1、 2023 21 14 4659 4665 MolecularPlantBreeding,2023,Vol.21,No.14,4659 4665 ResearchReport MeSAUR1 1,2 4 1,3 1,2 1,2 3 3 1,2*1,570228;2,570228;3,571101;4,571100*,MeAGPS1a AGPase MeSAUR1 MeAGPS1a。MeSAUR1 MeAGPS1a。MeSAUR1 pGBKT7 MeSAUR1 cDNA MeSAUR1 31。MePP2C MeSAUR1 MeSAUR1。;MeSAUR1;ScreeningandIdentifi
2、cationofMeSAUR1InteractingProteininCassavaZheng Wanru1,2Ding Kaixuan4Lu Xiaohua1,3Wang Rui1,2Chen Yinhua1,2Yao Yuan3Chen Xin3GengMengting1,2*1 College of Tropical Crops,Hainan University,Haikou,570228;2 Hainan Key Laboratory for Sustainable Utilization of Tropical Bioresources,Col-legeofTropicalCrop
3、s,HainanUniversity,Haikou,570228;3InstituteofTropicalBiotechnology,ChineseAcademyofTropical Agricultural Sciences,Haikou,571101;4HainanSeedStation,Haikou,571100*Correspondingauthor,DOI:10.13271/j.mpb.021.004659Abstract The small subunit encoded by MeAGPS1a gene is the catalytic center of AGPase in c
4、assava,andAGPase is a key enzyme in starch synthesis.Previous studies have found that MeSAUR1 transcription factor canbindtothepromoterof MeAGPS1a geneandregulateitsexpression.ScreeningtheinteractingproteinofMeSAUR1transcription factor by yeast two-hybrid technology can further analyze the molecular
5、 regulatory network ofMeAGPS1a gene expression.The yeast two-hybrid vector pGBKT7 MeSAUR1 of MeSAUR1 was constructed inthisstudy,and the candidate interactingproteinsofMeSAUR1 were screened from cassava cDNA librarybyyeasttwo-hybrid technology,and 31 positive clones were screened.The interaction bet
6、ween candidate interacting pro-tein MePP2C and MeSAUR1 was verified byyeast two-hybrid point-to-point experiment.The results ofthis studyarebeneficialtorevealtheinteractingnetworkofMeSAUR1proteinregulatingstarchsynthesisincassava.Keywords Manihot esculenta;MeSAUR1;Yeasttwo-hybrid;Interactprotein(No.
7、31960058)(No.Hys2020 140)Zheng W.R.,Ding K.X.,Lu X.H.,Wang R.,Chen Y.H.,Yao Y.,Chen X.,and Geng M.T.,2023,Screening and iden-tification of MeSAUR1 interacting protein in cassava,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),21(14):4659-4665.(,2023,MeSAUR1,21(14):4659-4665.)(Manihot esculenta Crantz)
8、(Eu-phorbiaceae)、(Zidenga et al.,2012)、MolecularPlantBreeding(Ceballos et al.,2004)(Zhangetal.,2010)。AGPase G 1 P ATP ADPG PPi ADPG(Lee et al.,2007)。MeAGPS1a AGPase。MeSAUR1 MeAGPS1a 201400bp MeAGPS1a(Ma etal.,2017)。(SantnerandEstelle,2009)SAUR(SmallAuxin-UpRNA)。Spartz(2012)SAUR。MeSAUR1 MeAGPS1a MeSA
9、UR1 MeSAUR1。1 1.1 pGBKT7 MeSAUR1 SC8 cDNA RT-PCR MeSAUR1。(318bp)。MeSAUR1 pGBKT7。7 PCR(1)pGBKT7 MeSAUR1。1.2 pGBKT7 MeSAUR1 pGBKT7 MeSAUR1(:BD-MeSAUR1)pGADT7(AD)AH109。SD/TL BD-MeSAUR1 AD(AD-T+BD-p53)SD/TLHA SD/TLHA+X-Gal BD-MeSAUR1 AD(AD-T+BD-lam)。MeSAUR1 cDNA(2)。1.3 MeSAUR1 BD-MeSAUR1 AH109。SD/THL+5m
10、mol/L3 AT 31。SD/TLHA+X-Gal PCR。PCR 5001000bp(3)。PCR phytozome BLAST Me-SAUR1 31(1)。2C(MePP2C)。1.4 MePP2C phytozome MePP2C(:Manes.04G071500)SC8 cDNA RT-PCR MePP2C(4)。MePP2C 1 PCR:M:DL2000DNAMarker;17:Figure1ColonyPCRscreeningforpositiveclonesNote:M:DL2000DNAMarker;17:Singlecolony 2pGBKT7 MeSAUR1:AD-T
