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Ukutholwa kanye nokusetshenziswa okuzuzisayo kwemikhiqizo yemvelo kungasiza ukuthuthukisa impilo yomuntu.Amakhemikhali avimbela ukukhula kwezitshalo asetshenziswa kakhulu njengemithi yokubulala ukhula ukulawula ukhula.Ngenxa yesidingo sokusebenzisa izinhlobo ezahlukene zemithi yokubulala ukhula, kunesidingo sokuhlonza izinhlanganisela nezindlela ezintsha zokusebenza.Kulolu cwaningo, sithole inhlanganisela yenoveli ye-N -alkoxypyrrole, i-coumamonamide, evela ku-Streptomyces werraensis MK493-CF1 futhi sasungula inqubo ephelele yokuhlanganisa.Ngokuhlola umsebenzi webhayoloji, sithole ukuthi i-urs-monoamic acid iyingxenye yokwenziwa ye-urs-monoamide futhi inamandla.inhibitor yokukhula kwezitshalo.Ngaphezu kwalokho, senze okuphuma ku-urbenonic acid okuhlukahlukene, okuhlanganisa okuphuma ku-urbenyloxy (UDA), okunomsebenzi omningi wokubulala ukhula ngaphandle kokuthikameze kabi ukukhula kwamaseli e-HeLa.Siphinde sathola ukuthi i-urmotonic acid derivatives iphazamisa ama-microtubules ezitshalo;ngaphezu kwalokho, i-KAND ithinta i-actin filaments futhi ibangele ukufa kwamaseli;Le miphumela enezici eziningi ihlukile kuleyo ye-microtubule inhibitor eyaziwayo futhi iphakamisa indlela entsha yokusebenza ye-ursonic acid, emele inzuzo ebalulekile ekuthuthukisweni kwemithi emisha yokubulala ukhula.
Ukutholwa kanye nokusetshenziswa okungokoqobo kwemikhiqizo yemvelo enenzuzo kanye nokuphuma kwayo kuyindlela yokuthuthukisa izinga lempilo yomuntu.Ama-metabolites esibili akhiqizwa ama-microorganisms, izitshalo nezinambuzane aholele ekuthuthukisweni okukhulu kwezokwelapha nezolimo.Imithi eminingi elwa namagciwane kanye nemithi elwa ne-leukemia yenziwe ngemikhiqizo yemvelo.Ngaphezu kwalokho, izinhlobo ezahlukeneizibulala-zinambuzane, ama-fungicides nama-herbicides akhishwa kule mikhiqizo yemvelo ukuze asetshenziswe kwezolimo.Ikakhulukazi, ama-herbicides okulawula ukhula angamathuluzi abalulekile okwandisa isivuno kwezolimo zesimanje, futhi izinhlobo ezahlukahlukene zezinhlanganisela sezivele zisetshenziswa kwezentengiso.Izinqubo eziningana zamangqamuzana ezitshalweni, njenge-photosynthesis, i-amino acid metabolism, i-cell wall synthesis, ukulawulwa kwe-mitosis, ukusayina kwe-phytohormone, noma ukuhlanganiswa kwamaprotheni, kubhekwa njengezinhloso ezijwayelekile zokubulala ukhula.Izithako ezivimbela ukusebenza kwe-microtubule ziyikilasi elivamile lemithi yokubulala ukhula ethinta ukukhula kwezitshalo ngokuthinta i-mitotic regulation2.
Ama-microtubules ayizingxenye ze-cytoskeleton futhi agcinwe kakhulu kumaseli e-eukaryotic.I-tubulin heterodimer iqukethe i-α-tubulin kanye ne-β-tubulin eyenza i-linear microtubule protofilaments, nama-protofilaments angu-13 akha isakhiwo se-cylindrical.Ama-Microtubules adlala izindima eziningi kumaseli ezitshalo, okubandakanya ukunquma ukuma kweseli, ukuhlukaniswa kwamaseli, kanye ne-intracellular transport3,4.Amaseli ezitshalo aqukethe ama-microtubules ngaphansi kwe-membrane ye-plasma ye-interphase, futhi lezi okuthiwa yi-cortical microtubules kucatshangwa ukuthi zilawula inhlangano ye-cellulose microfibrils ngokusebenzisa ukulawulwa kwe-cellulose synthase complexes4,5.Ama-microtubules angama-cortical amangqamuzana e-epidermal ezimpande, akhona endaweni yokwelulwa ngokushesha kwephuzu lempande, atholakala eceleni, futhi ama-microfibers e-cellulose alandela la ma-microtubules futhi anciphise ukuqondiswa kwamangqamuzana, ngaleyo ndlela akhuthaze ukunwetshwa kwamangqamuzana e-anisotropic.Ngakho-ke, umsebenzi we-microtubule uhlobene eduze ne-plant morphology.Ukushintshaniswa kwe-amino acid kuzakhi zofuzo ezifaka ikhodi ku-tubulin kubangela ukuhlehla kwemigqa ye-cortical microtubule kanye nokukhula kwesokunxele noma kwesokudla ku-Arabidopsis 6,7.Ngokufanayo, ukuguqulwa kwamaprotheni ahlobene ne-microtubule alawula i-microtubule dynamics kungase kuholele ekukhuleni kwezimpande ezihlanekezelwe8,9,10,11,12,13.Ukwengeza, ukwelashwa ngama-microtubule-disrupting herbicides afana ne-disopyramide, eyaziwa nangokuthi i-pretilachlor, iphinde ibangele ukukhula kwezimpande ze-oblique kwesokunxele14.Le datha ibonisa ukuthi ukulawulwa okunembile komsebenzi we-microtubule kubalulekile ekunqumeni isiqondiso sokukhula kwesitshalo.
Kutholwe izinhlobo ezahlukene ze-microtubule inhibitors, futhi le mithi ibe negalelo elikhulu ocwaningweni lwe-cytoskeletal, kanye nakwezolimo nemithi2.Ikakhulukazi, i-oryzalin, i-dinitroaniline compounds, i-disopyramide, i-benzamide-related compounds, kanye nama-analogs abo angavimbela umsebenzi we-microtubule futhi ngaleyo ndlela avimbele ukukhula kwezitshalo.Ngakho-ke, asetshenziswa kabanzi njengama-herbicides.Kodwa-ke, njengoba ama-microtubules eyingxenye ebalulekile yamangqamuzana ezitshalo nezilwane, ama-microtubule inhibitor amaningi ayi-cytotoxic kuzo zombili izinhlobo zamaseli.Ngakho-ke, naphezu kokusetshenziswa kwawo okuqashelwa njengama-herbicides, inani elilinganiselwe lama-antimicrotubule agents asetshenziselwa izinjongo ezingokoqobo.
