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Ho sibolloa le tšebeliso e ntle ea lihlahisoa tsa tlhaho ho ka thusa ho ntlafatsa bophelo ba motho.Lik'hemik'hale tse thibelang kholo ea limela li sebelisoa haholo joalo ka meriana e bolaeang limela ho laola lehola.Ka lebaka la tlhoko ea ho sebelisa mefuta e fapaneng ea litlama, ho hlokahala hore ho khethoe metsoako e nang le mekhoa e mecha ea ts'ebetso.Phuputsong ena, re fumane motsoako oa N -alkoxypyrrole, coumamonamide, ho tloha Streptomyces werraensis MK493-CF1 mme ra theha mokhoa o feletseng oa ho kopanya.Ka litlhahlobo tsa ts'ebetso ea likokoana-hloko, re fumane hore urs-monoamic acid ke motsoako oa maiketsetso oa urs-monoamide le monyetla oa ho ba teng.semela se thibelang ho hola.Ho feta moo, re entse lintho tse fapaneng tse tsoang ho urbenonic acid, ho kenyelletsa le urbenyloxy derivative (UDA), e nang le ts'ebetso e phahameng ea herbicide ntle le ho ama kholo ea lisele tsa HeLa hampe.Hape re fumane hore lihlahisoa tsa urmotonic acid li senya limela tsa microtubules;ho feta moo, KAND e ama li-actin filaments mme e baka lefu la lisele;Liphello tsena tse nang le mefuta e mengata li fapane le tsa li-microtubule inhibitors tse tsejoang 'me li fana ka maikutlo a mokhoa o mocha oa ts'ebetso bakeng sa asiti ea ursonic, e emelang molemo oa bohlokoa ho nts'etsopele ea likokoana-hloko tse ncha.
Ho sibolloa le tšebeliso e sebetsang ea lihlahisoa tsa tlhaho tse molemo le tse tsoang ho tsona ke mokhoa oa ho ntlafatsa boleng ba bophelo ba motho.Li-metabolite tsa bobeli tse hlahisoang ke likokoana-hloko, limela le likokoanyana li lebisitse tsoelo-peleng e kholo ea bongaka le temo.Lithibela-mafu tse ngata le lithethefatsi tse thibelang kankere ea mali li entsoe ka lihlahisoa tsa tlhaho.Ho feta moo, mefuta e fapaneng eachefo e bolaeang likokoanyana, li-fungicides le herbicides li ntšoa lihlahisoa tsena tsa tlhaho bakeng sa ho sebelisoa temong.Haholo-holo, libolaea-lehola tse laolang lehola ke lisebelisoa tsa bohlokoa bakeng sa ho eketsa chai ea lijalo temong ea sejoale-joale, 'me mefuta e fapaneng ea metsoako e se e sebelisoa khoebong.Mekhoa e mengata ea lisele tsa limela, tse kang photosynthesis, amino acid metabolism, cell wall synthesis, regulation of mitosis, phytohormone signing, kapa protheine synthesis, li nkoa e le liphofu tse tloaelehileng tsa likokoana-hloko.Metsoako e thibelang tšebetso ea microtubule ke sehlopha se tloaelehileng sa likokoana-hloko tse amang kholo ea limela ka ho ama mitotic regulation2.
Li-microtubules ke likarolo tsa cytoskeleton 'me li bolokiloe ka bongata liseleng tsa eukaryotic.Tubulin heterodimer e na le α-tubulin le β-tubulin e etsang linear microtubule protofilaments, e nang le li-protofilament tse 13 tse etsang sebopeho sa cylindrical.Li-microtubules li bapala likarolo tse ngata liseleng tsa limela, ho kenyelletsa le ho khetholla sebopeho sa sele, karohano ea lisele, le lipalangoang tsa intracellular3,4.Lisele tsa limela li na le li-microtubules ka tlas'a lesela la plasma la interphase, 'me ho nahanoa hore tsena tse bitsoang cortical microtubules li laola mokhatlo oa cellulose microfibrils ka ho laola cellulose synthase complexes4,5.Li-microtubules tsa cortical tsa lisele tsa epidermal tsa motso, tse teng sebakeng sa ho atolosoa ka potlako ha ntlha ea motso, li fumaneha ka thōko, 'me li-microfiber tsa cellulose li latela li-microtubules tsena le ho fokotsa tataiso ea ho atolosoa ha lisele, kahoo li khothalletsa ho lelefatsa lisele tsa anisotropic.Ka hona, mosebetsi oa microtubule o amana haufi-ufi le morphology ea limela.Li-substitutes tsa amino acid ho liphatsa tsa lefutso tse kenyang tubulin li baka skew ea cortical microtubule arrays le kholo ea leqele kapa le letona ho Arabidopsis 6,7.Ka mokhoa o ts'oanang, liphetoho tsa liprotheine tse amanang le microtubule tse laolang matla a microtubule li ka boela tsa etsa hore ho be le metso e sothehileng8,9,10,11,12,13.Ho phaella moo, phekolo e nang le likokoana-hloko tse senyang likokoana-hloko tse kang disopyramide, e tsejoang hape e le pretilachlor, e boetse e baka ho hōla ha metso ea oblique ka lehlakoreng le letšehali14.Lintlha tsena li bonts'a hore taolo e nepahetseng ea ts'ebetso ea microtubule e bohlokoa bakeng sa ho fumana tataiso ea kholo ea semela.
Ho fumanoe mefuta e fapaneng ea li-microtubule inhibitors, 'me lithethefatsi tsena li tlatselitse haholo lipatlisisong tsa cytoskeletal, hammoho le temo le meriana2.Haholo-holo, oryzalin, metsoako ea dinitroaniline, disopyramide, metsoako e amanang le benzamide, le li-analogues tsa tsona li ka thibela tšebetso ea microtubule 'me kahoo li thibela ho hōla ha limela.Ka hona, li sebelisoa haholo e le li-herbicides.Leha ho le joalo, kaha li-microtubules ke karolo ea bohlokoa ea lisele tsa limela le liphoofolo, boholo ba li-microtubule inhibitors ke cytotoxic ho mefuta e 'meli ea lisele.Ka hona, ho sa tsotellehe ts'ebeliso ea tsona e tsejoang e le li-herbicides, palo e lekanyelitsoeng ea li-antimicrotubule agents e sebelisetsoa merero e sebetsang.
