2018,27(1):38 42 犃犮狋犪犃犵狉犻犮狌犾狋狌狉犪犲犅狅狉犲犪犾犻 狅犮犮犻犱犲狀狋犪犾犻狊犛犻狀犻犮犪 doi:10.7606/j.issn.1004 1389.2018.01.006 :2018 01 12 :htp://kns.cnki.net/kcms/detail/61.1220.s.20180112.0934.014.html!"#$ 1, 2, 1 (1. <;2. => <,?@A 712100) B3CDE 01 pst 1374 pst 1178 pst 8713, FGHIJKLpET 32a PP pst 1374 pet 22b pst 1178/8713,MB N O BL21(DE3), P Q R S IPTG T U I J SDS PAGE VW XIY: 01 pst 1374 pst 8713 IJ 23 Z[\,] pst 1178 IJ^_`abc L6 *, HPLC 3, _ P6 01 ; 01;GHIJ;01 Q786 A 1004 1389(2018)01 0038 05 # P = ( 犘狌犮犮犻狀犻犪狊狋狉犻犳狅狉犿犻狊 f.sp. 狋狉犻狋犻犮,Pst) 6DE a[, a[ 6 P, a ' D E = 6 =,[ _b 10%~30%, 1!6, _J60%b, DE L 6 * 6, 6 [ G _b. [1], 2 6 01b 6, 6 Voegele78D QR 2 0 1 RTP1,a Y * IJ, ^O! 6 0 1 [2] " # 6 AvrM AvrL567 0 1 * G 6. O $ 6,M AvrM AvrL567* % & G O $ ' 6 `, 6()O$%& N N *6O$+,,-Y 6O$_`a. 6O$/ [3] 0 1 78 SIY * 23O$, 2 c3 01 6 O $ / \ / 4 5 6 0 * AvrL5677 8 9 : ; 6 +, < 6=>?@P* PDB 3<A ; [C_`B C`6DEDE 6 01 ToxA ] ToxA B [C Arg Gly Asp,_ b ) 6 @ C L45,S F, C X G H AvrL567 _ ` 6 O $ / [4] AvrM 6 78IH ` [CJ L01 B [DKLM6NOI,_b ), 01 * %& GO$ '6 ` PQR6S4 [5] ]T$ 78U2-Y01 = avw 01 `R 786 XY, IH 016O $/, 6\ / B,_b 01 = Z[ 016, \ 601 3<A(ProteinDataBank),;?@6, [] 016`, ^ = 6 Z[^ [6] _`ab3cde 0 1 GHIJ bc P78, H Z X L Z N 0 1 [7] 78DE / :2016 08 15 :2017 04 06 :!"#$%&(Z111021101);' &(21403168) : (,),*+,-,./01 2345678 E mail:543441552@qq.com :9 :,),;,./01 2345678 E mail:taohu@nwsuaf.edu.cn
1 (1:DE 01GHIJ cn P78 39 1 1.1! IJK L pet 22b(+) pet 32a pp (+) Turbo BL21(DE3) <01 ^ L M ; 01 =; ; 犜犪狇 PCR Mix P! ( 犖犱犲 Ⅰ 犖犮狅 Ⅰ 犡犺狅 Ⅰ) TA Q R T 4 DNA = ( ) ;DNA Marker(DM2000) DNA BCA 0 1 = ;0 1 Marker ThermoFisherScientific ;Ni NTAagarose ' GE ;HPLC 6, FL2200. 2 ; N ' 2 1.2 " # 1.2.1 [8] b =; 6, PCR 6 pst 1178 pst 1374 pst 8713,M TA Q R 犖犱犲 Ⅰ 犡犺狅 Ⅰ,2 5 B pst 1178 pst 8713 K L pet 22b, I J K L pet 22b pst 1178 pet 22b pst 8713 犖犮狅 Ⅰ 犡犺狅 Ⅰ,B pst 1374 KLpET 32a pp, IJKL pet 32a pp pst 1374 BW9 6 IJKLO BL21(DE3) 1.2.2 [9] 6 BL21 Q R,b L \ 1 500 O 1L 100mgAmp6 L LB,37 OD 600 0.6~0.8, ^, 1 mmol/l 6 (IPTG) pet 22b pst 8713 pet 32a pp pst 1374 a 20 T U 12h;pET 22b pst 1178 37 T U 5h 6000r/min, 4 5 min L ^ 8 (PBS,20 mmol/l KH 2PO 4,0.15 mol/l NaCl, ph7.9), 150μg/mL 6,1 104 (H8 