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Ukutholwa kanye nokusetshenziswa okunenzuzo kwemikhiqizo yemvelo kungasiza ekuthuthukiseni impilo yabantu. Amakhemikhali okuvimbela ukukhula kwezitshalo asetshenziswa kabanzi njengemithi yokubulala ukhula ukulawula ukhula. Ngenxa yesidingo sokusebenzisa izinhlobo ezahlukene zemithi yokubulala ukhula, kunesidingo sokuhlonza amakhemikhali anezindlela ezintsha zokusebenza. Kulesi sifundo, sithole inhlanganisela entsha ye-N-alkoxypyrrole, i-coumamonamide, evela ku-Streptomyces werraensis MK493-CF1 futhi sasungula inqubo ephelele yokwenziwa. Ngokusebenzisa izivivinyo zomsebenzi webhayoloji, sithole ukuthi i-urs-monoamic acid iyi-synthetic intermediate ye-urs-monoamide kanye ne-potentialisivimbeli sokukhula kwezitshaloNgaphezu kwalokho, sithuthukise izinhlobo ezahlukene ze-urbenonic acid derivatives, okuhlanganisa ne-urbenyloxy derivative (UDA), enomsebenzi ophezulu wokubulala ukhula ngaphandle kokuthinta kabi ukukhula kwamaseli e-HeLa. Sithole nokuthi i-urmotonic acid derivatives iphazamisa ama-microtubules ezitshalo; ngaphezu kwalokho, i-KAND ithinta ama-actin filaments futhi ibangela ukufa kwamaseli; Le miphumela enezici eziningi ihlukile kweye-microtubule inhibitors eyaziwayo futhi iphakamisa indlela entsha yokusebenza ye-ursonic acid, emele inzuzo ebalulekile ekuthuthukisweni kwemithi yokubulala ukhula emisha.
Ukutholwa kanye nokusetshenziswa okusebenzayo kwemikhiqizo yemvelo ezuzisayo kanye nemikhiqizo yayo kuyindlela yokuthuthukisa ikhwalithi yokuphila komuntu. Ama-metabolite esibili akhiqizwa yizinto ezincane, izitshalo nezinambuzane aholele ekuthuthukisweni okukhulu kwezokwelapha kanye nezolimo. Ama-antibiotic amaningi kanye nemithi yokulwa ne-leukemia kuye kwathuthukiswa ngemikhiqizo yemvelo. Ngaphezu kwalokho, izinhlobo ezahlukene zeizibulala-zinambuzane, ama-fungicides kanye nama-herbicides akhishwa kule mikhiqizo yemvelo ukuze asetshenziswe kwezolimo. Ikakhulukazi, ama-herbicides okulawula ukhula angamathuluzi abalulekile okwandisa isivuno sezitshalo kwezolimo zanamuhla, futhi izinhlobo ezahlukene zama-compounds sezivele zisetshenziswa kwezentengiselwano. Izinqubo eziningana zamaseli ezitshalweni, njenge-photosynthesis, i-amino acid metabolism, ukwakheka kodonga lwamaseli, ukulawulwa kwe-mitosis, i-phytohormone signaling, noma ukwakheka kwamaprotheni, kubhekwa njengezinhloso ezijwayelekile zama-herbicides. Ama-compounds avimbela umsebenzi we-microtubule ayisigaba esivamile sama-herbicides athinta ukukhula kwezitshalo ngokuthinta ukulawulwa kwe-mitotic2.
Ama-Microtubules ayizingxenye ze-cytoskeleton futhi agcinwa kabanzi kumaseli e-eukaryotic. I-tubulin heterodimer iqukethe i-α-tubulin kanye ne-β-tubulin ezakha ama-protofilaments e-microtubule aqondile, kanye nama-protofilaments ayi-13 akha isakhiwo esiyindilinga. Ama-Microtubules adlala indima eminingi kumaseli ezitshalo, okuhlanganisa nokunquma ukuma kweseli, ukuhlukaniswa kweseli, kanye nokuthuthwa kwangaphakathi kweseli3,4. Amaseli ezitshalo aqukethe ama-microtubules ngaphansi kwe-membrane ye-plasma ye-interphase, futhi lawa okuthiwa ama-cortical microtubules kucatshangwa ukuthi alawula ukuhlelwa kwama-microfibrils e-cellulose ngokulawula ama-complexes e-cellulose synthase4,5. Ama-microtubules e-cortical amaseli e-root epidermal, akhona endaweni yokwandiswa okusheshayo kwesihloko sempande, atholakala eceleni, kanti ama-microfiber e-cellulose alandela la ma-microtubules futhi akhawulela indlela yokwanda kweseli, ngaleyo ndlela akhuthaze ukwanda kweseli le-anisotropic. Ngakho-ke, umsebenzi we-microtubule uhlobene eduze nokwakheka kwesitshalo. Ukufakwa esikhundleni kwe-amino acid kuma-genes afaka ikhodi ye-tubulin kubangela ukugoba kwama-array e-cortical microtubule kanye nokukhula ohlangothini lwesobunxele noma lwesokudla ku-Arabidopsis 6,7. Ngokufanayo, ukuguqulwa kwamaprotheni ahlobene ne-microtubule alawula i-microtubule dynamics nakho kungaholela ekukhuleni kwezimpande okuphambene8,9,10,11,12,13. Ngaphezu kwalokho, ukwelashwa ngama-herbicides aphazamisa i-microtubule njenge-disopyramide, eyaziwa nangokuthi i-pretilachlor, nakho kubangela ukukhula kwezimpande ezigobile ohlangothini lwesobunxele14. Le datha ikhombisa ukuthi ukulawulwa okuqondile komsebenzi we-microtubule kubalulekile ekunqumeni indlela yokukhula kwezitshalo.
Kutholakale izinhlobo ezahlukene zezithibi ze-microtubule, futhi le mithi ibe negalelo elikhulu ocwaningweni lwe-cytoskeletal, kanye nezolimo kanye nezokwelapha2. Ikakhulukazi, i-oryzalin, ama-dinitroaniline compounds, i-disopyramide, ama-compounds ahlobene ne-benzamide, kanye nama-analogues awo angavimbela ukusebenza kwe-microtubule futhi ngaleyo ndlela avimbele ukukhula kwezitshalo. Ngakho-ke, asetshenziswa kabanzi njengemithi yokubulala ukhula. Kodwa-ke, njengoba ama-microtubule eyingxenye ebalulekile yamaseli ezitshalo nezilwane, iningi lama-microtubule inhibitors linobuthi kuzo zombili izinhlobo zamaseli. Ngakho-ke, naphezu kokusebenza kwawo njengemithi yokubulala ukhula, inani elilinganiselwe lama-antimicrotubule agents asetshenziselwa izinjongo ezisebenzayo.