11、+BD-p53:;AD-T+BD-lam:;1,1 10 1,1 10 2,1 10 3;TL:;TLHA:Figure2pGBKT7 MeSAUR1 selfactivationtestNote:AD-T+BD-p53:Growth condition of Positive control;AD-T+BD-lam:Growth condition of Negative control;1,1 10 1,1 10 2,1 10 3were the original solution and different dilutionmultiplesofyeastsolution,the sam
12、e as below;TL:Two deficientmedium:TLHA:Fourdeficientmedium4660 1194bp 397。1.5 MePP2C ExPASy(https:/web.expasy.org/protparam/)MePP2C C1955H3099N573O582S11 397 44295.26Da 6 220 pI 9.13。MePP2C Ser(9.8%)Leu(9.1%)Gly(7.8%)Val(7.8%)Arg(6.8%)Ala(6.3%)Gln(5.8%)Ile(5.5%)Lys(5.3%)Glu(5.3%)Asp(5.0%)。MePP2C(Asp
13、+Glu)41(Arg+Lys)48 85.92(II)42.32。MePP2C 0.376(,)(5)。1.6 MePP2C SOPMA MePP2C。MePP2C(Alpha helix)34.51%137(Random coil)44.33%176(Beta turn)5.54%22(Extended strand)15.62%62(6)。1.7 MePP2C MePP2C pGBKT7 BD-MePP2C。BD-MePP2C AD AH109。SD/TL(AD+BD-MePP2C)(AD-T+3 PCR:M:DL2000DNAMarker;+:MeSAUR1;:(ddH2O);131:
14、Figure3PCRidentificationofinteractingyeastcoloniesNote:M:DL2000 DNA Marker;+:Positive control with Me-SAUR1 plasmid as template;:Negative control(ddH2O);131:Positiveclone 4RT-PCR MePP2C:M:DL2000DNAMarker;1,2:MePP2C Figure4RT-PCRamplificationoftheMePP2C geneNote:M:DL2000DNAMarker;1,2:MePP2C gene 6 Me
15、PP2C Figure 6 Protein secondary structure analysis of MePP2C fromcassava 5 MePP2C:;Figure5HydrophilicanalysisofMePP2CproteinfromcassavaNote:Negative values indicate hydrophilic;Positive values indi-catehydrophobicity MeSAUR1 ScreeningandIdentificationofMeSAUR1InteractingProteininCassava4661 Molecula
16、rPlantBreedingBD-p53,AD-T+BD-lam)SD/TLHA SD/TLHA+X-Gal。MePP2C(7)。1.8 MeSAUR1 MePP2C MeSAUR1。AD-MeSAUR1+BD-Me-PP2C SD/TL、SD/TLHA SD/TLHA+X-Gal。SD/TLHA SD/TLHA+X-Gal。MePP2C MeSAUR1(8)。2。1MeSAUR1 Table1TheinformationofcandidateproteinsinteractwithMeSAUR1 No.123456789101112131415161718192021222324252627
17、28293031 FunctionalannotationPredictedyippee-typezinc-bindingproteinCalcium-bindingproteinCML24 relatedProteinphosphatase2C46 related(MePP2C)Elongationfactor1 alphaPhotosystemIsubunitV(psaG)Xyloglucanglycosyltransferase4RNA-bindingproteinrelated/subfamilyunnamedShikimatekinaseInactiveshikimicacidkin
18、aselike1,chloroplastic-relatedUDP-glycosyltransferase75B2D-cysteinedesulfhydraseOrnithineaminotransferase/oxo-acidtransaminaseBagfamilymolecularchaperoneregulator2 related26SproteassomeregulatorysubunitT2Peptidyl-prolyl cis-trans isomeraseFKBP17 2DNA-directedRNApolymeraseIIIsubunitRPC4SmallEDRK-rich
19、factor1Tetrapyrrole-bindingprotein,chloroplasticPeptidyl-prolyl cis-trans isomerase/subfamilynotnamedLargesubunitribosomalproteinL26e(RP-L26e,RPL26)Probablelipidtransfer(LTP_2)Microtubule-associatedprotein,RP/EBfamily(MAPRE)Alcoholdehydrogenase(E1.1.1.1,adh)/class-P(ADH1)Pthr24054:sf32 caseinkinaseI
