J.SHANXIAGRIC,UNIV.(NaturalScience Edition) ( )2015,35(4):342 003269 MATE 1,2,3 1,2,, 1 1 1,2,, (1., 810008;2., 650223; 3., 830011) : Zn, Sb04g031980,, MATE, SbMATE Zn, SbMATE,,, SbMATE,SbMATE Zn 2+, SbMATE Zn 2+ : ;MATE; ; :Q78;S514 :A :1671-8151(2015)04-0342-08 DOI:10.13842/j.cnki.issn1671-8151.2015.04.002 CloningofMATEGenefromSorghum andanalysisofheavymetaltoleranceinyeast ZhangPing 1,2,3,ZhangYuan 1,2,WangHuichun 1,Wang Wenying 1,XuJin 1,2 (1.Colege of Life Science and Geography,Qinghai Normal University,Xining Qinghai 810008,China; 2.Xishuangbanna Tropical Botanical Garden,Chinese Academy of Sciences,Kunming Yunnan 650223,China; myof Sciences,Urumqi,Xinjiang,830011,China) 3.State Key Laboratoryof Desertand OasisEcology,XinjiangInstituteof Ecology and Geography,Chinese Acade- Abstract:AZn-responsivegene,Sb04g031980,wasobtainedbythedeepsequencingofdiferentialgeneexpressionanal- ysisinsorghum bicolor.thesequenceanalysisshowedthatthegeneencodeda MATEsubfamilytransporter,named SbMATE.Forelucidatingthefunctionofthegene,theyeast-expressedplasmidswithSbMATE wereconstructedand transferredintoheavymetalsensitiveyeastmutantcels.thesubcelularlocalizationanalysisbythefused-gfpprotein expressionshowedthatsbmate waslocalizedinvacuolarofyeastcel.furtherstudyaboutthegrowthpaternofsb- MATE-expressedyeastindicatedthatoverexpressionofSbMATEimprovedZn 2+ toxicityresistanceintheyeastcels, suggestingthatsbmateplaysaroleinznaccumulationandtoleranceinplant. Keywords:Sorghum bicolor;mate;genecloning;yeastexpression. MATE (multidrugandtoxiccom-, poundextrusiontransporter),, [1] 56 Cu Fe Mn MATE, 50 Mo Ni Zn, MATE [2] [4,5] MATE,,, Zn,MATE Zn 200 mg kg -1 [3] :2015-03-30 :2015-04-30 : (1987-), ( ),,, : * :, Tel:0871-65140420;E-mail:1714565575@qq.com;, Tel:0871-65140420;E-mail:xujin@xtbg.ac.cn : (31170228,31272239); (C2013503042); (2013-01-01)
35(4) : MATE 343, Zn Zn [6] Zn 1.2.3 SbMATE, Zn pyes2-sbmate-gfp pyes2-, Zn GFP YK44,, GFP, SbMATE, Zn, 1.2.4, YK44-pYES2 YK44-pYES2- SbMATE, Zn MATE SbMATE 100uL 10 mlsd-ura (2%, ) + CdCl 2 (0.01~0.05 mmol L -1 ) Zn Zn ZnCl 2 (0.04~0.20mmol L -1 ) CoCl 2 (0.1~ 0.5mmol L -1 ),30,250r min -1, 36h 600 1 nm, 3, 1.1 3, BTx623 2 YK44 (ura3-52his3-200,δzrc,δcot1,matingtypeα) Zn/Cd/Ni/ 2.1 SbMATE Co, Dietrich H.Nies pyes2 GAL1, SbMATE 1.2 Sb04g031980, 1.2.1 SbMATE 1704bp, DNAman, ( 1), (DGE ), Zn 1704bp ORF, 567, MATE, 53.9kD MATE Sb04g031980 Zn,, 88~248bp 310~472bp SbMATE NCBI SbMATE, Treeview MATE, SbMATE 1.2.2, MATE, Hind I/BamHI,, 90% pyes2-sbmate pyes2-sbmate-,clustalw GFP pyes2-gfp ( 2) GRMZM2G135175 Si016794m MATE YK44, 98% MATE, 3,SbMATE PCR SD-ura 1d,OD600 0.2, Zn,,, Zn MATE (Sb04g031980) GenBankBlast (Sb04g031980) MATE YPD (1% 2% Sb06g025870 2%,pH5.8),
３４４ 山 西 农 业 大 学 学 报 自然科学版 图 １ 高粱 ＳｂＭＡＴＥ Ｓｂ０４ｇ０３１９８０ 基因及编码蛋白 Ｆ １ Ｇｎａｎｄｐ ｒ ｏ ｔ ｎｓ ｓｏ ｆｓｂｍａｔｅ Ｓｂ０４ｇ０３１９８０ ｑｕｎｃ ｇ ２０１５
