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Ukufunyanwa kunye nokusetyenziswa okuluncedo kweemveliso zendalo kunokunceda ukuphucula ubomi bomntu.Imichiza ethintela ukukhula kwezityalo isetyenziswa kakhulu njengemichiza yokutshabalalisa ukhula.Ngenxa yesidingo sokusebenzisa iintlobo ezahlukeneyo zemichiza yokutshabalalisa ukhula, kukho imfuneko yokuchonga iikhompawundi ezineendlela ezintsha zokusebenza.Kule sifundo, sifumene i-novel N -alkoxypyrrole compound, i-coumamonamide, esuka kwi-Streptomyces werraensis MK493-CF1 kwaye yaseka inkqubo epheleleyo yokudibanisa.Ngovavanyo lomsebenzi webhayoloji, sifumanise ukuba i-urs-monoamic acid sisixhobo esiphakathi sokwenziwa kwe-urs-monoamide kwaye sinokubakho.inhibitor yokukhula kwezityalo.Ukongeza, siye savelisa iintlobo ezahlukeneyo ze-urbenonic acid derivatives, kubandakanywa ne-urbenyloxy derivative (UDA), enomsebenzi ophezulu we-herbicidal ngaphandle kokuchaphazela kakubi ukukhula kweeseli ze-HeLa.Kwakhona sifumene ukuba i-urmotonic acid derivatives iphazamisa i-microtubules yezityalo;ukongeza, i-KAND ichaphazela i-actin filaments kwaye ibangela ukufa kweeseli;Ezi ziphumo zininzi ziyahluka kwezo zaziwa kwi-microtubule inhibitors kwaye zicebisa indlela entsha yokwenza i-ursonic acid, emele inzuzo ebalulekileyo ekuphuhliseni ama-herbicides amatsha.
Ukufunyanwa kunye nokusetyenziswa okusebenzayo kweemveliso zendalo ezinenzuzo kunye nezinto eziphuma kuzo yindlela yokuphucula umgangatho wobomi bomntu.I-metabolites yesibini eveliswe yi-microorganisms, izityalo kunye nezinambuzane ziye zakhokelela ekuphuculeni okukhulu kweyeza kunye nezolimo.Amachiza amaninzi okubulala iintsholongwane kunye ne-anti-leukemia aphuhliswe kwiimveliso zendalo.Ukongeza, iintlobo ezahlukeneyo zeizibulali zinambuzane, imichiza yokungunda kunye nemichiza yokutshabalalisa ukhula itsalwa kwezi mveliso zendalo ukuze zisetyenziswe kwezolimo.Ngokukodwa, imichiza yokutshabalalisa ukhula zizixhobo ezibalulekileyo zokwandisa izivuno zezityalo kulimo lwangoku, kwaye iintlobo ngeentlobo zeekhompawundi sele zisetyenziselwa urhwebo.Iinkqubo ezininzi zeselula kwizityalo, ezifana ne-photosynthesis, i-amino acid metabolism, i-cell wall synthesis, ukulawulwa kwe-mitosis, ukubonakaliswa kwe-phytohormone, okanye i-protein synthesis, zibhekwa njengeethagethi eziqhelekileyo ze-herbicides.Imichiza ethintela umsebenzi we-microtubule yiklasi eqhelekileyo ye-herbicides echaphazela ukukhula kwezityalo ngokuchaphazela i-mitotic regulation2.
I-Microtubules zizinto ze-cytoskeleton kwaye zigcinwe ngokubanzi kwiiseli ze-eukaryotic.I-tubulin heterodimer iqukethe i-α-tubulin kunye ne-β-tubulin eyenza i-microtubule protofilaments, kunye ne-13 protofilaments eyenza isakhiwo se-cylindrical.I-Microtubules idlala iindima ezininzi kwiiseli zezityalo, kubandakanywa nokumisela ukuma kweeseli, ukuhlukana kweeseli, kunye ne-intracellular transport3,4.Iiseli zezityalo ziqukethe i-microtubules phantsi kwe-membrane ye-plasma ye-interphase, kwaye ezi zibizwa ngokuba yi-cortical microtubules zicatshangelwa ukuba zilawule umbutho we-cellulose microfibrils ngokulawulwa kwe-cellulose synthase complexes4,5.I-Cortical microtubules yeeseli ze-epidermal yengcambu, ekhoyo kwindawo yokwandiswa ngokukhawuleza kwencam yengcambu, ibekwe ecaleni, kwaye i-cellulose microfibers ilandela ezi microtubules kwaye inciphisa icala lokwandiswa kweeseli, ngaloo ndlela ikhuthaza ukwanda kweseli ye-anisotropic.Ngoko ke, umsebenzi we-microtubule uhambelana ngokusondeleyo ne-morphology yezityalo.I-amino acid substitution in genes encoding tubulin ibangela i-skew ye-cortical microtubule arrays kunye nokukhula kwasekhohlo okanye ngasekunene kwi-Arabidopsis 6,7.Ngokufanayo, ukuguqulwa kweeprotheni ezihambelana ne-microtubule ezilawula i-microtubule dynamics nazo zingakhokelela ekukhuleni kweengcambu ezigqwesileyo8,9,10,11,12,13.Ukongezelela, unyango nge-microtubule-disrupting herbicides ezifana ne-disopyramide, eyaziwa ngokuba yi-pretilachlor, nayo ibangela ukukhula kweengcambu ze-oblique ezisekhohlo14.Ezi datha zibonisa ukuba ukulawulwa ngokuchanekileyo komsebenzi we-microtubule kubalulekile ekunqumeni indlela yokukhula kwezityalo.
Iindidi ezahlukeneyo ze-microtubule inhibitors zifunyenwe, kwaye la machiza enze igalelo elibalulekileyo kuphando lwe-cytoskeletal, kunye nezolimo kunye neyeza2.Ngokukodwa, i-oryzalin, i-dinitroaniline compounds, i-disopyramide, i-benzamide-related compounds, kunye ne-analogs yazo inokuthintela umsebenzi we-microtubule kwaye ngaloo ndlela inqande ukukhula kwezityalo.Ngoko ke, zisetyenziswa kakhulu njengeyeza lokutshabalalisa ukhula.Nangona kunjalo, ekubeni ii-microtubules ziyinxalenye ebalulekileyo yeeseli zezityalo kunye nezilwanyana, ezininzi i-microtubule inhibitors ziyi-cytotoxic kuzo zombini iindidi zeeseli.Ke ngoko, ngaphandle koncedo lwabo oluvunyiweyo njengeyeza lokutshabalalisa ukhula, inani elincinci lee-antimicrotubule agents lisetyenziselwa iinjongo ezisebenzayo.
