1、用ClustalX做多序列比對分析圖示1、打開程序如下圖所示:FontSize：lD|FiteEdtASgnmentTree?GdorfQualftyHelp|MultipleAlignmentModeT|112、LoadSequnce,載入序歹U如下圖所示：fasta格式的文件關(guān)鍵不在于文件名的后綴是什么，而是在于序列的格式fasta的格式是：1、第一行以>開頭，緊接著序列的注釋和描述。2、第二行是純序列atgcg.其他序列再起一行，如此下去就可以了。如：>seq1|thisisaexampleatgattggaacttgacgt.>seq2|thisisanotherex

2、amplettgagttgaccgtgacgtgag.3、選擇序列文件，F(xiàn)ASTA格式的如下圖所示:EditPlus或者Ultraedit4、用文本編輯器察看FASTA序列文件內(nèi)容，這里用的是記事本，推薦用如下圖所示:B為標(biāo)題-記事本文件（日編輯但）格式（愛看®幫助（中>gi1135067411gb|.4AK28313,111AF224703_1WRKYDNlAbindingprotein4Arabidopsisthaliana*MSEKEEAPSTSKSTGAPSRPTLSLPPRPFSEMFFNGGGFSPGPMTLVSNMFPDSDEFRSFSQLLAGAMISSPATA

3、AAAAAAATASDYQRLGEGTNSS5GDVDPRFKQNRPTAVLDLICNIVQGSYGMTHQQALAQVTAQAWNANMQPQTEYPPPSQVQSFSSGQAQIPTSAPLPAQRETSDVTIIEHRGQQPLNVDKPHOGYMWRk¥GQKQWGSEFPRSmCTNPGCPVKKKVERSLDGQVTEIIYKGQHNHEPPQNTKRGNKDNTANlIMGSSINNNRG55ELGASQFQTNSSNKTKREQHEAVSQATTTEHLSEASDGEEVGNGETDVREKDENEPDPKRRSTEVRI5EPAPAASHRTVTEPRIIVQTT

4、SEVDLLDDGYKWKYGQKWKGNPYPR5WKCTTPGCGVRKHVERAATDPKAWTTYEGKHNFDLPAAKSSSHAAAAAQLRPDNRPGGLANLNQQOQQQPVARLRLKEEQTT>gi113506737|gb|AAK28311.1|AF22470：l_lWRKYDNA-bindingprotenn3ArahidopsisthalianamaekeeikepsklksstgvsrptislpprpfgemffsggvgfspgpmtlvsnlfsdpdefksfsqllaswASPAAAAVAAAAW4TAHHQTPVSSVGDGGGSGGDVDPRF

5、KQ.SRPTGLMITQPlPGMFTVPPGLSPATLLD5PSFFGLFSPLQGTFCHWLAQVTAQAVQGNNVWQQSQQSEYP5STQQQQQQQ.QQASLTEIP5FS5APRSQIRASVQETSQGQRET5EI5VFEHRSQiPQN-ADKPjADDGYMWRKYGQKQVKGSDFPRSYYKCTHPACPVKKKVERSLDGQVTEIIYKGQHNHELPQKRGNNNGSCKS5DIANQFQTSNSSLNKSKFUDQETSQVT7TEQMQEASDSEEVGNAETSVGERHEDEPDPKRRNTEVR/SEPVASSHRTVTEPRIIVQTT

6、SEVDLLDDGYRWRKVGQKWKGNPYPRSYYKC7TPDCGVRKHVERMTD口KAWTTYEGKHNHDVPMJRTSSHQLRPNNQHNTSTVNFNHQQPVARLRLKEEQIT>gi|13506733|gb|AAK28309,l|AF224699_lWRKYDNAbindingprotein26ArabidopsisthalianaMGSFDRQRAVPKFKTATPSPLPL5PSPYFTMPPGLTPADFLDSPLLFT5SNILP5PTTGTFPAQSLNYNNNGLLIDKNEIKYEDTTPPLFLPSHVTQPLPQLDLFKSEIMSSNKT5

7、DDGYNWR.KYGQKQVKG5ENPRSYFKCTYPNCLTKKKVETSLVKGQMIIEIVYKGSIHINHPICPQSTKRSPSTAIAAHQNSSINGDGKDIGEDETEAKRWKREENVKEPRWVQTTSOIDILDDGYRWRKYGlKmGNPNPRSYYKCTFTGCFVRKHVERAFQPPKSVITTYEGKHKHQIPTPRRGPVLRLLGKTET>gi1159917461gb|AAL13050.ilAJF425837_1WRKYtranscriptionfactor20ArabidlopsisthalanaMNPQANDRKEFQOXSATGD