6 L \)H 8,1 mmol/l (PMSF), ^ 30min, 5min 1 30min(250 W, 5s, 5s)^ 12000r/min,4 30 min 5 2 SDS PAGE V W, ^ 0.22 μm 6 ) ^, 1.2.3! " 6 0 1 pst 1178 pst 8713 6 : 10 mmol/l 6 Tris (20mmol/LTris,0.15mol/LNaCl, 10mmol/L,pH7.9) (Ni NTA), B 6 5 b 1 ml/min C; ^ (20 mmol/l Tris,0.15 mol/l NaCl,20mmol/L,pH7.9) 3~5 L, ^ (20 mmol/l Tris,0.15 mol/lnacl,0.250mol/l,ph7.9) 601 ^ W 6 0 1, 6 bufera ( φ =0.1% TFA) bufer B( φ = 0.08% TFA, φ =70% )2 5 ^, 6 6 0 1 0.22μm ),* 10000r/min 2 min, HPLC S Tricine SDS PAGE VW01 X, 601 C,! "/" #, #$,-80 L 601pst 13746 : Z 01pst 1178 pst 8713[\, ^ 6 601 bl \1 100 6 Pres cissionprotease [10] (1 mg/ml) M 1L % (20 mmol/l Tris,0.15 mol/l NaCl,pH 7.9)% 12h,&' bufer A (20 mmol/l Tris,pH 7.9) HiTrap Q,B% 601 S0.22nm 6 ) ^ AKTA 0 1 ' b 0.8 ml/min, C ^ b bufera 10~ 15min, ^b bufera bufer B(20mmol/LTris,1mol/L NaCl,pH 7.9) P,S Tricine SDS PAGE V W 0 1 X ( 6 0 1 C 1mmol/L ) φ =0.02%6 NaN 3, * "^-80 L 2 2.1 $%&' (). I J 6 3 C Turbo 25 Q R + M 2 5, PCR VW (- 1-2 - 3). D Z [ \ M/N 0 W9, X 6 [\,IYIJKLpet 22b pst 1178 (- 1) pet 32a pp pst 1374 (- 2) pet 22b pst 8713 (-3) F
40 27 M.TaKaraDL2000marker;1~4.4 C Q R PCRprod uctsofrecombinant plasmidsfrom four diferent monoclonal cels;5. P Negativecontrol 1 *+,-./ 狆犈犜 22 犫 狆狊狋 1178 01 犘犆犚犉犻犵.1 犘犆犚狆狉狅犱狌犮狋狊狅犳狆犈犜 22 犫 狆狊狋 1178 1~2.2 C Q R PCRproductsofrecombinantplasmids fromtwodiferentmonoclonalcels;3. P Negativecon trol;m.takaradl2000marker. 3 *+,-./ 狆犈犜 22 犫 狆狊狋 8713 01 犘犆犚犉犻犵.3 犘犆犚狆狉狅犱狌犮狋狊狅犳狆犈犜 22 犫 狆狊狋 8713 M.TaKaraDL2000marker;1. P Negativecontrol; 2~3.2C QR PCRproductsofrecombinantplasmidsfrom twodiferentmonoclonalcels 2 *+,-./ 狆犈犜 32 犪 犘犘 狆狊狋 1374 01 犘犆犚犉犻犵.2 犘犆犚狆狉狅犱狌犮狋狊狅犳狆犈犜 32 犪 犘犘 狆狊狋 1374 2.2 &' 2.2.1 pst 1178! " B T U I J 6 L, ^ 5,B 6 0 1S HPLC, \ 6 601(-4) ^01. Z(13ku) [\, a Z63 <N ^ 01 pst 1178abc L * 2.2.2 pst 1374! " B T U I J 6 L, ^ 5,S Elutionbufer 6 01 27ku, PrescissionProte ase ^ 10ku 0 1 17.2ku 2 01 3,4b- 0 16. S HiTrapQ ^ [.6 56 601 23 Z[\(-5-6) 1~4.01I J 6 7 2 Thepartsofexpression;1.8 T U 6 Thecellysatebeforeinduction;2.IPTG T U ^ Thecellysateafterinduction;3. ^6 5 Thesuper natant;4. ^6 9 : Thesupernatantandpeletofcelly sate;m.0 1 2 3 ; Protein