、油菜菌核病菌(Sclerotinia sclerotiorum)、灰葡萄孢菌(Botrytis cinerea)、小麥穎枯病菌(Phaeosphaeria nodorum)、玉米瘤黑粉菌(Ustilago maydis)、粗球孢子菌(Coccidioidesimmitis)和新生隱球菌(Cryptococcus neoformans)等病原真菌的藥物。
全文摘要
本發(fā)明提供了絲狀真菌特異的一類新基因，該類基因編碼的蛋白質(zhì)具有序列表中SEQ ID №.3、4、5、6、7、8、9、10、11、12、13等所示的氨基酸序列。在梨孢菌中該基因被命名為MgPPF1，控制侵染釘?shù)男纬尚?，其缺失導致該菌對植物的致病力顯著下降。利用本發(fā)明的成果，可以設計和篩選相關化合物，破壞MgPPF1基因表達、剪切及其編碼蛋白的表達，或者阻斷MGPPF1蛋白的修飾和細胞定位，或者阻斷MgPPF1及其編碼蛋白MGPPF1參與的信號途徑；或者阻斷與MgPPF1蛋白在氨基酸序列一致性高于40％的同源蛋白及其基因的表達和相關的信號途徑，繼而從上述化合物中開發(fā)新型的抗真菌的藥物，用于病害的控制。
文檔編號C12N15/31GK1955191SQ200510117529
公開日2007年5月2日 申請日期2005年11月4日 優(yōu)先權日2005年10月24日
發(fā)明者彭友良, 黃俊麗, 李茂華, 楊俊 , 趙文生 申請人:中國農(nóng)業(yè)大學