Streptomyces ke leloko la lelapa la Streptomyces, le kenyeletsang libaktheria tsa aerobic, gram-positive, filamentous 'me li tsebahala haholo ka bokhoni ba tsona ba ho hlahisa mefuta e mengata ea metabolites ea bobeli.Ka hona, e nkoa e le e 'ngoe ea mehloli ea bohlokoa ka ho fetisisa ea lihlahisoa tse ncha tsa tlhaho tse sebetsang ka biologically.Phuputsong ea morao tjena, re fumane motsoako o mocha o bitsoang coumamonamide, o neng o arohane le Streptomyces werraensis MK493-CF1 le S. werraensis ISP 5486. Ho sebelisa tlhahlobo ea spectral le tlhahlobo e feletseng ea spectral, sebopeho sa coumamonamide se ne se khetholloa le skeleton ea eona e ikhethang ea N-alkoxypyrrole. e ne e ikemiselitse.tlhaho.Ursmonic acid, e leng motsoako oa maiketsetso oa ursmoamide le lihlahisoa tsa eona, e ile ea fumanoa e thibela ho hōla le ho mela ha semela se tummeng sa mohlala Arabidopsis thaliana.Phuputsong ea kamano ea ts'ebetso ea sebopeho, re fumane hore motsoako o nang le C9 o fetotsoeng ho ursonic acid, e bitsoang nonyloxy derivative of ursonic acid (KAND), e ntlafatsa haholo phello ea ho thibela ho hōla le ho mela.Ka ho hlakileng, inhibitor e sa tsoa sibolloa ea limela e boetse e ama kholo ea koae le sebeterwort mme e ne e se cytotoxic ho libaktheria kapa lisele tsa HeLa.Ho feta moo, lihlahisoa tse ling tsa urmotonic acid li etsa hore ho be le motsoako o sothehileng oa phenotype, e leng se bolelang hore lihlahisoa tsena li ama li-microtubules ka ho toba kapa ka tsela e sa tobang.Tumellanong le mohopolo ona, litebello tsa rona tsa li-microtubules tse ngotsoeng e ka ba immunohistochemically kapa ka liprotheine tsa fluorescent li bonts'a hore kalafo ea KAND e tlosa li-microtubules.Ntle le moo, kalafo e nang le li-derivatives tsa kumamotonic acid e sitisitse li-microfilaments tsa actin.Kahoo, re fumane inhibitor e ncha ea kholo ea limela eo mokhoa oa eona o ikhethang oa ts'ebetso o kenyelletsang ho timetsoa ha cytoskeleton.
Strain MK493-CF1 e ne e qheletsoe ka thoko ho mobu o Shinagawa-ku, Tokyo.Strain MK493-CF1 e thehile stromal mycelium e nang le makala a hantle.Tatelano e sa fellang ea 16S ribosomal RNA gene (1422 bp) e ile ea khethoa.Bothata bona bo tšoana haholo le S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: khatello e tloaelehileng, 99.93%).Ho itšetlehile ka sephetho sena, ho ile ha etsoa qeto ea hore mofuta ona o amana haufi-ufi le mofuta oa mofuta oa S. werraensis.Ka hona, re rehile mofuta ona ka nakoana S. werraensis MK493-CF1.S. werraensis ISP 5486T e boetse e hlahisa metsoako e tšoanang ea bioactive.Kaha ho ne ho e-na le lipatlisiso tse fokolang tsa pele tsa ho fumana lihlahisoa tsa tlhaho ho likokoana-hloko tsena, ho ile ha etsoa lipatlisiso tse ling tsa lik'hemik'hale.Ka mor'a ho lengoa S. werraensis MK493-CF1 sebakeng sa harese ka ho belisoa ha boemo bo tiileng ho 30 ° C ka matsatsi a 14, sehare se ile sa ntšoa ka 50% EtOH.60 ml ea sampole e ne e omisitsoe ho fumana 59.5 mg ea qotsulo e tala.Sehlahisoa sa HPLC se kentsoeng ka morao ho fana ka N-methoxy-1H-pyrrole-2-carboxamide (1, e bitsoang coumamonamide, 36.0 mg).Kakaretso ea 1 ke hoo e ka bang 60% ea qotsulo e tala.Ka hona, re nkile qeto ea ho ithuta ka ho qaqileng thepa ea kumamotoamide 1.
Coumamonamide 1 ke phofo e tšoeu ea amorphous le tekanyo e phahameng ea spectrometry (HRESIMS) e tiisa C6H8N2O2 (setšoantšo sa 1).Sekhechana sa pyrrole se nkeloang sebaka sa C2 sa motsoako ona se khetholloa ke δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH ho 1H NMR spectrum: 4. , H-5) le δH 6.78 (1H, d, J = 2.5 Hz, H-6), le spectrum ea 13C NMR e bonts'a boteng ba liathomo tse 'nè tsa sp2 carbon.Ho ba teng ha sehlopha sa amide sebakeng sa C2 ho ile ha hlahlojoa ke HMBC correlation ho tloha ho C-3 proton ho ea amide carbonyl carbon ho δC 161.1.Ho phaella moo, 1 H le 13 C NMR litlhōrō ho δH 4.10 (3H, S) le δC 68.3 li bontša boteng ba lihlopha tsa N-methoxy molek'hule.Le hoja boemo bo nepahetseng ba sehlopha sa methoxy bo ne bo e-s'o tsejoe ho sebelisoa tlhahlobo ea spectroscopic e kang spectroscopy e ntlafalitsoeng le nuclear Overhauser khutsufatso (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide e bile motsoako oa pele oa mokhethoa.
Ho fumana sebopeho se nepahetseng sa 1, ho entsoe kakaretso ea kakaretso (setšoantšo sa 2a).Kalafo ea 2-aminopyridine 2 e fumanehang khoebong e nang le m-CPBA e ile ea fella ka N-oxide 3 e lumellanang le lihlahisoa tse ngata.Kamora 2-aminoazidation ea 2, karabelo ea cyclocondensation e hlalositsoeng ke Abramovich e ile ea etsoa ka benzene ka 90 ° C ho fumana 1-hydroxy-1H-pyrrole-2-carbonitrile 5 e lakatsehang ka ligrama.Lebelo 60% (mehato e 'meli).15,16.Methylation le hydrolysis ea 4 e ntan'o fana ka 1-methoxy-1H-pyrrole-2-carboxylic acid (e bitsoang "cumotonic acid", 6) ka lihlahisoa tse ntle (70%, mehato e 'meli).Qetellong, amidation ka acid chloride intermediate 6 sebelisa ammonia aqueous e file Kumamoto amide 1 ho 98% chai.Lintlha tsohle tsa spectral tsa synthesized 1 li ne li tšoana le 1 e ka thōko, kahoo sebopeho sa 1 se ne se ikemiselitse;
Kakaretso ea tlhahiso le tlhahlobo ea ts'ebetso ea biological ea urbenamide le urbenic acid.(a) Kakaretso ea motsoako oa Kumamoto amide.(b) Lipeo tsa matsatsi a supileng tse hlaha tsa mofuta oa Arabidopsis Columbia (Col) li ne li lenngoe ka lipoleiti tsa Murashige le Skoog (MS) tse nang le coumamonamide 6 kapa coumamonamide 1 libakeng tse bontšitsoeng.Sekhahla sa bar = 1 cm.