molecular weight marker (Thermo #26617);5~8. Thepartsofnickel columnpurification;5. Flowthroughofsupernatantofcel lysateduringloading;6.10mmol/l 6 Thewashof 10mmol/Limidazoleafterloading;7 8.250mmol/L 6 Purificationproductelutedwith250 mmol/limidazolelysis bufer;9.hplc ^65 6 0 1 ResultsofHPLCpurifi cation 4 犜狉犻犮犻狀犲 犛犇犛 犘犃犌犈 狆狊狋 1178 犉犻犵.4 犈狓狆狉犲狊犻狅狀犪狀犱狆狌狉犻犳犻犮犪狋犻狅狀狅犳犎犻狊 狋犪犵犵犲犱狆狊狋 1178 狅狀犜狉犻犮犻狀犲 犛犇犛 犘犃犌犈 2.2.3 pst 8713! " B T U I J 6 L, ^ 5,S 0 1, S HPLC \ 6 6 0 1,N 23 Z(11ku)[\(-7)
1 (1:DE 01GHIJ cn P78 41 1~4.01IJ672 Thepartsofexpression;1.8 T U 6 Thecellysatebeforeinduction;2.IPTG T U ^ Thecellysateafterinduction;3. ^6 5 Thesuper natant;4. ^69: Thepeletofcellysate;5~8. Thepartsofnickelcolumnpurification;5. Flowthroughofsupernatantofcellysateduringloading;6.10 mmol/l 6 Thewashof10mmol/Limidazoleafter loading;7 8.250 mmol/l 6 Purificationproduct elutedwith250mmol/limidazolelysisbufer;m.0123 ; Proteinmolecularweightmarker(Thermo #26611) 5 犜狉犻犮犻狀犲 犛犇犛 犘犃犌犈 狆狊狋 1374 犉犻犵.5 犈狓狆狉犲狊犻狅狀犪狀犱狆狌狉犻犳犻犮犪狋犻狅狀狅犳犎犻狊 狋犪犵狆狊狋 1374 狅狀犜狉犻犮犻狀犲 犛犇犛 犘犃犌犈 M.0123 ; Proteinmolecularweightmarker(Thermo #26611);1.Prescissionprotease Prescissionprotease;2. ^6 0 1 Efectof His pst 1374digestedbyprescission protease;3~5. 2 ;3. Loading flowt10 mmol/limidazolehroughofprotease digestedproduct dialyzedinlowimidazolebufer;4.10mmol/l 10mmol/L imidazole;5.250 mmol/l 250 mmol/limidazole lysisbufer;6~7. 3 6 X ResultsofQcolumn; M.0123 ; Proteinmolecularweightmarker(Thermo #26630) 6 犜狉犻犮犻狀犲 犛犇犛 犘犃犌犈 狆狊狋 1374 犉犻犵.6 犈狓狆狉犲狊犻狅狀犪狀犱狆狌狉犻犳犻犮犪狋犻狅狀狅犳犎犻狊 狋犪犵犵犲犱狆狊狋 1374 狅狀犜狉犻犮犻狀犲 犛犇犛 犘犃犌犈 1~4.01IJ672 Thepartsofexpression;1.8TU6 Thecellysatebeforeinduction;2.IPTG TU^ Thecel lysateafterinduction;3. ^ 6 5 Thesupernatant;4. ^ 6 9 : Thepeletofcellysate;5~8. The partsofnickelcolumnpurification;5. Flowingthroughofsupernatantofcellysateduringloading;6.10mmol/L6 Thewashof10mmol/Limidazoleafterloading;7 8.250mmol/L Purificationproductelutedwith250mmol/Limidazole lysisbufer;m1.0123 ;(Thermo #26611) Proteinmolecularweightmarker(Thermo#26611);M2.0123 ;(Ther mo#26617) Proteinmolecularweightmarker(Thermo#26617);9 10.HPLC ^656 01 PresentresultsofHPLCpurifi cation 7 犜狉犻犮犻狀犲 犛犇犛 犘犃犌犈 狆狊狋 8713 犉犻犵.7 犈狓狆狉犲狊犻狅狀犪狀犱狆狌狉犻犳犻犮犪狋犻狅狀狅犳犎犻狊 狋犪犵犵犲犱狆狊狋 8713 狅狀犜狉犻犮犻狀犲 犛犇犛 犘犃犌犈 3 DE 01,a[ 2 P0 1, \ P? _ ^ 01678, b^iv <= _78 01*GHIJ ' _ PIJ >6 Z[,_ 56 601 ^3C 016O P?, @^ 8A=01 9:, [b H Z78 01 * pst 1178 6,A 1 Tricine SDS PAGE 5 6. 