I-Streptomyces uhlobo lomndeni we-Streptomyces, oluhlanganisa amabhaktheriya e-aerobic, gram-positive, filamentous futhi yaziwa kakhulu ngekhono layo lokukhiqiza izinhlobo eziningi ze-metabolites yesibili. Ngakho-ke, ibhekwa njengenye yemithombo ebaluleke kakhulu yemikhiqizo emisha yemvelo esebenzayo. Esifundweni samanje, sithole inhlanganisela entsha ebizwa ngokuthi i-coumamonamide, eyahlukaniswa ne-Streptomyces werraensis MK493-CF1 kanye ne-S. werraensis ISP 5486. Sisebenzisa ukuhlaziywa kwe-spectral kanye nokuhlaziywa okugcwele kwe-spectral, isakhiwo se-coumamonamide sachazwa futhi ukwakheka kwayo okuyingqayizivele kwe-N-alkoxypyrrole skeleton kwanqunywa. I-Ursmonic acid, i-synthetic intermediate ye-ursmonoamide kanye ne-derivatives yayo, yatholakala ivimbela ukukhula nokuhluma kwesitshalo esithandwayo semodeli i-Arabidopsis thaliana. Esifundweni sobudlelwano besakhiwo nomsebenzi, sithole ukuthi inhlanganisela ene-C9 eguqulwe yaba yi-ursonic acid, ebizwa ngokuthi i-nonyloxy derivative ye-ursonic acid (KAND), ithuthukisa kakhulu umphumela wokuvimbela ekukhuleni nasekuhlumeni. Okuphawulekayo ukuthi lesi sivimbeli sokukhula kwezitshalo esisanda kutholakala sithinte nokukhula kukagwayi kanye ne-liverwort futhi sasingenabo ubuthi kuma-bacteria noma kuma-HeLa cells. Ngaphezu kwalokho, ezinye izinto ezitholakala ku-urmotonic acid zibangela i-phenotype yezimpande ezisontekile, okusho ukuthi lezi zinto ezitholakala ku-derivatives zithinta ngqo noma ngokungaqondile ama-microtubules. Ngokuhambisana nalo mbono, ukuqaphela kwethu ama-microtubules abhalwe ngokuthi ama-immunohistochemically noma ngama-fluorescent proteins kubonisa ukuthi ukwelashwa kwe-KAND kususa i-polymerizes ama-microtubules. Ngaphezu kwalokho, ukwelashwa nge-kumamotonic acid derivatives kuphazamise ama-microfilaments e-actin. Ngakho-ke, sithole isivimbeli esisha sokukhula kwezitshalo esinendlela ehlukile yokusebenza ehilela ukubhujiswa kwe-cytoskeleton.
I-Stray MK493-CF1 yahlukaniswa enhlabathini eShinagawa-ku, eTokyo. I-Stray MK493-CF1 yakha i-stromal mycelium enamagatsha amahle. Uchungechunge oluyingxenye lwe-16S ribosomal RNA gene (1422 bp) lwatholakala. Lolu hlobo lufana kakhulu ne-S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: uhlobo olujwayelekile, 99.93%). Ngokusekelwe kulo mphumela, kwatholakala ukuthi lolu hlobo luhlobene eduze nohlobo lwe-S. werraensis. Ngakho-ke, saqamba lolu hlobo ngokuthi i-S. werraensis MK493-CF1 okwesikhashana. I-S. werraensis ISP 5486T nayo ikhiqiza ama-compounds afanayo e-bioactive. Njengoba kwakuncane ucwaningo lokuqala lokuthola imikhiqizo yemvelo evela kulesi silwanyana esincane, kwenziwa ucwaningo olwengeziwe lwamakhemikhali. Ngemva kokutshala i-S. werraensis MK493-CF1 endaweni yebhali ngokubilisa okuqinile ku-30°C izinsuku ezingu-14, indawo yakhishwa nge-50% EtOH. Ama-60 ml esampula omisiwe ukuze kutholakale ama-59.5 mg e-crude extract. I-crude extract yafakwa ku-reverse phase HPLC ukuze inikeze i-N-methoxy-1H-pyrrole-2-carboxamide (1, ebizwa ngokuthi i-coumamonamide, 36.0 mg). Inani eliphelele le-1 cishe liyi-60% ye-crude extract. Ngakho-ke, sinqume ukutadisha ngokuningiliziwe izakhiwo ze-kumamotoamide 1.
I-Coumamonamide 1 iyimpuphu emhlophe engaguquki kanye ne-high resolution mass spectrometry (i-HRESIMS) iqinisekisa i-C6H8N2O2 (Isithombe 1). Ingcezu ye-pyrrole eshintshiwe ye-C2 yale nhlanganisela ibonakaliswa yi-δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH ku-1H NMR spectrum: 4.5 Hz, H-5) kanye ne-δH 6.78 (1H, d, J = 2.5 Hz, H-6), kanti i-13C NMR spectrum ikhombisa ukuba khona kwama-athomu e-carbon amane e-sp2. Ukuba khona kweqembu le-amide endaweni ye-C2 kuhlolwe ngokuhlobana kwe-HMBC kusuka ku-C-3 proton kuya ku-amide carbonyl carbon ku-δC 161.1. Ngaphezu kwalokho, ukuphakama kwe-1 H kanye ne-13 C NMR ku-δH 4.10 (3H, S) kanye ne-δC 68.3 kubonisa ukuba khona kwamaqembu e-N-methoxy ku-molecule. Nakuba indawo efanele yeqembu le-methoxy yayingakatholakali kusetshenziswa ukuhlaziywa kwe-spectroscopic njenge-enhanced difference spectroscopy kanye nesifinyezo se-nuclear Overhauser (NOEDF), i-N-methoxy-1H-pyrrole-2-carboxamide yaba yinhlanganisela yokuqala engaba khona.
Ukuze kutholakale isakhiwo esifanele se-1, kwenziwa ukuhlanganiswa okuphelele (Isithombe 2a). Ukwelashwa kwe-2-aminopyridine 2 etholakala emakethe nge-m-CPBA kuholele ekuvuneni kwe-N-oxide 3 okuhambisanayo. Ngemva kwe-2-aminoazidation ye-2, ukusabela kwe-cyclocondensation okuchazwe ngu-Abramovich kwenziwa ku-benzene ku-90°C ukuthola i-1-hydroxy-1H-pyrrole-2-carbonitrile 5 ngamagremu. Isivinini 60% (izigaba ezimbili). 15,16. I-Methylation kanye ne-hydrolysis ye-4 kwabe sekunikeza i-1-methoxy-1H-pyrrole-2-carboxylic acid (ebizwa ngokuthi "i-cumotonic acid", 6) ekuvuneni okuhle (70%, izinyathelo ezimbili). Ekugcineni, ukuhlanganiswa nge-acid chloride ephakathi kwe-6 kusetshenziswa i-ammonia yamanzi kunikeze i-Kumamoto amide 1 ekuvuneni kwe-98%. Yonke idatha ye-spectral ye-1 eyenziwe yayifana ne-1 ehlukanisiwe, ngakho-ke isakhiwo se-1 sanqunywa;
Ukuhlanganiswa okuvamile kanye nokuhlaziywa komsebenzi webhayoloji we-urbenamide kanye ne-urbenic acid. (a) Ukuhlanganiswa okuphelele kwe-Kumamoto amide. (b) Izithombo ze-Arabidopsis Columbia (Col) zasendle ezinezinsuku eziyisikhombisa ubudala zitshalwe emapuletini e-Murashige kanye ne-Skoog (MS) aqukethe i-coumamonamide 6 noma i-coumamonamide 1 ngamanani abonisiwe. Ibha yesikali = 1 cm.