I-Streptomyces iwuhlobo lomndeni wakwa-Streptomyces, ohlanganisa amabhaktheriya e-aerobic, igram-positive, filamentous futhi yaziwa kabanzi ngekhono layo lokukhiqiza izinhlobonhlobo zama-metabolites esibili.Ngakho-ke, kubhekwa njengenye yemithombo ebaluleke kakhulu yemikhiqizo yemvelo emisha esebenzayo.Ocwaningweni lwamanje, sithole inhlanganisela entsha ebizwa ngokuthi i-coumamonamide, eyayihlukanisiwe ku-Streptomyces werraensis MK493-CF1 kanye ne-S. werraensis ISP 5486. Ngokusebenzisa ukuhlaziya okubonakalayo nokuhlaziywa okugcwele kwe-spectral, ukwakheka kwe-coumamonamide kwaphawulwa kanye ne-N-alkoxypyrrole skeleton yayo eyingqayizivele. kwanqunywa.ukuhlanganisa.I-Ursmonic acid, i-synthetic intermediate ye-ursmoamide kanye nokuphuma kwayo, kwatholakala ukuthi ivimbela ukukhula nokuhluma kwesitshalo esiyimodeli edumile i-Arabidopsis thaliana.Ocwaningweni lobudlelwano bomsebenzi wesakhiwo, sithole ukuthi inhlanganisela ene-C9 eguqulelwe ku-ursonic acid, ebizwa ngokuthi i-nonyloxy derivative ye-ursonic acid (KAND), ithuthukisa kakhulu umthelela ovimbayo ekukhuleni nasekuhlumeni.Ngokuphawulekayo, i-inhibitor yokukhula kwezitshalo esanda kutholwa nayo yathinta ukukhula kukagwayi kanye ne-liverwort futhi yayingeyona i-cytotoxic kubhaktheriya noma amaseli e-HeLa.Ngaphezu kwalokho, okunye okuphuma ku-urmotonic acid kubangela i-phenotype yempande ehlanekezelwe, okusho ukuthi lezi ziphuma zithinta ngokuqondile noma ngokungaqondile ama-microtubules.Ngokuvumelana nalo mbono, ukuqaphela kwethu ama-microtubules anelebulwe ngokuthi i-immunohistochemically noma amaprotheni e-fluorescent abonisa ukuthi ukwelashwa kwe-KAND kwenza ama-microtubules angasebenzi.Ngaphezu kwalokho, ukwelashwa nge-komamotonic acid derivatives kuphazamise i-actin microfilaments.Ngakho-ke, sithole inhibitor entsha yokukhula kwezitshalo indlela yayo ehlukile yokusebenza ibandakanya ukucekelwa phansi kwe-cytoskeleton.
I-Strain MK493-CF1 yahlukaniswa nenhlabathi yase-Shinagawa-ku, e-Tokyo.I-Strain MK493-CF1 yakha i-stromal mycelium enegatsha elihle.Ukulandelana kwengxenye yofuzo lwe-16S ribosomal RNA (1422 bp) kunqunyiwe.Lolu hlobo lufana kakhulu ne-S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: uhlobo olujwayelekile, 99.93%).Ngokusekelwe kulo mphumela, kwanqunywa ukuthi lolu hlobo lwaluhlobene eduze nohlobo lwe-S. werraensis.Ngakho-ke, siqambe okwesikhashana lolu hlobo S. werraensis MK493-CF1.I-S. werraensis ISP 5486T iphinda ikhiqize izinhlanganisela ze-bioactive ezifanayo.Njengoba beluncane ucwaningo lwakuqala lokuthola imikhiqizo yemvelo kule microorganism, ucwaningo olwengeziwe lwamakhemikhali lwenziwa.Ngemva kokutshalwa kwe-S. werraensis MK493-CF1 endaweni yebhali ngokuvutshelwa kwesimo esiqinile ku-30°C izinsuku ezingu-14, okuphakathi kwakhishwa ngo-50% EtOH.U-60 ml wesampula womiswa ukuze kutholwe u-59.5 mg we-extract crude.Ukukhishwa okungahluziwe kube ngaphansi kwesigaba sokuhlehla se-HPLC ukuze kunikezwe i-N-methoxy-1H-pyrrole-2-carboxamide (1, ebizwa nge-coumamonamide, 36.0 mg).Inani eliphelele lika-1 cishe lingama-60% okukhishwa okungahluziwe.Ngakho-ke, sinqume ukutadisha ngokuningiliziwe izakhiwo ze-kumamotoamide 1.
I-Coumamonamide 1 iyi-powder emhlophe e-amorphous kanye ne-high resolution mass spectrometry (HRESIMS) iqinisekisa i-C6H8N2O2 (Fig. 1).I-C2-substituted pyrrole fragment yale nhlanganisela ibonakala ngo-δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH ku-spectrum engu-1H NMR 5Hz:4. , H-5) kanye no-δH 6.78 (1H, d, J = 2.5 Hz, H-6), kanye ne-spectrum ye-13C NMR ibonisa ukuba khona kwama-athomu ekhabhoni e-sp2.Ukuba khona kweqembu le-amide endaweni ye-C2 kwahlolwa ukuhlobana kwe-HMBC kusuka ku-C-3 proton kuya ku-amide carbonyl carbon ku-δC 161.1.Ngaphezu kwalokho, i-1 H ne-13 C NMR ifinyelela isiqongo kokuthi δH 4.10 (3H, S) kanye no-δC 68.3 ibonisa ukuba khona kwamaqembu e-N-methoxy ku-molecule.Nakuba isikhundla esilungile seqembu le-methoxy sasingakanqunywa kusetshenziswa ukuhlaziywa kwe-spectroscopic okufana ne-spectroscopy yomehluko othuthukisiwe nesifinyezo se-nuclear Overhauser (NOEDF), i-N-methoxy-1H-pyrrole-2-carboxamide ibe inhlanganisela yekhandidethi lokuqala.
Ukuze unqume isakhiwo esifanele se-1, ukuhlanganiswa okuphelele kwenziwa (Fig. 2a).Ukwelashwa kwe-2-aminopyridine 2 etholakalayo kwezohwebo nge-m-CPBA kubangele i-N-oxide 3 ehambisanayo emkhiqizweni womthamo.Ngemuva kwe-2-aminoazidation ye-2, ukusabela kwe-cyclocondensation okuchazwe ngu-Abramovich kwenziwa ku-benzene ku-90 ° C ukuze kutholwe i-1-hydroxy-1H-pyrrole-2-carbonitrile 5 oyifunayo ngamagremu.Isivinini 60% (izigaba ezimbili).15,16.I-Methylation ne-hydrolysis ye-4 yabe inika i-1-methoxy-1H-pyrrole-2-carboxylic acid (ebizwa ngokuthi "cumotonic acid", 6) ngesivuno esihle (70%, izinyathelo ezimbili).Ekugcineni, ukusetshenziswa kwe-asidi chloride okuphakathi kwesi-6 kusetshenziswa i-ammonia enamanzi kwanikeza i-Kumamoto amide 1 kusivuno esingu-98%.Yonke idatha ye-spectral ye-synthesized 1 yayifana ne-1 ehlukile, ngakho-ke isakhiwo se-1 sinqunywe;
Ukuhlanganiswa okujwayelekile nokuhlaziywa komsebenzi webhayoloji we-urbenamide ne-urbenic acid.(a) Ukuhlanganiswa okuphelele kwe-Kumamoto amide.(b) Izithombo zezinsuku eziyisikhombisa ubudala zohlobo lwasendle i-Arabidopsis Columbia (Col) zitshalwe kumapuleti e-Murashige kanye ne-Skoog (MS) aqukethe i-coumamonamide 6 noma i-coumamonamide 1 ekugxiliseni okubonisiwe.Ibha yesikali = 1 cm.
