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Ukufunyanwa nokusetyenziswa okuluncedo kweemveliso zendalo kunokunceda ukuphucula ubomi babantu. Iikhemikhali ezithintela ukukhula kwezityalo zisetyenziswa kakhulu njengee-herbicides ukulawula ukhula. Ngenxa yesidingo sokusebenzisa iintlobo ezahlukeneyo ze-herbicides, kukho imfuneko yokuchonga ii-compounds ezineendlela ezintsha zokusebenza. Kolu phononongo, sifumene i-compound entsha ye-N-alkoxypyrrole, i-coumamonamide, evela kwi-Streptomyces werraensis MK493-CF1 kwaye saseka inkqubo epheleleyo yokwenziwa. Ngovavanyo lomsebenzi webhayoloji, sifumanise ukuba i-urs-monoamic acid sisixhobo esiphakathi se-urs-monoamide kunye ne-potentialisithinteli sokukhula kwezityaloUkongeza, siye saphuhlisa iintlobo ngeentlobo zee-urbenonic acid derivatives, kuquka i-urbenyloxy derivative (UDA), enomsebenzi ophezulu wokubulala ukhula ngaphandle kokuchaphazela kakubi ukukhula kweeseli zeHeLa. Sikwafumanise ukuba ii-urmotonic acid derivatives ziphazamisa ii-microtubules zezityalo; ukongeza, i-KAND ichaphazela ii-actin filaments kwaye ibangela ukufa kweeseli; Ezi ziphumo zininzi zahlukile kwezo ze-microtubule inhibitors ezaziwayo kwaye zibonisa indlela entsha yokusebenza kwe-ursonic acid, emele inzuzo ebalulekileyo kuphuhliso lwee-herbicides ezintsha.
Ukufunyanwa nokusetyenziswa okusebenzayo kweemveliso zendalo eziluncedo kunye neziphumo zazo yindlela yokuphucula umgangatho wobomi bomntu. Iimetabolites zesibini eziveliswa ziintsholongwane, izityalo kunye nezinambuzane zikhokelele kwinkqubela phambili enkulu kwezonyango nakwizolimo. Ii-antibiotics ezininzi kunye namayeza okulwa ne-leukemia zenziwe ngeemveliso zendalo. Ukongeza, iintlobo ezahlukeneyo zeizibulali-zinambuzane, iifungicides kunye ne-herbicides zikhutshwa kwezi mveliso zendalo ukuze zisetyenziswe kwezolimo. Ngokukodwa, ii-herbicides zokulawula ukhula zizixhobo ezibalulekileyo zokwandisa isivuno sezityalo kwezolimo zanamhlanje, kwaye iintlobo ezahlukeneyo zee-compounds sele zisetyenziswa kwizorhwebo. Iinkqubo ezininzi zeseli kwizityalo, ezifana ne-photosynthesis, i-amino acid metabolism, ukwenziwa kweendonga zeseli, ukulawulwa kwe-mitosis, i-phytohormone signaling, okanye ukwenziwa kweproteni, zithathwa njengezona zinto ziphambili kwii-herbicides. Ii-compounds ezithintela umsebenzi we-microtubule luhlobo oluqhelekileyo lwee-herbicides ezichaphazela ukukhula kwezityalo ngokuchaphazela ukulawulwa kwe-mitotic2.
Ii-Microtubules zizinto ze-cytoskeleton kwaye zigcinwa kakhulu kwiiseli ze-eukaryotic. I-tubulin heterodimer ine-α-tubulin kunye ne-β-tubulin ezenza ii-protofilaments ze-microtubule ezithe ngqo, kunye nee-protofilaments ezili-13 ezenza isakhiwo se-cylindrical. Ii-Microtubules zidlala indima ezininzi kwiiseli zezityalo, kubandakanya ukumisela imo yeseli, ulwahlulo lweseli, kunye nothutho lwangaphakathi3,4. Iiseli zezityalo ziqulethe ii-microtubules phantsi kwe-membrane ye-plasma ye-interphase, kwaye ezi zibizwa ngokuba zii-cortical microtubules kucingelwa ukuba zilawula ukulungelelaniswa kwee-cellulose microfibrils ngokulawula ii-cellulose synthase complexes4,5. Ii-cortical microtubules zeeseli ze-root epidermal, ezikhoyo kwindawo yokwandiswa ngokukhawuleza kwencam yengcambu, zifumaneka ecaleni, kwaye ii-cellulose microfibers zilandela ezi microtubules kwaye zinciphisa indlela yokwandiswa kweseli, ngaloo ndlela zikhuthaza ukwanda kweeseli ze-anisotropic. Ke ngoko, umsebenzi we-microtubule unxulumene kakhulu ne-morphology yesityalo. Ukutshintshwa kwe-amino acid kwiijini ezifaka i-tubulin kubangela ukugoba kwe-cortical microtubule arrays kunye nokukhula kwicala lasekhohlo okanye lasekunene kwi-Arabidopsis 6,7. Ngokufanayo, utshintsho kwiiproteni ezinxulumene ne-microtubule ezilawula i-microtubule dynamics lunokukhokelela ekukhuleni kweengcambu okugqwethekileyo8,9,10,11,12,13. Ukongeza, unyango nge-microtubule-disrupting herbicides ezifana ne-disopyramide, eyaziwa ngokuba yi-pretilachlor, lukwabangela ukukhula kweengcambu ezigobileyo kwicala lasekhohlo14. Ezi datha zibonisa ukuba ulawulo oluchanekileyo lomsebenzi we-microtubule lubalulekile ekumiseleni indlela yokukhula kwesityalo.
Kufunyenwe iintlobo ngeentlobo zee-microtubule inhibitors, kwaye la mayeza abe negalelo elikhulu kuphando lwe-cytoskeletal, kunye nezolimo kunye nezonyango2. Ngokukodwa, i-oryzalin, ii-dinitroaniline compounds, i-disopyramide, ii-benzamide-related compounds, kunye nee-analogs zazo zinokuthintela ukusebenza kwe-microtubule kwaye ngaloo ndlela zithintele ukukhula kwezityalo. Ke ngoko, zisetyenziswa kakhulu njenge-herbicides. Nangona kunjalo, ekubeni ii-microtubules ziyinxalenye ebalulekileyo yeeseli zezityalo nezilwanyana, uninzi lwee-microtubule inhibitors ziyingozi kwi-cytotoxic kuzo zombini iintlobo zeeseli. Ke ngoko, nangona ziyaziwa njenge-herbicides, inani elincinci lee-antimicrotubule agents zisetyenziselwa iinjongo ezisebenzayo.