Okokuqala, sihlole imisebenzi yezinto eziphilayo ye-urbenamide kanye nezingxenye zayo ukuze sibone ikhono layo lokulawula ukukhula kwezitshalo. Singeze amazinga ahlukahlukene e-ursmonamide 1 noma i-ursmonic acid 6 ku-MS agar medium kanye nezithombo ze-Arabidopsis thaliana ezikhuliswe kule ndawo. Lokhu kuhlola kubonise ukuthi amazinga aphezulu (500 μM) ka-6 avimbele ukukhula kwezimpande (Isithombe 2b). Okulandelayo, sikhiqize ama-derivatives ahlukahlukene ngokufaka esikhundleni sendawo ye-N1 ka-6 futhi senza izifundo zobudlelwano besakhiwo nomsebenzi kuzo (inqubo yokwenziwa kwe-analogue ichazwe ku-Supporting Information (SI)). Izithombo ze-Arabidopsis zikhuliswe endaweni equkethe ama-derivatives e-ursonic acid angu-50 μM, futhi ubude bezimpande balinganiswa. njengoba kuboniswe esithombeni. Njengoba kuboniswe kuZithombe 3a, b, kanye no-S1, ama-coumamo acid anobude obuhlukene bezintambo ze-alkoxy eziqondile (9, 10, 11, 12, kanye no-13) noma izintambo ezinkulu ze-alkoxy (15, 16, kanye no-17) endaweni ye-N1. Ama-derivatives abonise ukuvimbela okukhulu ukukhula kwezimpande. Ngaphezu kwalokho, sithole ukuthi ukusetshenziswa kwe-200 μM 10, 11, noma 17 kuvimbele ukuhluma (Izithombe 3c kanye no-S2).
Ucwaningo lobudlelwano besakhiwo nomsebenzi we-Kumamoto amide kanye namakhemikhali ahlobene. (a) Uhlelo lwesakhiwo kanye nokuhlanganiswa kwama-analogue. (b) Ukulinganiswa kobude bezimpande zezithombo zezinsuku eziyi-7 ezikhule endaweni ephakathi ye-MS ezine-coumamonamide derivatives engu-50 μM noma ezingenayo. Ama-asterisk abonisa umehluko omkhulu ngokwelashwa okungamanga (t test, p< 0.05). n>18. Idatha ikhonjiswa njengesilinganiso ± SD. nt isho ukuthi “ayihlolwanga” ngoba imbewu engaphezu kuka-50% ayizange ihlume. (c) Ukulinganiswa kwesilinganiso sokuhluma kwembewu ephathwe kahle efakwe izinsuku ezingu-7 endaweni ephakathi ne-MS ene-coumamonamide engu-200 μM noma engenayo kanye namakhemikhali ahlobene nayo. Izinkanyezi zibonisa umehluko omkhulu ngokwelashwa ngamanga (ukuhlolwa kwesikwele se-chi). n=96.
Ngokuthakazelisayo, ukungezwa kwamaketanga aseceleni e-alkyl amade kune-C9 kunciphisa umsebenzi wokuvimbela, okuphakamisa ukuthi amakhemikhali ahlobene ne-kumamotoic acid adinga amaketanga aseceleni osayizi othile ukuze abonise umsebenzi wawo webhayoloji.
Ngenxa yokuthi ukuhlaziywa kobudlelwano besakhiwo nomsebenzi kubonise ukuthi i-C9 iguqulwe yaba yi-ursonic acid kanye ne-nonyloxy derivative ye-ursonic acid (ebizwa ngokuthi i-KAND 11) kwakuyiyona evimbela ukukhula kwezitshalo ephumelela kakhulu, senze ukucaciswa okuningiliziwe kwe-KAND 11. Ukwelashwa kwe-Arabidopsis nge-50 μM KAND 11 cishe kwavimbela ngokuphelele ukuhluma, kanti amazinga aphansi (40, 30, 20, noma 10 μM) e-KAND 11 avimbele ukukhula kwezimpande ngendlela encike kumthamo (Isithombe 4a, b). Ukuze sihlole ukuthi i-KAND 11 iyakuthinta yini ukuphila kwezimpande, sihlole izimpande ze-meristems ezifakwe i-propidium iodide (PI) futhi salinganisa usayizi wendawo ye-meristem. Usayizi we-meristem yezithombo ezikhuliswe endaweni equkethe i-25 μM KAND-11 wawungu-151.1 ± 32.5 μm, kanti usayizi we-meristem yezithombo ezikhuliswe endaweni yokulawula equkethe i-DMSO wawungu-264.7 ± 30.8 μm (Isithombe 4c, d), okubonisa ukuthi i-KAND-11 ibuyisela umsebenzi weseli. ukusabalala. I-Root meristem. Ngokuvumelana nalokhu, ukwelashwa kwe-KAND 11 kunciphise inani lesignali yokuhlukaniswa kweseli i-CDKB2;1p::CDKB2;1-GUS ku-root meristem (Isithombe 4e) 17. Le miphumela ikhombisa ukuthi i-KAND 11 ivimbela ukukhula kwezimpande ngokunciphisa umsebenzi wokwanda kweseli.
Ukuhlaziywa komphumela wokuvimbela we-urbenonic acid derivatives (i-urbenyloxy derivatives) ekukhuleni. (a) Izithombo ze-wild-type Col ezinezinsuku ezingu-7 ubudala ezikhuliswe kuma-MS plates ezinamazinga abonisiwe e-KAND 11. Ibha yesikali = 1 cm. (b) Ukulinganiswa kobude bempande. Izinhlamvu zibonisa umehluko obalulekile (ukuhlolwa kwe-Tukey HSD, p< 0.05). n>16. Idatha iboniswa njengesilinganiso ± SD. (c) I-Confocal microscopy yezimpande ze-propidium iodide-stained wild-type Col ezikhule kuma-MS plates ane-25 μM KAND noma angenayo 11. Ama-bracket amhlophe abonisa i-root meristem. Ibha yesikali = 100 µm. (d) Ukulinganiswa kosayizi we-root meristem (n = 10 kuya ku-11). Umehluko wezibalo unqunywe kusetshenziswa i-t-test (p< 0.05). Amabha amelela usayizi ojwayelekile we-meristem. (e) I-Differential interference contrast (DIC) microscopy ye-root meristem equkethe ukwakheka kwe-CDKB2; 1pro: CDKB2; 1-GUS efakwe umbala futhi yafakwa umbala ezitshalweni zezinsuku ezi-5 ezikhule kuma-MS plate ane-25 µM KAND assay noma angenayo.
Ubuthi be-KAND 11 buhlolwe kabanzi kusetshenziswa esinye isitshalo esinama-dicotyledonous, ugwayi (i-Nicotiana tabacum), kanye nesitshalo esikhulu sezitshalo zomhlaba, i-liverwort (i-Marchantia polymorpha). Njengasendabeni ye-Arabidopsis, izithombo ze-SR-1 zikagwayi ezikhuliswe endaweni ephakathi equkethe i-25 μM KAND 11 zikhiqize izimpande ezimfushane (Isithombe 5a). Ngaphezu kwalokho, imbewu engu-40 kwezingu-48 zahluma emapuletini aqukethe i-200 μM KAND 11, kanti zonke imbewu ezingu-48 zahluma ezindaweni eziphathwe ngendlela yokulinganisa, okubonisa ukuthi amazinga aphezulu e-KAND ayebalulekile (p).< 0.05; ukuhlolwa kwe-chi -square) kuvimbele ukuhluma kukagwayi. (Isithombe 5b). Ngaphezu kwalokho, ukuhlushwa kwe-KAND 11 okuvimbela ukukhula kwamagciwane e-liverwort kwakufana nokuhlushwa okusebenzayo ku-Arabidopsis (Isithombe 5c). Le miphumela ikhombisa ukuthi i-KAND 11 ingavimbela ukukhula kwezitshalo ezahlukahlukene. Sabe sesihlola ubuthi obungaba khona bamakhemikhali ahlobene ne-bear monoamide kwezinye izinto eziphilayo, okungukuthi amangqamuzana e-HeLa abantu kanye nohlobo lwe-Escherichia coli DH5α, njengabamele amangqamuzana aphezulu ezilwane namagciwane, ngokulandelana. Ochungechungeni lokuhlolwa kokwanda kwamaseli, sabona ukuthi i-coumamonamide 1, i-coumamonamidic acid 6, kanye ne-KAND 11 azizange zithinte ukukhula kwamaseli e-HeLa noma e-E. coli ekuhlushweni okungu-100 μM (Isithombe 5d,e).