Taba ea pele, re ile ra lekola mesebetsi ea tlhaho ea urbenamide le li-intermediate tsa eona bakeng sa bokhoni ba tsona ba ho fetola kholo ea semela.Re kentse mefuta e fapaneng ea ursmonamide 1 kapa ursmonic acid 6 ho MS agar medium le lipeo tsa Arabidopsis thaliana tse hlahisitsoeng ka mokhoa ona.Litlhahlobo tsena li bontšitse hore likhahla tse phahameng (500 μM) tsa 6 li thibela kholo ea metso (setšoantšo sa 2b).Ka mor'a moo, re ile ra hlahisa lihlahisoa tse fapa-fapaneng ka ho fetola boemo ba N1 ba 6 le ho etsa lithuto tsa kamano ea sebopeho-ts'ebetso ho tsona (ts'ebetso ea analogue synthesis e hlalositsoe ho Tšehetso ea Boitsebiso (SI)).Lipeo tsa Arabidopsis li ne li lengoa ka seaplane se nang le 50 μM derivatives ea ursonic acid, 'me bolelele ba metso bo ile ba lekanngoa.joalokaha e bontšitsoe setšoantšong.Joalokaha ho bontšitsoe litšoantšong tsa 3a, b, le S1, li-acid tsa coumamo li na le bolelele bo fapaneng ba liketane tsa alkoxy (9, 10, 11, 12, le 13) kapa liketane tse kholo tsa alkoxy (15, 16, le 17) sebakeng sa N1.Li-derivatives li bontšitse tšitiso e kholo ea kholo ea metso.Ho phaella moo, re fumane hore kopo ea 200 μM 10, 11, kapa 17 e thibela ho mela (Figs. 3c le S2).
Boithuto ba kamano ea sebopeho-ts'ebetso ea Kumamoto amide le metsoako e amanang le eona.(a) Sebopeho le moralo oa li-analogues.(b) Palo ea bolelele ba metso ea lipeo tsa matsatsi a 7 tse hōlileng ka MS medium e nang le 50 μM coumamonamide derivatives kapa ntle le eona.Linaleli li bontša phapang e kholo le kalafo ea sham (teko ea t, leq<0.05).n>18. Lintlha li bontšoa e le tse bolelang ± SD.nt e bolela "ho se hlahlojoe" hobane peō e fetang 50% ha ea ka ea mela.(c) Palo ea sekhahla sa ho mela ha peo e phekotsoeng e ekeletsoeng matsatsi a 7 ho MS medium e nang le kapa ntle le 200 μM coumamonamide le metsoako e amanang le eona.Linaleli li bontša phapang e khōlō ka phekolo ea sham (teko ea chi-square).n=96.
Hoa thahasellisa hore ho eketsoa ha liketane tsa mahlakoreng a alkyl nako e telele ho feta C9 ho fokolitse mosebetsi oa ho thibela, ho fana ka maikutlo a hore metsoako e amanang le kumamotoic acid e hloka liketane tsa mahlakoreng a boholo bo itseng ho bontša mosebetsi oa bona oa likokoana-hloko.
Hobane tlhahlobo ea likamano tsa sebopeho le ts'ebetso e bontšitse hore C9 e fetotsoe ho ursonic acid le motsoako oa nonyloxy oa ursonic acid (eo ka mor'a moo e bitsoang KAND 11) e ne e le inhibitor e atlehang ka ho fetisisa ea kholo ea limela, re ile ra etsa tlhaloso e qaqileng haholoanyane ea KAND 11. Phekolo ea Arabidopsis le 50 μM KAND 11 hoo e ka bang ka ho feletseng thibela tsoele pele ho mela, athe concentrations tlaase (40, 30, 20, kapa 10 μM) ea KAND 11 thibela kgolo ya metso ka mokhoa o itšetlehileng ka lethal dose (sa feiga. 4a, b).Ho lekola hore na KAND 11 e ama ho sebetsa ha metso, re ile ra hlahloba metso e nang le propidium iodide (PI) le boholo ba sebaka sa meristem.Boholo ba meristem ea lipeo tse hōlileng ka mokhoa o nang le 25 μM KAND-11 e ne e le 151.1 ± 32.5 μm, ha boholo ba meristem ea lipeo tse hōlileng ka mokhoa oa ho laola o nang le DMSO e ne e le 264.7 ± 30.8 μm (sa feiga. 4c, d) , e bontšang hore KAND-11 e tsosolosa mosebetsi oa cellular.jala.Motso oa meristem.Tumellanong le sena, phekolo ea KAND 11 e ile ea fokotsa palo ea letšoao la karohano ea lisele CDKB2; 1p :: CDKB2; 1-GUS pontšo ka motso oa meristem (Setšoantšo sa 4e) 17.Liphetho tsena li bonts'a hore KAND 11 e thibela kholo ea metso ka ho fokotsa ts'ebetso ea ho ata ha lisele.
Tlhahlobo ea phello ea inhibitory ea urbenonic acid derivatives (urbenyloxy derivatives) kholong.(a) Lipeo tsa Cold tsa matsatsi a 7 tse hōlileng ka lipoleiti tsa MS tse nang le likhahla tse bontšitsoeng tsa KAND 11. Scale bar = 1 cm.(b) Palo ea bolelele ba metso.Mangolo a bontša phapang e kholo (Teko ea Tukey HSD, leq<0.05).n>16. Lintlha li bontšoa e le tse bolelang ± SD.(c) Confocal microscopy ea propidium iodide-stained wild-type Col roots e hōlileng holim'a lipoleiti tsa MS tse nang le 25 μM KAND 11 kapa ntle le 11. Masakaneng a masoeu a bontša hore ho na le motso oa meristem.Scale bar = 100 µm.(d) Palo ea boholo ba metso ea meristem (n = 10 ho isa ho 11).Liphapang tsa lipalo-palo li ile tsa khethoa ho sebelisoa tlhahlobo ea t (leq<0.05).Li-bar li emela boholo bo tloaelehileng ba meristem.(e) Phapang ea litšitiso tse fapaneng (DIC) microscopy ea motso oa meristem o nang le moaho oa CDKB2;1pro: CDKB2;1-GUS e silafalitsoeng le matheba holim'a lipeo tsa matsatsi a 5 tse hōlileng ka lipoleiti tsa MS tse nang le tlhahlobo ea 25 µM KAND kapa ntle le eona.