I-Streptomyces luhlobo lwentsapho ye-Streptomyces, equka i-aerobic, i-gram-positive, ibhaktheriya ye-filamentous kwaye iyaziwa ngokubanzi ngokukwazi ukuvelisa uluhlu olubanzi lwee-metabolites zesibini.Ke ngoko, ithathwa njengenye yezona mithombo zibalulekileyo zemveliso yendalo esebenzayo.Kuphononongo lwangoku, sifumene i-compound entsha ebizwa ngokuba yi-coumamonamide, eyayihlukanisiwe kwi-Streptomyces werraensis MK493-CF1 kunye ne-S. werraensis ISP 5486. Ukusebenzisa uhlalutyo lwe-spectral kunye nohlalutyo olupheleleyo lwe-spectral, isakhiwo se-coumamonamide sibonakaliswe kunye ne-N-alkoxypyrrole skeleton. yayizimisele.udibaniso.I-Ursmonic acid, i-synthetic intermediate ye-ursmonoamide kunye nezinto eziphuma kuyo, yafunyanwa inqanda ukukhula kunye nokuntshula kwesityalo esithandwayo se-Arabidopsis thaliana.Kuphononongo lobudlelwane bomsebenzi wesakhiwo, sifumene ukuba i-compound ene-C9 eguqulelwe kwi-ursonic acid, ebizwa ngokuba yi-nonyloxy derivative ye-ursonic acid (KAND), iphucula kakhulu isiphumo sokuthintela ukukhula kunye nokuhluma.Ngokucacileyo, i-inhibitor yokukhula kwezityalo esanda kufunyanwa ichaphazele ukukhula kwecuba kunye ne-liverwort kwaye yayingekho i-cytotoxic kwiibhaktheriya okanye iiseli ze-HeLa.Ngaphezu koko, ezinye i-urmotonic acid derivatives zibangela i-phenotype yengcambu egqwethekileyo, ebonisa ukuba ezi ziphuma ngokuthe ngqo okanye ngokungathanga zichaphazela i-microtubules.Ngokuhambelana nale ngcamango, ukuqwalasela kwethu i-microtubules ebhalwe mhlawumbi i-immunohistochemically okanye ngeeprotheni ezikhanyayo zibonisa ukuba unyango lwe-KAND lukhupha ii-microtubules.Ukongezelela, unyango nge-kumamotonic acid derivatives luphazamise i-actin microfilaments.Ke, sifumene inhibitor entsha yokukhula kwezityalo enendlela eyodwa yokusebenza ibandakanya ukutshatyalaliswa kwe-cytoskeleton.
Uxinzelelo lwe-MK493-CF1 lwabekwa lodwa kumhlaba waseShinagawa-ku, eTokyo.Uxinzelelo lwe-MK493-CF1 lwenze i-stromal mycelium enesebe elihle.Ukulandelelana kwenxalenye ye-16S ribosomal RNA gene (1422 bp) yamiselwa.Olu hlobo lufana kakhulu no-S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: uhlobo oluqhelekileyo, 99.93%).Ngokusekelwe kwesi siphumo, kwagqitywa ukuba olu hlobo luhlobene ngokusondeleyo nohlobo lwe-S. werraensis.Ke ngoko, siluthiye okwethutyana olu hlobo S. werraensis MK493-CF1.I-S. werraensis ISP 5486T iphinda ivelise i-bioactive compounds efanayo.Ekubeni beluncinci uphando lwangethuba lokufumana iimveliso zendalo kule microorganism, uphando olongezelelweyo lwemichiza lwenziwa.Emva kokulinywa kwe-S. werraensis MK493-CF1 kumbindi webhali ngokuvundiswa kombuso oqinileyo kuma-30°C kangangeentsuku ezili-14, okuphakathi kwatsalwa nge-50% EtOH.I-60 ml yesampuli yomiswa ukufumana i-59.5 mg yesicatshulwa esikrwada.Isicatshulwa esikrwada sasiphantsi kwe-HPLC yesigaba esibuyisela umva ukunika i-N-methoxy-1H-pyrrole-2-carboxamide (1, ebizwa ngokuba yi-coumamonamide, 36.0 mg).Isixa esipheleleyo se-1 simalunga ne-60% yesicatshulwa esikrwada.Ke ngoko, siye sagqiba kwelokuba sifunde ngokweenkcukacha iipropathi ze-kumamotoamide 1.
I-Coumamonamide 1 i-white amorphous powder kunye nesisombululo esiphezulu se-mass spectrometry (HRESIMS) iqinisekisa i-C6H8N2O2 (umzobo 1).I-C2-substituted pyrrole fragment yale khompawundi ibonakaliswe ngu-δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH kwi-spectrum ye-1H NMR 5H: 4. , H-5) kunye no-δH 6.78 (1H, d, J = 2.5 Hz, H-6), kunye ne-13C NMR spectrum ibonisa ubukho bee-athom ze-carbon ze-sp2.Ubukho beqela le-amide kwindawo ye-C2 yavavanywa yi-HMBC yokulungelelanisa ukusuka kwi-C-3 proton ukuya kwi-amide carbonyl carbon kwi-δC 161.1.Ukongezelela, i-1 H kunye ne-13 C NMR iphakamileyo kwi-δH 4.10 (3H, S) kunye ne-δC 68.3 ibonisa ubukho bamaqela e-N-methoxy kwi-molecule.Nangona indawo echanekileyo yeqela le-methoxy yayingekaqinisekiswa kusetyenziswa uhlalutyo lwe-spectroscopic olufana ne-spektroscopy eyandisiweyo kunye ne-nuclear Overhauser abbreviation (NOEDF), i-N-methoxy-1H-pyrrole-2-carboxamide ibe yikhompawundi yomviwa wokuqala.
Ukumisela isakhiwo esichanekileyo se-1, i-synthesis epheleleyo yenziwa (umzobo 2a).Ukunyangwa kwe-2-aminopyridine 2 ekhoyo ngokurhweba nge-m-CPBA kubangele i-N-oxide 3 ehambelanayo kwisivuno sobungakanani.Emva kwe-2-aminoazidation ye-2, i-cyclocondensation reaction echazwe ngu-Abramovich yenziwa kwi-benzene kwi-90 ° C ukuze ifumane i-1-hydroxy-1H-pyrrole-2-carbonitrile efunwayo ye-5 ngamagremu.Isantya 60% (izigaba ezibini).15,16.I-Methylation kunye ne-hydrolysis ye-4 emva koko yanika i-1-methoxy-1H-pyrrole-2-carboxylic acid (ebizwa ngokuba yi "cumotonic acid", 6) kwisivuno esihle (70%, amanyathelo amabini).Ekugqibeleni, i-amidation nge-acid chloride ephakathi kwe-6 usebenzisa i-ammonia enamanzi yanika i-Kumamoto amide 1 kwi-98% yesivuno.Yonke idatha ye-spectral ye-synthesized 1 yayifana ne-1 eyodwa, ngoko ke isakhiwo se-1 sinqunywe;
Ukwenziwa ngokubanzi kunye nohlalutyo lomsebenzi webhayoloji we-urbenamide kunye ne-urbenic acid.(a) I-Total synthesis ye-Kumamoto amide.(b) Izithole zeentsuku ezisixhenxe zasendle zohlobo lwase-Arabidopsis Columbia (Col) zikhuliswe kwiipleyiti ze-Murashige kunye ne-Skoog (MS) eziqulethe i-coumamonamide 6 okanye i-coumamonamide 1 kugxininiso olubonisiweyo.Isikali bar = 1 cm.
Okokuqala, siye savavanya imisebenzi yebhayoloji ye-urbenamide kunye neziphakathi zayo ukuze zikwazi ukulungelelanisa ukukhula kwezityalo.Songeze iintlobo ezahlukeneyo ze-ursmonamide 1 okanye i-ursmonic acid 6 kwi-MS agar medium kunye nezithole ze-Arabidopsis thaliana ezikhuliswe kule medium.Olu vavanyo lubonise ukuba ugxininiso oluphezulu (500 μM) lwe-6 luthintele ukukhula kweengcambu (umzobo 2b).Emva koko, siye savelisa iintlobo ezahlukeneyo ze-derivatives ngokubeka indawo ye-N1 ye-6 kwaye senza izifundo zobudlelwane bomsebenzi-umsebenzi kuzo (inkqubo ye-analogue synthesis ichazwe kwiNkxaso yoLwazi (SI)).Izithole ze-Arabidopsis zakhuliswa kumbindi oqulethe i-50 μM ye-ursonic acid derivatives, kwaye ubude beengcambu balinganiswa.njengoko kubonisiwe emfanekisweni.Njengoko kubonisiwe kwiMifanekiso 3a, b, kunye ne-S1, i-coumamo acids inobude obuhlukeneyo bomgca we-alkoxy chain (9, 10, 11, 12, kunye ne-13) okanye i-alkoxy chain ezinkulu (15, 16, kunye ne-17) kwindawo ye-N1.Izinto eziphuma kuzo zibonise uthintelo olukhulu lokukhula kweengcambu.Ukongeza, sifumene ukuba ukusetyenziswa kwe-200 μM 10, 11, okanye i-17 inqanda ukuhluma (iiFig. 3c kunye ne-S2).