Ukuvinjelwa kokukhula kwe-KAND 11 ezintweni eziphilayo ezingezona eze-Arabidopsis. (a) Izithombo zikagwayi we-SR-1 wasendle ezinamasonto amabili ubudala zikhuliswe kumapuleti e-MS abekwe phezulu aqukethe i-25 μM KAND 11. (b) Izithombo zikagwayi we-SR-1 wasendle ezinamasonto amabili ubudala zikhuliswe kumapuleti e-MS abekwe ngokuvundlile aqukethe i-200 μM KAND 11. (c) Amahlumela e-liverwort we-Tak-1 wasendle anamasonto amabili ubudala akhuliswe kumapuleti e-Gamborg B5 anamazinga abonisiwe e-KAND 11. Imicibisholo ebomvu ikhombisa ama-spores ayeka ukukhula phakathi nesikhathi sokufukamela samasonto amabili. (d) Ukuhlolwa kokwanda kwamaseli kwamaseli e-HeLa. Inani lamaseli asebenzayo lalinganiswa ngezikhathi ezimisiwe kusetshenziswa ikhithi yokubala amaseli 8 (i-Dojindo). Njengokulawula, amaseli e-HeLa aphathwe nge-actinomycin D D engu-5 μg/ml, evimbela ukubhalwa kwe-RNA polymerase futhi ibangele ukufa kwamaseli. Ukuhlaziywa kwenziwe kathathu. (e) Ukuhlolwa kokwanda kwamaseli e-E. coli. Ukukhula kwe-E. coli kwahlaziywa ngokulinganisa i-OD600. Njengokulawula, amaseli aphathwe nge-ampicillin (Amp) engu-50 μg/ml, evimbela ukwakheka kodonga lwamaseli ebhaktheriya. Ukuhlaziywa kwenziwe kathathu.
Ukuze siqonde indlela yokusebenza kwe-cytotoxicity ebangelwa amakhemikhali ahlobene ne-uramide, sihlaziye kabusha ama-derivatives e-urbenic acid ngemiphumela yokuvimbela emaphakathi. njengoba kuboniswe esithombeni. Njengoba kuboniswe kuZithombe 2b, 6a, izithombo ezikhulele kuma-agar plates aqukethe amazinga aphezulu (200 μM) e-urmotonic acid 6 zikhiqize izimpande ezimfushane nezigobile ngakwesobunxele (θ = – 23.7 ± 6.1), kanti izithombo ezikhulele endaweni yokulawula, izithombo zikhiqize izimpande eziqondile (θ = – 3.8 ± 7.1). Lokhu kukhula kwe-oblique okuphawulekayo kwaziwa ukuthi kubangelwa ukungasebenzi kahle kwama-microtubules e-cortical14,18. Ngokuhambisana nalokhu okutholakele, izidakamizwa eziqeda amandla ama-microtubule i-disopyramide ne-oryzalin zibangele ukuthambekela okufanayo kwezimpande ngaphansi kwezimo zethu zokukhula (Izithombe 2b, 6a). Ngesikhathi esifanayo, sihlole ama-derivatives e-urmotonic acid futhi sakhetha eziningana zazo, ngamanani athile, ezibangele ukukhula kwezimpande ze-oblique. Ama-Compounds 8, 9, kanye no-15 ashintshe indlela yokukhula kwezimpande ku-75 μM, 50 μM, kanye no-40 μM, ngokulandelana, okubonisa ukuthi la ma-compound angawaphazamisa ngempumelelo ama-microtubules (Isithombe 2b, 6a). Siphinde sahlola i-ursolic acid derivative enamandla kakhulu, i-KAND 11, ngesilinganiso esiphansi (15 µM) futhi sathola ukuthi ukusetshenziswa kwe-KAND 11 kuvimbele ukukhula kwezimpande nokuthi indlela yokukhula kwezimpande yayingalingani, yize ayethambekele ekuthambekeni ngakwesobunxele (Isithombe C3). . Ngenxa yokuthi amazinga aphezulu emithi yokuqeda amandla ama-microtubule ngezinye izikhathi avimbela ukukhula kwezitshalo kunokubangela ukuthambekela kwezimpande, kamuva sahlola ukuthi kungenzeka ukuthi i-KAND 11 ithinta ama-microtubules ngokubuka ama-cortical microtubules kumaseli e-epidermal ezimpande. I-immunohistochemistry esebenzisa ama-anti-β-tubulin antibodies kumaseli e-epidermal ezimpande zezithombo eziphathwe nge-25 μM KAND 11 ibonise ukunyamalala cishe kwawo wonke ama-microtubules e-cortical kumaseli e-epidermal endaweni yokwelulwa (Isithombe 6b). Le miphumela ikhombisa ukuthi i-kumamotonic acid kanye nezinto eziphuma kuyo zisebenza ngokuqondile noma ngokungaqondile kuma-microtubules ukuze ziwaphazamise nokuthi lawa ma-compounds ayizivimbeli ezintsha zama-microtubule.
I-Ursonic acid kanye ne-derivatives yayo ishintsha ama-microtubules e-cortical ku-Arabidopsis thaliana. (a) I-angle yokuthambekela kwezimpande elinganiswa lapho kukhona ama-derivatives ahlukahlukene e-urmotonic acid kumazinga abonisiwe. Imiphumela yamakhemikhali amabili aziwa ngokuvimbela ama-microtubules: i-disopyramide ne-oryzalin nawo ahlaziywe. I-insert ikhombisa indinganiso esetshenziswa ukukala i-angle yokukhula kwezimpande. Ama-Asterisk akhombisa umehluko omkhulu ngokwelashwa okungamanga (t test, p< 0.05). n>19. Ibha yesikali = 1 cm. (b) Ama-microtubules e-cortical kumaseli e-epidermal endaweni yokwelula. Ama-Microtubules ezimpandeni ze-Arabidopsis Col zohlobo lwasendle ezikhule kuma-plate e-MS ane-25 μM KAND 11 noma angenayo abonwe ngokudaya kwe-immunohistochemical kusetshenziswa ama-antibodies e-β-tubulin primary kanye nama-antibodies e-Alexa Fluor-conjugated secondary. Ibha yesikali = 10 µm. (c) Isakhiwo se-mitotic sama-microtubules e-root meristem. Ama-Microtubules abonwe kusetshenziswa ukudaya kwe-immunohistochemical. Izakhiwo ze-mitotic, kufaka phakathi izindawo ze-prophase, ama-spindle, nama-phragmoplast, zabalwa kusukela ezithombeni ze-confocal. Imicibisholo ikhombisa izakhiwo ze-mitotic microtubule. Ama-Asterisk akhombisa umehluko omkhulu ngokwelashwa okungamanga (t test, p< 0.05). n>9. Ibha yesikali = 50 µm.