2 3 D Z B, pst 1178615N HSQC J C YC01_`a [11], [b_ DE 1 Z[WN23 M b [10] ^,B 016 bc 6L L!601 ^,R 016FG H, I;() JY /,cq K LDE M
42 27 ; 犚犲犳犲狉犲狀犮犲 : [1] STRUCK C,HAHN M,MENDGEN K.Plasmamembrane H + ATPaseactivityinspores,germtubes,andhaustoriaof therustfungus 犝狉狅犿狔犮犲狊狏犻犮犻犪犲 犳犪犫犪犲 [J]. 犉狌狀犵犪犾犌犲狀犲狋 犻犮狊犪狀犱犅犻狅犾狅犵狔 : 犉犌牔犅,1996,20:30 35. [2] KEMEN E,KEMEN A C,RAFIQIM, 犲狋犪犾.Identification ofaproteinfromrustfungitransferredfromhaustoriainto infectedplantcels [J]. 犕狅犾犲犮狌犾犪狉犘犾犪狀狋 犿犻犮狉狅犫犲犐狀狋犲狉犪犮 狋犻狅狀狊,2005,18:1130 1139. [3] RAFIQIM,GANP H,RAVENSDALE M, 犲狋犪犾.Internali zationofflaxrustavirulenceproteinsintoflaxandtobacco celscanoccurintheabsenceofthepathogen[j]. 犜犺犲犘犾犪狀狋犆犲犾,2010,22:2017 2032. [4] WANG CI,GUNCAR G,FORWOODJK, 犲狋犪犾.Crystal structuresofflaxrustavirulenceproteinsavrl567 Aand Drevealdetailsofthestructuralbasisforflaxdiseasere sistancespecificity[j]. 犜犺犲犘犾犪狀狋犆犲犾,2007,19:2898 2912. [5] VET,WILLIAMSSJ,CATANZARITIA M, 犲狋犪犾.Struc turesoftheflax rustefectoravrmrevealinsightsintothe molecularbasisofplant celentryandefector triggeredim munity[j]. 犘狉狅犮犲犲犱犻狀犵狊狅犳狋犺犲犖犪狋犻狅狀犪犾犃犮犪犱犲犿狔狅犳犛犮犻 犲狀犮犲狊狅犳狋犺犲犝狀犻狋犲犱犛狋犪狋犲狊狅犳犃犿犲狉犻犮犪,2013,110:17594 17599. [6] NO. DE 01cTU 6 `2 [D].? @ A:,2013. TANG CH L.Characterizationandfunctionalanalysesof efectorsandhostceldeathinducinggenesin wheatand 犘狌犮犮犻狀犻犪狊狋狉犻犳狅狉犿犻狊 interactions[d].yangling Shaanxi: NorthwestA&FUniversity,2013. [7],P O,QRR,1. JS T8O 6 U" 6 V[J]. '= 23=,2015,6:624 629. GONGZ M,QIU SH,WANG Y Y, 犲狋犪犾.Solutionconfor mationofperipheralsubunit bindingdomainof 犈. 犮狅犾犻 di hydrolipoyltransacetylase[j]. 犆犺犻狀犲狊犲犑狅狌狉狀犪犾狅犳犅犻狅犮犺犲犿 犻狊狋狉狔犪狀犱犕狅犾犲犮狌犾犪狉犅犻狅犾狅犵狔,2015,6:624 629. [8] WXY Q J, D W. Z.23QR ^[\ [M].3. : ],2002:611 633. SAMBROOKJ,RUSSELL D W,HUANG P T,tran.Mo lecularcloning:a Laboratory Manual[M].3edition.Bei jing:sciencepress,2002:611 633. [9] COLIGANJE,DUNNB M,PLPEGH H L,1.^_ `Z. ab01 ^ [ \ [M]. : ],2007: 90 96. COLIGANJE,DUNN B M,PLPEGH H L, 犲狋犪犾.LISH T,tran.ShortProtocolsinProteinScience[M].Beijing:Sci encepress,2007:90 96. [10]. c8s JS T8O 6? / ab78[d].?