53 2 ( ) Vol.53 No JournalofXiamenUniversity (NaturalScience) Mar.2014 doi: /j.issn Hsp20 Adcs 1, 1, 2, 1* (1., 3

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1 53 2 ( ) Vol53 No JournalofXiamenUniversity (NaturalScience) Mar2014 doi:106043/jissn Hsp20 Adcs * (1,361102; ) : 2 (Hsp20) (Adcs) (Agrobacterium) (Arabidopsisthaliana), (PCR) (Westernblot),Hsp20 Adcs, : 45 1h,Hsp20, ;Adcs,Hsp20,Adcs : ; ;; : Q812 : A : (2014) [AgaricusbisporusLange(Imbach)], 02,, , (Adcs)02 [1-2], 20 (Hsp20),,, :02, 25~28,,, ; ,, 16~ [3], pearleygate201 (Escherichiacoli) DB3 02 cdna [4], atl atr (LR) (Arabidopsisthaliana) [5-7] ( ) ; Marker ECL ; : : ( ) * :sysong@xmueducn, 1 Adcs (Agrobacterium) [4] ATEG5(GV3101) ; pdonr207 1 ; (Col-0) ;atb atp (BP) Invitrogen ;TaqDNA,DNA Marker ; (JacksonImmuno Research Laboratories, INC); Origene ; DNA ;MurashigeandSkoog Sigma

268 ( ) 2014 Aldrich 13 ATEG2 12 ATEG2 1μL ATEG2 (01μg)50μL Gateway ATEG2 GV3101, 5min; 1 PCR BP atb Hsp20 ORF, ExTaq PCR, atb1-2f111:5 -AAAAAGCAGGCT- TCATGTCTACTTTCTTCTACGA -3 atb2-2f111:5 -

2 268 ( ) 2014 Aldrich 13 ATEG2 12 ATEG2 1μL ATEG2 (01μg)50μL Gateway ATEG2 GV3101, 5min; 1 PCR BP atb Hsp20 ORF, ExTaq PCR, atb1-2f111:5 -AAAAAGCAGGCT- TCATGTCTACTTTCTTCTACGA -3 atb2-2f111:5 - AGAAAGCTGGGTTTCAATGCCTCAGCTGGGCATC- 3, 95 2min;95 30s,68 30s,15 ; 72 2min; PCR, atb1- :5 -GGGGACAAGTTTGTA- CAAAAAAGCAGGCTTC-3 atb2- :5 -GGG- GACCACTTTGTACAAGAAAGCTGGGTT-3, 95 1 min;94 30s,55 (GV3101) ATEG5(GV3101) 30s,68 30s,25 ; 68 2 min;pcr, 500bp BP (5μL):atB-PCR (25fmol/L),pDONR-,T 3 PCR Western 207 (25fmol/L),BP 1μL, blot (5 μl): (25fmol/L),pEarleyg- ate201 (25fmol/L),LR 1μL, 5μL;25 ;1μL K,37 1 ATEG2 Fig1 AflowchartilustratingconstructionofATEG2vector 2mm, ( :25 μf :247kV :400Ω, :9ms) ;, 5 min 10 ml LB,28 1h; LB ( 50μg/mL 10μg/mL),28 2d PCR Hsp20 ATEG2(GV3101) 14 ATEG2 [8] T 1 30mg/L, 1,3, T 3 5μL;25 ;1μL K, min; 10 μl,1 μl T 3, 10 mg/l DB31, LB ( 10 1/2 MS T 3 μg/ml), PCR, T 2, (Entryclone), ;LR 1/4, T 3, 10 min; 10μL,1μL 16 PCR DB31, LB ( 50 SDS μg/ml), PCR, DNA PCR (expressionclone) pearleygate201 atb1- :5 -GGGGA- ATEG2,-20 CAAGTTTGTACAAAAAAGCAGGCTTC-3 atb2- :5 -GGGGACCACTTTGTACAAGAAAGCTGGGTT- 3 ATEG2 rtaq; 95 5min;95 30s,57 35s,72 30s,35 ; min; 500bp ATEG5 LaTaq ; 95 5min;95 30s,57 35s,72 2 min,32 ; 72 10min; 2200bp 17 Westernblot,10 mg 40μL (10 mg/l 20 mg/lβ- ), 10 min; g

2 : Hsp20 Adcs 269 15 min, 30μL 10μL 4,100 10 minateg2 ATEG5 15%10% SDS- (PAGE),40μg/ (PVDF), Western blot (HA) 1 2000( ); 1 5000( ) (ECL), Bio-RAD 18 :1/2 MS 21 DB31 500 T 1 pearleyg- bp