Nakuba i-Ursa inamandla okuphazamisa umsebenzi we-microtubule, indlela esebenza ngayo kulindeleke ukuthi ihluke kuma-agent ajwayelekile e-microtubule depolymerizing. Isibonelo, amazinga aphezulu ama-agent e-microtubule depolymerizing njenge-disopyramide ne-oryzalin abangela ukwanda kwamaseli e-epidermal a-anisotropic, kanti i-KAND 11 ayikwenzi lokho. Ngaphezu kwalokho, ukusetshenziswa ngokubambisana kwe-KAND 11 kanye ne-disopyramide kuholele ekuphenduleni kokukhula kwezimpande okubangelwa yi-disopyramide kanye nokuvinjelwa kokukhula okubangelwa yi-KAND 11 kwabonwa (Isithombe S4). Siphinde sahlaziya impendulo ye-mutant ye-disopyramide 1-1 (phs1-1) ezwela kakhulu ku-KAND 11. i-phs1-1 inokuguqulwa kwephuzu le-tubulin kinase okungeyona i-canonical futhi ikhiqiza izimpande ezimfushane uma iphathwa nge-disopyramide9,20. Izithombo ze-phs1-1 mutant ezikhuliswe ku-agar medium equkethe i-KAND 11 zazinezimpande ezimfushane ezifana nalezo ezikhuliswe ku-disopyramid (Isithombe S5).
Ngaphezu kwalokho, sibone izakhiwo ze-mitotic microtubule, njengezindawo ze-prophase, ama-spindle, nama-phragmoplast, ezimpandeni zezitshalo eziphathwe nge-KAND 11. Ngokuhambisana nokubonwa kwe-CDKB2;1p::CDKB2;1-GUS, kwabonakala ukwehla okukhulu kwenani lama-mitotic microtubules (Umfanekiso .6c).
Ukuze sichaze ubuthi be-KAND 11 ekuxazululweni kwe-subcellular, selaphe amaseli okumiswa kwe-BY-2 kagwayi nge-KAND 11 futhi sabona impendulo yawo. Siqale sengeza i-KAND 11 kumaseli e-BY-2 aveza i-TagRFP-TUA6, ebhala ama-microtubules ngokukhanya, ukuze sihlole umphumela we-KAND 11 kuma-microtubules e-cortical. Ubuningi be-cortical microtubule buhlolwe kusetshenziswa ukuhlaziywa kwesithombe, okwalinganisa iphesenti lama-pixel e-cytoskeletal phakathi kwama-pixel e-cytoplasmic. Imiphumela yokuhlolwa ibonise ukuthi ngemva kokwelashwa nge-50 μM noma i-100 μM KAND 11 ihora eli-1, ubuningi behle kakhulu baba ngu-0.94 ± 0.74% noma u-0.23 ± 0.28%, ngokulandelana, kuyilapho ubuningi bamaseli aphathwe nge-DMSO, baba ngu-1.61 ± 0.34% (Isithombe 7a). Le miphumela ihambisana nokubonwa ku-Arabidopsis ukuthi ukwelashwa kwe-KAND 11 kubangela ukususwa kwe-polymerization yama-microtubules e-cortical (Isithombe 6b). Siphinde sahlola umugqa we-BY-2 one-GFP-ABD-labeled actin filaments ngemva kokwelashwa nge-concentration efanayo ye-KAND 11 futhi sabona ukuthi ukwelashwa kwe-KAND 11 kuphazamise ama-filaments e-actin. Ukwelashwa nge-50 μM noma i-100 μM KAND 11 ihora eli-1 kunciphisa kakhulu ukuminyana kwe-actin filament kwaba ngu-1.20 ± 0.62% noma u-0.61 ± 0.26%, ngokulandelana, kanti ukuminyana kumaseli aphathwe yi-DMSO kwakungu-1.69 ± 0.51% (Isithombe 2). 7b). Le miphumela iphikisana nemiphumela ye-propyzamide, engathinti ama-filaments e-actin, kanye ne-latrunculin B, i-actin depolymerizer engathinti ama-microtubules (Isithombe S6 se-SI). Ngaphezu kwalokho, ukwelashwa nge-coumamonamide 1, i-coumamonamide acid 6, noma i-KAND 11 akuzange kuthinte ama-microtubules kumaseli e-HeLa (Isithombe se-SI S7). Ngakho-ke, indlela yokusebenza kwe-KAND 11 kukholakala ukuthi ihlukile kuneye-cytoskeleton disruptors eyaziwayo. Ngaphezu kwalokho, ukubuka kwethu okuncane kwamaseli e-BY-2 aphathwe nge-KAND 11 kwembule ukuqala kokufa kwamaseli ngesikhathi sokwelashwa kwe-KAND 11 futhi kwabonisa ukuthi isilinganiso samaseli afile anombala oluhlaza okwesibhakabhaka e-Evans asizange sikhule kakhulu ngemva kwemizuzu engama-30 yokwelashwa kwe-KAND 11, kanti ngemva kwemizuzu engama-90 yokwelashwa nge-50 μM noma i-100 μM KAND, inani lamaseli afile lenyuke laya ku-43.7% noma ku-80.1%, ngokulandelana (Isithombe 7c). Uma kuhlanganiswa, le mininingwane ikhombisa ukuthi i-ursolic acid derivative KAND 11 entsha iyisivimbeli se-cytoskeletal esithile sesitshalo esinendlela yokusebenza engaziwa ngaphambilini.
I-KAND ithinta ama-microtubules e-cortical, ama-actin filaments, kanye nokuphila kwamaseli e-BY-2 kagwayi. (a) Ukubona ngeso lengqondo ama-microtubules e-cortical kumaseli e-BY-2 lapho kukhona i-TagRFP-TUA6. Amaseli e-BY-2 aphathwe nge-KAND 11 (50 μM noma i-100 μM) noma i-DMSO ahlolwe nge-confocal microscopy. Ubuningi be-cortical microtubule babalwe kusukela kuma-micrographs amaseli angu-25 azimele. Izinhlamvu zibonisa umehluko omkhulu (ukuhlolwa kwe-Tukey HSD, p.< 0.05). Ibha yesikali = 10 µm. (b) Ama-filament e-cortical actin kumaseli e-BY-2 abonwe phambi kwe-GFP-ABD2. Amaseli e-BY-2 aphathwe nge-KAND 11 (50 μM noma 100 μM) noma i-DMSO ahlolwe nge-confocal microscopy. Ubuningi be-cortical actin filaments babalwe kusukela kuma-micrograph amaseli angu-25 azimele. Izinhlamvu zibonisa umehluko obalulekile (ukuhlolwa kwe-Tukey HSD, p< 0.05). Ibha yesikali = 10 µm. (c) Ukubonwa kwamaseli e-BY-2 afile yi-Evans blue staining. Amaseli e-BY-2 aphathwe nge-KAND 11 (50 μM noma 100 μM) noma i-DMSO ahlolwe nge-bright-field microscopy. n=3. Ibha yesikali = 100 µm.