8、LTAKHDSAGGNGGGGARYKLMSPAKLPISRSTDITIPPGLSPTSFLESPVFISNIKPEPSPTTGSLFKPRPVHIISASSSSYTGRGFHQMTFTEQKSSEFEFRPPASNMVYAELGKIRSEPPVHFQGQGHGSSHSPSSISDMGSSSELSRPTPPCQMTPTSSDIPAGSDQEESIQTSQNOSRGSTPSILADDGMNWRKYQ例HVKGSEFPRSYYKCTHPNCEVKKLFERSrtDGQITDIIYKGTHDHPKPQPGRRNSGGMAAQEERLDKVPS5TGRDEKGSGVYNLSNPMEQTGNPEVPPI

9、5,A5DDGGEAAA5NRNKDEFWOOPFSKRRRMEGAMEITPLVKPIREPRWVQTL5EVDILDDGYRWRK¥GQI<WRGNPNPRSYYKCTAHGCPVRKHVERASHDPKAVITTYEGKHDKJVPTSKSSSMHEIQPRFRPDETDTISLNLGVGISSDGPNHASNEHQHQMqQLVNQTHPNGVNFRFVHASPMSSYYASLNSGMNQVGQRETKNETQ.MGDISSLNNSSYPVPPNMGFIVQ9GP>gi115991742|gbIML1304S+l|AF425835_lWRKYtranscripti

10、onfactor4Arabidopsiisthal1anaM5EKEEAPSTSKSTGAPSRPTL5LPPRPFSEMFFNGGVGFSPGPHrTLVSNMFPD5DEFR5FSQLL.AGyW5色5、序列Load進去之后如下圖所示：MChstaK(1.81)FontSize:|lD|FiteEdtAbgnmentTreesColorsQualityHelp|MultipleAlignmentMode|TH-11i-1-i-1-1-1°1gggggggmmg1350674113S06737135D67331E3917461E991742159917261599171599059

11、215384227/gbgt»gbgbgbqb曲gb1064803|emb17sHMFPDSLEFM三忖FERFTjGIKNEDNfQANDRKEFQGD'：GEKHAFSTSKSTGSSTSFTCfLGSSGVGFHN7MGEUVIGQTTCIEEDTSSNTFQESSRGtLRERIIYGKSVIQGTHRlESLEEfDCKELEKEAPbl-KbTGAPgRPTlSLPP|KEPSKIKS5TGVSFFilSlP：|RQE<VPKFKTA|PSPLFLSPS：AGIDIA|7MGFU-nCSTTNHRZR;SCID|SDSRNYWVKFKiiKljslaTAEV

12、GKVSDMELDHSNETllVDD?iDPDEFKSFTTGTiPPGI1DEFRS7rLEHI405060FileD:MyDocumcn1sscquencEsWRKYWRKY_gioup1,aaloaded.6、DoCompleteAlignment,通常情況下直接選這個即可，無須修改比對參數(shù)如下圖所示:,|D|x|FileEdtAignmeritTreesColors令溫yHelpDoCompleteAlignment12345678901-IProduceGuideTreeOnlyDoAigrmnentfromGuideJr®RealignSefectedSequences

13、RealignSefectedResidkjeRangeAl:gnProfile2toProfile1AlgnProfilesfromGuideTrMSAlgnSequencestoProfile1Al:gnSequencestoProfile1fromTreAlnmentParametersSaveLogHieCXjtputFormatOptionsIDjJEPSPLPLSPYFT:tFPGLZFQGDCGFSF'31'lllVlNMFPLilDEFRSVGFSPGFiniVSNDPDEFKIDFLD9F1I1TISNILPSFTTGTI'LTAKLHSPAHLFIB

14、RETDIIIFPQ工GEEKSSKEflLEREL：GF|T|V|NMFDEFRSLPKFKTAQPFPLFISQSSPT|gL?FPFBSEMFFNGGVTEDDEDLP79GSSFGCTVPERVIGfEEDTSSNTFQESSRGiLRER三二師1長距口！_RH¥WYKFKAKI|SKlTVS|L4NM|QGNRQC|URQSElVfYGKS71QGTHRBDMELDHSHETllVDDVD睡干IPl4循E?"一ILEEIDCKELEKFileD:MyDocumEnl3scqueiiicEsWRIKYWRICY_gfoup1.aaloaded.7、點DoComple

15、teAlignment之后彈出的文件對話框，.dnd的是輸出的指導(dǎo)樹文件，.aln的是序列比對結(jié)果，它們都是純文本文件如下圖所示:ClustalX(1.81)-laixiFileEditAlignmentTreesColorsQualityHrfp1234567B9O1-l6737135067331539174615917421599172615991715990592153342271064S33|1lCompleteAlignmentOutputGuideTreeFile：D:WRKY_groijp1.dndOutpulAlignmentFiles：止SEMTlV