Uphononongo lobudlelwane bomsebenzi we-Kumamoto amide kunye neekhompawundi ezinxulumeneyo.(a) Ulwakhiwo kunye nenkqubo yokudityaniswa kwee-analogu.(b) Ubungakanani beengcambu ubude beentsuku ezi-7 zezithole ezikhuliswe kwi-MS medium kunye okanye ngaphandle kwe-50 μM ye-coumamonamide derivatives.Iinkwenkwezi zibonisa umahluko omkhulu ngonyango lwe-sham (uvavanyo lwe-t, iphe<0.05).n >18. Idatha iboniswa njengentsingiselo ± SD.nt ithetha "ayivavanywanga" kuba ngaphezu kwe-50% yembewu ayizange intshule.(c) Ubungakanani besantya sokuntshula kwembewu enyangweyo efukanywe iintsuku ezisi-7 kwi-MS medium kunye okanye ngaphandle kwama-200 μM coumamonamide kunye neekhompawundi ezinxulumeneyo.Iinkwenkwezi zibonisa ukungafani okuphawulekayo ngonyango lwe-sham (uvavanyo lwe-chi-square).n=96.
Okubangel 'umdla kukuba, ukongezwa kwamatyathanga e-alkyl emide kune-C9 kuncitshiswe umsebenzi wokuthintela, ecebisa ukuba iikhompawundi ezinxulumene ne-komamotoic acid zifuna amatyathanga asecaleni obungakanani obuthile ukubonisa umsebenzi wabo webhayoloji.
Ngenxa yokuba uhlalutyo lobudlelwane besakhiwo-umsebenzi lubonise ukuba i-C9 yatshintshwa ibe yi-ursonic acid kunye ne-nonyloxy derivative ye-ursonic acid (emva koku kubhekiselwa kuyo njenge-KAND 11) yayiyeyona inhibitor yokukhula kwezityalo esebenzayo, senze i-KAND 11. Unyango lwe-Arabidopsis. nge 50 μM KAND 11 phantse ithintele ngokupheleleyo ukuntshula, kanti iqondo elisezantsi (40, 30, 20, okanye 10 μM) ye KAND 11 yanqanda ukukhula kweengcambu ngendlela exhomekeke kwithamo (Fig. 4a, b).Ukuvavanya ukuba ingaba i-KAND 11 iyakuchaphazela na ukusebenza kweengcambu ze-meristem, siye savavanya iingcambu ze-meristems ezinepropidium iodide (PI) kunye nobungakanani bendawo yemeristem.Ubungakanani bemeristem yezithole ezikhuliswe kumbindi oqulathe i-25 μM KAND-11 yayiyi-151.1 ± 32.5 μm, ngelixa ubungakanani bemeristem yezithole ezikhuliswe kwindawo yolawulo equlethe i-DMSO yayingama-264.7 ± 30.8 μm (Fig. 4c, d) , ebonisa ukuba i-KAND-11 ibuyisela umsebenzi weselula.ukusasazeka.Ingcambu meristem.Ngokuhambelana nale nto, unyango lwe-KAND 11 lunciphise inani le-cell division marker CDKB2; 1p:: CDKB2; i-1-GUS isignali kwi-root meristem (Umfanekiso 4e) 17.Ezi ziphumo zibonisa ukuba i-KAND 11 inqanda ukukhula kweengcambu ngokunciphisa umsebenzi wokwandisa iiseli.
Uhlalutyo lwempembelelo yokuthintela i-urbenonic acid derivatives (i-urbenyloxy derivatives) ekukhuleni.(a) Izithole zeCold ezineentsuku ezisi-7 ezikhuliswe kwiipleyiti zeMS ezinophawu lweKAND 11. Isikali sebha = 1 cm.(b) Ubungakanani bobude beengcambu.Iileta zibonisa umahluko omkhulu (uvavanyo lwe-Tukey HSD, iphe<0.05).n >16. Idatha iboniswa njengentsingiselo ± SD.(c) I-Confocal microscopy yepropidium iodide-stained wild-type Iingcambu zeCol ezikhule kwiipleyiti zeMS ezino okanye ngaphandle kwama-25 μM KAND 11. Izibiyeli ezimhlophe zibonisa ukumila kwengcambu.Ibar yesikali = 100 µm.(d) Ubungakanani bobungakanani bengcambu yemeristem (n = 10 ukuya kwi-11).Umahluko ngokwamanani wamiselwa kusetyenziswa uvavanyo-t (iphe<0.05).Imivalo imele ubungakanani obuqhelekileyo bemeristem.(e) Umahluko wongenelelo olwahlukileyo (DIC) imakroskopu yengcambu yemeristem equlathe ulwakhiwo lwe-CDKB2;1pro: CDKB2;I-1-GUS idyojwe kwaye inebala kwizithole ezineentsuku ezi-5 ubudala ezikhuliswe kwiipleyiti zeMS ezinovavanyo okanye ngaphandle kwama-25 µM KAND.
I-phytotoxicity ye-KAND 11 yavavanywa ngakumbi kusetyenziswa esinye isityalo se-dicotyledonous, icuba (i-Nicotiana tabacum), kunye neyona nto ibalulekileyo yesityalo somhlaba, i-liverwort (i-Marchantia polymorpha).Njengakwimeko ye-Arabidopsis, izithole ze-SR-1 zecuba ezikhule phakathi eziqulathe i-25 μM KAND 11 zivelise iingcambu ezimfutshane (Fig. 5a).Ukongeza, iimbewu ezingama-40 kwezingama-48 zantshula kwiipleyiti ezine-200 μM KAND 11, ngelixa zonke iimbewu ezingama-48 zantshula kumajelo aphathwa kakubi, ebonisa ukuba ugxininiso oluphezulu lwe-KAND lwalubalulekile (p.< 0.05;chi test -square) yanqanda ukuntshula kwecuba.(Umfanekiso 5b).Ukongezelela, i-concentration ye-KAND 11 evimbela ukukhula kwebhaktheriya kwi-liverwort yayifana nokugxininiswa okusebenzayo kwi-Arabidopsis (Umfanekiso 5c).Ezi ziphumo zibonisa ukuba i-KAND 11 inokuthintela ukukhula kwezityalo ezahlukeneyo.Emva koko siphanda i-cytotoxicity enokwenzeka ye-bear monoamide-related compounds kwezinye izinto eziphilayo, ezizezi iiseli ze-HeLa zabantu kunye ne-Escherichia coli strain DH5α, njengabameli bezilwanyana eziphezulu kunye neeseli zebhaktheriya, ngokulandelanayo.Kuluhlu lwee-cell proliferation assays, siye sabona ukuba i-coumamonamide 1, i-coumamonamidic acid 6, kunye ne-KAND 11 ayizange ichaphazele ukukhula kweeseli ze-HeLa okanye ze-E. coli ekugxininiseni kwe-100 μM (Umfanekiso 5d, e).