3 2 : Hsp20 Adcs min, 30μL 10μL 4, minateg2 ATEG5 15%10% SDS- (PAGE),40μg/ (PVDF), Western blot (HA) ( ); ( ) (ECL), Bio-RAD 18 :1/2 MS 21 DB T 1 pearleyg- bp ;3(b),ATEG-5(T 3 ) 5F-1 5F-2 ate201 (phosphinothri- 5L-1 5L-2 5G-1 5G-2atB1- atb2- [9] cinacetyltransferase) (bar), PCR 2200bp, T 1 2~3, 5F-1 5F-2 5L-1 5L-2, 7d, 4 5G-1 5G-2 Adcs,, 2 ATEG5 DB31 T 1, bp mg/l 3, Westernblot HA Westernblot : 25 22,4 3d,23 T 3 ATEG- --, 2d, 80% (T 3 )( geneline-strain), 08~1cm 36 1/2 MS (10mg/L ),25 7d, h 23, 3d (1)ATEG2 3/4 2M- ; 3 [4] 2 2S-3, ATEG5 4 :,1/2 5F-1 5F-2 5G-1 5G-2 MS,4 3d,25 23 PCR 7d 45 1h, 3(a),ATEG-2(T 3 ) 2M-1 2M-2 2S- 25, 7d 1 2S-2 2S-3 2M(2M ATEG2T 2 ) ; 3 [5] 2 2 Fig2 Bastaresistanceselectionoftransgenic Athalianaseedlings, DNA,atB1- atb2- PCR 500bp, 2M-1 2M-2 2S-1 2S-2 2S-3 2M Hsp20, ATEG2 Tab1 1 T3 HomozygoteidentificationfortransgenicAthalianaT3generationseedlings Col-0 2M-1 2M-2 2S-1 2S-2 2S-3 5F-1 5F-2 5L-1 5L-2 5G-1 5G :Col-0: ;2M-1 2M-2 2S-1 2S-2 2S-3: ATEG2 ;5F-1 5F-2 5G-1 5G-2 5L-1 5L-2:ATEG5

270 ( ) 2014 M2:DNA Marker2000;M15:DNA Marker15000; 2M:ATEG2 (T2)DNA PCR ;ATEG2 ATEG5: ATEG2 ATEG5 DB31 PCR,23 3d,

PCRidentificationof transgenicathalianaseedlings ATEG-2(T 3 )2M-1 2M-2 2M,2S-1 2S-2 2S-3 ( )(4(a)), ATEG-2M-2 ; ATEG-5(T

6:2S-3;7:2M;8:Col-0;9:5F-1;10:5F-2; 11:5G-1;12:5G-2;13:5L-1;14:5L-2 4, Fig4 Westernblotverification

4 270 ( ) 2014 M2:DNA Marker2000;M15:DNA Marker15000; 2M:ATEG2 (T2)DNA PCR ;ATEG2 ATEG5: ATEG2 ATEG5 DB31 PCR,23 3d, ATEG-2M-2, ATEG-5L-2 ; HSP20, (ADCS) (5(a)) : 45 1h, ATEG-2M-2, ATEG-5L-2, HSP20, ADCS (5(b)) 3 PCR Fig3 PCRidentificationof transgenicathalianaseedlings ATEG-2(T 3 )2M-1 2M-2 2M,2S-1 2S-2 2S-3 ( )(4(a)), ATEG-2M-2 ; ATEG-5(T 3 )5L-2 5G- 2,5F-2 5L-1 5G-1,5F-1 (4(b)), ATEG- 5L-2 ATEG-5G-2, 3 :1:Col-0;2:2M-1;3:2M-2;4:2S-1;5:2S-2; 6:2S-3;7:2M;8:Col-0;9:5F-1;10:5F-2; 11:5G-1;12:5G-2;13:5L-1;14:5L-2 4, Fig4 Westernblotverification oftransgenicathalianaseedlings,, 45 1h, 25 5 Fig5 Thermotolerancephenotypeidentification oftransgenicathalianaseedlings 2, [4] Ⅰ( : 8213 : 02 ) ; Ⅱ( : 02 : 02 ), 02,,

2 : Hsp20 Adcs 271, I I AdcsHsp20,, 002% ( ), T 3 ATEG2ATEG5 PCR Westernblot ATEG2M-2ATEG5L-2 (heatshockproteins,hsp), (heatstressproteins),,, HSP100 HSP90 HSP70 HSP60 HSP,, [7,10]