Ukutholakala nokusetshenziswa kwemikhiqizo emisha yemvelo kuholele ekuthuthukisweni okukhulu ezicini ezahlukene zokuphila kwabantu, okuhlanganisa nezokwelapha kanye nezolimo. Ucwaningo lomlando luye lwenziwa ukuthola amakhemikhali awusizo avela ezinsizeni zemvelo. Ikakhulukazi, ama-actinomycetes aziwa ukuthi awusizo njengama-antibiotic alwa nezinambuzane kuma-nematode ngenxa yamandla awo okukhiqiza ama-metabolites ahlukahlukene esibili njenge-avermectin, inhlanganisela eholayo ye-ivermectin kanye ne-bleomycin kanye nemikhiqizo yayo, esetshenziswa kwezokwelapha njenge-ejenti yokulwa nomdlavuza21,22. Ngokufanayo, izinhlobo ezahlukene zamakhemikhali abulala ukhula zitholakale kuma-actinomycetes, amanye awo asevele esetshenziswa kwezentengiselwano1,23. Ngakho-ke, ukuhlaziywa kwama-metabolites e-actinomycete ukuhlukanisa imikhiqizo yemvelo enemisebenzi yebhayoloji efiselekayo kubhekwa njengesu eliphumelelayo. Kulesi sifundo, sithole inhlanganisela entsha, i-coumamonamide, evela ku-S. werraensis futhi sayihlanganisa ngempumelelo. I-Ursonic acid iyi-intermediate yokwenziwa ye-urbenamide kanye nemikhiqizo yayo. Ingabangela ukugoba kwezimpande okuphawulekayo, ibonise umsebenzi ophakathi kuya koqinile wokubulala ukhula, futhi ilimaze ngqo noma ngokungaqondile ama-microtubules ezitshalo. Kodwa-ke, indlela yokusebenza kwe-urmotonic acid ingase ihluke kweye-microtubule inhibitors ekhona, njengoba i-KAND 11 nayo iphazamisa ama-actin filaments futhi ibangele ukufa kwamaseli, okuphakamisa indlela yokulawula lapho i-urmotonic acid kanye nezinto eziphuma kuyo zithonya khona uhla olubanzi lwezakhiwo ze-cytoskeletal.
Ukucaciswa okwengeziwe kwe-urbenonic acid kuzosiza ukuqonda kangcono indlela yokusebenza kwe-urbenonic acid. Ikakhulukazi, umgomo olandelayo ukuhlola ikhono le-ursonic acid lokubopha kuma-microtubules ancishisiwe ukuze kunqunywe ukuthi i-ursonic acid kanye nezinto eziphuma kuyo zisebenza ngqo kuma-microtubules futhi ziwasuse, noma ukuthi isenzo sazo siphumela ekuwohlokeni kwe-microtubule. Ngaphezu kwalokho, esimweni lapho ama-microtubules engeyona into eqondile, ukuhlonza indawo yesenzo kanye nezinhloso zama-molecule ze-ursonic acid kumaseli ezitshalo kuzosiza ukuqonda kabanzi izakhiwo zamakhemikhali ahlobene nezindlela ezingaba khona zokuthuthukisa umsebenzi wokubulala ukhula. Ukuhlolwa kwethu kwe-bioactivity kwembule ikhono eliyingqayizivele le-cytotoxic le-ursonic acid ekukhuleni kwezitshalo ezifana ne-Arabidopsis thaliana, ugwayi kanye ne-liverwort, kuyilapho amaseli e-E. coli noma i-HeLa engathintekanga. Ubuthi obuncane noma obungekho nhlobo kumaseli ezilwane buyinzuzo ye-ursonic acid derivatives uma ithuthukiswa njengemithi yokubulala ukhula ukuze isetshenziswe emasimini avulekile ezolimo. Ngempela, njengoba ama-microtubules ayizakhiwo ezivamile kuma-eukaryotes, ukuvimbela kwawo okukhethayo ezitshalweni kuyisidingo esiyinhloko semithi yokubulala ukhula. Isibonelo, i-propyzamide, i-microtubule depolymerizing agent ebopha ngqo kwi-tubulin futhi ivimbele i-polymerization, isetshenziswa njenge-herbicide ngenxa yobuthi bayo obuphansi kumaseli ezilwane. Ngokungafani ne-disopyramide, ama-benzamide ahlobene anezinhlobo ezahlukene zezinhloso. Ngaphezu kwama-microtubules ezitshalo, i-RH-4032 noma i-benzoxamide iphinde ivimbele ama-microtubules amaseli ezilwane noma ama-oomycetes, ngokulandelana, kanti i-zalilamide isetshenziswa njenge-fungicide ngenxa yobuthi bayo obuphansi be-phyto25,26,27. Ibhere elisanda kutholakala kanye nezinhlobo zalo zibonisa ubuthi obukhethekile ezitshalweni, kodwa kubalulekile ukuqaphela ukuthi izinguquko ezengeziwe zingashintsha ukucaciswa kwazo, okungenzeka zinikeze ezinye izinhlobo zezithako zokulawula isikhunta noma ama-oomycetes abangela izifo.
Izakhiwo eziyingqayizivele ze-urbenonic acid kanye nezinto ezitholakala kuyo ziwusizo ekuthuthukisweni kwazo njengemithi yokubulala ukhula kanye nokusetshenziswa njengamathuluzi ocwaningo. Ukubaluleka kwe-cytoskeleton ekulawuleni ukuma kweseli lesitshalo kuqashelwa kabanzi. Izifundo zangaphambilini zibonise ukuthi izitshalo zithuthukise izindlela eziyinkimbinkimbi zokuhlela i-cortical microtubule ngokulawula i-microtubule dynamics ukuze zilawule kahle i-morphogenesis. Inani elikhulu lama-molecule abhekene nokulawulwa komsebenzi we-microtubule selitholiwe, futhi ucwaningo oluhlobene nalo lusaqhubeka3,4,28. Ukuqonda kwethu kwamanje nge-microtubule dynamics kumaseli ezitshalo akuchazi ngokugcwele izindlela zokuhlela i-cortical microtubule. Isibonelo, yize i-disopyramide kanye ne-oryzalin zingasusa i-polymerize microtubules, i-disopyramide ibangela ukuphambuka okukhulu kwezimpande kuyilapho i-oryzalin inomphumela omncane. Ngaphezu kwalokho, izinguquko ku-tubulin, eziqinisa ama-microtubules, nazo zibangela i-dextrorotation ezimpandeni, kanti i-paclitaxel, eqinisa futhi i-microtubule dynamics, ayizinzisi. Ngakho-ke, ukufunda nokuhlonza izinhloso zama-molecule ze-ursolic acid kufanele kunikeze ukuqonda okusha ngokulawulwa kwama-microtubules e-cortical ezitshalo. Ngokufanayo, ukuqhathaniswa kwesikhathi esizayo kwamakhemikhali asebenza kahle ekukhuthazeni ukukhula okuphambene, njenge-disopyramide, kanye namakhemikhali angasebenzi kahle, njenge-oryzalin noma i-kumamotoric acid, kuzohlinzeka ngezinkomba zokuthi ukukhula okuphambene kwenzeka kanjani.