Ukuvinjelwa kokukhula kwe-KAND 11 kwizinto eziphilayo ezingenayo i-Arabidopsis.(a) Izithole zecuba ze-SR-1 ezineeveki ezimbini ubudala zakhuliswa kwiipleyiti zeMS ezibekwe nkqo eziqulathe 25 μM KAND 11. (b) Izithole eziziiveki ezimbini ubudala zohlobo lwasendle lwe-SR-1 zakhuliswa kwindawo ethe tye. Iipleyiti zeMS eziqulathe 200 μM KAND 11. ( c) Iiveki ezimbini ubudala zohlobo lwasendle lweTak-1 liverwort buds ezikhuliswe kwiipleyiti zeGamborg B5 ezinogxininiso olubonisiweyo lwe-KAND 11. Iintolo ezibomvu zibonisa iinkozo eziye zayeka ukukhula kwisithuba seeveki ezimbini zokufukamela. ixesha.(d) Uvavanyo lokwanda kweeseli zeeseli ze-HeLa.Inani leeseli ezisebenzayo lilinganiswa ngamaxesha amisiweyo kusetyenziswa ikiti yokubala iseli 8 (Dojindo).Njengolawulo, iiseli ze-HeLa zaphathwa nge-5 μg / ml actinomycin D (uMthetho D), evimbela ukubhalwa kwe-RNA polymerase kwaye ibangele ukufa kweeseli.Uhlalutyo lwenziwa kathathu.(e) Uvavanyo lokwanda kweeseli ze-E.Ukukhula kwe-E. coli kwahlalutywa ngokulinganisa i-OD600.Njengolawulo, iiseli zaphathwa nge-50 μg / ml ampicillin (Amp), evimbela ukuhlanganiswa kodonga lwe-bacterial cell.Uhlalutyo lwenziwa kathathu.
Ukucacisa indlela yesenzo se-cytotoxicity ebangelwa yi-uramide-related compounds, siphinde sahlalutya i-urbenic acid derivatives kunye nemiphumo ephakathi yokuthintela.njengoko kubonisiwe emfanekisweni.Njengoko kubonisiwe kwiMizobo 2b, 6a, izithole ezikhuliswe kwiipleyiti zeagar eziqulathe ugxininiso oluphezulu (200 μM) yeurmotonic acid 6 zavelisa iingcambu ezimfutshane nezigophe ekhohlo (θ = – 23.7 ± 6.1), kanti izithole ezikhuliswe kwindlela yokulawula, izithole zavelisa phantse iingcambu ezithe tye (θ = – 3.8 ± 7.1).Olu phawu lokukhula kwe-oblique luyaziwa ngenxa yokungasebenzi kakuhle kwe-cortical microtubules14,18.Ngokuhambelana noku kufunyaniswayo, i-microtubule-destabilizing drug disopyramide kunye ne-oryzalin yenze ingcambu efanayo iyancipha phantsi kweemeko zethu zokukhula (Imifanekiso 2b, 6a).Kwangaxeshanye, siye savavanya izinto eziphuma kwi-urmotonic acid saza sakhetha uninzi lwazo oluthi, kwiindawo ezithile, lubangele ukukhula kweengcambu ezigudileyo.I-Compounds 8, 9, kunye ne-15 yatshintsha indlela yokukhula kweengcambu kwi-75 μM, 50 μM, kunye ne-40 μM, ngokulandelanayo, ebonisa ukuba ezi zixhobo ziyakwazi ukuphazamisa ngokufanelekileyo i-microtubules (Umfanekiso 2b, 6a).Siphinde savavanya eyona ursolic acid ephuma kuyo, i-KAND 11, kugxininiso olusezantsi (15 µM) kwaye safumanisa ukuba ukusetyenziswa kwe-KAND 11 kuthintele ukukhula kweengcambu kwaye indlela yokukhula kweengcambu yayingalingani, nangona ithande ukuthambeka ukuya ngasekhohlo. Umzobo C3)..Ngenxa yokuba iindawo eziphakamileyo ze-microtubule-destabilizing drug ngamanye amaxesha zithintela ukukhula kwezityalo kunokuba zibangele ukuthambeka kweengcambu, siye savavanya ukuba kungenzeka ukuba i-KAND 11 ichaphazela i-microtubules ngokujonga i-cortical microtubules kwiiseli ze-epidermal zengcambu.I-Immunohistochemistry isebenzisa i-anti-β-tubulin antibodies kwiiseli ze-epidermal zeengcambu zezithole eziphathwe nge-25 μM KAND 11 ibonise ukunyamalala phantse zonke ii-microtubules ze-cortical kwiiseli ze-epidermal kwindawo yokwandisa (Umfanekiso 6b).Ezi ziphumo zibonisa ukuba i-kumamotonic acid kunye ne-derivatives yayo yenza ngokuthe ngqo okanye ngokungathanga ngqo kwi-microtubules ukuphazamisa kwaye ezi zixhobo ziyi-novel microtubule inhibitors.
I-Ursonic acid kunye ne-derivatives yayo iguqula i-cortical microtubules kwi-Arabidopsis thaliana.(a) I-engile yokuthambekela kwengcambu ilinganiswe kubukho beentlobo ezahlukeneyo ze-urmotonic acid eziphuma kugxininiso olubonisiweyo.Imiphumo yeekhompawundi ezimbini ezaziwa ngokuthintela i-microtubules: i-disopyramide kunye ne-oryzalin nazo zahlaziywa.I-inset ibonisa umgangatho osetyenziswayo ukulinganisa i-engile yokukhula kweengcambu.Iinkwenkwezi zibonisa umahluko omkhulu ngonyango lwe-sham (uvavanyo lwe-t, iphe<0.05).n >19. Isikali bar = 1 cm.(b) I-Cortical microtubules kwiiseli ze-epidermal kwindawo yobude.I-Microtubules kwi-wild-type Arabidopsis Col iingcambu ezikhule kwiiplate ze-MS kunye okanye ngaphandle kwe-25 μM KAND 11 zabonwa nge-immunohistochemical staining usebenzisa i-β-tubulin antibodies eziphambili kunye ne-Alexa Fluor-conjugated antibodies yesibini.Ibar yesikali = 10 µm.(c) Ubume beMitotic yeemicrotubules kwingcambu yemeristem.I-Microtubules yabonwa ngokusebenzisa i-immunohistochemical staining.Izakhiwo zeMitotic, ezibandakanya imimandla yeprophase, i-spindles, kunye ne-phragmoplasts, zibalwe kwimifanekiso edibeneyo.Iintolo zibonisa izakhiwo ze-mitotic microtubule.Iinkwenkwezi zibonisa umahluko omkhulu ngonyango lwe-sham (uvavanyo lwe-t, iphe<0.05).n >9. Isikali sebha = 50 µm.
Nangona i-Ursa inamandla okuphazamisa umsebenzi we-microtubule, indlela yokusebenza kwayo kulindeleke ukuba yahluke kwi-microtubule depolymerizing agents.Ngokomzekelo, ugxininiso oluphezulu lwe-microtubule depolymerizing agents ezifana ne-disopyramide kunye ne-oryzalin zenza ukwanda kwe-anisotropic yeeseli ze-epidermal, kanti i-KAND 11 ayifuni.Ukongezelela, ukusetyenziswa ngokubambisana kwe-KAND 11 kunye ne-disopyramide kubangele impendulo yokukhula kweengcambu ezidibeneyo kunye ne-KAND 11-induced growth inhibition (Fig. S4).Siphinde sahlalutya impendulo ye-hypersensitive disopyramide 1-1 (phs1-1) eguquguqukayo kwi-KAND 11. I-phs1-1 ine-non-canonical tubulin kinase point mutation kwaye ivelise iingcambu ezimfutshane xa iphathwa nge-disopyramide9,20.phs1-1 izithole eziguqukayo ezikhuliswe kwi-agar medium equlethe i-KAND 11 zineengcambu ezimfutshane ezifana nezo zikhuliswe kwi-disopyramid (fig. S5).