I-Streptomyces luhlobo losapho lwe-Streptomyces, oluquka i-aerobic, i-gram-positive, i-filamentous bacteria kwaye yaziwa kakhulu ngokukwazi kwayo ukuvelisa uluhlu olubanzi lwee-metabolites zesibini. Ke ngoko, ithathwa njengenye yezona mithombo zibalulekileyo zeemveliso zendalo ezintsha ezisebenzayo kwi-biologically. Kolu phononongo lwangoku, sifumene ikhompawundi entsha ebizwa ngokuba yi-coumamonamide, eyahlulwa kwi-Streptomyces werraensis MK493-CF1 kunye ne-S. werraensis ISP 5486. Sisebenzisa uhlalutyo lwe-spectral kunye nohlalutyo olupheleleyo lwe-spectral, ulwakhiwo lwe-coumamonamide lwachazwa kwaye ulwakhiwo lwayo olukhethekileyo lwe-N-alkoxypyrrole skeleton lwachongwa. I-Ursmonic acid, i-synthetic intermediate ye-ursmonoamide kunye ne-derivatives yayo, yafunyanwa ithintela ukukhula kunye nokuhluma kwesityalo esithandwayo semodeli i-Arabidopsis thaliana. Kwisifundo sobudlelwane besakhiwo-umsebenzi, sifumanise ukuba ikhompawundi ene-C9 eguqulwe kwi-ursonic acid, ebizwa ngokuba yi-nonyloxy derivative ye-ursonic acid (KAND), iphucula kakhulu isiphumo sokuthintela ekukhuleni nasekuhlumeni. Okuphawulekayo kukuba, i-inhibitor yokukhula kwezityalo esandul’ ukufunyanwa nayo ichaphazele ukukhula kwecuba kunye ne-liverwort kwaye yayingenabungozi kwiibhaktheriya okanye kwiiseli ze-HeLa. Ngaphezu koko, ezinye izinto eziphuma kwi-urmotonic acid zibangela i-phenotype yeengcambu ezigqwethekileyo, nto leyo ethetha ukuba ezi zinto ziphuma kwi-microtubules zichaphazela ngokuthe ngqo okanye ngokungathanga ngqo ii-microtubules. Ngokuhambelana nale ngcamango, ukuqaphela kwethu ii-microtubules ezibhalwe nge-immunohistochemically okanye ngeeproteni ze-fluorescent kubonisa ukuba unyango lwe-KAND luyawasusa ama-microtubules. Ukongeza, unyango olusebenzisa i-kumamotonic acid derivatives luphazamise ii-microfilaments ze-actin. Ke ngoko, sifumene i-inhibitor entsha yokukhula kwezityalo enendlela yayo yokusebenza eyahlukileyo ebandakanya ukutshatyalaliswa kwe-cytoskeleton.
I-Strein MK493-CF1 yahlulwa emhlabeni eShinagawa-ku, eTokyo. I-Strein MK493-CF1 yenze i-stromal mycelium enamasebe amahle. Ulandelelwano oluncinci lwe-16S ribosomal RNA gene (1422 bp) lufunyenwe. Olu hlobo lufana kakhulu ne-S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: uhlobo oluqhelekileyo, 99.93%). Ngokusekelwe kwesi siphumo, kwafunyaniswa ukuba olu hlobo lunxulumene kakhulu nohlobo lwe-S. werraensis. Ke ngoko, salibiza okwethutyana olu hlobo lwe-S. werraensis MK493-CF1. I-S. werraensis ISP 5486T ikwavelisa iikhompawundi ezifanayo ze-bioactive. Ekubeni bekukho uphando oluncinci ekuqaleni lokufumana iimveliso zendalo kule microorganism, kwenziwe uphando olongezelelweyo lweekhemikhali. Emva kokulinywa kwe-S. werraensis MK493-CF1 kwindawo yerhasi ngokubilisa i-solid-state kwi-30°C kangangeentsuku ezili-14, indawo yokusetyenziswa yakhutshwa nge-50% EtOH. I-60 ml yesampulu yomiswa ukuze kufunyanwe i-59.5 mg ye-crude extract. I-crude extract yafakwa kwi-reverse phase HPLC ukuze inike i-N-methoxy-1H-pyrrole-2-carboxamide (1, ebizwa ngokuba yi-coumamonamide, 36.0 mg). Isixa sisonke se-1 simalunga ne-60% ye-crude extract. Ke ngoko, sigqibe ekubeni sifunde ngokweenkcukacha iipropati ze-kumamotoamide 1.
I-Coumamonamide 1 yipowder emhlophe e-amorphous kunye ne-high resolution mass spectrometry (HRESIMS) iqinisekisa i-C6H8N2O2 (Umzobo 1). Iqhekeza le-pyrrole elifakwe kwi-C2 lale compound libonakaliswa yi-δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH kwi-1H NMR spectrum: 4.5 Hz, H-5) kunye ne-δH 6.78 (1H, d, J = 2.5 Hz, H-6), kwaye i-13C NMR spectrum ibonisa ubukho bee-athomu ezine ze-sp2 carbon. Ubukho beqela le-amide kwindawo ye-C2 buvavanywe yi-HMBC correlation ukusuka kwi-C-3 proton ukuya kwi-amide carbonyl carbon kwi-δC 161.1. Ukongeza, i-1 H kunye ne-13 C NMR peaks kwi-δH 4.10 (3H, S) kunye ne-δC 68.3 zibonisa ukuba kukho amaqela e-N-methoxy kwimolekyuli. Nangona indawo echanekileyo yeqela le-methoxy yayingekamiselwa kusetyenziswa uhlalutyo lwe-spectroscopic olufana ne-enhanced difference spectroscopy kunye ne-nuclear Overhauser abbreviation (NOEDF), i-N-methoxy-1H-pyrrole-2-carboxamide yaba yi-compound yokuqala.
Ukufumanisa isakhiwo esichanekileyo se-1, kwenziwa ukwenziwa kwe-total synthesis (Umzobo 2a). Unyango lwe-2-aminopyridine 2 ethengiswayo nge-m-CPBA luphumele kwi-N-oxide 3 ehambelanayo kwisivuno sobuninzi. Emva kwe-2-aminoazidation ye-2, impendulo ye-cyclocondensation echazwe ngu-Abramovich yenziwa kwi-benzene kwi-90°C ukuze kufunyanwe i-1-hydroxy-1H-pyrrole-2-carbonitrile 5 kwiigrama. Isantya 60% (amanqanaba amabini). 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 kusetyenziswa i-ammonia yamanzi inike i-Kumamoto amide 1 kwisivuno se-98%. Yonke idatha ye-spectral ye-synthesized 1 yayifana ne-isolated 1, ngoko ke isakhiwo se-1 samiselwa;
Ukwenziwa ngokubanzi kunye nohlalutyo lomsebenzi webhayoloji we-urbenamide kunye ne-urbenic acid. (a) Ukwenziwa ngokupheleleyo kwe-Kumamoto amide. (b) Izithole ze-Arabidopsis Columbia (Col) zasendle ezineentsuku ezisixhenxe zikhuliswe kwiipleyiti zeMurashige kunye neSkoog (MS) eziqulethe i-coumamonamide 6 okanye i-coumamonamide 1 kumanqanaba abonisiwe. Ibha yesikali = 1 cm.
Okokuqala, sivavanye imisebenzi yebhayoloji ye-urbenamide kunye nezinto ezingaphakathi kwayo ukuze sibone amandla ayo okulawula ukukhula kwezityalo. Songeze amazinga ahlukeneyo e-ursmonamide 1 okanye i-ursmonic acid 6 kwi-MS agar medium kunye nezithole ze-Arabidopsis thaliana ezikhuliswe kule medium. Olu vavanyo lubonise ukuba amazinga aphezulu (500 μM) e-6 athintele ukukhula kweengcambu (Umzobo 2b). Okulandelayo, sivelise iintlobo ngeentlobo zeemveliso ngokufaka indawo ye-N1 ye-6 kwaye senza izifundo zobudlelwane bolwakhiwo-umsebenzi kuzo (inkqubo yokwenziwa kwe-analogue ichazwe kwiNkcazo yoNcedo (SI)). Izithole ze-Arabidopsis zikhuliswe kwindawo equlethe i-50 μM i-ursonic acid derivatives, kwaye ubude beengcambu bulinganiswe. njengoko kubonisiwe kumfanekiso. Njengoko kubonisiwe kwiMifanekiso 3a, b, kunye ne-S1, ii-coumamo acids zinobude obahlukeneyo bee-linear alkoxy chains (9, 10, 11, 12, kunye ne-13) okanye ii-alkoxy chains ezinkulu (15, 16, kunye ne-17) kwindawo ye-N1. Ii-derivatives zibonise ukuthintela okukhulu ukukhula kweengcambu. Ukongeza, sifumanise ukuba ukusetyenziswa kwe-200 μM 10, 11, okanye 17 kuthintele ukuhluma (IiFig. 3c kunye ne-S2).