5 2 : Hsp20 Adcs 271, I I AdcsHsp20,, 002% ( ), T 3 ATEG2ATEG5 PCR Westernblot ATEG2M-2ATEG5L-2 (heatshockproteins,hsp), (heatstressproteins),,, HSP100 HSP90 HSP70 HSP60 HSP,, [7,10] :1:Col-0;2:2M-2;3:5L-2;4:Col-0;5:2M-2;6:5L-2 1~3:NC ;4~6:HS, Hsp20,Hsp20 6 Adcs ( Fig6 Rootsandhypocotylselongationlengthoftransgenic 6(a) 3 NC ) Arabidopsisthalianaafterheat-stress Hsp20, Hsp20 ( 6(a) ATEG2 ( 6(b) 3 NC ), 3HS ) Pab ( Adcs )02, ADCS, ATEG5 ( 6(a)(b),, HS ) :1) , ADCS, 23 Adcs 02 ( ADCS, [4],Adcs 25, 30 ADCS,ADCS, );2)ADCS ADC, ( ), Pab (Adcs ) [4] 3 :, : 332, (, (1~8 ), ) Pab ; (45 ), ( : ) [11], (ATEG-5A AT25-L7) 10~, 13mm,,5mm, - 10~13 mm [12] 45 1 ATEG5 h 3d,

6 272 ( ) 2014 ATEG2 5 mm turestress[j]proceedingsofthe NationalAcademyof ( ATEG-5L),,, HSP, : [8] CloughSJ,BentA FFloraldip:asimplifiedmethodfor Agrobacterium-mediatedtransformation of Arabidopsis : [1],, blevectorsforplantfunctionalgenomicsandproteomics [M] :,2003 lecularapproachesingeneticimprovementofcultivated buton mushroom (Agaricusbisporus)[J]Symbiotic Fungi,Berlin Heidelberg,Germany:Springer,2009: [3] RomaineCTransgenic breedingofagaricusbisporus:the nextfrontier[j]actaedulisfungi,2005,12: [12] JabrinS,RavanelS,GambonnetB,etalOne-carbonme- [4] tabolisminplantsregulationoftetrahydrofolatesyn- [D] :,2010 [5] HongS W,VierlingEMutantsofArabidopsisthaliana Sciences,2000,97(8): [2] GoltapehE M,DaneshY,KamalS,etalBiologyandmo- defectiveintheacquisitionoftolerancetohightempera- [6],, AtHs- fa2 [J] :C,2005,35(5): [7] PFsHSP17-1 [J],2011(11): thaliana[j]theplantjournal,1998,16(6): [J]PlantJournal,2006,45(4): [10] Sanmiya K,Suzuki K,Egawa Y,etalMitochondrial [9] EarleyK W,HaagJR,PontesO,etalGateway-compati- smalheat-shockproteinenhancesthermotoleranceinto- baccoplants[j]febsleters,2004,557(1): [11] ScotJ,RébeiléF,FletcherJFolicacidandfolates:the feasibilityfornutritionalenhancementinplantfoods[j] JSciFoodAgric,2000,80(7): thesisduringgerminationandseedlingdevelopment[j] PlantPhysiol,2003,131(3): IdentificationThermotoleranceFunctionofHsp20andAdcs GenesfromAgaricusbisporusbyArabidopsisthaliana DENGLi-xin 1,LUZhong-lei 1,SHEN Yue-mao 2,SONGSi-yang 1* (1SchoolofLifeSciences,XiamenUniversity,Xiamen361102,China; 2SchoolofPharmaceuticalSciences,ShandongUniversity,Jinan250012,China) Abstract: Astwodiferentiatedexpressinggenes,heatshockprotein20 (Hsp20)and4-amino-4-deoxychorismatesynthase(Adcs) weretransformedintowildtypearabidopsisplantsbyagrobacterium tumefaciens-mediatedfloraldip method,afterbastascreen- ing,pcrand WesternblotdetectionindicatedHsp20andAdcsgenehadbeenintegratedintothegenomeofArabidopsisandoverex- pressedtheperformanceofthetransformedplantsandthewildtypeunderheatstresswasanalyzedtheresultsshowedthat,after 1hourstressof45,thehypocotylofHsp20transgenicplantscanrecoverextending,what smore,someseedlingsoftransgenic plantscansurvive;however,likewildtype,thehypocotylofadcstransgenicplantscan trecoverextendingandtheirseedlingscan t surviveconsequently,theexperimentalresultsdisplayedthathsp20genecontributesthermotolerancetoagaricusbisporusdirectly whereasadcsgeneprobablynot Keywords: heatshockprotein20 ;4-amino-4-deoxychorismatesynthase;Arabidopsisthaliana;Agaricusbisporus