Ukongezelela, siye saqwalasela izakhiwo ze-microtubule ze-mitotic, ezifana neendawo zeprophase, i-spindles, kunye ne-phragmoplasts, kwingcambu ye-meristem yezithole eziphathwe nge-KAND 11. Ngokuhambelana nokuqwalaselwa kwe-CDKB2; 1p:: CDKB2; 1-GUS, ukuncipha okukhulu inani le-mitotic microtubules labonwa (Umfanekiso .6c).
Ukubonakalisa i-cytotoxicity ye-KAND 11 kwisisombululo esincinci, siye saphatha icuba BY-2 iiseli zokumiswa nge-KAND 11 kwaye sajonga impendulo yazo.Siqale songeza i-KAND 11 kwiiseli ze-BY-2 ezibonisa i-TagRFP-TUA6, ebhala ngokucacileyo i-microtubules, ukuvavanya umphumo we-KAND 11 kwi-cortical microtubules.Uxinaniso lweCortical microtubule luye lwavavanywa kusetyenziswa uhlalutyo lwemifanekiso, olulinganise ipesenti yeepikseli zecytoskeletal phakathi kweepikseli zecytoplasmic.Iziphumo zovavanyo zibonise ukuba emva konyango nge-50 μM okanye i-100 μM KAND 11 ngeyure ye-1, ukuxinana kwehle kakhulu ukuya ku-0.94 ± 0.74% okanye 0.23 ± 0.28%, ngokulandelanayo, ngelixa ubuninzi beeseli eziphathwa nge-DMSO, zibalelwa kwi-1.63 ± 1.63 % (Umfanekiso 7a).Ezi ziphumo zihambelana nokuqwalaselwa kwi-Arabidopsis ukuba unyango lwe-KAND 11 lubangela i-depolymerization ye-cortical microtubules (Umfanekiso we-6b).Siphinde sahlola umgca we-BY-2 kunye ne-GFP-ABD-ebhalwe i-actin filaments emva konyango kunye noxinzelelo olufanayo lwe-KAND 11 kwaye sabona ukuba unyango lwe-KAND 11 luphazamise i-actin filaments.Unyango nge-50 μM okanye i-100 μM KAND 11 ye-1 h yanciphisa kakhulu ingxinano ye-actin filament ukuya kwi-1.20 ± 0.62% okanye i-0.61 ± 0.26%, ngokulandelelana, kanti ubuninzi beeseli eziphathwa nge-DMSO yi-1.69 ± 0.51% (Fig 0.51).7b).Ezi ziphumo zichasene nemiphumo yepropyzamide, engachaphazeli i-actin filaments, kunye ne-latrunculin B, i-actin depolymerizer engachaphazeli i-microtubules (SI Umfanekiso we-S6).Ukongezelela, unyango nge-coumamonamide 1, i-coumamonamide acid 6, okanye i-KAND 11 ayizange ichaphazele i-microtubules kwiiseli ze-HeLa (i-SI Umfanekiso we-S7).Ke, indlela yokusebenza ye-KAND 11 kukholelwa ukuba yahlukile kuleyo yaziwayo abaphazamisi be-cytoskeleton.Ukongeza, ukujonga kwethu nge-microscopic yeeseli ze-BY-2 eziphathwe nge-KAND 11 zibonakalise ukuqala kokufa kweseli ngexesha lonyango lwe-KAND 11 kwaye yabonisa ukuba inani leeseli ezifileyo eziluhlaza okwesibhakabhaka alizange linyuke kakhulu emva kwe-30 min ye-KAND 11 unyango, ngelixa. emva kwemizuzu engama-90 yonyango kunye ne-50 μM okanye i-100 μM KAND, inani leeseli ezifileyo landa ukuya kwi-43.7% okanye i-80.1%, ngokulandelanayo (umzobo 7c).Idityaniswe kunye, ezi datha zibonisa ukuba inoveli ye-ursolic acid ephuma kwi-KAND 11 sisityalo esithe ngqo se-cytoskeletal inhibitor enendlela yokusebenza eyayingaziwa ngaphambili.
I-KAND ichaphazela i-cortical microtubules, i-actin filaments, kunye nokusebenza kwecuba BY-2 iiseli.(a) Umbono we-cortical microtubules kwiiseli ze-BY-2 phambi kwe-TagRFP-TUA6.Iiseli ze-BY-2 eziphathwe nge-KAND 11 (50 μM okanye i-100 μM) okanye i-DMSO zihlolwe nge-microscopy edibeneyo.I-Cortical microtubule density yabalwa ukusuka kwi-micrographs yeeseli ezizimeleyo ze-25.Iileta zibonisa umahluko omkhulu (uvavanyo lwe-Tukey HSD, iphe<0.05).Ibar yesikali = 10 µm.(b) I-cortical actin filaments kwiiseli ze-BY-2 ezibonwayo phambi kwe-GFP-ABD2.Iiseli ze-BY-2 eziphathwe nge-KAND 11 (50 μM okanye i-100 μM) okanye i-DMSO zihlolwe nge-microscopy edibeneyo.Ubuninzi be-cortical actin filaments bubalwe kwi-micrographs ye-25 iiseli ezizimeleyo.Iileta zibonisa umahluko omkhulu (uvavanyo lwe-Tukey HSD, iphe<0.05).Ibar yesikali = 10 µm.(c) Ukuqwalaselwa kweeseli ezifileyo ze-BY-2 yi-Evans blue staining.Iiseli ze-BY-2 eziphathwe nge-KAND 11 (50 μM okanye i-100 μM) okanye i-DMSO zihlolwe ngemakroskopu ekhanyayo.n=3.Ibar yesikali = 100 µm.
Ukufunyanwa kunye nokusetyenziswa kweemveliso zendalo ezintsha kukhokelele kwinkqubela phambili ebalulekileyo kwiinkalo ezahlukeneyo zobomi bomntu, kubandakanywa amayeza kunye nezolimo.Uphando lwembali luye lwenziwa ukufumana iikhompawundi eziluncedo kwimithombo yendalo.Ngokukodwa, i-actinomycetes iyaziwa ngokuba luncedo njenge-antibiotics ye-antiparasitic ye-nematodes ngenxa yokukwazi ukuvelisa i-metabolites ezahlukeneyo zesibini ezifana ne-avermectin, i-compound ekhokelayo ye-ivermectin kunye ne-bleomycin kunye ne-derivatives yayo, esetyenziswa ngamayeza njenge-anticancer agent21,22.Ngokukwanjalo, iintlobo ngeentlobo zemixube ye-herbicidal zifunyenwe kwi-actinomycetes, ezinye zazo sele zisetyenziselwa urhwebo1,23.Ke ngoko, uhlalutyo lwe-actinomycete metabolites ukwahlula iimveliso zendalo kunye nezinto ezifunwayo zebhayoloji kuthathwa njengesicwangciso esisebenzayo.Kule sifundo, sifumene i-compound entsha, i-coumamonamide, esuka kwi-S. werraensis kwaye yayenza ngempumelelo.Iasidi ye-Ursonic yinto ephakathi yokwenziwa ye-urbenamide kunye nezinto eziphuma kuyo.Inokubangela ukugoba kweengcambu, ibonise umsebenzi ophakathi ukuya kowomeleleyo wokutshabalalisa ukhula, kwaye yonakalise ngokuthe ngqo okanye ngokungathanga ngqo ii-microtubules zezityalo.Nangona kunjalo, indlela yokusebenza ye-urmotonic acid inokwahluka kuleyo ye-microtubule inhibitors ekhoyo, ekubeni i-KAND 11 iphinda iphazamise i-actin filaments kwaye ibangele ukufa kweeseli, iphakamisa indlela yokulawula apho i-urmotonic acid kunye ne-derivatives yayo ichaphazela uluhlu olubanzi lwezakhiwo ze-cytoskeletal..