Okokuqala, sihlole imisebenzi yebhayoloji ye-urbenamide nezimaphakathi zayo ukuze ikwazi ukulinganisa ukukhula kwezitshalo.Sengeze ukugxila okuhlukahlukene kwe-ursmonamide 1 noma i-ursmonic acid 6 ku-MS agar medium kanye nezithombo ze-Arabidopsis thaliana ezikhulisiwe kule ndlela.Lezi zivivinyo zibonise ukuthi ukugxila okuphezulu (500 μM) kwe-6 kuvimbele ukukhula kwezimpande (Fig. 2b).Okulandelayo, sikhiqize okuphuma kokunye okuhlukile ngokushintsha indawo ye-N1 yesi-6 futhi senza izifundo zobudlelwane besakhiwo–umsebenzi kuzo (inqubo yokuhlanganisa i-analogue ichazwa Olwazini Olusekelayo (SI)).Izithombo ze-Arabidopsis zatshalwa endaweni equkethe okuphuma ku-ursonic acid engu-50 μM, futhi ubude bezimpande balinganiswa.njengoba kuboniswe esithombeni.Njengoba kuboniswe ku-Figure 3a, b, kanye ne-S1, ama-coumamo acid anobude obuhlukene bamaketanga e-alkoxy aqondile (9, 10, 11, 12, kanye ne-13) noma amaketanga amakhulu e-alkoxy (15, 16, kanye ne-17) endaweni ye-N1.I-derivatives ibonise ukuvinjelwa okukhulu kokukhula kwezimpande.Ngaphezu kwalokho, sithole ukuthi ukusetshenziswa kwe-200 μM 10, 11, noma 17 kuvimbele ukuhluma (Amakhiwane 3c kanye ne-S2).
Ucwaningo lobudlelwano bomsebenzi wesakhiwo se-Kumamoto amide nezinhlanganisela ezihlobene.(a) Isakhiwo kanye nohlelo lokuhlanganisa lwe-analogue.(b) Ukulinganisa ubude bezimpande zezithombo ezinezinsuku ezingu-7 ezitshalwe ku-MS medium noma ngaphandle kokuphuma kokuphuma kwe-coumamonamide okungu-50 μM.Izinkanyezi zikhombisa umehluko omkhulu ngokwelashwa mbumbulu (t test, p<0.05).n >18. Idatha ikhonjiswa njengesho ukuthi ± SD.nt isho ukuthi “ayihloliwe” ngoba imbewu engaphezu kuka-50% ayizange imile.(c) Ukulinganisa izinga lokumila kwezimbewu ezilashiwe ezifukanyelwe izinsuku ezingu-7 ku-MS medium noma ngaphandle kwama-200 μM coumamonamide nezinhlanganisela ezihlobene.Izinkanyezi zibonisa umehluko omkhulu ngokwelashwa kwe-sham (ukuhlolwa kwe-chi-square).n=96.
Kuyathakazelisa ukuthi ukungezwa kwamaketanga e-alkyl side isikhathi eside kune-C9 kunciphise umsebenzi ovimbelayo, okuphakamisa ukuthi ama-compounds ahlobene ne-komamotoic acid adinga amaketanga aseceleni osayizi othile ukuze abonise umsebenzi wabo wezinto eziphilayo.
Ngenxa yokuthi ukuhlaziywa kobudlelwane besakhiwo nomsebenzi kubonise ukuthi i-C9 ishintshelwe ku-ursonic acid kanye nokuphuma kwe-nonyloxy kwe-ursonic acid (ngemuva kwalokhu okubizwa ngokuthi i-KAND 11) kwakuyi-inhibitor ephumelela kakhulu yokukhula kwezitshalo, senze ukucaciswa okunemininingwane okwengeziwe kwe-KAND 11. Ukwelashwa kwe-Arabidopsis nge 50 μM KAND 11 cishe kwavinjelwa ngokuphelele ukuhluma, kuyilapho ukugxila okuphansi (40, 30, 20, noma 10 μM) kwe-KAND 11 kuvimbele ukukhula kwezimpande ngendlela encike kumthamo (Fig. 4a, b).Ukuhlola ukuthi ingabe i-KAND 11 iyakuthinta yini ukusebenza kwezimpande, sihlole ama-root meristem angcoliswe i-propidium iodide (PI) kanye nosayizi wendawo ye-meristem elinganisiwe.Usayizi we-meristem yezithombo ezitshalwe endaweni equkethe u-25 μM KAND-11 wawungu-151.1 ± 32.5 μm, kuyilapho ubukhulu be-meristem yezithombo ezitshalwe endaweni yokulawula equkethe i-DMSO yayingu-264.7 ± 30.8 μm (Fig. 4c, d) , okubonisa ukuthi i-KAND-11 ibuyisela umsebenzi weselula.ukusabalala.Impande meristem.Ngokuvumelana nalokhu, ukwelashwa kwe-KAND 11 kwehlise inani lomaka wokuhlukaniswa kweseli CDKB2;1p::CDKB2;1-GUS isignali kumpande meristem (Fig. 4e) 17.Le miphumela ibonisa ukuthi i-KAND 11 ivimbela ukukhula kwezimpande ngokunciphisa umsebenzi wokwanda kwamaseli.
Ukuhlaziywa komphumela ovimbelayo wokuphuma kwe-urbenonic acid (okuphuma ku-urbenyloxy) ekukhuleni.(a) Izithombo zohlobo lwasendle ezinezinsuku ezingu-7 ezitshalwe kumapuleti e-MS anokugxila okubonisiwe kwe-KAND 11. Ibha yesikali = 1 cm.(b) Ukulinganisa ubude bezimpande.Izinhlamvu zikhombisa umehluko omkhulu (ukuhlolwa kwe-Tukey HSD, p<0.05).n >16. Idatha ikhonjiswa njengesho ukuthi ± SD.(c) I-Confocal microscopy ye-propidium iodide-stained wild-type roots Col etshalwe kumapuleti e-MS anoma-25 μM KAND noma ngaphandle kwawo 11. Abakaki abamhlophe babonisa i-root meristem.Ibha yesikali = 100 µm.(d) Ukulinganisa usayizi we-root meristem (n = 10 kuya ku-11).Umehluko wezibalo wanqunywa kusetshenziswa i-t-test (p<0.05).Amabha amelela usayizi we-meristem omaphakathi.(e) I-Differential interference Confusion (DIC) microscopy ye-root meristem equkethe i-CDKB2 construct;1pro: CDKB2;I-1-GUS ingcolisiwe futhi inezithombo ezinezinsuku ezingu-5 ubudala ezitshalwe kumapuleti e-MS noma ngaphandle kokuhlolwa okungu-25 µM KAND.
I-phytotoxicity ye-KAND 11 yaphinde yavivinywa kusetshenziswa esinye isitshalo se-dicotyledonous, ugwayi (i-Nicotiana tabacum), kanye nesitshalo esikhulu sesitshalo sezwe, i-liverwort (i-Marchantia polymorpha).Njengasendabeni ye-Arabidopsis, izithombo zikagwayi SR-1 ezitshalwe endaweni ephakathi equkethe 25 μM KAND 11 zikhiqize izimpande ezimfushane (Fig. 5a).Ukwengeza, imbewu engama-40 kwangu-48 imila emapuletini aqukethe u-200 μM KAND 11, kuyilapho zonke izimbewu ezingama-48 zimila emithonjeni yezindaba ebhuqwayo, okubonisa ukuthi ukugxila okuphezulu kwe-KAND kwakubalulekile (p.< 0.05;chi test -square) ivimbele ukuhluma kukagwayi.(Umdwebo 5b).Ngaphezu kwalokho, ukuhlushwa kwe-KAND 11 okuvimbele ukukhula kwebhaktheriya ku-liverwort kwakufana nokuhlushwa okuphumelelayo ku-Arabidopsis (Fig. 5c).Le miphumela ikhombisa ukuthi i-KAND 11 ingavimbela ukukhula kwezinhlobonhlobo zezitshalo.Sabe sesiphenya ukuthi kungenzeka yini i-cytotoxicity yezinhlanganisela ezihlobene nebhere monoamide kwezinye izinto eziphilayo, okungukuthi amaseli e-HeLa womuntu kanye nohlobo lwe-Escherichia coli lwe-DH5α, njengabameleli bezilwane eziphakeme namaseli ebhaktheriya, ngokulandelana.Ochungechungeni lwezilinganiso zokwanda kwamaseli, sabona ukuthi i-coumamonamide 1, i-coumamonamidic acid 6, ne-KAND 11 ayizange ithinte ukukhula kwamaseli we-HeLa noma we-E. coli ekugxilweni kwe-100 μM (Fig. 5d, e).