Uphononongo lobudlelwane bolwakhiwo nomsebenzi we-Kumamoto amide kunye neekhompawundi ezinxulumene noko. (a) Isicwangciso solwakhiwo kunye nokuhlanganiswa kwee-analogues. (b) Ukulinganiswa kobude beengcambu zezityalo ezineentsuku ezisi-7 ezikhuliswe kwi-MS medium kunye okanye ngaphandle kwe-50 μM coumamonamide derivatives. Ii-asterisks zibonisa umahluko omkhulu ngonyango lwe-fake (t test, p< 0.05). n>18. Idatha iboniswa njenge-mean ± SD. nt ithetha "ayivavanywanga" kuba ngaphezulu kwe-50% yembewu ayizange ikhule. (c) Ukulinganiswa kwesantya sokuhluma kwembewu enyangiweyo efakwe kwiintsuku ezi-7 kwi-MS medium ene-200 μM coumamonamide okanye engenayo kunye neekhompawundi ezinxulumene nayo. Ii-asterisk zibonisa umahluko omkhulu ngonyango lwe-sham (uvavanyo lwe-chi-square). n=96.
Okubangel’ umdla kukuba, ukongezwa kwee-alkyl side chains ezinde kune-C9 kunciphisa umsebenzi wokuthintela, nto leyo ebonisa ukuba ii-compounds ezinxulumene ne-kumamotoic acid zifuna ii-side chains zobukhulu obuthile ukuze zibonise umsebenzi wazo webhayoloji.
Ngenxa yokuba uhlalutyo lobudlelwane bolwakhiwo nomsebenzi lubonise ukuba i-C9 iguqulwe yaba yi-ursonic acid kwaye i-nonyloxy derivative ye-ursonic acid (ebizwa ngokuba yi-KAND 11) yayiyeyona nto isebenzayo ekuthinteleni ukukhula kwezityalo, senze uhlalutyo oluneenkcukacha ngakumbi lwe-KAND 11. Unyango lwe-Arabidopsis nge-50 μM KAND 11 luphantse lwathintela ukuhluma, ngelixa ubuninzi obuphantsi (40, 30, 20, okanye 10 μM) be-KAND 11 buthintele ukukhula kweengcambu ngendlela exhomekeke kumthamo (Umzobo 4a, b). Ukuvavanya ukuba i-KAND 11 iyayichaphazela na impilo yeengcambu, sihlolisise iingcambu ze-meristems ezidaywe yi-propidium iodide (PI) kwaye salinganisa ubungakanani bendawo ye-meristem. Ubungakanani be-meristem yezithole ezikhuliswe kwindawo equlethe i-25 μM KAND-11 yayiyi-151.1 ± 32.5 μm, ngelixa ubukhulu be-meristem yezithole ezikhuliswe kwindawo yokulawula equlethe i-DMSO yayiyi-264.7 ± 30.8 μm (Umzobo 4c, d), nto leyo ebonisa ukuba i-KAND-11 ibuyisela umsebenzi weseli. Ukusasazeka. I-Root meristem. Ngokuhambelana noku, unyango lwe-KAND 11 lunciphise ubungakanani bophawu lokwahlulahlula iseli i-CDKB2;1p::CDKB2;1-GUS signal kwi-root meristem (Umzobo 4e) 17. Ezi ziphumo zibonisa ukuba i-KAND 11 ithintela ukukhula kweengcambu ngokunciphisa umsebenzi wokwanda kweseli.
Uhlalutyo lwempembelelo yokuthintela i-urbenonic acid derivatives (i-urbenyloxy derivatives) ekukhuleni. (a) Izithole ze-wild-type Col ezineentsuku ezisi-7 ubudala ezikhuliswe kwiiplate ze-MS ezinoxinzelelo olubonisiweyo lwe-KAND 11. I-Scale bar = 1 cm. (b) Ubungakanani bobude beengcambu. Oonobumba babonisa umahluko obalulekileyo (uvavanyo lwe-Tukey HSD, p.< 0.05). n>16. Idatha iboniswa njenge-avareji ± SD. (c) I-Confocal microscopy yeengcambu ze-propidium iodide-stained wild-type Col ezikhuliswe kwiiplate ze-MS ezine-25 μM KAND okanye ezingenazo 11. Izibiyeli ezimhlophe zibonisa i-root meristem. I-Scale bar = 100 µm. (d) Ubungakanani bobukhulu be-root meristem (n = 10 ukuya kwi-11). Umahluko wezibalo umiselwe kusetyenziswa uvavanyo lwe-t (p< 0.05). Iibhari zimele ubungakanani obuqhelekileyo be-meristem. (e) I-Differential interference contrast (DIC) microscopy ye-root meristem equlethe i-CDKB2 construct; 1pro: CDKB2; 1-GUS efakwe idayi kwaye yafakwa idayi kwizithole ezineentsuku ezi-5 ubudala ezikhuliswe kwiiplate ze-MS ezinovavanyo lwe-25 µM KAND okanye ezingenalo.
Ubuthi be-KAND 11 buvavanywe ngakumbi kusetyenziswa esinye isityalo esine-dicotyledonous, icuba (iNicotiana tabacum), kunye nesityalo esibalulekileyo somzekelo wezityalo zomhlaba, i-liverwort (iMarchantia polymorpha). Njengakwimeko ye-Arabidopsis, izithole zecuba ze-SR-1 ezikhuliswe kwindawo ephakathi equlethe i-25 μM KAND 11 zivelise iingcambu ezimfutshane (Umzobo 5a). Ukongeza, imbewu ezingama-40 kwezingama-48 zikhule kwiipleyiti eziqulethe i-200 μM KAND 11, ngelixa zonke imbewu ezingama-48 zikhule kwimidiya ephathwe ngendlela ethile, nto leyo ebonisa ukuba amazinga aphezulu e-KAND ayebalulekile (p.< 0.05; uvavanyo lwe-chi -square) luthintele ukuhluma kwecuba. (Umzobo 5b). Ukongeza, uxinzelelo lwe-KAND 11 oluthintele ukukhula kwebhaktheriya kwi-liverwort lwalufana noxinzelelo olusebenzayo kwi-Arabidopsis (Umzobo 5c). Ezi ziphumo zibonisa ukuba i-KAND 11 inokuthintela ukukhula kwezityalo ezahlukeneyo. Emva koko siphande nge-cytotoxicity enokubakho yeekhompawundi ezinxulumene ne-bear monoamide kwezinye izinto eziphilayo, ezizezi iiseli ze-HeLa zabantu kunye ne-Escherichia coli strain DH5α, njengabameli beeseli zezilwanyana eziphezulu kunye neebhaktheriya, ngokwahlukeneyo. Kuluhlu lweemvavanyo zokwanda kweeseli, siqaphele ukuba i-coumamonamide 1, i-coumamonamidic acid 6, kunye ne-KAND 11 azizange zichaphazele ukukhula kweeseli ze-HeLa okanye ze-E. coli kumanqanaba e-100 μM (Umzobo 5d,e).