20、IsubunitalphaWD40 likebetapropellerrepeat(PD40)CTDsmallphosphatase-likeprotein2(CTDSPL2)Acylcarrierprotein/zincfingerprotein593 relatedProbablelipidtransfer(LTP_2)BTB/POZdomain-containgproteindot3 relatedBagfamilymolecularchaperoneregulator2 relatedAuxin-inducedprotein-related GeneIDManes.05G192500M
21、anes.01G116700Manes.04G071500Manes.03G146700Manes.01G006400Manes.S064700Manes.10G116300Manes.11G079700Manes.11G079700Manes.13G056900Manes.12G019600Manes.15G123600Manes.14G029000Manes.13G043200Manes.14G170200Manes.14G037500Manes.15G054000Manes.03G108000Manes.01G161300Manes.12G113600Manes.02G034500Man
22、es.14G076900Manes.18G086800Manes.05G180400Manes.08G075000Manes.12G011700Manes.09G065800Manes.02G034500Manes.12G134700Manes.14G029000Manes.05G1494004662 8:AD-MeSAUR1+BD-MePP2C:pGBKT7 MePP2C pGADT7 MeSAUR1 AH109;AD-T+BD-p53:;AD-T+BD-lam:;1,1 10 1,1 10 2,1 10 3;TL:;TLHA:Figure8Yeasttwo-hybridverificati
23、onNote:AD-MeSAUR1+BD-MePP2C:Growth condition of baitvector pGBKT7 MePP2C and pGADT7 MeSAUR1 co-transfor-mation yeast AH109;AD-T+BD-p53:Growth condition of posi-tive control;AD-T+BD-lam:Growth condition of negative con-trol;1,1 10 1,1 10 2,1 10 3were the original solution and dif-ferent dilution mult
24、iples of yeast solution;The same as below;TL:Twodeficientmedium:TLHA:Fourdeficientmedium 7MePP2C:AD+BD-MePP2C:pGBKT7 MePP2C pGADT7 AH109;AD-T+BD-p53:;AD-T+BD-lam:;1,1 10 1,1 10 2,1 10 3;TL:;TLHA:Figure7Self-activationdetectionofMePP2CNote:AD+BD-MePP2C:Growth condition of bait vector pG-BKT7 MePP2C a
25、nd pGADT7 co-transformation yeast AH109;AD-T+BD-p53:Growth condition of positive control;AD-T+BD-lam:Growth condition of negative control;1,1 10 1,1 10 2,1 10 3were the original solution and different dilution multiplesof yeast solution;TL:Two deficient medium:TLHA:Four defi-cientmedium。MeSAUR1 MePP
26、2C MeSAUR1 MePP2C。2C(PP2C)/。SAUR 2C AtPP2C-D2、AtPP2C-D5、AtPP2C-D5 H-ATP AHA1/2 SAURs PP2C.D1 AtSARK(Renetal.,2018)。SAUR PP2C-D PMH+-ATPase(Sunetal.,2016)。PP2C-D1 AHA2 PMH-ATPase(Spartzetal.,2014)。SAUR PP2C(StortenbekerandBe-mer,2019)。MeSAUR1 MePP2C MeAGPS1a。、MeSAUR1 MePP2C MeAGPS1a MePP2C。MeSAUR1。3
27、3.1(SD)TaKaRa(Nico-tiana benthamiana)、X-Gal、Trans5、AH109、GV3101(pSoup-p19)、pGBKT7 lam、pGADT7 T、pGBKT7、pGADT7、pGBKT7 p53、cDNA。3.2 pGBKT7 SAUR1 pGBKT7(BD)MeSAUR1 Trans5 PCR BD-Me-SAUR1。BD-MeSAUR1 pGADT7 AH109 SD/TL PCR AH109/pGBKT7 MeSAUR1。SD/TL 10 1、10 2、10 3 SD/TL、SD/TLHA、SD/TLHA+X-Gal 28 24 d(AD+BD
28、-SAUR1)(AD-T+BD-p53、(AD-T+BD-lam)MeSAUR1 ScreeningandIdentificationofMeSAUR1InteractingProteininCassava4663 MolecularPlantBreeding SD/TL、SD/TLHA、SD/TLHA+X-Gal SAUR1。3.3 MeSAUR1 SD/-Trp pGBKT7 MeSAUR1 AH109 50 mLSD/-Trp 28 OD 600=0.60.8 ddH2O 0.1mol/LLiAc AH109。SD/THL+5mmol/L3 AT 28 30 d。1 mLSD/TL dd
29、H2O SD/TL SD/TLHA SD/THL+X-Gal 28 35d。SD/TLHA+X-Gal SD/TL Trans5 LB(Amp+)PCR Phytozome BLAST MeSAUR1。3.4 MePP2C phytozome MePP2C Primer Premier 5.0 MePP2C。SC8 cDNA RT-PCR MePP2C。1%、MePP2C。ExPASy MePP2C SOPMA MePP2C(2)。3.5 37 pGADT7(Eco R,Bam H)、pGBKT7(Eco R,Sal)。T4 DNA MePP2C MeSAUR1 pGBKT7、pGADT7。p