Ukuvinjelwa kokukhula kwe-KAND 11 ezintweni ezingezona ze-Arabidopsis.(a) Izithombo zikagwayi wasendle we-SR-1 ezinamaviki amabili ubudala zitshalwe kumapuleti e-MS abekwe mpo aqukethe 25 μM KAND 11. (b) Izithombo zikagwayi zohlobo lwasendle ze-SR-1 ezinamaviki amabili ubudala zatshalwa endaweni evundlile. Amapuleti e-MS aqukethe u-200 μM KAND 11. ( c) Amahlamvu e-Tak-1 liverwort enamasonto amabili ubudala atshalwe kumapuleti e-Gamborg B5 anenani elibonisiwe le-KAND 11. isikhathi.(d) Ukuhlolwa kokwanda kwamaseli kwamaseli e-HeLa.Inombolo yamaseli asebenzayo ikalwe ngezikhathi ezithile kusetshenziswa ikhithi yokubala yamaseli 8 (Dojindo).Njengokulawula, amaseli e-HeLa aphathwe nge-5 μg/ml actinomycin D (Act D), evimbela ukulotshwa kwe-RNA polymerase futhi ibangele ukufa kweseli.Ukuhlaziya kwenziwa ngokuphindwe kathathu.(e) Ukuhlolwa kokwanda kwamaseli e-coli.Ukukhula kwe-E. coli kwahlaziywa ngokulinganisa i-OD600.Njengokulawula, amaseli aphathwe nge-50 μg/ml ampicillin (Amp), evimbela ukwakheka kodonga lwamaseli ebhaktheriya.Ukuhlaziya kwenziwa ngokuphindwe kathathu.
Ukuze siqonde indlela yokusebenza ye-cytotoxicity ebangelwa izinhlanganisela ezihlobene ne-uramide, sihlaziye kabusha okuphuma ku-urbenic acid okunemiphumela emaphakathi yokuvimbela.njengoba kuboniswe esithombeni.Njengoba kuboniswe kuMfanekiso 2b, 6a, izithombo ezitshalwe kumapuleti e-agar aqukethe ukugxila okuphezulu (200 μM) we-urmotonic acid 6 zikhiqize izimpande ezimfushane nezigobe kwesokunxele (θ = – 23.7 ± 6.1), kanti izithombo ezitshalwe endaweni yokulawula, izithombo zakhiqiza izimpande ezicishe ziqonde (θ = – 3.8 ± 7.1).Lesi sici sokukhula kwe-oblique kwaziwa ukuthi sibangelwa ukungasebenzi kwama-microtubules e-cortical14,18.Ngokuvumelana nalokhu okutholakele, izidakamizwa ze-microtubule-destabilizing i-disopyramide ne-oryzalin zenze izimpande ezifanayo zitsheke ngaphansi kwezimo zethu zokukhula (Amakhiwane 2b, 6a).Ngasikhathi sinye, sahlola okuphuma ku-urmotonic acid futhi sakhetha ezimbalwa zazo okuthi, ekugxiliseni okuthile, okubangele ukukhula kwezimpande ezitshekile.Ama-Compounds 8, 9, kanye ne-15 ashintshe isiqondiso sokukhula kwezimpande ku-75 μM, 50 μM, no-40 μM, ngokulandelana, okubonisa ukuthi lezi zinhlanganisela zingakwazi ukuphazamisa ngempumelelo ama-microtubules (Fig. 2b, 6a).Siphinde sahlola okuphuma kokuphuma kwe-ursolic acid okunamandla kakhulu, i-KAND 11, ekugxiliseni okuphansi (15 µM) futhi sathola ukuthi ukusetshenziswa kwe-KAND 11 kuvimbele ukukhula kwezimpande nokuthi isiqondiso sokukhula kwezimpande sasingalingani, nakuba ijwayele ukutshekela kwesokunxele ( Umfanekiso C3)..Ngenxa yokuthi ukugxiliswa okuphezulu kwezidakamizwa eziqeda ukuqina kwe-microtubule ngezinye izikhathi kuvimbela ukukhula kwesitshalo kunokuba kubangele ukutsheka kwezimpande, kamuva sahlola ukuthi kungenzeka yini ukuthi i-KAND 11 ithinta ama-microtubules ngokubheka ama-microtubule e-cortical kumaseli e-epidermal ezimpande.I-Immunohistochemistry isebenzisa ama-anti-β-tubulin antibodies kumaseli e-epidermal ezimpande zesithombo eziphathwe nge-25 μM KAND 11 ibonise ukunyamalala cishe kwawo wonke ama-microtubules e-cortical kumaseli e-epidermal endaweni ye-elongation (Fig. 6b).Le miphumela ibonisa ukuthi i-kumamotonic acid kanye nokuphuma kwayo kusebenza ngokuqondile noma ngokungaqondile kuma-microtubules ukuze iwaphazamise nokuthi lezi zinhlanganisela ziyi-novel microtubule inhibitors.
I-Ursonic acid kanye nokuphuma kwayo kushintsha ama-cortical microtubules ku-Arabidopsis thaliana.(a) I-engeli yokuthambekela kwezimpande ikalwa phambi kokuphuma kokunye okuhlukile kwe-urmotonic acid ekugxiliseni okubonisiwe.Imiphumela yezinhlanganisela ezimbili ezaziwa ukuvimbela ama-microtubules: i-disopyramide ne-oryzalin nayo yahlaziywa.Isingeniso sibonisa izinga elisetshenziswa ukukala i-engeli yokukhula kwezimpande.Izinkanyezi zikhombisa umehluko omkhulu ngokwelashwa mbumbulu (t test, p<0.05).n >19. Ibha yesikali = 1 cm.(b) Ama-cortical microtubules kumaseli e-epidermal endaweni yokwelula.Ama-Microtubules ohlotsheni lwasendle lwezimpande ze-Arabidopsis Col ezikhule kumapuleti e-MS ane-25 μM KAND 11 noma angenayo abonwa nge-immunohistochemical staining kusetshenziswa amasosha omzimba ayinhloko e-β-tubulin kanye namasosha omzimba e-Alexa Fluor-conjugated yesibili.Ibha yesikali = 10 µm.(c) Isakhiwo se-Mitotic sama-microtubules ku-meristem yempande.Ama-Microtubules abonwa ngeso lengqondo kusetshenziswa i-immunohistochemical staining.Izakhiwo ze-Mitotic, ezihlanganisa izindawo ze-prophase, ama-spindles, nama-phragmoplasts, zibalwe kusukela ezithombeni ezihlangene.Imicibisholo ibonisa izakhiwo ze-mitotic microtubule.Izinkanyezi zikhombisa umehluko omkhulu ngokwelashwa mbumbulu (t test, p<0.05).n >9. Ibha yesikali = 50 µm.