Ukuthintela ukukhula kwe-KAND 11 kwizinto eziphilayo ezingezizo eze-Arabidopsis. (a) Izithole zecuba ze-SR-1 zasendle ezineeveki ezimbini ubudala zikhuliswe kwiipleyiti ze-MS ezibekwe ngokuthe nkqo eziqulethe i-25 μM KAND 11. (b) Izithole zecuba ze-SR-1 zasendle ezineeveki ezimbini ubudala zikhuliswe kwiipleyiti ze-MS ezibekwe ngokuthe nkqo eziqulethe i-200 μM KAND 11. (c) Iintyatyambo ze-Tak-1 zasendle ezineeveki ezimbini ubudala ezikhuliswe kwiipleyiti ze-Gamborg B5 ezinomlinganiselo obonisiweyo we-KAND 11. Iintolo ezibomvu zibonisa ii-spores eziye zayeka ukukhula kwixesha lokufukama leeveki ezimbini. (d) Uvavanyo lokwanda kweeseli zeeseli ze-HeLa. Inani leeseli eziphilayo lilinganiswe ngamaxesha athile kusetyenziswa ikhithi yokubala iiseli 8 (iDojindo). Njengolawulo, iiseli ze-HeLa ziphathwe nge-5 μg/ml ye-actinomycin D (uMthetho D), ethintela ukubhalwa kwe-RNA polymerase kwaye ibangela ukufa kweeseli. Uhlalutyo lwenziwe kathathu. (e) Uvavanyo lokwanda kweeseli ze-E. coli. Ukukhula kwe-E. coli kuhlalutywe ngokulinganisa i-OD600. Njengolawulo, iiseli ziphathwe nge-50 μg/ml ampicillin (Amp), ethintela ukwenziwa kodonga lweeseli zebhaktheriya. Uhlalutyo lwenziwe kathathu.
Ukuze siqonde indlela esebenza ngayo i-cytotoxicity ebangelwa ziikhompawundi ezinxulumene ne-uramide, siphinde sahlalutya ii-urbenic acid derivatives ngeziphumo eziphakathi zokuthintela. njengoko kubonisiwe kumfanekiso. Njengoko kubonisiwe kwiMifanekiso 2b, 6a, izithole ezikhuliswe kwiiplate ze-agar eziqulathe ubuninzi obuphezulu (200 μM) be-urmotonic acid 6 zivelise iingcambu ezimfutshane nezigobileyo ngasekhohlo (θ = – 23.7 ± 6.1), ngelixa kwizithole ezikhuliswe kwindawo yokulawula, izithole zivelise iingcambu ezithe ngqo (θ = – 3.8 ± 7.1). Olu khula olucacileyo lwe-oblique luyaziwa ukuba lubangelwa kukungasebenzi kakuhle kwe-cortical microtubules14,18. Ngokuhambelana nolu fumaniso, iziyobisi ezithintela i-microtubule-destabilizing disopyramide kunye ne-oryzalin zibangele ukuthambekela okufanayo kweengcambu phantsi kweemeko zethu zokukhula (IiFig. 2b, 6a). Kwangaxeshanye, sivavanye ii-urmotonic acid derivatives kwaye sikhethe ezininzi zazo, kumanqanaba athile, ezibangele ukukhula kweengcambu ezigobileyo. Iikhompawundi 8, 9, kunye ne-15 zitshintshe indlela yokukhula kweengcambu kwi-75 μM, 50 μM, kunye ne-40 μM, ngokulandelelana, nto leyo ebonisa ukuba ezi khompawundi zinokuzinzisa ngempumelelo ii-microtubules (Umzobo 2b, 6a). Sikwavavanye i-ursolic acid derivative enamandla kakhulu, i-KAND 11, kwi-concentration ephantsi (15 µM) saza safumanisa ukuba ukusetyenziswa kwe-KAND 11 kuthintele ukukhula kweengcambu kwaye indlela yokukhula kweengcambu yayingalingani, nangona zazithambekele ekuthambekeni ngasekhohlo (Umfanekiso C3). . Ngenxa yokuba ubuninzi beziyobisi ezithintela ukukhula kwezityalo ngamanye amaxesha zithintela ukukhula kwezityalo kunokuba zibangele ukuthambekela kweengcambu, emva koko sivavanye ukuba kungenzeka ukuba i-KAND 11 ichaphazela ii-microtubules ngokujonga ii-cortical microtubules kwiiseli ze-epidermal zeengcambu. I-Immunohistochemistry esebenzisa ii-anti-β-tubulin antibodies kwiiseli ze-epidermal zeengcambu zezithole eziphathwe nge-25 μM KAND 11 ibonise ukunyamalala phantse kwazo zonke ii-cortical microtubules kwiiseli ze-epidermal kwindawo yokwandiswa (Umzobo 6b). Ezi ziphumo zibonisa ukuba i-kumamotonic acid kunye nezinto eziphuma kuyo zisebenza ngokuthe ngqo okanye ngokungathanga ngqo kwi-microtubules ukuze ziphazamise kwaye ezi zinto zi-compounds zizithinteli ezintsha ze-microtubule.
I-Ursonic acid kunye ne-derivatives yayo itshintsha ii-cortical microtubules kwi-Arabidopsis thaliana. (a) I-angle yokuthambekela kweengcambu ilinganiswe phambi kweentlobo ezahlukeneyo ze-urmotonic acid derivatives kumanqanaba abonakalisiweyo. Iziphumo zeekhompawundi ezimbini ezaziwa ngokuba zithintela ii-microtubules: i-disopyramide kunye ne-oryzalin nazo zihlalutywe. I-inset ibonisa umgangatho osetyenziswayo ukulinganisa i-angle yokukhula kweengcambu. Ii-asterisks zibonisa umahluko omkhulu ngonyango lwe-sham (t test, p< 0.05). n>19. Ibha yesikali = 1 cm. (b) Ii-microtubules ze-cortical kwiiseli ze-epidermal kwindawo yobude. Ii-Microtubules kwiingcambu ze-Arabidopsis Col ezikhule kwiiplate ze-MS ezine-25 μM KAND 11 okanye ezingenazo zibonwe ngokudaya kwe-immunohistochemical kusetyenziswa ii-antibodies eziphambili ze-β-tubulin kunye nee-antibodies ze-Alexa Fluor-conjugated secondary. Ibha yesikali = 10 µm. (c) Ulwakhiwo lwe-mitotic lwee-microtubules kwi-root meristem. Ii-Microtubules zibonwe kusetyenziswa ukudaya kwe-immunohistochemical. Izakhiwo ze-mitotic, kubandakanya iindawo ze-prophase, ii-spindles, kunye ne-phragmoplasts, zibalwa kwimifanekiso ye-confocal. Iintolo zibonisa izakhiwo ze-mitotic microtubule. Ii-asterisk zibonisa umahluko omkhulu ngonyango lwe-sham (uvavanyo lwe-t, p< 0.05). n>9. Ibha yesikali = 50 µm.