Ukucaciswa okungakumbi kwe-urbenonic acid kuya kunceda ukuqonda ngcono indlela yokusebenza kwe-urbenonic acid.Ngokukodwa, injongo elandelayo kukuvavanya amandla e-ursonic acid ukubopha kwii-microtubules ezincitshisiweyo ukugqiba ukuba i-ursonic acid kunye ne-derivatives yayo isebenza ngokuthe ngqo kwi-microtubules kwaye i-depolymerize, okanye ukuba isenzo sabo siphumela kwi-microtubule destabilization.Ukongezelela, kwimeko apho i-microtubules ingekho ekujoliswe kuyo ngokuthe ngqo, ukuchonga indawo yesenzo kunye neethagethi ze-molecular ye-ursonic acid kwiiseli zezityalo ziya kunceda ukuqonda ngakumbi iipropati zee-compounds ezinxulumene kunye neendlela ezinokwenzeka zokuphucula umsebenzi we-herbicidal.Uvavanyo lwethu lwe-bioactivity lubonise isakhono esisodwa se-cytotoxic se-ursonic acid ekukhuleni kwezityalo ezifana ne-Arabidopsis thaliana, icuba kunye ne-liverwort, ngelixa i-E. coli kunye neeseli ze-HeLa zichaphazeleka.Ubuncinci obuncinci okanye akukho tyhefu kwiiseli zezilwanyana luncedo lwezinto eziphuma kwi-ursonic acid ukuba ziphuhlisiwe njengemichiza yokutshabalalisa ukhula ukuze zisetyenziswe kumasimi ezolimo avulekileyo.Enyanisweni, ekubeni i-microtubules zizakhiwo eziqhelekileyo kwi-eukaryotes, ukuthintela kwabo okukhethiweyo kwizityalo kuyimfuno ephambili ye-herbicides.Ngokomzekelo, ipropyzamide, i-microtubule depolymerizing agent ebophelela ngokuthe ngqo kwi-tubulin kwaye inqanda i-polymerization, isetyenziswe njenge-herbicide ngenxa yetyhefu ephantsi kwiiseli zezilwanyana24.Ngokwahlukileyo kwi-disopyramid, i-benzamides ehambelanayo ineenkalo ezahlukeneyo ekujoliswe kuzo.Ukongeza kwi-microtubules yezityalo, i-RH-4032 okanye i-benzoxamide iphinda inqanda i-microtubules yeeseli zezilwanyana okanye i-oomycetes, ngokulandelanayo, kwaye i-zalilamide isetyenziswe njenge-fungicide ngenxa ye-phytotoxicity25,26,27 ephantsi.Ibhere esandula ukubhaqwa kunye nezinto eziphuma kuyo zibonisa i-cytotoxicity ekhethiweyo ngokuchasene nezityalo, kodwa kubalulekile ukuqaphela ukuba uhlengahlengiso olongezelelweyo lunokuguqula izinto ekujoliswe kuzo, ezinokuthi zinike izinto eziphuma kuzo ezongezelelweyo kulawulo lwe-pathogenic fungi okanye i-oomycetes.
Iimpawu ezikhethekileyo ze-urbenonic acid kunye ne-derivatives zayo ziluncedo ekuphuhlisweni kwazo njengama-herbicides kunye nokusetyenziswa njengezixhobo zophando.Ukubaluleka kwe-cytoskeleton ekulawuleni ukumila kweeseli zesityalo kubonwa ngokubanzi.Uphononongo lwangaphambili lubonise ukuba izityalo ziye zavelisa iindlela eziyinkimbinkimbi zentlangano ye-cortical microtubule ngokulawula i-microtubule dynamics ukulawula ngokufanelekileyo i-morphogenesis.Inani elikhulu leeamolekyu ezijongene nokulawulwa komsebenzi we-microtubule zichongiwe, kwaye uphando oluhambelanayo lusaqhubeka3,4,28.Ukuqonda kwethu kwangoku kwe-microtubule dynamics kwiiseli zezityalo azichazi ngokupheleleyo iindlela zombutho we-cortical microtubule.Ngokomzekelo, nangona zombini i-disopyramide kunye ne-oryzalin inokukhupha i-microtubules, i-disopyramide ibangela ukugqwesa kweengcambu ezinzima ngelixa i-oryzalin inesiphumo esincinci.Ngaphezu koko, ukuguqulwa kwe-tubulin, okuzinzisa i-microtubules, kwakhona kubangela i-dextrorotation kwiingcambu, kanti i-paclitaxel, ephinda izinze i-microtubule dynamics, ayifuni.Ke ngoko, ukufunda kunye nokuchonga iithagethi zemolekyuli ye-ursolic acid kufuneka kubonelele ngemibono emitsha kulawulo lwezityalo ze-cortical microtubules.Ngokufanayo, ukuthelekiswa kweekhemikhali kwixesha elizayo ezisebenzayo ekukhuthazeni ukukhula okugqwethekileyo, njenge-disopyramide, kunye neekhemikhali ezingasebenzi kakuhle, njenge-oryzalin okanye i-kumamotoric acid, ziya kunika ingcaciso malunga nendlela ukukhula okugqwethekileyo okwenzeka ngayo.
Kwelinye icala, uhlengahlengiso olunxulumene nokhuselo lwe-cytoskeletal yenye indlela yokuchaza i-cytotoxicity ye-ursonic acid.Usulelo lwe-pathogen okanye ukuqaliswa kwe-elicitor kwiiseli zezityalo ngamanye amaxesha kubangela ukutshatyalaliswa kwe-cytoskeleton kunye nokufa kweseli elandelayo29.Ngokomzekelo, i-cryptoxanthin evela kwi-oomycete iye yabikwa ukuba iphazamise i-microtubules kunye ne-actin filaments ngaphambi kokufa kwecuba, kufana noko kwenzekayo ngonyango lwe-KAND30,31.Ukufana phakathi kweempendulo zokukhusela kunye neempendulo zeselula ezibangelwa yi-ursonic acid zenze ukuba sicinge ukuba zibangela iinkqubo eziqhelekileyo zeselula, nangona umphumo okhawulezayo kunye namandla we-ursonic acid kune-cryptoxanthin ibonakala.Nangona kunjalo, uphando lubonise ukuba ukuphazamiseka kwe-actin filaments kukhuthaza ukufa kweeseli ngokukhawuleza, okungahlali kukhatshwa yi-microtubule disruption29.Ukongeza, kuya kuhlala kubonakala ukuba i-pathogen okanye i-elicitor ibangela ukukhula kweengcambu ezigqwethekileyo, njengoko i-ursonic acid derivatives yenza.Ke, ulwazi lwemolekyuli oludibanisa iimpendulo zokhuselo kunye ne-cytoskeleton yingxaki enomtsalane ekufuneka iqwalaselwe.Ngokuxhaphaza ubukho bezinto eziphantsi ze-molecular weight compounds ezinxulumene ne-ursonic acid, kunye noluhlu lwezinto eziphuma kuzo ezinamandla ahlukeneyo, zinokubonelela ngamathuba okujolisa kwiindlela ezingaziwayo zeselula.