Nakuba i-Ursa inamandla okuphazamisa ukusebenza kwe-microtubule, indlela yokusebenza kwayo kulindeleke ukuthi yehluke kuma-ejenti avamile wokuqeda ama-microtubule.Isibonelo, ukugxila okuphezulu kwama-microtubule depolymerizing agents afana ne-disopyramide ne-oryzalin kudala ukwanda kwe-anisotropic yamaseli e-epidermal, kanti i-KAND 11 ayikwenzi lokho.Ukwengeza, ukusetshenziswa ngokubambisana kwe-KAND 11 kanye ne-disopyramide kubangele impendulo yokukhula kwezimpande ehlanganisiwe ye-disopyramide kanye nokuvimbela ukukhula okubangelwa i-KAND 11 (Fig. S4).Siphinde sahlaziya impendulo ye-hypersensitive disopyramide 1-1 (phs1-1) eguquguqukayo ku-KAND 11. I-phs1-1 ine-non-canonical tubulin kinase point mutation futhi ikhiqiza izimpande ezimfushane lapho iphathwa nge-disopyramide9,20.phs1-1 izithombo eziguquguqukayo ezitshalwe ku-agar medium equkethe i-KAND 11 zinezimpande ezimfushane ezifana nalezo ezitshalwe ku-disopyramid (fig. S5).
Ngaphezu kwalokho, sabona izakhiwo ze-mitotic microtubule, njengezindawo ze-prophase, ama-spindles, nama-phragmoplasts, empandeni yezimpande zezithombo eziphathwe nge-KAND 11. Ngokuvumelana nokubhekwa kwe-CDKB2;1p::CDKB2;1-GUS, ukwehla okukhulu inani lama-microtubules emitotic labonwa (Fig. .6c).
Ukuze sibonise i-cytotoxicity ye-KAND 11 ekulungisweni kwe-subcellular, siphathe amaseli amisiwe kagwayi BY-2 nge-KAND 11 futhi sabona impendulo yawo.Siqale sengeza i-KAND 11 kumaseli e-BY-2 aveza i-TagRFP-TUA6, elebula ngokukhanyayo ama-microtubules, ukuze ahlole umphumela we-KAND 11 kuma-cortical microtubules.Ukuminyana kwe-Cortical microtubule kwahlolwa kusetshenziswa ukuhlaziywa kwesithombe, okulinganise iphesenti lamaphikseli e-cytoskeletal phakathi kwamaphikseli e-cytoplasmic.Imiphumela yokuhlolwa ibonise ukuthi ngemva kokwelashwa nge-50 μM noma 100 μM KAND 11 ngehora elingu-1, ukuminyana kwehle kakhulu kuya ku-0.94 ± 0.74% noma 0.23 ± 0.28%, ngokulandelana, kuyilapho ukuminyana kwamaseli aphathwa nge-DMSO, kwaba ngu-10.63 ± 1. % (Umdwebo 7a).Le miphumela ihambisana nokubhekwa ku-Arabidopsis ukuthi ukwelashwa kwe-KAND 11 kubangela ukuchithwa kwe-microtubules ye-cortical (Fig. 6b).Siphinde sahlola ulayini we-BY-2 one-actin filaments enelebula ye-GFP-ABD ngemva kokwelashwa nge-concentration efanayo ye-KAND 11 futhi sabona ukuthi ukwelashwa kwe-KAND 11 kuphazamise imicu ye-actin.Ukwelashwa nge-50 μM noma 100 μM KAND 11 ngehora elingu-1 kwehlise ngokuphawulekayo ukuminyana kwe-actin filament kuya ku-1.20 ± 0.62% noma 0.61 ± 0.26%, ngokulandelana, kuyilapho ukuminyana kumaseli alashwe yi-DMSO kwakungu-1.69 ± 0.51% (Fig 0.51).7b).Le miphumela ihluke nemiphumela ye-propyzamide, engathinti i-actin filaments, kanye ne-latrunculin B, i-actin depolymerizer engathinti ama-microtubules (SI Figure S6).Ukwengeza, ukwelashwa nge-coumamonamide 1, i-coumamonamide acid 6, noma i-KAND 11 ayizange ithinte ama-microtubules kumaseli we-HeLa (SI Figure S7).Ngakho-ke, indlela yokusebenza ye-KAND 11 kukholakala ukuthi ihlukile kuleyo yabaphazamisi be-cytoskeleton abaziwayo.Ngaphezu kwalokho, ukubhekwa kwethu okuncanyana kwamaseli e-BY-2 aphathwe nge-KAND 11 kwembula ukuqala kokufa kweseli ngesikhathi sokwelashwa kwe-KAND 11 futhi kwabonisa ukuthi ingxenye yamaseli afile e-Evans anebala elihlaza okwesibhakabhaka ayizange ikhule kakhulu ngemva kwemizuzu engama-30 yokwelashwa kwe-KAND 11, kuyilapho. ngemva kwemizuzu engu-90 yokwelashwa nge-50 μM noma i-100 μM KAND, inani lamaseli afile lenyuke laya ku-43.7% noma 80.1%, ngokulandelana (Fig. 7c).Ihlanganiswe ndawonye, le datha ibonisa ukuthi okuphuma ku-ursolic acid yenoveli KAND 11 iyisivimbeli se-cytoskeletal esiqondene nesitshalo esinomshini wokusebenza owawungaziwa ngaphambili.
I-KAND ithinta ama-cortical microtubules, ama-actin filaments, nokusebenza kwamaseli kagwayi BY-2.(a) Ukubukwa kwe-cortical microtubules kumaseli angu-BY-2 phambi kwe-TagRFP-TUA6.Amaseli angu-BY-2 aphathwe nge-KAND 11 (50 μM noma 100 μM) noma i-DMSO ahlolwe nge-confocal microscopy.Ukuminyana kwe-Cortical microtubule kubalwe kusuka kuma-micrographs amaseli azimele angama-25.Izinhlamvu zikhombisa umehluko omkhulu (ukuhlolwa kwe-Tukey HSD, p<0.05).Ibha yesikali = 10 µm.(b) Ama-cortical actin filaments kumaseli angu-BY-2 aboniswe phambi kwe-GFP-ABD2.Amaseli angu-BY-2 aphathwe nge-KAND 11 (50 μM noma 100 μM) noma i-DMSO ahlolwe nge-confocal microscopy.Ukuminyana kwama-cortical actin filaments kubalwe kusukela kuma-micrographs amaseli azimele angama-25.Izinhlamvu zikhombisa umehluko omkhulu (ukuhlolwa kwe-Tukey HSD, p<0.05).Ibha yesikali = 10 µm.(c) Ukubhekwa kwamaseli e-BY-2 afile yi-Evans staining eluhlaza okwesibhakabhaka.Amaseli angu-BY-2 aphathwe nge-KAND 11 (50 μM noma 100 μM) noma i-DMSO ahlolwe nge-microscope yenkundla ekhanyayo.n=3.Ibha yesikali = 100 µm.