Phytotoxicity ea KAND 11 e ile ea lekoa ka ho eketsehileng ho sebelisoa semela se seng sa dicotyledonous, koae (Nicotiana tabacum), le sebōpuoa se seholo sa semela sa mobu, liverwort (Marchantia polymorpha).Joaloka tabeng ea Arabidopsis, lipeo tsa koae tsa SR-1 tse hōlileng ka seaplane tse nang le 25 μM KAND 11 li hlahisitse metso e khutšoanyane (sa feiga. 5a).Ho feta moo, lipeo tse 40 ho tse 48 li ile tsa mela lipoleiting tse nang le 200 μM KAND 11, athe lipeo tsohle tse 48 li ile tsa mela mecheng ea litaba e somang, e bonts'ang hore likhakanyo tse phahameng tsa KAND li bohlokoa (leq.<0.05;chi test -square) thibela ho mela ha koae.(Setšoantšo sa 5b).Ho phaella moo, mahloriso a KAND 11 a neng a thibela kholo ea baktheria ka har'a sebete e ne e tšoana le mahloriso a atlehang ho Arabidopsis (Fig. 5c).Liphetho tsena li bonts'a hore KAND 11 e ka thibela kholo ea mefuta e fapaneng ea limela.Ka mor'a moo re ile ra batlisisa hore na cytotoxicity e ka 'na ea e-ba teng ea lik'hemik'hale tse amanang le bere monoamide linthong tse ling, e leng lisele tsa HeLa tsa motho le mofuta oa Escherichia coli DH5α, e le baemeli ba lisele tse phahameng tsa liphoofolo le baktheria, ka ho latellana.Letotong la litlhahlobo tsa ho ata ha lisele, re hlokometse hore coumamonamide 1, coumamonamidic acid 6, le KAND 11 ha ea ka ea ama kholo ea lisele tsa HeLa kapa E. coli ka lihlopha tsa 100 μM (Fig. 5d, e).
Thibelo ea kholo ea KAND 11 linthong tse se nang Arabidopsis.(a) Lipeo tsa koae ea mofuta oa SR-1 ea libeke tse peli li ile tsa lengoa holim'a lipoleiti tsa MS tse nang le 25 μM KAND 11. (b) Lipeo tsa mofuta oa SR-1 tsa mofuta oa SR-1 tsa libeke tse peli li ne li lengoa ka mokhoa o otlolohileng. Lipoleiti tsa MS tse nang le 200 μM KAND 11. ( c) Li-buds tsa mofuta oa Tak-1 liverwort tse nang le libeke tse peli tse hōlileng ka lipoleiti tsa Gamborg B5 tse nang le likhahla tse bontšitsoeng tsa KAND 11. Metsu e khubelu e bontša likokoana-hloko tse ileng tsa khaotsa ho hōla ka hare ho libeke tse peli. nako.(d) Teko ea ho ata ha lisele tsa lisele tsa HeLa.Palo ea lisele tse sebetsang e ile ea lekanyetsoa ka linako tse behiloeng ho sebelisoa lisebelisoa tsa ho bala lisele 8 (Dojindo).E le taolo, lisele tsa HeLa li ile tsa phekoloa ka 5 μg/ml actinomycin D (Act D), e thibelang ho ngoloa ha RNA polymerase le ho baka lefu la sele.Litlhahlobo li entsoe ka makhetlo a mararo.(e) Teko ea ho ata ha lisele tsa E. coli.Khōlo ea E. coli e ile ea hlahlojoa ka ho lekanya OD600.E le taolo, lisele li ile tsa phekoloa ka 50 μg/ml ampicillin (Amp), e thibelang motsoako oa lerako la baktheria.Litlhahlobo li entsoe ka makhetlo a mararo.
Ho utloisisa mokhoa oa ts'ebetso ea cytotoxicity e bakoang ke metsoako e amanang le uramide, re ile ra boela ra hlahloba lihlahisoa tsa urbenic acid tse nang le liphello tse itekanetseng tsa ho thibela.joalokaha e bontšitsoe setšoantšong.Joalokaha ho bontšitsoe ho Lipapiso 2b, 6a, lipeo tse hōlileng ka lipoleiti tsa agar tse nang le metsoako e phahameng (200 μM) ea urmotonic acid 6 e ile ea hlahisa metso e khutšoanyane le e kobehileng ka ho le letšehali (θ = - 23.7 ± 6.1), athe lipeo tse hōlileng sebakeng sa taolo, lipeo li ile tsa hlahisa metso e batlang e otlolohile (θ = - 3.8 ± 7.1).Khōlo ena e ikhethang ea oblique e tsejoa ka lebaka la ho se sebetse ha li-microtubules tsa cortical14,18.Tumellanong le se fumanoeng sena, lithethefatsi tse senyang li-microtubule-destabilizing disopyramide le oryzalin li ile tsa etsa hore metso e ts'oanang e sisinyehe tlas'a maemo a rona a ho hōla (Figs. 2b, 6a).Ka nako e ts'oanang, re ile ra leka li-derivatives tsa urmotonic acid 'me ra khetha tse' maloa tsa tsona, tseo ka linako tse itseng li ileng tsa baka kholo ea metso e oblique.Lik'hemik'hale 8, 9, le 15 li fetotse tataiso ea ho hōla ha metso ka 75 μM, 50 μM, le 40 μM, ka ho latellana, e bontšang hore metsoako ena e ka senya microtubules ka katleho (setšoantšo sa 2b, 6a).Hape re ile ra leka motsoako o matla ka ho fetisisa oa ursolic acid, KAND 11, boemong bo tlaase (15 µM) mme ra fumana hore ts'ebeliso ea KAND 11 e thibela ho hōla ha metso le hore tsela ea ho hōla ha metso e ne e sa lekane, le hoja e ne e sekametse ho le letšehali. Setšoantšo sa C3)..Hobane ka linako tse ling li-microtubule-destabilizing lithethefatsi tse ngata li thibela kholo ea semela ho e-na le ho etsa hore metso e sekame, re ile ra hlahloba monyetla oa hore KAND 11 e ama li-microtubules ka ho shebella li-microtubules tsa cortical liseleng tsa epidermal.Immunohistochemistry e sebelisa li-anti-β-tubulin antibodies liseleng tsa epidermal tsa metso ea sethopo e tšoaroang ka 25 μM KAND 11 e bontšitse ho nyamela hoo e batlang e le li-microtubules tsohle tsa cortical liseleng tsa epidermal sebakeng sa elongation (Setšoantšo sa 6b).Liphetho tsena li bontša hore kumamotonic acid le lihlahisoa tsa eona li sebetsa ka ho toba kapa ka tsela e sa tobang ho li-microtubules ho li senya le hore metsoako ena ke li-inhibitors tse ncha tsa microtubule.