30、GBKT7 MePP2C AD AH109 SD/TL 24d PCR SD/TL 10 1、10 2、10 3 SD/TL SD/TLHA SD/TLHA+X-Gal。pGBKT7 MePP2C pGADT7 MeSAUR1 AH109 SD/TL 10 1、10 2、10 3 SD/TL SD/TLHA SD/TLHA+X-Gal AD-T+BD-p53、AD-T+BD-lam 28 35d。、。(No.31960058)(No.Hys2020 140)。Ceballos H.,Iglesias C.A.,P rez A.C.,and Dixon A.G.O.,2004,Cassava b
31、reeding:opportunities and challenges,Plant Mol.Biol.,56(4):503-516.Lee S.K.,Hwang S.K.,Han M.,Eom J.S.,Kang H.G.,Han Y.,Choi S.B.,Cho M.H.,Bhoo S.H.,An G.,Hahn T.R.,OkitaT.W.,andJeonJ.S.,2007,IdentificationoftheADP-glucosepyrophosphorylase isoforms essential for starch synthesis inthe leaf and seed
32、endosperm of rice(Oryza sativa L.),PlantMol.Biol.,65(4):531-546.Ma P.A.,Chen X.,Liu C.,Meng Y.H.,Xia Z.Q.,Zeng C.Y.,Lu C.,and Wang W.Q.,2017,MeSAUR1 encoded by a 2 Table2Primerdesign PrimernamepGBKT7 MeSAUR1pGBKT7 MePP2C(5 3)Primersequence(5 3)F:GGAATTCCATATGATGGCGATCAGGAAATCAACCR:CGGAATTCTCATCTGAGC
33、ATGGATGTTAGAGF:GGAATTCCATATGATGTTATCTGGGTTAATGR:CGGAATTCTCAGGTACCGCCAGCTTC4664SMALLAUXIN-UP RNA gene acts as a transcription reg-ulator to positively regulate ADP-glucose pyrophosphorylase small subunit1 geneincassava,Front.PlantSci.,8:1315.Ren H.,Park M.Y.,Spartz A.K.,Wong J.H.,and Gray W.M.,2018,A
34、 subset of plasma membrane-localized PP2C.Dphosphatases negatively regulate SAUR-mediated cell ex-pansioninArabidopsis,PLoSGenet.,14(6):e1007455.Santner A.,and Estelle M.,2009,Recent advances and emergingtrends in plant hormone signalling,Nature,459(7250):1071-1078.SpartzA.K.,Lee S.H.,WengerJ.P.,Gon
35、zalezN.,Itoh H.,Inz D.,Peer W.A.,Murphy A.S.,Overvoorde P.J.,and Gray W.M.,2012,The SAUR19 subfamily of SMALL AUXIN UP RNA genespromotecellexpansion,PlantJ.,70(6):978-990.Spartz A.K.,Ren H.,Park M.Y.,Grandt K.N.,Lee S.H.,MurphyA.S.,SussmanM.R.,Overvoorde P.J.,and GrayW.M.,2014,SAUR inhibition of PP2
36、C-D phosphatases activates plasmamembrane H+-ATPases to promote cell expansion in Ara-bidopsis,PlantCell,26(5):2129-2142.Stortenbeker N.,and Bemer M.,2019,The SAUR gene family:the plants toolbox for adaptation of growth and develop-ment,J.Exp.Bot.,70(1):17-27.Sun N.,Wang J.J.,Gao Z.X.,Dong J.,Chen H
37、.,Terzaghi W.,Wei N.,Deng X.W.,and Chen H.D.,2016,ArabidopsisSAURs are critical for differential light regulation of the de-velopment of various organs,Proc.Natl.Acad.Sci.USA,113(21):6071-6076.Zhang P.,Wang W.Q.,Zhang G.L.,Kaminek M.,Dobrev P.,XuJ.,andGruissemW.,2010,Senescence-inducible expressiono
38、fisopentenyltransferaseextendsleaflife,increasesdroughtstress resistance and alters cytokinin metabolism in cassava,J.Integr.PlantBiol.,52(7):653-669.ZidengaT.,Leyva-GuerreroE.,MoonH.,SiritungaD.,and SayreS.R.,2012,Extendingcassavarootshelflife via reduction ofreactive oxygen species production,Plant Physiol.,159(4):1396-1407.MeSAUR1 ScreeningandIdentificationofMeSAUR1InteractingProteininCassava4665