Ukutholwa kanye nokusetshenziswa kwemikhiqizo emisha yemvelo kuye kwaholela ekuthuthukisweni okuphawulekayo ezicini ezihlukahlukene zokuphila komuntu, okuhlanganisa imithi nezolimo.Ucwaningo olungokomlando lwenziwe ukuze kutholwe izinhlanganisela eziwusizo ezivela emithonjeni yemvelo.Ikakhulukazi, ama-actinomycetes ayaziwa ukuthi awusizo njengama-antiparasitic antibiotics we-nematodes ngenxa yekhono lawo lokukhiqiza ama-metabolite esibili ahlukahlukene njenge-avermectin, isakhi esiholayo se-ivermectin ne-bleomycin kanye nokuphuma kwayo, okusetshenziselwa ukwelapha njenge-agent anticancer21,22.Ngokunjalo, kutholwe izinhlobonhlobo zezithako ezibulala ukhula kuma-actinomycetes, ezinye zazo esezivele zisetshenziswa ngokwentengiso1,23.Ngakho-ke, ukuhlaziywa kwama-metabolites e-actinomycete ukuhlukanisa imikhiqizo yemvelo nemisebenzi efiselekayo yezinto eziphilayo kuthathwa njengesu eliphumelelayo.Kulolu cwaningo, sithole inhlanganisela entsha, i-coumamonamide, evela ku-S. werraensis futhi sayihlanganisa ngempumelelo.I-Ursonic acid iwukwenziwa okuphakathi kwe-urbenamide nokuphuma kwayo.Ingabangela ukugoqa kwezimpande, ibonise ukusebenza kokubulala ukhula okumaphakathi kuya kokuqinile, futhi ilimaze ngokuqondile noma ngokungaqondile ama-microtubule ezitshalo.Kodwa-ke, indlela yokusebenza ye-urmotonic acid ingase ihluke kuleyo ye-microtubule inhibitors ekhona, njengoba i-KAND 11 iphinde iphazamise i-actin filaments futhi ibangele ukufa kweseli, iphakamisa indlela yokulawula lapho i-urmotonic acid kanye nokuphuma kwayo kuthonya 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 ukubopha kuma-microtubules ancishisiwe ukuze kutholakale ukuthi i-ursonic acid kanye nokuphuma kwayo kusebenza ngokuqondile kuma-microtubules futhi iwakhiphe e-polymerize, noma ukuthi isenzo sawo siphumela ekuqothulweni kwe-microtubule.Ukwengeza, esimweni lapho ama-microtubules engeyona inhloso eqondile, ukukhomba indawo yesenzo kanye nokuhlosiwe kwamangqamuzana e-ursonic acid kumaseli ezitshalo kuzosiza ngokuqhubekayo ukuqonda izakhiwo zamakhompiyutha ahlobene nezindlela ezingenzeka zokuthuthukisa umsebenzi we-herbicidal.Ukuhlolwa kwethu kwe-bioactivity kwembula ikhono eliyingqayizivele le-cytotoxic le-ursonic acid ekukhuleni kwezitshalo ezifana ne-Arabidopsis thaliana, ugwayi kanye ne-liverwort, kuyilapho engazange athinteke amaseli e-E. coli noma e-HeLa.Ubuthi obuncane noma obungekho emangqamuzaneni ezilwane kuyinzuzo yezinto eziphuma ku-ursonic acid uma zithuthukiswa njengezibulala-khula ukuze zisetshenziswe emasimini ezolimo avulekile.Ngempela, njengoba ama-microtubules eyizakhiwo ezivamile kuma-eukaryote, ukuvimbela kwawo okukhethekile ezitshalweni kuyisidingo esiyinhloko semithi yokubulala ukhula.Isibonelo, i-propyzamide, i-microtubule depolymerizing agent ebophezela ngokuqondile ku-tubulin futhi ivimbele ukwenziwa kwe-polymerization, isetshenziswa njengesibulala-khula ngenxa yobuthi bayo obuphansi kumaseli ezilwane24.Ngokungafani ne-disopyramide, i-benzamides ehlobene inokucaciswa okuhlukile okuhlosiwe.Ngaphezu kwama-microtubules ezitshalo, i-RH-4032 noma i-benzoxamide iphinde ivimbele ama-microtubules amangqamuzana ezilwane noma ama-oomycetes, ngokulandelana, futhi i-zalilamide isetshenziswa njengesibulala-fungali ngenxa ye-phytotoxicity ephansi25,26,27.Ibhere elisanda kutholwa kanye nokuphuma kwalo kubonisa i-cytotoxicity ekhethiwe ngokumelene nezitshalo, kodwa kubalulekile ukuqaphela ukuthi ukulungiswa okwengeziwe kungase kuguqule ukucaciswa kwalo okuhlosiwe, okungase kunikeze okuphuma kokunye okwengeziwe kokulawulwa kwesikhunta se-pathogenic noma i-oomycetes.
Izici eziyingqayizivele ze-urbenonic acid kanye nokuphuma kwayo ziwusizo ekuthuthukisweni kwazo njengama-herbicides futhi zisetshenziswa njengamathuluzi ocwaningo.Ukubaluleka kwe-cytoskeleton ekulawuleni ukuma kweseli lesitshalo kuqashelwa kabanzi.Ucwaningo lwangaphambili lubonise ukuthi izitshalo ziguqule izindlela eziyinkimbinkimbi zenhlangano ye-cortical microtubule ngokulawula amandla e-microtubule ukulawula kahle i-morphogenesis.Inani elikhulu lama-molecule anesibopho sokulawula umsebenzi we-microtubule likhonjwe, futhi ucwaningo oluhlobene lusaqhubeka3,4,28.Ukuqonda kwethu kwamanje kwe-microtubule dynamics kumaseli ezitshalo akuchazi ngokugcwele izindlela zenhlangano ye-cortical microtubule.Isibonelo, nakuba kokubili i-disopyramide ne-oryzalin zingakhipha ama-microtubules, i-disopyramide ibangela ukuhlanekezela kwezimpande okunamandla kuyilapho i-oryzalin inomphumela omncane ngokuqhathaniswa.Ngaphezu kwalokho, ukuguqulwa kwe-tubulin, okuzinzisa ama-microtubules, nakho kubangela i-dextrorotation ezimpandeni, kanti i-paclitaxel, ebuye iqinise i-microtubule dynamics, ayikwenzi.Ngakho-ke, ukutadisha nokuhlonza okuhlosiwe kwamangqamuzana e-ursolic acid kufanele kunikeze ulwazi olusha ekulawuleni ama-microtubules angama-cortical plant.Ngokufanayo, ukuqhathanisa esikhathini esizayo kwamakhemikhali aphumelelayo ekukhuthazeni ukukhula okuhlanekezelwe, njenge-disopyramide, namakhemikhali angasebenzi kahle, njenge-oryzalin noma i-kumamotoric acid, azonikeza izinkomba zokuthi ukukhula okuhlanekezelwe kwenzeka kanjani.
Ngakolunye uhlangothi, ukuhlelwa kabusha kwe-cytoskeletal okuhlobene nokuzivikela kungenye indlela yokuchaza i-cytotoxicity ye-ursonic acid.Ukutheleleka kwe-pathogen noma ukwethulwa kwe-elicitor kumaseli ezitshalo kwesinye isikhathi kubangela ukubhujiswa kwe-cytoskeleton kanye nokufa kweseli okulandelayo29.Isibonelo, i-cryptoxanthin etholakala ku-oomycete kubikwe ukuthi iphazamisa ama-microtubules nama-actin filaments ngaphambi kokufa kweseli likagwayi, okufana nalokho okwenzeka ngokwelashwa kwe-KAND30,31.Ukufana phakathi kwezimpendulo zokuzivikela kanye nezimpendulo zamaselula okubangelwa i-ursonic acid kusiholele ekucabangeni ukuthi aqala izinqubo ezivamile zamaselula, nakuba umphumela osheshayo nonamandla we-ursonic acid kune-cryptoxanthin ibonakala.Kodwa-ke, ucwaningo luye lwabonisa ukuthi ukuphazamiseka kwe-actin filaments kukhuthaza ukufa kweseli okuzenzakalelayo, okungahlali kuhambisana nokuphazamiseka kwe-microtubule29.Ngaphezu kwalokho, kusazobonakala ukuthi i-pathogen noma i-elicitor idala ukukhula kwezimpande okusontekile, njengoba kwenza okuphuma ku-ursonic acid.Ngakho-ke, ulwazi lwamangqamuzana oluxhumanisa izimpendulo zokuzivikela kanye ne-cytoskeleton kuyinkinga ekhangayo okufanele ixazululwe.Ngokuxhaphaza ukuba khona kwezinhlanganisela eziphansi zesisindo samangqamuzana ezihlobene ne-ursonic acid, kanye nohlu lokuphuma kokunye okunamandla ahlukahlukene, zingase zinikeze amathuba okukhomba izindlela zamaselula ezingaziwa.