Nangona i-Ursa inako ukuphazamisa umsebenzi we-microtubule, indlela esebenza ngayo kulindeleke ukuba yahluke kwii-arhente eziqhelekileyo ze-microtubule depolymerizing. Umzekelo, ubuninzi be-arhente ze-microtubule depolymerizing ezifana ne-disopyramide kunye ne-oryzalin zibangela ukwanda kwe-anisotropic kweeseli ze-epidermal, ngelixa i-KAND 11 ingenalo. Ukongeza, ukusetyenziswa ngokubambisana kwe-KAND 11 kunye ne-disopyramide kubangele impendulo yokukhula kweengcambu ezibangelwa yi-disopyramide kunye nokuthintelwa kokukhula okubangelwa yi-KAND 11 kwabonwa (Umzobo S4). Sikwahlalutye impendulo ye-hypersensitive disopyramide 1-1 (phs1-1) mutant kwi-KAND 11. i-phs1-1 ine-non-canonical tubulin kinase point mutation kwaye ivelisa iingcambu ezimfutshane xa inyangwa nge-disopyramide9,20. Izithole ze-phs1-1 mutant ezikhuliswe kwi-agar medium equlethe i-KAND 11 zineengcambu ezimfutshane ezifana nezo zikhuliswe kwi-disopyramid (Umzobo S5).
Ukongeza, sibone izakhiwo ze-mitotic microtubule, ezifana neendawo ze-prophase, ii-spindles, kunye ne-phragmoplasts, kwi-root meristem yezithole eziphathwe nge-KAND 11. Ngokuhambelana nokuqwalaselwa kwe-CDKB2;1p::CDKB2;1-GUS, kubonwe ukwehla okukhulu kwinani le-mitotic microtubules (Umzobo .6c).
Ukuze sichaze ubuthi be-KAND 11 kwisisombululo se-subcellular, sinyange iiseli ze-BY-2 suspension zecuba nge-KAND 11 saza sajonga impendulo yazo. Okokuqala songeze i-KAND 11 kwiiseli ze-BY-2 eziveza i-TagRFP-TUA6, ebhala ii-microtubules nge-fluorescently, ukuvavanya isiphumo se-KAND 11 kwii-microtubules ze-cortical. Uxinano lwe-microtubule ye-cortical luhlolwe kusetyenziswa uhlalutyo lomfanekiso, olulinganise ipesenti yee-pixels ze-cytoskeletal phakathi kwee-pixels ze-cytoplasmic. Iziphumo zovavanyo zibonise ukuba emva konyango nge-50 μM okanye i-100 μM KAND 11 iyure e-1, uxinano lwehle kakhulu lwaya kwi-0.94 ± 0.74% okanye i-0.23 ± 0.28%, ngokulandelanayo, ngelixa uxinano lweeseli ezinyangwe nge-DMSO, lwalufikelela kwi-1.61 ± 0.34% (Umzobo 7a). Ezi ziphumo zihambelana nokubonwa kwi-Arabidopsis ukuba unyango lwe-KAND 11 lubangela ukususwa kwe-polymerization ye-cortical microtubules (Umzobo 6b). Sikwahlolisise umgca we-BY-2 one-GFP-ABD-labeled actin filaments emva konyango olune-concentration efanayo ye-KAND 11 kwaye sabona ukuba unyango lwe-KAND 11 luphazamise i-actin filaments. Unyango olusebenzisa i-50 μM okanye i-100 μM KAND 11 iyure e-1 lunciphise kakhulu uxinano lwe-actin filament ukuya kwi-1.20 ± 0.62% okanye i-0.61 ± 0.26%, ngokulandelanayo, ngelixa uxinano kwiiseli eziphathwe yi-DMSO yayiyi-1.69 ± 0.51% (Umzobo 2). 7b). Ezi ziphumo zichasene nemiphumo ye-propyzamide, engachaphazeli i-actin filaments, kunye ne-latrunculin B, i-actin depolymerizer engachaphazeli i-microtubules (Umfanekiso we-SI S6). Ukongeza, unyango nge-coumamonamide 1, i-coumamonamide acid 6, okanye i-KAND 11 aluzange luchaphazele ii-microtubules kwiiseli ze-HeLa (uMfanekiso we-SI S7). Ngoko ke, indlela yokusebenza kwe-KAND 11 kukholelwa ukuba yahlukile kuneye-cytoskeleton disruptors ezaziwayo. Ukongeza, ukujonga kwethu iiseli ze-BY-2 ezinyangwe nge-KAND 11 kutyhile ukuqala kokufa kweeseli ngexesha lonyango lwe-KAND 11 kwaye kwabonisa ukuba inani leeseli ezifileyo eziluhlaza okwesibhakabhaka ze-Evans alizange linyuke kakhulu emva kwemizuzu engama-30 yonyango lwe-KAND 11, ngelixa emva kwemizuzu engama-90 yonyango nge-50 μM okanye i-100 μM KAND, inani leeseli ezifileyo landa laya kwi-43.7% okanye i-80.1%, ngokulandelanayo (Umzobo 7c). Xa zidibene, ezi datha zibonisa ukuba i-ursolic acid derivative KAND 11 entsha yi-cytoskeletal inhibitor yezityalo enendlela yokusebenza engaziwayo ngaphambili.
I-KAND ichaphazela ii-cortical microtubules, ii-actin filaments, kunye nokuphila kweeseli ze-BY-2 zecuba. (a) Ukubona ii-cortical microtubules kwiiseli ze-BY-2 phambi kweTagRFP-TUA6. Iiseli ze-BY-2 ezinyangwe nge-KAND 11 (50 μM okanye 100 μM) okanye i-DMSO zihlolwe nge-confocal microscopy. Uxinano lwe-cortical microtubule lubalwe kwii-micrographs zeeseli ezingama-25 ezizimeleyo. Oonobumba babonisa umahluko obalulekileyo (uvavanyo lwe-Tukey HSD, p.< 0.05). Ibha yesikali = 10 µm. (b) Iifilamenti ze-cortical actin kwiiseli ze-BY-2 ezibonwe phambi kwe-GFP-ABD2. Iiseli ze-BY-2 ezinyangwe nge-KAND 11 (50 μM okanye 100 μM) okanye i-DMSO zihlolwe nge-confocal microscopy. Ubuninzi beefilamenti ze-cortical actin bubalwe kwii-micrographs zeeseli ezingama-25 ezizimeleyo. Oonobumba babonisa umahluko obalulekileyo (Uvavanyo lwe-Tukey HSD, p< 0.05). Ibha yesikali = 10 µm. (c) Ukujongwa kweeseli ze-BY-2 ezifileyo yi-Evans blue staining. Iiseli ze-BY-2 ezinyangwe nge-KAND 11 (50 μM okanye 100 μM) okanye i-DMSO zihlolwe nge-bright-field microscopy. n=3. Ibha yesikali = 100 µm.