Ngakolunye uhlangothi, ukuhlelwa kabusha kwe-cytoskeletal okuhlobene nokuzivikela kungenye indlela yokuchaza ubuthi be-ursonic acid. Ukutheleleka kwe-pathogen noma ukufakwa kwe-ecitor kumaseli ezitshalo ngezinye izikhathi kubangela ukubhujiswa kwe-cytoskeleton kanye nokufa kweseli okulandelayo29. Isibonelo, i-cryptoxanthin etholakala ku-oomycete ibikwe ukuthi iphazamisa ama-microtubules nama-filaments e-actin ngaphambi kokufa kweseli likagwayi, okufana nalokho okwenzeka ngokwelashwa kwe-KAND30,31. Ukufana phakathi kwezimpendulo zokuzivikela kanye nezimpendulo zeseli ezibangelwa yi-ursonic acid kusenze sacabanga ukuthi ziqala izinqubo zeseli ezivamile, yize umphumela osheshayo nonamandla we-ursonic acid kune-cryptoxanthin ubonakala. Kodwa-ke, izifundo zikhombisile ukuthi ukuphazamiseka kwe-actin filaments kukhuthaza ukufa kweseli okuzenzakalelayo, okungahlali kuhambisana nokuphazamiseka kwe-microtubule29. Ngaphezu kwalokho, kusazobonakala ukuthi i-pathogen noma i-ecitor ibangela ukukhula kwezimpande okuphambene, njengoba kwenza i-ursonic acid derivatives. Ngakho-ke, ulwazi lwama-molecule oluxhumanisa izimpendulo zokuzivikela kanye ne-cytoskeleton luyinkinga ekhangayo okufanele ixazululwe. Ngokusebenzisa ukuba khona kwamakhemikhali anesisindo esiphansi sama-molecule ahlobene ne-ursonic acid, kanye nohlu lwezinhlobo zezinto ezitholakalayo ezinamandla ahlukahlukene, zingase zinikeze amathuba okuhlosa izindlela ezingaziwa zamaseli.
Uma kuhlanganiswa, ukutholakala nokusetshenziswa kwamakhemikhali amasha aguqula amandla e-microtubule kuzohlinzeka ngezindlela ezinamandla zokubhekana nezinqubo eziyinkimbinkimbi zama-molecule eziyisisekelo sokunqunywa komumo weseli lesitshalo. Kulesi simo, i-compound urmotonic acid esanda kuthuthukiswa, ethinta ama-microtubules kanye nama-actin filaments futhi ibangele ukufa kwamangqamuzana, inganikeza ithuba lokuqonda ukuxhumana phakathi kokulawulwa kwe-microtubule kanye nalezi ezinye izindlela. Ngakho-ke, ukuhlaziywa kwamakhemikhali kanye ne-biological kusetshenziswa i-urbenonic acid kuzosisiza siqonde izindlela zokulawula ama-molecule ezilawula i-cytoskeleton yesitshalo.
Faka i-S. werraensis MK493-CF1 ebhodleleni le-Erlenmeyer elingama-500 mL elixutshwe kahle eliqukethe i-110 mL yembewu ephakathi equkethe 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 kanye ne-0.2% CaCO3 emanzini acwengekile. (pH 7.4 ngaphambi kokubulala inzalo). Izitshalo zembewu zafakwa ku-rotary shaker (180 rpm) ku-27°C izinsuku ezi-2. Ukutshala ngokukhiqiza ngokubilisa okuqinile. Ukukhuliswa kwembewu (7 ml) kudluliselwe ebhodleleni elingu-500 ml le-K-1 eliqukethe amagremu angu-40 okukhiqiza okuqukethe amagremu angu-15 ebhali elicindezelwe (MUSO Co., Ltd., eJapane) kanye namagremu angu-25 amanzi akhishwe i-ion (i-pH ayilungiswanga ngaphambi kokuhlanza). Ukuvutshelwa kwenziwa ku-30°C ebumnyameni izinsuku ezingu-14. Izinto zokuvutshelwa zikhishwe nge-40 ml/ibhodlela le-EtOH futhi zafakwa ku-centrifuge (1500 g, 4°C, imizuzu eyi-10). I-supernatant yokukhuliswa (60 ml) ikhishwe ngengxube ye-10% MeOH/EtOAc. Ingqimba yezinto eziphilayo yahwamuka ngaphansi kwengcindezi encishisiwe ukuze kutholakale insalela (59.5 mg), eyafakwa ku-HPLC nge-gradient elution (imizuzu engu-0–10: 90%) kukholomu yesigaba esingemuva (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × ubude 250 mm) H2O/CH3CN, imizuzu eyi-10–35: 90% H2O/CH3CN kuya ku-70% H2O/CH3CN (i-gradient), imizuzu engama-35–45: 90% H2O/EtOH, imizuzu engama-45–155: 90% H2O/EtOH kuya ku-100% EtOH (i-gradient (i-gradient), imizuzu engama-155–200: 100% EtOH) ngesilinganiso sokugeleza esingu-1.5 ml/min, i-coumamonamide (1, 36.0 mg) yahlukaniswa njengempuphu emhlophe engaguquki.
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]+ inani elibaliwe: 141.0659, inani elilinganisiwe: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
Imbewu yaseColumbia (Col-0) itholakale e-Arabidopsis Biological Resource Center (ABRC) ngemvume yokusetshenziswa kocwaningo. Imbewu ye-Col-0 yasakazwa futhi yagcinwa ngaphansi kwezimo zethu zelebhu futhi yasetshenziswa njengezitshalo zasendle ze-Arabidopsis. Imbewu ye-Arabidopsis yahlanzwa phezulu futhi yatshalwa endaweni enamandla e-Murashige kanye ne-Skoog medium equkethe i-2% sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino)ethanesulfonic acid (MES) (Fujifilm Wako Pure Chemical). ) kanye ne-1.5% agar (Fujifilm Wako Pure Chemical), i-pH 5.7, ku-23 °C kanye nokukhanya okungaguquki. Imbewu ye-phs1-1 mutant yanikezwa yi-T. Hashimoto (Nara Institute of Science and Technology).
Imbewu yohlobo lwe-SR-1 yanikezwa yi-T. Hashimoto (Nara Institute of Science and Technology) futhi yasetshenziswa njengezitshalo zikagwayi zasendle. Imbewu kagwayi yahlanzwa ngamanzi ahlanzekile ubusuku obuthathu ukuze kukhuthazwe ukuhluma, yabe isifakwa esixazululweni samandla ayingxenye equkethe i-2% sucrose, i-0.05% (w/v) MES, kanye ne-0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige. kanye ne-Skoog medium) ene-pH engu-5.7 futhi yafakwa ku-23°C ngaphansi kokukhanya okungaguquki.
I-Strain Tak-1 yanikezwa yi-T. Kohchi (eYunivesithi yaseKyoto) futhi yasetshenziswa njengeyunithi yokuhlola ejwayelekile yocwaningo lwe-liverwort. I-Gemma yatholakala ezitshalweni ezikhuliswe ngokubulala amagciwane yabe isifakwa ku-Gamborg B5 medium (Fujifilm Wako Pure Chemical) equkethe i-1% sucrose kanye ne-0.3% gellan gum futhi yafakwa ku-23°C ngaphansi kokukhanya okuqhubekayo.
Amaseli e-BY-2 kagwayi (i-Nicotiana tabacum L. cv. Bright Yellow 2) ahlinzekwe yi-S. Hasezawa (eNyuvesi yaseTokyo). Amaseli e-BY-2 ancibilikiswa izikhathi ezingu-95 ku-Linsmeier eguquliwe kanye ne-Skoog medium futhi afakwa njalo ngeviki nge-2,4-dichlorophenoxyacetic acid 32. Ukumiswa kweseli kwaxutshwa ku-rotary shaker ku-130 rpm ku-27°C ebumnyameni. Geza amaseli ngevolumu ephindwe kayishumi kunomthamo we-medium entsha bese uphinde uxutshwe ku-medium efanayo. Imigqa yamaseli e-BY-2 transgenic eveza ngokuzinzile uphawu lwe-microtubule TagRFP-TUA6 noma uphawu lwe-actin filament GFP-ABD2 ngaphansi kwe-cauliflower mosaic virus 35S promoter yakhiqizwa njengoba kuchaziwe33,34,35. Le migqa yamaseli ingagcinwa futhi ivumelaniswe kusetshenziswa izinqubo ezifana nalezo ezisetshenziswe kumugqa wamaseli we-BY-2 wokuqala.