Kuhlanganiswe ndawonye, ukutholakala kanye nokusetshenziswa kwezinhlanganisela ezintsha eziguqula amandla e-microtubule kuzohlinzeka ngezindlela ezinamandla zokubhekana nezinqubo eziyinkimbinkimbi zamangqamuzana angaphansi kokunqunywa kokuma kweseli yesitshalo.Kulo mongo, i-urmotonic acid esanda kuthuthukiswa, ethinta ama-microtubules kanye ne-actin filaments futhi ibangele ukufa kweseli, ingase inikeze ithuba lokucacisa ukuxhumana phakathi kokulawulwa kwe-microtubule kanye nalezi ezinye izindlela.Ngakho-ke, ukuhlaziya kwamakhemikhali nokwebhayoloji kusetshenziswa i-urbenonic acid kuzosisiza siqonde izindlela zokulawula amangqamuzana alawula i-cytoskeleton yesitshalo.
Chaza i-S. werraensis MK493-CF1 eflaskini engu-500 mL edidekile ye-Erlenmeyer equkethe u-110 mL wembewu ephakathi nendawo ehlanganisa u-2% (w/v) we-galactose, 2% (w/v) Essence paste, 1% ( w/v) Ukwakheka kwe-Bacto .-soyton (Thermo Fisher Scientific, Inc.), 0.5% (w/v) i-corn extract (KOGOSTCH Co., Ltd., Japan), 0.2% (w/v) (NH4)2SO4 kanye ne-0.2% CaCO3 emanzini angcolile.(pH 7.4 ngaphambi kokuvalwa inzalo).Amasiko embewu afukanyelwa ku-rotary shaker (180 rpm) ku-27°C izinsuku ezimbili.Ukutshala ukukhiqizwa ngokuvutshelwa kwesimo esiqinile.Imbewu yesiko (7 ml) yadluliselwa ku-500 ml ye-K-1 flask equkethe u-40 g wokukhiqiza ohlanganisa u-15 g webhali elicindezelwe (i-MUSO Co., Ltd., Japan) kanye nama-25 g amanzi akhishwe i-deionized (i-pH ayilungiswanga). ngaphambi kokuvala inzalo).).Ukuvutshelwa kwenziwa ku-30 ° C ebumnyameni izinsuku ezingu-14.Izinto zokuvutshelwa zikhishwe nge-40 ml/ibhodlela EtOH futhi i-centrifuged (1500 g, 4°C, 10 min).I-culture supernatant (60 ml) ikhishwe ngengxube engu-10% ye-MeOH/EtOAc.Isendlalelo sezinto eziphilayo sahwamuka ngaphansi kwengcindezi encishisiwe ukuze kutholwe insalela (59.5 mg), eyayingaphansi kwe-HPLC ene-gradient elution (imizuzu engu-0–10: 90%) kukholomu yesigaba sokuhlehla (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × ubude 250 mm) H2O/CH3CN, 10–35 amaminithi: 90% H2O/CH3CN kuya ku-70% H2O/CH3CN (igradient), 35–45 amaminithi: 90% H2O/EtOH, 45–155 imizuzu: 90% H2O / EtOH kuya ku-100% EtOH (i-gradient (i-gradient), i-155-200 min: 100% EtOH) ngesilinganiso sokugeleza kwe-1.5 ml / min, i-coumamonamide (1, 36.0 mg) yahlukaniswa 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]+ inani elibaliwe: 141.0659, inani elilinganiselwe: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
Imbewu ye-Columbia (Col-0) itholwe kwa-Arabidopsis Biological Resource Centre (ABRC) ngemvume yokusebenzisa ucwaningo.Imbewu ye-Col-0 yasakazwa futhi yagcinwa ngaphansi kwezimo zethu zaselabhorethri futhi yasetshenziswa njengezitshalo zasendle ze-Arabidopsis.Imbewu ye-Arabidopsis yafakwa inzalo ebusweni futhi yakhuliswa ku-Murashige yokuqina okuyingxenye kanye ne-Skoog medium equkethe u-2% we-sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino) ethanesulfonic acid (MES) (Fujifilm Wako Pure Chemical ).) kanye ne-agar engu-1.5% (i-Fujifilm Wako Pure Chemical), i-pH 5.7, ku-23 °C nokukhanya okuqhubekayo.Imbewu ye-phs1-1 mutant yanikezwa ngu-T. Hashimoto (Nara Institute of Science and Technology).
Imbewu yohlobo lwe-SR-1 yanikezwa ngu-T. Hashimoto (Nara Institute of Science and Technology) futhi yasetshenziswa njengezitshalo zikagwayi zasendle.Imbewu kagwayi yafakwa inzalo ebusweni futhi yacwiliswa emanzini angenalutho ubusuku obuthathu ukuze ikhuthaze ukuhluma, yabe isifakwa kusixazululo esinamandla uhhafu esiqukethe u-2% we-sucrose, u-0.05% (w/v) MES, kanye no-0.8% we-gellan gum (Fujifilm Wako Pure Chemical) Murashige.kanye ne-Skoog medium) ene-pH 5.7 futhi ifakwe ku-23°C ngaphansi kokukhanya okungaguquki.
I-Strain Tak-1 yanikezwa ngu-T. Kohchi (Kyoto University) futhi yasetshenziswa njengeyunithi yokuhlola evamile yocwaningo lwe-liverwort.I-Gemma yatholwa ezitshalweni ezikhuliswe inzalo futhi yafakwa ku-Gamborg B5 medium (Fujifilm Wako Pure Chemical) equkethe u-1% we-sucrose kanye no-0.3% we-gellan gum futhi yafakwa ku-23°C ngaphansi kokukhanya okuqhubekayo.
Amaseli kagwayi BY-2 (Nicotiana tabacum L. cv. Okuphuzi Okukhanyayo 2) anikezwe ngu-S. Hasezawa (University of Tokyo).Amaseli angu-BY-2 ahlanjululwe ngokuphindwe izikhathi ezingu-95 endaweni eshintshiwe ye-Linsmeier ne-Skoog futhi engezwa masonto onke nge-2,4-dichlorophenoxyacetic acid 32.Ukumiswa kweseli kwakuxutshwe ku-rotary shaker ngo-130 rpm ngo-27°C ebumnyameni.Geza amaseli ngevolumu ephindwe ka-10 ye-medium entsha futhi uphinde umise ngendlela efanayo.Imigqa yeseli ye-BY-2 transgenic eveza kahle umaka we-microtubule i-TagRFP-TUA6 noma umaka we-actin filament GFP-ABD2 ngaphansi komgqugquzeli we-cauliflower mosaic virus 35S wenziwa njengoba kuchaziwe33,34,35.Le migqa yeseli ingagcinwa futhi ivunyelaniswe kusetshenziswa izinqubo ezifanayo nalezo ezisetshenziselwa ulayini weseli we-BY-2 wangempela.