Ursonic acid le lihlahisoa tsa eona li fetola cortical microtubules ho Arabidopsis thaliana.(a) Lekhalo la ho sekamela ha metso le lekanyetsoa ka boteng ba li-derivatives tse fapaneng tsa urmotonic acid maemong a bontšitsoeng.Liphello tsa metsoako e 'meli e tsejoang ho thibela li-microtubules: disopyramide le oryzalin le tsona li ile tsa hlahlojoa.Sets'oants'o se bonts'a maemo a sebelisoang ho metha sekhahla sa kholo ea metso.Linaleli li bontša phapang e kholo le kalafo ea sham (teko ea t, leq<0.05).n>19. Sekhahla sa bar = 1 cm.(b) Cortical microtubules liseleng tsa epidermal sebakeng sa bolelele.Li-microtubules tsa mefuta e hlaha ea Arabidopsis Col metso e hōlileng ka lipoleiti tsa MS tse nang le 25 μM KAND 11 kapa tse se nang 25 li ne li bonoa ka ho silafala ha immunohistochemical ho sebelisa li-antibodies tsa mantlha tsa β-tubulin le li-antibodies tsa Alexa Fluor-conjugated.Sekhahla = 10 µm.(c) Sebopeho sa mitotic sa li-microtubules ka har'a metso ea meristem.Li-microtubules li ne li bonoa ho sebelisoa litšila tsa immunohistochemical.Mehaho ea Mitotic, ho kenyeletsoa libaka tsa prophase, spindles, le phragmoplasts, li ne li baloa ho tsoa litšoantšong tse kopaneng.Metsu e bontša mehaho ea mitotic microtubule.Linaleli li bontša phapang e kholo le kalafo ea sham (teko ea t, leq<0.05).n>9. Scale bar = 50 µm.
Le hoja Ursa e na le bokhoni ba ho senya ts'ebetso ea microtubule, mokhoa oa eona oa ts'ebetso o lebeletsoe ho fapana le li-microtubule depolymerizing agents tse tloaelehileng.Mohlala, likhakanyo tse phahameng tsa li-microtubule depolymerizing agents tse kang disopyramide le oryzalin li etsa hore ho be le keketseho ea anisotropic ea lisele tsa epidermal, athe KAND 11 ha e etse joalo.Ho phaella moo, tšebeliso e kopanetsoeng ea KAND 11 le disopyramide e ile ea fella ka karabelo e kopantsoeng ea ho hōla ha metso e entsoeng ke disopyramide le KAND 11-induced inhibition inhibition e ile ea hlokomeloa (Fig. S4).Re ile ra boela ra hlahloba karabelo ea hypersensitive disopyramide 1-1 (phs1-1) mutant ho KAND 11. phs1-1 e na le phetoho ea ntlha e seng ea canonical tubulin kinase 'me e hlahisa metso e khutšoanyane ha e phekoloa ka disopyramide9,20.phs1-1 lipeo tse fetotsoeng tse hōlileng ka har'a agar medium e nang le KAND 11 li ne li e-na le metso e khutšoanyane e tšoanang le e hōlileng ka disopyramid (fig. S5).
Ho phaella moo, re ile ra hlokomela mehaho ea mitotic microtubule, e kang libaka tsa prophase, spindles, le phragmoplasts, motso oa lipeo tse tšoaroang ka KAND 11. Ho lumellana le litlhaloso tsa CDKB2; 1p:: CDKB2; 1-GUS, ho fokotseha ho hoholo ha palo ea mitotic microtubules ile a hlokomela (sa feiga. .6c).
Ho tsebahatsa cytotoxicity ea KAND 11 ho qeto ea subcellular, re ile ra tšoara lisele tse emisitsoeng tsa koae BY-2 ka KAND 11 mme ra hlokomela karabelo ea bona.Re kentse pele KAND 11 ho BY-2 lisele tse hlalosang TagRFP-TUA6, e ngolang ka fluorescent ka li-microtubules, ho lekola phello ea KAND 11 ho li-cortical microtubules.Cortical microtubule density e ile ea hlahlojoa ho sebelisoa tlhahlobo ea litšoantšo, e ileng ea lekanya liperesente tsa lipikselse tsa cytoskeletal har'a lipikselse tsa cytoplasmic.Liphetho tsa tlhahlobo li bonts'itse hore kamora kalafo ka 50 μM kapa 100 μM KAND 11 bakeng sa hora ea 1, letsoalo le fokotsehile haholo ho 0.94 ± 0.74% kapa 0.23 ± 0.28%, ka ho latellana, ha boima ba lisele tse tšoaroang ka DMSO, bo fihlile ho ± 1.63 ± 1.63% % (Setšoantšo sa 7a).Liphetho tsena li lumellana le tlhokomeliso ea Arabidopsis hore kalafo ea KAND 11 e etsa hore ho be le depolymerization ea cortical microtubules (setšoantšo sa 6b).Re ile ra boela ra hlahloba mohala oa BY-2 o nang le GFP-ABD-labeled actin filaments ka mor'a phekolo e nang le mahloriso a tšoanang a KAND 11 'me ra hlokomela hore phekolo ea KAND 11 e sitisitse li-actin filaments.Kalafo e nang le 50 μM kapa 100 μM KAND 11 bakeng sa 1 h e fokolitse haholo matla a actin filament ho 1.20 ± 0.62% kapa 0.61 ± 0.26%, ka ho latellana, athe boima ba lisele tse tšoaroang ke DMSO e ne e le 1.69 ± 0.51% (fig51%).7b).Liphetho tsena li fapane le liphello tsa propyzamide, tse sa ameng li-actin filaments, le latrunculin B, e leng actin depolymerizer e sa ameng microtubules (SI Figure S6).Ho phaella moo, phekolo ea coumamonamide 1, coumamonamide acid 6, kapa KAND 11 ha ea ka ea ama microtubules liseleng tsa HeLa (SI Figure S7).Ka hona, ho lumeloa hore mokhoa oa ts'ebetso oa KAND 11 o fapane le oa bahlaseli ba cytoskeleton ba tsejoang.Ntle le moo, tlhahlobo ea rona ea microscopic ea lisele tsa BY-2 tse tšoaroang ka KAND 11 e senotse ho qaleha ha lefu la sele nakong ea kalafo ea KAND 11 mme e bonts'a hore karolo ea lisele tse shoeleng tse nang le boputsoa ba Evans ha ea ka ea eketseha haholo kamora 30 min ea kalafo ea KAND 11, athe ka mor'a metsotso e 90 ea phekolo le 50 μM kapa 100 μM KAND, palo ea lisele tse shoeleng e eketsehile ho 43,7% kapa 80,1%, ka ho latellana (sa feiga. 7c).Ha li kopane, lintlha tsena li bonts'a hore buka e ncha ea ursolic acid e tsoang ho KAND 11 ke semela se ikhethileng sa cytoskeletal inhibitor se nang le ts'ebetso e neng e sa tsejoe pele.