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35(4) : MATE 347 3 MATE Fig.3 PhylogenetictreeofSorghum MATEprotein 2.2 pyes2-sbmate pyes2-sbmate-gfp MATE Hind I/BamHI, pyes2 P GAL1, pyes2-sbmate- GFP pyes2-sbmate, SbMATE 4, PCR,,, Sb- 4 pyes2-sbmate pyes2-sbmate-gfp pyes2-gfp Fig.4 VectorprofileofpYES2-SbMATE,pYES2-SbMATE-GFP andpyes2-gfp
348 ( ) 2015 2.3 SbMATE 5,SbMATE YK44-pYES2-SbMATE-GFP, SbMATE 5 SbMATE Fig.5 SubcelularlocalizationofSbMATEinyeastcel 2.4 SbMATE pyes2, 0.05~0.10 mmol L -1 ZnCl 2 Zn Co Ni Cd SbMATE 6,, Ni Co Cd SbMATE,SbMATE,SbMATE Zn 6 SbMATE Fig.6 HeavymetaltoleranceanalysisofSbMATEinyeastcels 3 TE ALF5 (At3g23560) [1] ; TT12(At3g59030),MATE, [2], MA-,
35(4) : MATE 349,MATE,, FRD3 (At3g08040), YK44 Zn, Fe, Fe [3] MATE, SbMATE,,,OsFRDL4 Al Zn,, Al Zn, MATE AltSB, Al, [7~10] Zn, Zn, Zn MATE SbMATE, GFP Zn [1]DienerAC,GaxiolaRA,FinkGR.ArabidopsisALF5,amultidrugefluxtransportergenefamily member,confersresistancetotoxins [J].PlantCel,2001,13(7):1625-1638. 871. [3],,,.MATE [J].,2014,29(4):398-405. [4],,,. [J].,2007,18(7):1631-1639. [5],,,. [J].,2007,27(2):1003-2630. [6],,,. [J].,1993,25(6):319-322. [2]DebeaujonI,PeetersAJ,Leon-KloosterzielKM,etal.TheTransparentTesta12 geneofarabidopsisencodesa multidrugsecondary transporter-likeproteinrequiredforflavonoidsequestrationinvacuolesoftheseedcoatendothelium [J].PlantCel,2001,13(4):853- [7]MagalhaesJV,LiuJ,Guimar esct,etal.ageneinthemultidrugandtoxiccompoundextrusion(mate)familyconfersaluminumtol- eranceinsorghum [J].NatGenet,2007,39(9):1156-1161. [8]XuJ,SunJH,DuLG,etal.ComparativetranscriptomeanalysisofcadmiumresponsesinSolanum nigrum andsolanum torvum [J].New Phytol,2012a,196:110-124. [9]XuJ,ZhuYY,GeQ,etal.ComparativephysiologicalresponsesofSolanum nigrum andsolanum torvum tocadmiumstress [J].New Phytol,2012b,196:125-138. quencing[j].jintegrativeagriculture,2013,12(11):2036-2044. ( : ) ( 341 ) [12]BaconE,MorreDJ,PlasmamembraneNADH oxidaseofmaizerootsrespondstogravityandimposedcentrifugalforces [J].Plant physiologyandbiochemistry,2001,39(6):487-494. 8):551-558. [10]ShiDQ,ZhangY,MaJH,etal.IdentificationofZincDeficiency-Responsive MicroRNAsinBrassicajuncea RootsbySmalRNA Se- [13]GarciaC,HicksC,MorreDJ,PlasmamembraneNADHoxidaseisgravi-responsive[J].Plantphysiologyandbiochemistry,1999,37(7- [14]PoovaiahBW,YangT,vanLoonJJ,Calcium/calmodulin-mediatedgravitropicresponseinplants[J].Journalofgravitationalphysiolo- gy,2002,9(1):211-214. [15]TauchiH,MatsumotoE,IijimaK,etal,EfectofgravitystressonfidelityofDNAdouble-strandbreakrepair[J].Biologicalsciences inspace,2003,17(3):255-256. [16]Pedroso MC,DurzanDJ,EfectofdiferentgravityenvironmentsonDNAfragmentationandceldeathinKalanchoeleaves[J].Annals ofbotany,2000,86(5):983-994. ( : )