Kuthatyathwe kunye, ukufunyanwa kunye nokusetyenziswa kweekhompawundi ezintsha ezimodareyitha i-microtubule dynamics ziya kubonelela ngeendlela ezinamandla zokujongana neendlela eziyinkimbinkimbi zemolekyuli eziphantsi kokuzimisela kobume beeseli zesityalo.Kulo mongo, i-compound urmotonic acid esandul 'ukuphuhliswa, echaphazela i-microtubules kunye ne-actin filaments kwaye ibangela ukufa kweeseli, inokunika ithuba lokucacisa uxhulumaniso phakathi kokulawulwa kwe-microtubule kunye nezinye iindlela.Ke, uhlalutyo lweekhemikhali kunye nebhayoloji usebenzisa i-urbenonic acid iya kusinceda siqonde iindlela zokulawula iimolekyuli ezilawula i-cytoskeleton yesityalo.
Inoculate S. werraensis MK493-CF1 kwi-500 mL edidekileyo ye-Erlenmeyer flask ene-110 mL yembewu ephakathi equka i-2% (w/v) yegalactose, 2% (w/v) Essence paste, 1% ( w/v) Ukwakheka kwe-Bacto .-soyton (i-Thermo Fisher Scientific, Inc.), 0.5% (w / v) i-corn extract (KOGOSTCH Co., Ltd., Japan), 0.2% (w / v) (NH4) 2SO4 kunye ne-0.2% CaCO3 emanzini ahlanjululweyo.(pH 7.4 phambi kokuvalwa inzala).Iinkcubeko zembewu zifukanywe kwi-rotary shaker (180 rpm) kwi-27 ° C iintsuku ezi-2.Ukulinywa kwemveliso ngokuvundiswa kwemeko eqinileyo.Imveliso yembewu (7 ml) idluliselwe kwi-500 ml ye-K-1 yeflask equlethe i-40 g yemveliso ephakathi equka i-15 g yebhali ecinezelweyo (i-MUSO Co., Ltd., eJapan) kunye ne-25 g yamanzi e-deionized (pH ayilungiswanga phambi kokuvalwa inzala).).Ukuvundiswa kwenziwa kwi-30 ° C ebumnyameni iintsuku ezili-14.Izinto ezivuthayo zikhutshwe nge-40 ml / ibhotile ye-EtOH kunye ne-centrifuged (1500 g, 4 ° C, 10 min).I-culture supernatant (60 ml) yatsalwa ngomxube we-10% ye-MeOH/EtOAc.Uluhlu lwezinto eziphilayo luye lwaba ngumphunga phantsi koxinzelelo oluncitshisiweyo lokufumana i-residu (59.5 mg), eyayiphantsi kwe-HPLC nge-gradient elution (imizuzu eyi-0-10: 90%) kwikholamu yesigaba esingasemva (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × ubude 250 mm) H2O/CH3CN, 10–35 imizuzu: 90% H2O/CH3CN ukuya 70% H2O/CH3CN (gradient), 35–45 imizuzu: 90% H2O/EtOH, 45–155 imizuzu: 90% H2O / EtOH ukuya kwi-100% EtOH (i-gradient (i-gradient), i-155-200 min: i-100% EtOH) kwinqanaba lokuhamba kwe-1.5 ml / min, i-coumamonamide (1, 36.0 mg) yodwa njenge-amorphous powder emhlophe.
I-Kumamotoamide(1);1H-NMR (500 MHz, CDCl3) δ 6.93 (t, J = 2.5 Hz, 1H), 6.76 (dd, J = 4.3, 1.8 Hz 1H), 6.05 (t, J = 3.8 Hz, 1H).), 4.08 (s, 3H);13C-NMR (125 MHz, CDCl3) δ 161.1, 121.0, 119.9, 112.2, 105.0, 68.3;ESI-HRMS [M+H]+: [C6H9N2O2]+ ixabiso elibaliweyo: 141.0659, ixabiso elilinganisiweyo: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
Imbewu yase-Columbia (Col-0) yafunyanwa kwi-Arabidopsis Biological Resource Centre (ABRC) ngemvume yokusetyenziswa kophando.Imbewu ye-Col-0 yasasazwa kwaye yagcinwa phantsi kweemeko zethu zebhubhoratri kwaye isetyenziswe njengezityalo zasendle ze-Arabidopsis.Imbewu ye-Arabidopsis yafakwa inzalo kumphezulu kwaye yakhuliswa kwisiqingatha sokuqina kwe-Murashige kunye ne-Skoog medium equlethe i-2% ye-sucrose (i-Fujifilm Wako I-Chemical ecocekileyo), 0.05% (w/v) 2-(4-morpholino) ethanesulfonic acid (MES) (Fujifilm Wako Chemical Pure Chemical ).) kunye ne-1.5% ye-agar (i-Fujifilm Wako I-Chemical ecocekileyo), i-pH 5.7, kwi-23 ° C kunye nokukhanya okuqhubekayo.Imbewu ye-phs1-1 ye-mutant yanikezelwa ngu-T. Hashimoto (Nara Institute of Science and Technology).
Imbewu yoxinzelelo lwe-SR-1 yanikezelwa nguT. Hashimoto (Nara Institute of Science and Technology) kwaye isetyenziswe njengezityalo zecuba zasendle.Imbewu yecuba yacolwa phezu komhlaba yaza yafakwa emanzini angenazintsholongwane kangangeentsuku ezintathu ukukhuthaza ukuntshula, emva koko yafakwa kwisisombululo esinesiqingatha samandla esine-2% ye-sucrose, 0.05% (w/v) MES, kunye ne-0.8% ye-gellan gum (Fujifilm Wako Chemical Pure Chemical) Murashige.kunye ne-Skoog medium) ene-pH 5.7 kunye ne-incubated kwi-23°C phantsi kokukhanya okungaguqukiyo.
I-Strain Tak-1 yanikezelwa ngu-T. Kohchi (iYunivesithi yaseKyoto) kwaye isetyenziswe njengeyunithi yovavanyo oluqhelekileyo lwesifundo se-liverwort.I-Gemma yafunyanwa kwizityalo ezikhuliswe inzalo emva koko yafakwa kwiGamborg B5 medium (Fujifilm Wako Pure Chemical) equlethe i-1% ye-sucrose kunye ne-0.3% ye-gellan gum kwaye ifakwe kwi-23 ° C phantsi kokukhanya okuqhubekayo.
Icuba BY-2 iiseli (Nicotiana tabacum L. cv. Bright Yellow 2) zanikwa S. Hasezawa (University of Tokyo).Iiseli ze-BY-2 zahlanjululwa i-95-fold in modified Linsmeier kunye ne-Skoog medium kwaye zongezwa ngeveki nge-2,4-dichlorophenoxyacetic acid 32.Ukumiswa kweeseli kwakuxutywe kwi-rotary shaker kwi-130 rpm kwi-27 ° C ebumnyameni.Geza iiseli kunye namaxesha angama-10 umthamo we-medium entsha kwaye uphinde umise kwindawo efanayo.I-BY-2 imigca yeeseli eziguquguqukayo ebonisa ngokuzinzileyo i-microtubule marker TagRFP-TUA6 okanye i-actin filament marker GFP-ABD2 phantsi kwe-cauliflower mosaic virus 35S umgqugquzeli zenziwa njengoko kuchaziwe33,34,35.Le migca yeeseli inokugcinwa kwaye ihambelane ngokusebenzisa iinkqubo ezifanayo nezo zisetyenziselwa umgca weseli we-BY-2 wokuqala.