Amaseli e-HeLa akhuliswe ku-Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) engezwe nge-serum ye-fetal bovine engu-10%, i-penicillin engu-1.2 U/ml, kanye ne-streptomycin engu-1.2 μg/ml ku-incubator engu-37°C ene-5% CO2.
Zonke izivivinyo ezichazwe kulo mbhalo wesandla zenziwe ngokuhambisana nemithethonqubo kanye neziqondiso zokuphepha kwezinto eziphilayo zaseJapan.
Ama-compound ancibilikiswa ku-dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) njengezixazululo zesitoko futhi ancibilikiswa ku-MS medium ye-Arabidopsis kanye ne-tobacco noma i-Gamborg B5 medium ye-liverwort. Ukuze kuhlolwe ukuvimbela ukukhula kwezimpande, imbewu engaphezu kwe-10 ngepuleti ngalinye yahlwanyelwa ku-agar medium equkethe ama-compound abonisiwe noma i-DMSO. Imbewu yafakwa ekamelweni lokukhula izinsuku eziyi-7. Izithombo zathathwa izithombe futhi ubude bezimpande balinganiswa. Ukuze kuhlolwe ukuhluma kwe-Arabidopsis, imbewu engu-48 ngepuleti ngalinye yahlwanyelwa ku-agar medium equkethe i-200 μM compound noma i-DMSO. Imbewu ye-Arabidopsis yakhuliswa ekamelweni lokukhula futhi inani lezithombo ezihlumile labalwa ezinsukwini eziyi-7 ngemva kokuhluma (dag). Ukuze kuhlolwe ukuhluma kwe-tobacco, imbewu engu-24 ngepuleti ngalinye yahlwanyelwa ku-agar medium equkethe i-200 μM KAND noma i-DMSO. Imbewu kagwayi yakhuliswa ekamelweni lokukhula futhi inani lezithombo ezihlumile labalwa ngemva kwezinsuku eziyi-14. Ukuze kuhlolwe ukuvimbela ukukhula kwe-liverwort, ama-embryo angu-9 avela epuletini ngalinye afakwe ku-agar medium equkethe amazinga abonisiwe e-KAND noma i-DMSO futhi afakwa ekamelweni lokukhula izinsuku ezingu-14.
Sebenzisa izithombo ezifakwe i-5 mg/ml ye-propidium iodide (PI) ukuze ubone ngeso lengqondo ukuhlelwa kwe-root meristem. Izimpawu ze-PI zibonwe nge-fluorescence microscopy kusetshenziswa i-TCS SPE confocal laser scanning microscope (Leica Microsystems).
Ukufakwa kwe-histochemical kwezimpande nge-β-glucuronidase (GUS) kwenziwa ngokwenqubo echazwe nguMalami noBenfey36. Izithombo zafakwa ku-90% acetone ubusuku bonke, zafakwa i-0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid ku-GUS buffer ihora eli-1 futhi zafakwa kwisisombululo se-chloraldehyde esimanzi. (8 g chloral hydrate, 2 ml amanzi kanye ne-1 ml glycerol) futhi kwabonwa nge-differential interference contrast microscopy kusetshenziswa i-Axio Imager M1 microscope (Carl Zeiss).
Ama-engeli ezimpande alinganiswa ezithonjeni ezinezinsuku ezingu-7 ubudala ezikhuliswe emapuletini abekwe phezulu. Kala i-engeli yempande kusukela ohlangothini lwe-vector yamandla adonsela phansi njengoba kuchaziwe esinyathelweni sesi-6.
Ukuhleleka kwama-microtubules e-cortical kwabonwa njengoba kuchaziwe, kanye nokuguqulwa okuncane kuphrothokholi 37. I-anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) kanye ne-Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) kwasetshenziswa njengama-antibodies ayisisekelo kanye nawesibili ku-dilutions engu-1:1000 kanye no-1:100, ngokulandelana. Izithombe ze-Fluorescence zatholakala kusetshenziswa i-TCS SPE confocal laser scanning microscope (Leica Microsystems). Thola izithombe ze-Z-stack bese udala ama-projections aphezulu kakhulu ngokwemiyalelo yomenzi.
Ukuhlolwa kokwanda kwamaseli e-HeLa kwenziwe kusetshenziswa i-Cell Counting Kit 8 (Dojindo) ngokwemiyalelo yomenzi.
Ukukhula kwe-E. coli DH5α kwahlaziywa ngokulinganisa ubuningi bamaseli ekukhuleni kusetshenziswa i-spectrophotometer ku-600 nm (OD600).
Ukuhleleka kwe-cytoskeletal kumaseli e-BY-2 aguqulwe izakhi zofuzo kwabonwa kusetshenziswa imakroskopu ye-fluorescence efakwe idivayisi yokuskena ye-CSU-X1 confocal (Yokogawa) kanye nekhamera ye-sCMOS (Zyla, Andor Technology). Ubuningi be-cytoskeletal buhlolwe ngokuhlaziywa kwesithombe, okwalinganisa iphesenti lamaphikseli e-cytoskeletal phakathi kwamaphikseli e-cytoplasmic ezithombeni ze-confocal kusetshenziswa isofthiwe ye-ImageJ njengoba kuchaziwe38,39.
Ukuze kutholakale ukufa kweseli kumaseli e-BY-2, ingxenye yeseli elimisiwe yafakwa nge-Evans blue engu-0.05% imizuzu eyi-10 ekushiseni kwegumbi. Ukufakwa kwe-Evans blue okukhethiwe kwamaseli afile kuncike ekukhishweni kwedayi emaseli aphilayo yi-plasma membrane engaguquki40. Amaseli anemibala abonwe kusetshenziswa i-bright-field microscope (BX53, Olympus).
Amaseli e-HeLa akhuliswe ku-DMEM afakwe i-10% FBS ku-incubator enomswakama ku-37°C kanye ne-5% CO2. Amaseli aphathwe nge-100 μM KAND 11, i-kumamonamic acid 6, i-kumamonamide 1, i-100 ng/ml colcemid (Gibco), noma i-100 ng/ml Nocodmaze (Sigma) amahora ayi-6 ku-37°C. Amaseli aqiniswa nge-MetOH imizuzu eyi-10 bese kuba ne-acetate imizuzu emi-5 ekushiseni kwegumbi. Amaseli aqinisiwe afakwa i-incubator nge-β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) exutshwe ku-0.5% BSA/PBS amahora ama-2, agezwa izikhathi ezi-3 nge-TBST, bese efakwa i-incubator nge-Alexa Fluor goat antibody. 488 Ihora eli-1. – I-Mouse IgG (Thermo Fisher Scientific: A11001) kanye ne-15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) exutshwe ku-0.5% BSA/PBS. Ngemva kokugezwa nge-TBST kathathu, amangqamuzana anombala abonakala kuma-microscope aguquliwe e-Nikon Eclipse Ti-E. Izithombe zithathwe ngekhamera epholile ye-Hamamatsu ORCA-R2 CCD kusetshenziswa isofthiwe ye-MetaMorph (Amadivayisi E-Molecular).
Isikhathi sokuthunyelwe: Juni-17-2024