Ukufunyanwa nokusetyenziswa kweemveliso ezintsha zendalo kukhokelele kwinkqubela phambili ebalulekileyo kwiinkalo ezahlukeneyo zobomi babantu, kuquka amayeza kunye nezolimo. Uphando lwembali luye lwenziwa ukuze kufunyanwe iikhompawundi eziluncedo kwizixhobo zendalo. Ngokukodwa, ii-actinomycetes zaziwa ngokuba luncedo njengee-antibiotics zokulwa ne-parasitic kwi-nematodes ngenxa yokukwazi kwazo ukuvelisa ii-metabolites ezahlukeneyo zesibini ezifana ne-avermectin, ikhompawundi ekhokelayo ye-ivermectin kunye ne-bleomycin kunye ne-derivatives zayo, esetyenziswa ngamayeza njenge-arhente yokulwa nomhlaza21,22. Ngokufanayo, iintlobo ngeentlobo zeekhompawundi zokubulala ukhula zifunyenwe kwi-actinomycetes, ezinye zazo sele zisetyenziswa kurhwebo1,23. Ke ngoko, uhlalutyo lwee-metabolites ze-actinomycete zokwahlula iimveliso zendalo ezinemisebenzi yebhayoloji efunekayo luthathwa njengecebo elisebenzayo. Kolu phononongo, sifumene ikhompawundi entsha, i-coumamonamide, evela kwi-S. werraensis kwaye sayivelisa ngempumelelo. I-Ursonic acid sisixhobo esiphakathi se-urbenamide kunye ne-derivatives zayo. Inokubangela ukugoba kweengcambu okubonakalayo, ibonise umsebenzi ophakathi ukuya kowomeleleyo we-herbicidal, kwaye yonakalisa ngokuthe ngqo okanye ngokungathanga ngqo ii-microtubules zezityalo. Nangona kunjalo, indlela esebenza ngayo i-urmotonic acid ingahluka kweye-microtubule inhibitors ezikhoyo, kuba i-KAND 11 ikwaphazamisa ii-actin filaments kwaye ibangele ukufa kweeseli, nto leyo ebonisa indlela yokulawula apho i-urmotonic acid kunye ne-derivatives zayo zichaphazela uluhlu olubanzi lwezakhiwo ze-cytoskeletal.
Ukuchazwa ngakumbi kwe-urbenonic acid kuya kunceda ukuqonda ngcono indlela esebenza ngayo i-urbenonic acid. Ngokukodwa, injongo elandelayo kukuvavanya amandla e-ursonic acid okubophelela kwi-microtubules ezinciphileyo ukuze kuchongwe ukuba i-ursonic acid kunye ne-derivatives zayo zisebenza ngokuthe ngqo kwi-microtubules kwaye ziwasuse, okanye ukuba isenzo sazo siphumela ekuwohlokeni kwe-microtubule. Ukongeza, kwimeko apho ii-microtubules zingengomjongo ngqo, ukuchonga indawo yokusebenza kunye neenjongo ze-molecular ze-ursonic acid kwiiseli zezityalo kuya kunceda ukuqonda ngakumbi iipropati zeekhompawundi ezinxulumene noko kunye neendlela ezinokwenzeka zokuphucula umsebenzi wokubulala ukhula. Uvavanyo lwethu lwe-bioactivity lutyhile amandla akhethekileyo e-cytotoxic e-ursonic acid ekukhuleni kwezityalo ezifana ne-Arabidopsis thaliana, icuba kunye ne-liverwort, ngelixa iiseli ze-E. coli kunye ne-HeLa zingachaphazelekanga. Ubuthi obuncinci okanye obungekhoyo kwiiseli zezilwanyana buluncedo kwi-ursonic acid derivatives ukuba ziphuhliswa njenge-herbicides zokusetyenziswa kumasimi avulekileyo ezolimo. Enyanisweni, ekubeni ii-microtubules zizakhiwo eziqhelekileyo kwi-eukaryotes, ukuthintela kwazo okukhethwayo kwizityalo yimfuneko ephambili kwi-herbicides. Umzekelo, ipropyzamide, iarhente yokususa ipolymerizing ye-microtubule ebopha ngokuthe ngqo kwi-tubulin kwaye ithintele i-polymerization, isetyenziswa njenge-herbicide ngenxa yokuba inetyhefu ephantsi kwiiseli zezilwanyana. Ngokungafaniyo ne-disopyramide, ii-benzamides ezinxulumene nazo zinezinto ezahlukeneyo ekujoliswe kuzo. Ukongeza kwi-microtubules zezityalo, i-RH-4032 okanye i-benzoxamide ikwathintela ii-microtubules zeeseli zezilwanyana okanye ii-oomycetes, ngokwahlukeneyo, kwaye i-zalilamide isetyenziswa njenge-fungicide ngenxa yokuba inetyhefu ephantsi ye-phyto25,26,27. Ibhere esandul' ukufunyanwa kunye nezinto eziphuma kuyo zibonisa i-cytotoxicity ekhethiweyo kwizityalo, kodwa kubalulekile ukuqaphela ukuba utshintsho olongezelelweyo lunokutshintsha ukucaciswa kwazo ekujoliswe kuzo, okunokubonelela ngezinye izinto eziphuma kuzo ukulawula i-fungus okanye ii-oomycetes ezibangela izifo.
Iimpawu ezizodwa ze-urbenonic acid kunye nezinto eziphuma kuyo ziluncedo ekuphuhlisweni kwazo njenge-herbicides kunye nokusetyenziswa njengezixhobo zophando. Ukubaluleka kwe-cytoskeleton ekulawuleni imo yeseli yesityalo kuyaqatshelwa ngokubanzi. Izifundo zangaphambili zibonise ukuba izityalo ziye zavelisa iindlela ezintsonkothileyo zokuhlelwa kwe-cortical microtubule ngokulawula i-microtubule dynamics ukulawula ngokufanelekileyo i-morphogenesis. Inani elikhulu lee-molecules ezinoxanduva lokulawula umsebenzi we-microtubule lichongiwe, kwaye uphando oluhambelana nalo lusaqhubeka3,4,28. Ukuqonda kwethu ngoku i-microtubule dynamics kwiiseli zezityalo akuchazi ngokupheleleyo iindlela zokuhlelwa kwe-cortical microtubule. Umzekelo, nangona zombini i-disopyramide kunye ne-oryzalin zinokususa i-polymerize microtubules, i-disopyramide ibangela ukugqwetheka okukhulu kweengcambu ngelixa i-oryzalin inesiphumo esincinci. Ngaphezu koko, utshintsho kwi-tubulin, oluzinzisa ii-microtubules, lukwabangela i-dextrorotation kwiingcambu, ngelixa i-paclitaxel, ekwazinzisa i-microtubule dynamics, ayikwenzi oko. Ke ngoko, ukufunda nokuchonga iinjongo ze-molecular ze-ursolic acid kufuneka kunike ulwazi olutsha malunga nokulawulwa kwee-microtubules ze-cortical zezityalo. Ngokufanayo, ukuthelekiswa kwexesha elizayo kweekhemikhali ezisebenzayo ekukhuthazeni ukukhula okugqwethekileyo, njenge-disopyramide, kunye neekhemikhali ezingasebenzi kakuhle, ezifana ne-oryzalin okanye i-kumamotoric acid, kuya kunika izikhokelo zendlela ukukhula okugqwethekileyo okwenzeka ngayo.
Kwelinye icala, ukuhlelwa kwakhona kwe-cytoskeletal enxulumene nokuzikhusela yenye indlela yokuchaza ubuthi be-ursonic acid. Ukosuleleka yi-pathogen okanye ukungeniswa kwe-ecitor kwiiseli zezityalo ngamanye amaxesha kubangela ukutshatyalaliswa kwe-cytoskeleton kunye nokufa kweseli okulandelayo29. Umzekelo, i-cryptoxanthin evela kwi-oomycete ibikwe ukuba iphazamisa ii-microtubules kunye ne-actin filaments ngaphambi kokufa kweseli yecuba, okufana noko kwenzekayo ngonyango lwe-KAND30,31. Ukufana phakathi kweempendulo zokuzikhusela kunye neempendulo zeseli ezibangelwa yi-ursonic acid kusenze sacinga ukuba zibangela iinkqubo zeseli eziqhelekileyo, nangona isiphumo esikhawulezayo nesinamandla se-ursonic acid kune-cryptoxanthin siyabonakala. Nangona kunjalo, izifundo zibonise ukuba ukuphazamiseka kwe-actin filaments kukhuthaza ukufa kweseli okuzenzekelayo, okungasoloko kuhamba nokuphazamiseka kwe-microtubule29. Ukongeza, kusafuneka kubonwe ukuba ngaba i-pathogen okanye i-ecitor ibangela ukukhula kweengcambu okugqwethekileyo, njengoko i-ursonic acid derivatives isenza. Ke ngoko, ulwazi lweemolekyuli oludibanisa iimpendulo zokuzikhusela kunye ne-cytoskeleton yingxaki ekhangayo ekufuneka ijongwane nayo. Ngokusebenzisa ubukho beekhompawundi ezinobunzima obuphantsi beemolekyuli ezinxulumene ne-ursonic acid, kunye noluhlu lwee-derivatives ezineempawu ezahlukeneyo, zinokubonelela ngamathuba okujolisa kwiindlela zeseli ezingaziwayo.