KAND e ama cortical microtubules, actin filaments, le ho sebetsa ha koae BY-2 lisele.(a) Ponahalo ea li-microtubules tsa cortical ka liseleng tsa BY-2 ka boteng ba TagRFP-TUA6.Lisele tsa BY-2 tse tšoaroang ka KAND 11 (50 μM kapa 100 μM) kapa DMSO li ile tsa hlahlojoa ka microscopy ea confocal.Cortical microtubule density e ne e baloa ho tloha ho li-micrographs tsa lisele tse ikemetseng tsa 25.Mangolo a bontša phapang e kholo (Teko ea Tukey HSD, leq<0.05).Sekhahla = 10 µm.(b) Cortical actin filaments ka BY-2 lisele tse bonahalang ka pel'a GFP-ABD2.Lisele tsa BY-2 tse tšoaroang ka KAND 11 (50 μM kapa 100 μM) kapa DMSO li ile tsa hlahlojoa ka microscopy ea confocal.Boima ba cortical actin filaments bo ne bo baloa ho tloha ho micrographs ea lisele tse 25 tse ikemetseng.Mangolo a bontša phapang e kholo (Teko ea Tukey HSD, leq<0.05).Sekhahla = 10 µm.(c) Ho shebella lisele tse shoeleng tsa BY-2 ka Evans blue staining.BY-2 lisele tse tšoaroang ka KAND 11 (50 μM kapa 100 μM) kapa DMSO li ile tsa hlahlojoa ka microscope ea tšimo e khanyang.n=3.Scale bar = 100 µm.
Ho sibolloa le ho sebelisoa ha lihlahisoa tse ncha tsa tlhaho ho entse hore ho be le tsoelo-pele e kholo likarolong tse sa tšoaneng tsa bophelo ba batho, ho akarelletsa le meriana le temo.Patlisiso ea nalane e entsoe ho fumana metsoako ea bohlokoa ho tsoa mehloling ea tlhaho.Haholo-holo, actinomycetes e tsejoa e le ea bohlokoa e le lithibela-mafu tse thibelang likokoana-hloko bakeng sa li-nematode ka lebaka la bokhoni ba tsona ba ho hlahisa metabolite e sa tšoaneng ea bobeli e kang avermectin, motsoako o ka sehloohong oa ivermectin le bleomycin le lihlahisoa tsa eona, tse sebelisoang meriana e le moemeli oa anticancer21,22.Ka mokhoa o ts'oanang, mefuta e fapaneng ea metsoako ea herbicide e fumanoe ho tsoa ho actinomycetes, e meng ea eona e seng e sebelisoa khoebong1,23.Ka hona, tlhahlobo ea metabolites ea actinomycete ho arola lihlahisoa tsa tlhaho ka mesebetsi e lakatsehang ea likokoana-hloko e nkoa e le leano le sebetsang.Thutong ena, re ile ra fumana motsoako o mocha, coumamonamide, ho tloha S. werraensis 'me ra o etsa ka katleho.Ursonic acid ke motsoako oa maiketsetso oa urbenamide le lihlahisoa tsa eona.E ka etsa hore metso e kobehe ka mokhoa o itekanetseng, 'me e senye li-microtubules tsa semela ka ho toba kapa ka tsela e sa tobang.Leha ho le joalo, mokhoa oa ts'ebetso oa urmotonic acid o ka fapana le oa li-microtubule inhibitors tse teng, kaha KAND 11 e boetse e senya li-filaments tsa actin 'me e baka lefu la lisele, e fana ka maikutlo a mokhoa oa ho laola oo urmotonic acid le lihlahisoa tsa eona li susumetsang mefuta e mengata ea cytoskeletal structures..
Tlhaloso e eketsehileng ea urbenonic acid e tla thusa ho utloisisa hamolemo mokhoa oa ts'ebetso ea urbenonic acid.Haholo-holo, sepheo se latelang ke ho hlahloba bokhoni ba ursonic acid ho tlama ho fokotsa microtubules ho fumana hore na ursonic acid le lihlahisoa tsa eona li sebetsa ka ho toba ho li-microtubules le ho li senya, kapa hore na ketso ea bona e fella ka ho senyeha ha microtubule.Ho phaella moo, tabeng eo li-microtubules e seng sepheo se tobileng, ho khetholla sebaka sa ts'ebetso le lipakane tsa limolek'hule tsa ursonic acid ka lisele tsa limela li tla thusa ho utloisisa haholoanyane thepa ea metsoako e amanang le litsela tse ka khonehang tsa ho ntlafatsa mosebetsi oa herbicidal.Tekolo ea rona ea bioactivity e senotse bokhoni bo ikhethang ba cytotoxic ea asiti ea ursonic kholong ea limela tse joalo ka Arabidopsis thaliana, koae le sebeterwort, athe lisele tsa E. coli kapa tsa HeLa ha lia ameha.Chefo e nyane kapa e se nang chefo ea lisele tsa liphoofolo ke monyetla oa ho tsoa ho ursonic acid haeba e entsoe e le meriana e bolaeang limela bakeng sa ho sebelisoa masimong a bulehileng a temo.Ha e le hantle, kaha li-microtubules ke mehaho e tloaelehileng ea li-eukaryote, ho thibela ha tsona limela ke ntho e ka sehloohong e hlokahalang bakeng sa likokoana-hloko.Ka mohlala, propyzamide, microtubule depolymerizing agent e tlamang ka ho toba ho tubulin le ho thibela polymerization, e sebelisoa e le sebolayalehola ka lebaka la chefo ea eona e tlaase ho lisele tsa liphoofolo24.Ho fapana le disopyramide, benzamide e amanang le eona e na le lintlha tse fapaneng tsa sepheo.Ho phaella ho li-microtubules tsa limela, RH-4032 kapa benzoxamide e boetse e thibela li-microtubules tsa lisele tsa liphoofolo kapa oomycetes, ka ho latellana, 'me zalilamide e sebelisoa e le fungicide ka lebaka la phytotoxicity25,26,27 e tlaase.Bere e sa tsoa sibolloa le lintho tse tsoang ho eona li bonts'a cytotoxicity e ikhethileng khahlano le limela, empa ho bohlokoa ho hlokomela hore liphetoho tse ling li ka fetola sepheo sa tsona, tse ka fanang ka litlatsetso tse ling bakeng sa taolo ea li-fungus tsa pathogenic kapa oomycete.