Amaseli e-HeLa akhuliswe ku-Eagle's medium (DMEM) eshintshiwe ye-Eagle (DMEM) (Life Technologies) ehlanganiswe ne-serum ye-fetus ye-fetus, 1.2 U/ml penicillin, kanye ne-1.2 μg/ml streptomycin ku-incubator engu-37°C eno-5% CO2.
Konke ukuhlola okuchazwe kulo mbhalo wesandla kwenziwa ngokuhambisana nemithetho nemihlahlandlela ye-biosafety yaseJapane.
Amakhompiyutha ahlakazwa ku-dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) njengezixazululo zesitoko futhi ahlanjululwa nge-MS medium ye-Arabidopsis nogwayi noma i-Gamborg B5 medium ye-liverwort.Ukuze kuhlolwe ukuvimbela ukukhula kwezimpande, imbewu engaphezu kwe-10 ipuleti ngalinye ihlwanyelwe endaweni ye-agar equkethe izinhlanganisela ezibonisiwe noma i-DMSO.Imbewu ifakwe ekamelweni lokukhula izinsuku ezingu-7.Izithombo zathwetshulwa kwalinganiswa nobude bezimpande.Ukuze kuhlolwe ukuhluma kwe-Arabidopsis, izimbewu ezingama-48 ipuleti ngalinye zahlwanyelwa endaweni ye-agar equkethe i-200 μM compound noma i-DMSO.Imbewu ye-Arabidopsis yatshalwa egumbini lokukhula futhi inani lezithombo ezimile labalwa ezinsukwini eziyi-7 ngemuva kokuhluma (dag).Ukuze kuhlolwe ukuhluma kukagwayi, izimbewu ezingama-24 ipuleti ngalinye zihlwanyelwe endaweni ye-agar equkethe u-200 μM KAND noma i-DMSO.Imbewu kagwayi yayitshalwa ekamelweni lokukhula futhi isibalo sezithombo ezimile sasibalwa ngemva kwezinsuku ezingu-14.Ukuze kuhlolwe inhibition ye-liverwort growth, imibungu engu-9 epuletini ngalinye yayifakwe ku-agar medium equkethe ukugxilisa okubonisiwe kwe-KAND noma i-DMSO futhi yafakwa egumbini lokukhula izinsuku ezingu-14.
Sebenzisa izithombo ezingcoliswe 5 mg/ml propidium iodide (PI) ukubona ngeso lengqondo ukuhlelwa kwezimpande.Amasignali e-PI abonwe nge-fluorescence microscopy kusetshenziswa isibonakhulu se-TCS SPE confocal laser scanning (Leica Microsystems).
I-histochemical staining yezimpande nge-β-glucuronidase (GUS) yenziwa ngokuvumelana nephrothokholi echazwe nguMalami no-Benfey36.Izithombo zilungiswe ku-90% i-acetone ngobusuku obubodwa, ingcoliswe nge-0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid ku-GUS buffer ngehora le-1 futhi ifakwe kusisombululo se-hydrated chloraldehyde.(8 g chloral hydrate, 2 ml amanzi kanye no-1 ml glycerol) futhi ibonwe ngokuphazamiseka kokuhlukanisa imakroskopu kusetshenziswa isibonakhulu se-Axio Imager M1 (Carl Zeiss).
Ama-engeli ezimpande alinganiswa ngezithombo ezinezinsuku ezingu-7 ezitshalwe emapuletini abekwe phezulu.Linganisa i-engeli yempande ukusuka ohlangothini lwevekhtha yamandla adonsela phansi njengoba kuchazwe esinyathelweni sesi-6.
Ukuhlelwa kwama-microtubules e-cortical kwabonwa njengoba kuchaziwe, ngokuguqulwa okuncane ku-protocol 37.I-anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) kanye ne-Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) zisetshenziswe njengezivikelamzimba eziyinhloko nezesibili ku-1:1000 naku-1:100 dilution, ngokulandelana.Izithombe ze-fluorescence zitholwe kusetshenziswa isibonakhulu se-TCS SPE confocal laser scanning (Leica Microsystems).Thola izithombe ze-Z-stack futhi udale ukuqagela kokuqina okukhulu ngokuya ngemiyalelo yomkhiqizi.
Ukuhlolwa kokwanda kwamaseli e-HeLa kwenziwa kusetshenziswa Ikhithi Yokubala Yeseli 8 (Dojindo) ngokuvumelana nemiyalelo yomkhiqizi.
Ukukhula kwe-E. coli DH5α kwahlaziywa ngokulinganisa ukuminyana kwamaseli kusiko kusetshenziswa i-spectrophotometer ku-600 nm (OD600).
Inhlangano ye-Cytoskeletal kumaseli e-transgenic BY-2 yabonwa kusetshenziswa isibonakhulu se-fluorescence efakwe idivayisi yokuskena ye-CSU-X1 (Yokogawa) kanye nekhamera ye-sCMOS (Zyla, Andor Technology).Ubuningi be-cytoskeletal buhlolwe ngokuhlaziywa kwesithombe, okulinganise iphesenti lamaphikseli e-cytoskeletal phakathi kwamaphikseli e-cytoplasmic ezithombeni ezihlangene kusetshenziswa isofthiwe ye-ImageJ njengoba kuchazwe38,39.
Ukuze kutholwe ukufa kweseli kumaseli e-BY-2, i-aliquot yokumiswa kweseli yafakwa ku-0.05% Evans eluhlaza okwesibhakabhaka imizuzu eyi-10 ekamelweni lokushisa.Ukukhethwa kwe-Evans okuluhlaza okwesibhakabhaka kwamaseli afile kuncike ekukhishweni kukadayi kumaseli asebenzayo nge-plasma membrane40 eqinile.Amaseli anebala abonwa kusetshenziswa isibonakhulu senkundla ekhanyayo (BX53, Olympus).
Amaseli e-HeLa atshalwe ku-DMEM engezwe ngo-10% FBS ku-incubator enomswakama ongu-37°C kanye no-5% CO2.Amaseli aphathwe nge-100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), noma 100 ng/ml Nocodmaze (Sigma) amahora angu-6 ku-37°C.Amaseli ahlanganiswe ne-MetOH ye-10 min bese nge-acetate ye-5 min ekamelweni lokushisa.Amaseli angaguquki ahlanganiswe ne-β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) ehlanjululwe ku-0.5% BSA/PBS amahora angu-2, ahlanjululwe izikhathi ezingu-3 nge-TBST, bese ehlanganiswa ne-Alexa Fluor antibody yembuzi.4881 ihora.– I-Mouse IgG (Thermo Fisher Scientific: A11001) kanye ne-15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) ehlanjululwe ku-0.5% BSA/PBS.Ngemva kokugeza nge-TBST izikhathi ezintathu, amaseli anamabala abonwa kusibonakhulu esihlanekezelwe se-Nikon Eclipse Ti-E.Izithombe zithwetshulwe ngekhamera epholile ye-Hahamatsu ORCA-R2 CCD kusetshenziswa isofthiwe ye-MetaMorph (Amadivayisi Emangqamuzana).
Isikhathi sokuthumela: Jun-17-2024