Xa zizonke, ukufunyanwa nokusetyenziswa kweekhompawundi ezintsha eziguqula amandla e-microtubule kuya kubonelela ngeendlela ezinamandla zokujongana neendlela ezintsonkothileyo zeemolekyuli ezisisiseko sokumisela ukuma kweseli yesityalo. Kule meko, i-compound urmotonic acid esandul’ ukuphuhliswa, echaphazela ii-microtubules kunye ne-actin filaments kwaye ibangele ukufa kweeseli, inokubonelela ngethuba lokufumanisa unxibelelwano phakathi kolawulo lwe-microtubule kunye nezi ndlela ezinye. Ngoko ke, uhlalutyo lweekhemikhali kunye nebhayoloji olusebenzisa i-urbenonic acid luya kusinceda siqonde iindlela zokulawula iimolekyuli ezilawula i-cytoskeleton yesityalo.
Faka i-S. werraensis MK493-CF1 kwi-flask ye-Erlenmeyer engama-500 mL exutywe ne-baffled equlethe i-110 mL yembewu ephakathi equlathe i-2% (w/v) galactose, i-2% (w/v) Essence paste, i-1% (w/v) Bacto composition. -soyton (Thermo Fisher Scientific, Inc.), i-0.5% (w/v) corn extract (KOGOSTCH Co., Ltd., Japan), i-0.2% (w/v) (NH4)2SO4 kunye ne-0.2% CaCO3 emanzini acocekileyo. (pH 7.4 ngaphambi kokubulala iintsholongwane). Iimbewu ezikhuliswe kwi-rotary shaker (180 rpm) kwi-27°C kangangeentsuku ezi-2. Ukulima ngemveliso ngokubilisa okuqinileyo. Imbewu ekhuliswe (7 ml) idluliselwe kwi-flask ye-K-1 engama-500 ml equlethe i-40 g yemveliso equlathe i-15 g yerhasi ecinezelweyo (MUSO Co., Ltd., eJapan) kunye ne-25 g yamanzi anyibilikisiweyo (i-pH ayilungiswanga ngaphambi kokuba ibulawe iintsholongwane). Ukubiliswa kwenziwe kwi-30°C ebumnyameni kangangeentsuku ezili-14. Izinto zokubilisa zikhutshwe nge-40 ml/ibhotile ye-EtOH kwaye zifakwe i-centrifuged (1500 g, 4°C, imizuzu eli-10). I-culture supernatant (60 ml) ikhutshwe ngomxube we-10% MeOH/EtOAc. Umaleko wendalo wapheliswa phantsi koxinzelelo oluncitshisiweyo ukuze kufunyanwe intsalela (59.5 mg), eyathi yafakwa kwi-HPLC nge-gradient elution (imizuzu eyi-0–10: 90%) kwikholamu yesigaba esingasemva (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × ubude obuyi-250 mm) H2O/CH3CN, imizuzu eyi-10–35: 90% H2O/CH3CN ukuya kwi-70% H2O/CH3CN (i-gradient), imizuzu engama-35–45: 90% H2O/EtOH, imizuzu engama-45–155: 90% H2O/EtOH ukuya kwi-100% EtOH (i-gradient (i-gradient), imizuzu eyi-155–200: 100% EtOH) ngesantya sokuhamba se-1.5 ml/min, i-coumamonamide (1, 36.0 mg) yahlulwa njengomgubo omhlophe ongaguqukiyo.
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; I-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 yaseColumbia (Col-0) ifunyenwe kwiZiko leeMveliso zeBiological Arabidopsis (ABRC) ngemvume yokusetyenziswa kuphando. Imbewu yeCol-0 yasasazwa yaza yagcinwa phantsi kweemeko zethu zelebhu kwaye yasetyenziswa njengezityalo ze-Arabidopsis zasendle. Imbewu ye-Arabidopsis yahlanjululwa phezulu yaza yatyalwa kwi-Murashige kunye ne-Skoog medium enamandla angaphantsi kwesiqingatha equlethe i-2% ye-sucrose (iFujifilm Wako Pure Chemical), i-0.05% (w/v) i-2-(4-morpholino)ethanesulfonic acid (MES) (iFujifilm Wako Pure Chemical). ) kunye ne-1.5% ye-agar (iFujifilm Wako Pure Chemical), i-pH 5.7, kwi-23 °C kunye nokukhanya okungaguqukiyo. Imbewu ye-phs1-1 mutant inikwe ngu-T. Hashimoto (iNara Institute of Science and Technology).
Imbewu ye-SR-1 yohlobo lwesityalo inikwe yi-T. Hashimoto (Nara Institute of Science and Technology) kwaye isetyenziswa njengezityalo zecuba zasendle. Imbewu yecuba yacocwa yaza yafakwa emanzini angenazintsholongwane ubusuku obuthathu ukuze kukhuthazwe ukuhluma, emva koko yafakwa kwisisombululo esiqinileyo esine-2% ye-sucrose, i-0.05% (w/v) MES, kunye ne-0.8% ye-gellan gum (Fujifilm Wako Pure Chemical) Murashige. kunye ne-Skoog medium) ene-pH 5.7 kwaye yafakwa kwi-23°C phantsi kokukhanya okungaguqukiyo.
I-Strain Tak-1 yabonelelwa yi-T. Kohchi (iYunivesithi yaseKyoto) kwaye yasetyenziswa njengeyunithi yovavanyo eqhelekileyo yophando lwe-liverwort. I-Gemma ifunyenwe kwizityalo ezikhuliswe ngokucoca iintsholongwane yaza yabekwa kwi-Gamborg B5 medium (iFujifilm Wako Pure Chemical) equlethe i-1% ye-sucrose kunye ne-0.3% ye-gellan gum yaza yafakwa kwi-23°C phantsi kokukhanya okuqhubekayo.
Iiseli ze-BY-2 zecuba (iNicotiana tabacum L. cv. Bright Yellow 2) zibonelelwe yi-S. Hasezawa (iYunivesithi yaseTokyo). Iiseli ze-BY-2 zixutywe kayi-95 kwi-modified Linsmeier kunye ne-Skoog medium kwaye zongezwa veki nganye nge-2,4-dichlorophenoxyacetic acid 32. I-cell suspension ixutywe kwi-rotary shaker kwi-130 rpm kwi-27°C ebumnyameni. Hlamba iiseli nge-volume ephindwe kalishumi kune-medium entsha kwaye uphinde uxutywe kwi-medium efanayo. Imigca ye-BY-2 transgenic cell lines eveza ngokuzinzileyo uphawu lwe-microtubule TagRFP-TUA6 okanye uphawu lwe-actin filament GFP-ABD2 phantsi kwe-cauliflower mosaic virus 35S promoter zenziwe njengoko kuchaziwe33,34,35. Le migca yeseli inokugcinwa kwaye ihambelaniswe kusetyenziswa iinkqubo ezifana nezo zisetyenziselwe umgca weseli we-BY-2 wokuqala.