Thepa e ikhethang ea urbenonic acid le lihlahisoa tsa eona li na le thuso bakeng sa nts'etsopele ea tsona e le litlama le ho sebelisoa e le lisebelisoa tsa lipatlisiso.Bohlokoa ba cytoskeleton ho laola sebopeho sa lisele tsa semela bo tsebahala haholo.Liphuputso tsa pejana li bontšitse hore limela li fetohile mekhoa e rarahaneng ea mokhatlo oa cortical microtubule ka ho laola microtubule dynamics ho laola morphogenesis hantle.Palo e kholo ea limolek'hule tse ikarabellang bakeng sa taolo ea ts'ebetso ea microtubule li fumanoe, 'me lipatlisiso tse amanang le tsona li ntse li tsoela pele3,4,28.Kutloisiso ea rona ea hona joale ea matla a microtubule liseleng tsa limela ha e hlalose ka botlalo mekhoa ea mokhatlo oa cortical microtubule.Mohlala, le hoja disopyramide le oryzalin ka bobeli li ka senya li-microtubules, disopyramide e baka khopamo e matla ea metso ha oryzalin e na le phello e batlang e fokola.Ho feta moo, liphetoho tsa tubulin, tse tsitsang li-microtubules, li boetse li baka dextrorotation ka metso, athe paclitaxel, e boetse e tsitsisa microtubule dynamics, ha e etse joalo.Ka hona, ho ithuta le ho khetholla lipakane tsa limolek'hule tsa ursolic acid ho lokela ho fana ka lintlha tse ncha mabapi le taolo ea limela tsa cortical microtubules.Ka ho tšoanang, lipapiso tsa nakong e tlang tsa lik’hemik’hale tse atlehang ho khothalletsa khōlo e sothehileng, tse kang disopyramide, le lik’hemik’hale tse sa sebetseng hantle, tse kang oryzalin kapa kumamotoric acid, li tla fana ka leseli la hore na khōlo e sothehileng e etsahala joang.
Ka lehlakoreng le leng, litlhophiso tse amanang le ts'ireletso ea cytoskeletal ke monyetla o mong oa ho hlalosa cytotoxicity ea ursonic acid.Tšoaetso ea kokoana-hloko kapa ho kenngoa ha selikalikoe ka har'a lisele tsa limela ka linako tse ling ho baka tšenyo ea cytoskeleton le lefu le latelang la sele29.Ka mohlala, cryptoxanthin e entsoeng ka oomycete e tlalehiloe hore e senya li-microtubules le actin filaments pele ho lefu la lisele tsa koae, tse tšoanang le tse etsahalang ka phekolo ea KAND30,31.Ho tšoana pakeng tsa likarabo tsa ts'ireletso le likarabo tsa cellular tse bakoang ke ursonic acid li ile tsa etsa hore re nahane hore li baka mekhoa e tloaelehileng ea lisele, le hoja phello e potlakileng le e matla ea ursonic acid ho feta cryptoxanthin e bonahala.Leha ho le joalo, liphuputso li bontšitse hore tšitiso ea li-actin filaments e khothalletsa ho shoa ha lisele ka tšohanyetso, e seng kamehla e tsamaeang le ho senyeha ha microtubule29.Ho feta moo, ho sa ntse ho tla bonahala hore na kokoana-hloko kapa li-elicitor li baka kholo e sothehileng ea metso, joalo ka ha li-deronic acid li etsa.Kahoo, tsebo ea limolek'hule e hokahanyang likarabo tsa ts'ireletso le cytoskeleton ke bothata bo khahlang bo lokelang ho rarolloa.Ka ho sebelisa hampe boteng ba lik'hemik'hale tse tlaase tsa boima ba limolek'hule tse amanang le ursonic acid, hammoho le mefuta e mengata ea lihlahisoa tse nang le matla a sa tšoaneng, li ka fana ka menyetla ea ho lebisa mekhoa e sa tsejoeng ea lisele.
Ha li kopantsoe, ho sibolloa le ho sebelisoa ha metsoako e mecha e tsamaisang matla a microtubule ho tla fana ka mekhoa e matla ea ho sebetsana le mekhoa e rarahaneng ea limolek'hule e thehiloeng ho qeto ea sebopeho sa lisele tsa semela.Boemong bona, motsoako o sa tsoa etsoa oa urmotonic acid, o amang li-microtubules le actin filaments le ho etsa hore lisele li shoe, li ka fana ka monyetla oa ho hlalosa kamano pakeng tsa taolo ea microtubule le mekhoa ena e meng.Kahoo, tlhahlobo ea lik'hemik'hale le ea baeloji e sebelisang urbenonic acid e tla re thusa ho utloisisa mekhoa ea taolo ea limolek'hule e laolang semela sa cytoskeleton.