@a:,2015. GONGZ M.Preliminarystudyontheinteraction mecha nism between 犘狔狉狌狏犪狋犲犱犲犺狔犱狉狅犵犲狀犪狊犲 and dihydroli poamide acetyltransferase of 犈. 犮狅犾犻 [D ].Yangling Shaanxi:NorthwestA&FUniversity,2015. [11] ARBELY E,NEUWEILER H,SHARPE T D, 犲狋犪犾.The human peripheralsubunit binding domain folds rapidly whileovercomingrepulsivecoulombforces[j]. 犘狉狅狋犲犻狀犛犮犻犲狀犮犲,2010,19(9):1704 1713. 犘狉狅犽犪狉狔狅狋犻犮犈狓狆狉犲狊狊犻狅狀, 犘狌狉犻犳犻犮犪狋犻狅狀犪狀犱犛狅犾狌犫犻犾犻狋狔狅犳犜犺狉犲犲犈犳犳犲犮狋狅狉狊犳狉狅犿犠犺犲犪狋犛狋狉犻狆犲犚狌狊狋犉狌狀犵狌狊 ZHAO Haibin 1,WANG Yuanyuan 2 andtao Hu 1 (1.ColegeofChemistry & Pharmacy,NorthwestA&FUniversity,YanglingShaanxi 712100,China; 2.ColegeofLifeSciences,NorthwestA&FUniversity,YanglingShaanxi 712100,China) 犃犫狊狋狉犪犮狋 Haustoriumistheonlyspecializedinfectionstructureofwheatstriperustfungus( 犘狌犮犮犻狀犻犪狊狋狉犻犳狅狉犿犻狊 f.sp. 狋狉犻狋犻犮 ).Thefungustakesnutrientsfromthehostbyhaustoriumanddeliversefec torsdirectlyintohostcelsinordertoestablishanintimatefeedingrelationshipduringinfection.in thisstudy,threeefectors(pst 1374,pst 1178andpst 8713 )wereclonedtopet 32a PP pst 1374, pet 22b pst 1178/8713vectorsandtransformedinto 犈. 犮狅犾犻 BL21(DE3)cels.Thepositiverecom binantcloneswereinducedbyiptgandtheexpressedproteinswereanalyzedbysds PAGE.There sultsindicatedtheefectorspst 1178andpst 1374 wereexpressedcorrectlyandthemolecularmassof efectorsestimatedwereinconsentwiththetheoreticalvalues.meanwhile,preliminarystudiessug gestedtheefectorpst 1178 existedaspolymerinsolution.thetargetproteinswerepurifiedbyni NTA,HPLCandion exchangecolumnandthesolubleproteinwereobtained. 犓犲狔狑狅狉犱狊 Striperust;Efectorproteins;Expression;Purification 犚犲犮犲犻狏犲犱 2016 08 15 犚犲狋狌狉狀犲犱 2017 04 06 犉狅狌狀犱犪狋犻狅狀犻狋犲犿 SpecialTalentRecruitmentFundofNorthwestA&F University(No.Z111021101); thenationalnaturalsciencefoundationofchina(no.21403168). 犉犻狉狊狋犪狌狋犺狅狉 ZHAO Haibin,male,masterstudent.Researcharea:protein proteininteractionand structuralresolution.e mail:543441552@qq.com 犆狅狉狉犲狊狆狅狀犱犻狀犵犪狌狋犺狅狉 TAO Hu,male,professor.Researcharea:protein proteininteractionandstruc turalresolution.e mail:taohu@nwsuaf.edu.cn ( : < 犚犲狊狆狅狀狊犻犫犾犲犲犱犻狋狅狉 : 犌犝犗犅犪犻狊犺狅狌 )