Iiseli zeHeLa zakhuliswa kwi-Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) eyongezwe yi-10% ye-fetal bovine serum, i-1.2 U/ml penicillin, kunye ne-1.2 μg/ml streptomycin kwi-incubator engama-37°C ene-5% CO2.
Zonke iimvavanyo ezichazwe kulo mbhalo wesandla zenziwe ngokuhambelana nemigaqo nezikhokelo zokhuseleko lwezinto eziphilayo zaseJapan.
Iikhompawundi zanyibilikiswa kwi-dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) njengezisombululo zesitokhwe zaza zaxutywa kwi-MS medium ye-Arabidopsis kunye necuba okanye i-Gamborg B5 medium ye-liverwort. Kwi-assay yokuthintela ukukhula kweengcambu, imbewu engaphezulu kwe-10 kwipleyiti nganye yahlwayelwa kwi-agar medium equlethe iikhompawundi ezibonisiweyo okanye i-DMSO. Imbewu yafakwa kwigumbi lokukhula kangangeentsuku ezisi-7. Izithole zafotwa kwaye ubude beengcambu balinganiswa. Kwi-assay yokuhluma kwe-Arabidopsis, imbewu ezingama-48 kwipleyiti nganye zahlwayelwa kwi-agar medium equlethe i-200 μM compound okanye i-DMSO. Imbewu ye-Arabidopsis yakhuliswa kwigumbi lokukhula kwaye inani lezithole ezihlumileyo labalwa emva kweentsuku ezisi-7 emva kokuhluma (dag). Kwi-assay yokuhluma kwecuba, imbewu ezingama-24 kwipleyiti nganye zahlwayelwa kwi-agar medium equlethe i-200 μM KAND okanye i-DMSO. Imbewu yecuba yakhuliswa kwigumbi lokukhula kwaye inani lezithole ezihlumileyo labalwa emva kweentsuku ezili-14. Kwivavanyo lokuthintela ukukhula kwe-liverwort, ii-embryos ezili-9 ezivela kwipleyiti nganye zafakwa kwi-agar medium equlethe ubuninzi obubonisiweyo be-KAND okanye i-DMSO zaza zafakwa kwigumbi lokukhula iintsuku ezili-14.
Sebenzisa izithole ezifakwe i-5 mg/ml ye-propidium iodide (PI) ukuze ubone ulungelelwaniso lweengcambu ze-meristem. Imiqondiso ye-PI ibonwe nge-fluorescence microscopy kusetyenziswa i-TCS SPE confocal laser scanning microscope (Leica Microsystems).
Ukufakwa kweengcambu nge-β-glucuronidase (GUS) ngokwemigaqo echazwe nguMalami noBenfey36. Izithole zifakwe kwi-90% ye-acetone ubusuku bonke, zafakwa i-0.5 mg/ml ye-5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid kwi-GUS buffer kangangeyure e-1 zaza zafakwa kwisisombululo se-chloraldehyde esimanzi. (8 g ye-chloral hydrate, 2 ml yamanzi kunye ne-1 ml ye-glycerol) kwaye zajongwa nge-differential interference contrast microscopy kusetyenziswa i-Axio Imager M1 microscope (Carl Zeiss).
Ii-engile zeengcambu zilinganiswe kwizithole ezineentsuku ezisi-7 ubudala ezikhuliswe kwiipleyiti ezibekwe ngokuthe nkqo. Linganisa i-engile yengcambu ukusuka kwicala le-gravity vector njengoko kuchaziwe kwinyathelo lesi-6.
Ulungiselelo lwee-cortical microtubules lubonwe njengoko kuchaziwe, kunye notshintsho oluncinci kwiprotokholi 37. I-anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) kunye ne-Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) zisetyenziswe njenge-primary antibodies kunye ne-secondary kwi-1:1000 kunye ne-1:100 dilutions, ngokulandelanayo. Imifanekiso ye-Fluorescence ifunyenwe kusetyenziswa i-TCS SPE confocal laser scanning microscope (Leica Microsystems). Fumana imifanekiso ye-Z-stack kwaye wenze ii-maximum intensity projections ngokwemiyalelo yomenzi.
Uvavanyo lokwanda kweeseli zeHeLa lwenziwe kusetyenziswa iCell Counting Kit 8 (Dojindo) ngokwemiyalelo yomenzi.
Ukukhula kwe-E. coli DH5α kuhlalutywe ngokulinganisa uxinano lweeseli kwinkcubeko kusetyenziswa i-spectrophotometer kwi-600 nm (OD600).
Ulungelelwaniso lwe-cytoskeletal kwiiseli ze-BY-2 eziguqulweyo lubonwe kusetyenziswa imakroskopu ye-fluorescence exhotyiswe ngesixhobo sokuskena se-CSU-X1 confocal (Yokogawa) kunye nekhamera ye-sCMOS (Zyla, Andor Technology). Uxinano lwe-cytoskeletal luhlolwe ngohlalutyo lomfanekiso, olulinganise ipesenti yee-cytoskeletal pixels phakathi kwee-cytoplasmic pixels kwimifanekiso ye-confocal kusetyenziswa isoftware ye-ImageJ njengoko kuchaziwe38,39.
Ukuze kufunyanwe ukufa kweeseli kwiiseli ze-BY-2, i-aliquot ye-cell suspension yafakwa kwi-0.05% Evans blue imizuzu eli-10 kubushushu begumbi. Ukufakwa kwedayi eluhlaza okwesibhakabhaka kwe-Evans kwiiseli ezifileyo kuxhomekeke ekukhutshweni kwedayi kwiiseli eziphilayo yi-plasma membrane engaphelelanga40. Iiseli ezinemibala zabonwa kusetyenziswa i-bright-field microscope (BX53, Olympus).
Iiseli zeHeLa zikhuliswe kwiDMEM zongezwe yi-10% FBS kwi-incubator efumileyo kwi-37°C kunye ne-5% CO2. Iiseli ziphathwe nge-100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), okanye i-100 ng/ml Nocodmaze (Sigma) iiyure ezi-6 kwi-37°C. Iiseli zilungisiwe ngeMetOH imizuzu eli-10 zaza emva koko zafakwa i-acetate imizuzu emi-5 kubushushu begumbi. Iiseli ezizinzileyo zafakwa i-β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) yaxutywa kwi-0.5% BSA/PBS iiyure ezi-2, zahlanjwa izihlandlo ezi-3 nge-TBST, zaza zafakwa i-Alexa Fluor goat antibody. 488 iyure e-1. – I-Mouse IgG (Thermo Fisher Scientific: A11001) kunye ne-15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) exutywe kwi-0.5% BSA/PBS. Emva kokuhlamba nge-TBST kathathu, iiseli ezinemibala zabonwa kwi-microscope eguqulweyo ye-Nikon Eclipse Ti-E. Imifanekiso yathathwa ngekhamera epholileyo ye-Hamamatsu ORCA-R2 CCD kusetyenziswa isoftware ye-MetaMorph (iiMolecular Devices).
Ixesha leposi: Juni-17-2024