16、StfHFFESDEFES15PGFin.VSNIfSI'PDEFK，網(wǎng)口琢;二jNIirSPTTGTSFAKL?IBRSTDllIFPGIPGPlTllIMFFESDEFRSGLPKFKTiQPFFLFISQSS!ELS|NHG07TJEE口TG二Keds|ottfqessrgaLRERWRQSEABYGKSVPQGTHRRTE1VIE5LEEIDCKELEKClustal：FontSize：FileD:MyDocuments3equEncEsWRK.YWRKY_grDup1,aaloaded.點“ALIGN”之后開始等待，如果序列不多，很快就可以算完，如果數(shù)據(jù)很多，可能要等一段時間

17、，這時候可以用眼睛盯著ClustalX的狀態(tài)欄，那里會有程序運行狀態(tài)和現(xiàn)在正在比對那兩條序列的提示信息，看看可以消磨時間。8、比對結(jié)束之后，我們可以看到這個結(jié)果如下圖所示：MChstaK(1.81)FontSi2e:|lD|FiteEtfcASgnmentTree?ColorsQualityHelp|MultipleAlignmentMode|ggggongggmg1559172415990592135D67411E59174213EQ6737135067331599172615991746Igb15334227Igb1064S83|embGlDlVMGEW.TkSFSFGTLF上內(nèi)工GEE氏

18、VMGEWFDgSTTMKRKiRFTL3LPPR|HHG0VI(4EBDT55BtlifKhIdslqtVSN：iDEflE；PP1I5LPPR-fssTMNPQAKDEKEFQGD®yr-IAZHHtSDMEflD昨M405060CLUSTAL-Alignmentfilecreated|9、這時候我們可以發(fā)現(xiàn)ClustalX已經(jīng)生成了.dnd和.aln兩個文件，仍然用文本編輯器打開來看，這時.aln文件，這個文件可以用Mega2做進一步的bootstrap進化樹分析如下圖所示:文件（日編輯（白格式（愛看®幫助（小CLUSTALX(1.S1)multiplesequenc

19、ealignmemt丁丁方9?91丁q：y：甲丁q：q：91丁9':g：歹甲15991724l5gge1591350674115991742135067371350673315991726159917461538422710648E3|emb1599172415990TO1350674115991742L3S06737135067331599172615991746153842271064BE3|embgi115991724gi15990592gi1135067411115991742g-i113506737gi113506733hi115991726空蹙gbAAL13O39二|好41

20、ML11010+1|AAK2S313,1|AF22AAL130-1S,lAF42AAK28311.LAF22AAK28309.1IAF22AAL13040.1IAF41AAL130504l|AF42AAK96200.ilAMOCAA6355-1.1AAL13039*1AAL1101Q.1AAK2S313,1AAL13D48.1AAK28311.1MK283g.iAAL13040.1AALL3050.1AAK96200.1CAA63554.1AF4LfiF22AF42AF2.2AF22AF41AF42AF40AALL3039.1|AFlAAL11010.ilAAK2S313.LAF22AAL13

21、CH8.1AF42AAK23311.1AF22AAK23309.1IAF22AAL13CH0.1IAF4L10、這是.dnd文件（指導(dǎo)樹）如下圖所示:MAGFDEMVAVMGEWVPRSPSPGTLFSSAIGEEK5SKRVLERELS_MHGQVIHRKR"占卜1MnK-H5EKEE-APST5K5TGAPSRPTLSLPPRPFSEMFFNGGVGFSPGPMTLVSM5EKEE-APSTSK5TGAPSRPTL5LPPRPFSEMFFNGGVGF5PGPMTLVSMAEKEEKEPSKLK55-G¥SRPTISLPPRPFGEMFFSGGVGF5PGPMTLVSMS

22、5TSFTDLLGSSGVDCVECOEDLR-MNPQArORKFFQGDCSATQDLT研必NGGGGARYKLMSPAKLPIMSCmaevigkvlasdIGLEEDT$SNHNkDSSQSNVFHGZLSERIMRJ4GFNAPRLNTbMrRTLJTD-一MEDSLQTTFQESSRALRERIAARSGFNAPWLNTEDIF11FPD5DEFRSF5QLLAG-AMS5PATAAAAAAAATA5DY1QRr>lFPD5DEFR5T5QLLAG-AMSSPATAAAAAA4ATASDYQRNLF5DPDEFK5F5QLLAG-AMASPAAAAVAAMWATAHHQT-PV帕SFDrqr_AVPKFKTATPSPLPLSPSPYFT-VSGSSFGGVYPERTGSGLPKFKTWPPLPISQSSRSTOITIPPGL5PTSFLESPVFISNIKPEPSPTTG5LFKPRPVHDODSDSRMYWYKPKAKLVSKATVSALWILQGNRQQTWRQMELDHSMETTKAVDDWATTDKA