Iiseli ze-HeLa zikhuliswe kwi-Dulbecco's modified Eagle's medium (DMEM) (i-Life Technologies) eyongezwa nge-10% ye-fetal bovine serum, i-1.2 U / ml penicillin, kunye ne-1.2 μg/ml streptomycin kwi-incubator ye-37 ° C ene-5% CO2.
Zonke iimvavanyo ezichazwe kulo mbhalo-ngqangi zenziwa ngokuhambelana nemigaqo yokhuseleko lwebhayoloji yaseJapan kunye nezikhokelo.
Iikhompawundi zachithwa kwi-dimethyl sulfoxide (i-DMSO; i-Fujifilm Wako Pure Chemical) njengezisombululo zesitokhwe kwaye ihlanjululwe kwi-MS medium ye-Arabidopsis kunye necuba okanye i-Gamborg B5 medium ye-liverwort.Kwi-assay ye-root inhibition assay, ngaphezu kwe-10 imbewu ngeplate nganye yahlwayelwa kwi-agar medium equlethe iikhompawundi ezibonisiweyo okanye i-DMSO.Imbewu yafukanyelwa kwigumbi lokukhula kangangeentsuku ezisi-7.Izithole zafotwa kwaza kwalinganiswa ubude beengcambu.Kwi-Arabidopsis germination assay, iimbewu ezingama-48 kwipleyiti nganye zahlwayelwa kwi-agar medium equlethe i-200 μM ikhompawundi okanye i-DMSO.Imbewu ye-Arabidopsis yayikhuliswe kwigumbi lokukhula kwaye inani lezithole ezintshulisiweyo labalwa kwiintsuku ezisi-7 emva kokuntshula (dag).Kuvavanyo lokuntshula kwecuba, iimbewu ezingama-24 kwipleyiti nganye zahlwayelwa kwi-agar medium equlethe i-200 μM KAND okanye i-DMSO.Imbewu yecuba yayikhuliswe kwigumbi lokukhula kwaye inani lezithole ezintshulisiweyo labalwa emva kweentsuku ezilishumi elinesine.Kwi-liverwort ukukhula kwe-inhibition assay, ii-embryo ze-9 ezivela kwipleyiti nganye zifakwe kwi-agar medium equlethe i-concentrations ebonakalisiweyo ye-KAND okanye i-DMSO kwaye ifakwe kwigumbi lokukhula iintsuku ze-14.
Sebenzisa izithole ezidyojwe nge-5 mg/ml yepropidium iodide (PI) ukuze ube nomfanekiso ngqondweni wentlangano yokufaneleka kweengcambu.Imiqondiso ye-PI yabonwa ngemakroskopu ye-fluorescence kusetyenziswa i-TCS SPE confocal laser scanning microscope (Leica Microsystems).
I-Histochemical staining yeengcambu kunye ne-β-glucuronidase (GUS) yenziwa ngokweprotocol echazwe nguMalami noBenfey36.Izithole zilungiswe kwi-90% ye-acetone ngobusuku, i-0.5 mg / ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid kwi-GUS buffer ye-1 iyure kwaye ifakwe kwisisombululo se-hydrated chloraldehyde.(I-8 g ye-chloral hydrate, i-2 ml yamanzi kunye ne-1 ml ye-glycerol) kwaye ibonwe ngokuphazamiseka kokuphazamiseka kwe-microscope usebenzisa i-Axio Imager M1 microscope (Carl Zeiss).
Iingcambu zalinganiswa kwizithole ezineentsuku ezi-7 ubudala ezikhuliswe kwiipleyiti ezibekwe ngokuthe nkqo.Ukulinganisa i-engile yengcambu ukusuka kwicala le vector yomxhuzulane njengoko kuchaziwe kwinyathelo lesi-6.
Ukulungiswa kwee-microtubules ze-cortical kwabonwa njengoko kuchazwe, kunye nokuguqulwa okuncinci kwiprotocol ye-37.I-anti-β-tubulin antibody (i-KMX-1, i-Merk Millipore: i-MAB3408) kunye ne-Alexa Fluor 488-conjugated anti-mouse IgG (i-Thermo Fisher Scientific: A32723) yayisetyenziswe njenge-antibodies ephambili kunye neyesibini kwi-1: 1000 kunye ne-1: 100 dilutions, ngokulandelelanayo.Imifanekiso yeFluorescence yafunyanwa kusetyenziswa i-TCS SPE confocal laser scanning microscope (Leica Microsystems).Fumana imifanekiso ye-Z-stack kwaye wenze uqikelelo olukhulu kakhulu ngokwemiyalelo yomenzi.
I-HeLa i-cell proliferation assay yenziwa ngokusebenzisa i-Cell Counting Kit 8 (i-Dojindo) ngokwemiyalelo yomenzi.
Ukukhula kwe-E. coli DH5α kwahlalutywa ngokulinganisa ubuninzi beeseli kwinkcubeko usebenzisa i-spectrophotometer kwi-600 nm (OD600).
Umbutho we-Cytoskeletal kwiiseli ze-transgenic BY-2 zabonwa kusetyenziswa i-microscope ye-fluorescence exhotyiswe nge-CSU-X1 isixhobo sokuskena se-confocal (Yokogawa) kunye nekhamera ye-sCMOS (Zyla, Andor Technology).Ubuninzi be-Cytoskeletal density buhlolwe ngokuhlalutya komfanekiso, oye wachaza ipesenti yeepikseli ze-cytoskeletal phakathi kweepikseli ze-cytoplasmic kwimifanekiso ye-confocal usebenzisa i-software ye-ImageJ njengoko ichazwe38,39.
Ukubona ukufa kweeseli kwiiseli ze-BY-2, i-aliquot yokumiswa kweseli yafakwa kwi-0.05% ye-Evans eluhlaza okwesibhakabhaka imizuzu eyi-10 kwiqondo lokushisa.Ukukhethwa kwe-Evans eluhlaza okwesibhakabhaka kweeseli ezifileyo kuxhomekeke kwi-extrusion yedayi evela kwiiseli ezisebenzayo nge-plasma membrane40.Iiseli ezinamabala zabonwa kusetyenziswa i-microscope-field field (BX53, Olympus).
Iiseli ze-HeLa zakhuliswa kwi-DMEM zongezwa nge-10% ye-FBS kwi-incubator e-humidified kwi-37 ° C kunye ne-5% CO2.Iiseli zaphathwa nge-100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng / ml colcemid (Gibco), okanye 100 ng / ml Nocodmaze (Sigma) kwi-6 h kwi-37 ° C.Iiseli zilungiswe kunye ne-MetOH ye-10 min kwaye emva koko nge-acetate ye-5 min kwiqondo lokushisa.Iiseli ezilungisiweyo zifakwe kwi-β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) ehlanjululwe kwi-0.5% ye-BSA / PBS kwiiyure ze-2, ihlanjwe ngamaxesha e-3 nge-TBST, kwaye emva koko ifakwe kwi-Alexa Fluor yebhokhwe yebhokhwe.488 1 iyure.- I-Mouse IgG (i-Thermo Fisher Scientific: A11001) kunye ne-15 ng / ml 4′, 6-diamidino-2-phenylindole (DAPI) ihlanjululwe kwi-0.5% BSA / PBS.Emva kokuvasa nge-TBST amaxesha amathathu, iiseli ezinebala zajongwa kwi-Nikon Eclipse Ti-E yemakroskopu eguqulweyo.Imifanekiso ibanjwe ngekhamera ye-CCD epholileyo ye-Hamamatsu ORCA-R2 kusetyenziswa isoftware ye-MetaMorph (iZixhobo zeMolekyuli).
Ixesha lokuposa: Jun-17-2024