Inoculate S. werraensis MK493-CF1 ka har'a botlolo ea 500 mL e makatsang ea Erlenmeyer e nang le 110 mL ea sebaka sa peo se nang le 2% (w/v) galactose, 2% (w/v) Essence paste, 1% ( w/v) Bacto composition .-soyton (Thermo Fisher Scientific, Inc.), 0.5% (w / v) motsoako oa poone (KOGOSTCH Co., Ltd., Japan), 0.2% (w / v) (NH4) 2SO4 le 0.2% CaCO3 metsing a hloekisitsoeng.(pH 7.4 pele ho sterilization).Limela tsa peo li ile tsa kenngoa ka mochine oa rotary (180 rpm) ka 27 ° C ka matsatsi a mabeli.Temo ea tlhahiso ka ho belisoa ha boemo bo tiileng.Semela sa peo (7 ml) se ile sa fetisetsoa ka botlolong ea 500 ml K-1 e nang le 40 g ea tlhahiso ea tlhahiso e nang le 15 g ea harese e hatelitsoeng (MUSO Co., Ltd., Japan) le 25 g ea metsi a deionized (pH e sa fetoloang. pele ho sterilization).).Ho belisoa ho ne ho etsoa ka 30 ° C lefifing ka matsatsi a 14.Thepa ea ho belisoa e ne e ntšoa ka 40 ml / botlolo EtOH le centrifuged (1500 g, 4 ° C, 10 min).Sehlahisoa sa supernatant (60 ml) se ntšitsoe ka motsoako oa 10% MeOH/EtOAc.Lera la lintho tse phelang le ile la fetoha mouoane tlas'a khatello e fokotsehileng ea ho fumana masala (59.5 mg), a neng a le tlas'a HPLC e nang le gradient elution (metsotso e 0-10: 90%) holim'a kholomo ea reverse phase (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID. 10 mm × bolelele 250 mm) H2O/CH3CN, 10–35 metsotso: 90% H2O/CH3CN ho 70% H2O/CH3CN (gradient), 35–45 metsotso: 90% H2O/EtOH, 45–155 metsotso: 90% H2O / EtOH ho 100% EtOH (gradient (gradient), 155-200 min: 100% EtOH) ka tekanyo ea phallo ea 1.5 ml / min, coumamonamide (1, 36.0 mg) e ne e arotsoe e le phofo e tšoeu ea amorphous.
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]+ boleng bo baliloeng: 141.0659, boleng bo lekantsoeng: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
Lipeo tsa Columbia (Col-0) li fumanoe ho Arabidopsis Biological Resource Center (ABRC) ka tumello ea tšebeliso ea lipatlisiso.Lipeo tsa Col-0 li ile tsa phatlalatsoa le ho hlokomeloa tlas'a maemo a laboratori ea rona 'me tsa sebelisoa e le limela tse hlaha tsa Arabidopsis.Peo ea Arabidopsis e ne e hloekisitsoe 'me ea lengoa ka Murashige le Skoog medium e nang le 2% sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino)ethanesulfonic acid (MES) (Fujifilm Wako Pure Chemical ).) le 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, ho 23 °C le khanya e sa feleng.Peo ea phetoho ea phs1-1 e fanoe ke T. Hashimoto (Nara Institute of Science and Technology).
Peo ea khatello ea SR-1 e fanoe ke T. Hashimoto (Nara Institute of Science and Technology) 'me e sebelisoa e le limela tsa koae tse hlaha.Lipeo tsa koae li ile tsa hloekisoa 'me tsa kolobisoa ka metsing a sa hloekang ka masiu a mararo ho khothalletsa ho mela, ebe li kenngoa ka har'a motsoako oa halofo ea matla e nang le 2% sucrose, 0.05% (w/v) MES, le 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige.le Skoog medium) e nang le pH 5.7 mme e ekeditswe ho 23°C tlasa kganya e sa feleng.
Strain Tak-1 e fanoe ke T. Kohchi (Univesithi ea Kyoto) 'me e ne e sebelisoa e le tekanyo e tloaelehileng ea liteko bakeng sa thuto ea liverwort.Gemma e fumanwe ho tswa dimeleng tse hlahisitsweng ka sterilized mme ya bewa ka Gamborg B5 medium (Fujifilm Wako Pure Chemical) e nang le 1% sucrose le 0.3% gellan gum mme e eketswe ho 23 ° C tlasa kganya e sa feleng.
Lisele tsa koae BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) li fanoe ke S. Hasezawa (Univesithi ea Tokyo).Lisele tsa BY-2 li ile tsa hlapolloa ka makhetlo a 95 ho Linsmeier le Skoog tse fetotsoeng 'me tsa tlatsetsoa beke le beke ka 2,4-dichlorophenoxyacetic acid 32.Ho emisoa ha sele ho ne ho kopantsoe le shaker ea rotary ka 130 rpm ho 27 ° C lefifing.Hlatsoa lisele ka makhetlo a 10 ho feta bophahamo ba seaplane se foreshe ebe u emisa ka mokhoa o ts'oanang.BY-2 transgenic cell lines e hlalosang ka mokhoa o tsitsitseng letšoao la microtubule TagRFP-TUA6 kapa letšoao la actin filament GFP-ABD2 tlas'a cauliflower mosaic virus 35S promotor e hlahisitsoe joalo ka ha ho hlalositsoe33,34,35.Mehala ena ea lisele e ka bolokoa le ho hokahanngoa ho sebelisoa mekhoa e ts'oanang le e sebelisoang bakeng sa mohala oa sele oa BY-2 oa mantlha.
Lisele tsa HeLa li ne li entsoe ka Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) e tlatsitsoeng ka 10% ea fetal bovine serum, 1.2 U/ml penicillin, le 1.2 μg/ml streptomycin ka 37°C incubator e nang le 5% CO2.
Liteko tsohle tse hlalositsoeng bukeng ena e ngotsoeng ka letsoho li entsoe ho latela melao le litataiso tsa Majapane tsa biosafety.
Lik'hemik'hale li ile tsa qhibilihisoa ka dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) e le litharollo tsa stock 'me tsa hlapolloa ka MS medium bakeng sa Arabidopsis le koae kapa Gamborg B5 medium bakeng sa sebeterwort.Bakeng sa tlhahlobo ea thibelo ea kholo ea metso, lipeo tse fetang 10 ka poleiti ka 'ngoe li ile tsa jaloa holim'a agar medium e nang le metsoako e bontšitsoeng kapa DMSO.Peo e ile ea kenngoa ka kamoreng ea kholo ka matsatsi a 7.Lipeo li ile tsa nkoa senepe 'me ha lekanngoa bolelele ba metso.Bakeng sa tlhahlobo ea ho mela ha Arabidopsis, lipeo tse 48 ka poleiti ka 'ngoe li ile tsa jaloa holim'a agar medium e nang le motsoako oa 200 μM kapa DMSO.Lipeo tsa Arabidopsis li ne li lengoa ka phaposing ea kholo mme palo ea lipeo tse melang e ne e baloa matsatsi a 7 kamora ho mela (dag).Bakeng sa tlhahlobo ea ho mela ha koae, lipeo tse 24 ka poleiti ka 'ngoe li ile tsa jaloa holim'a agar medium e nang le 200 μM KAND kapa DMSO.Lipeo tsa koae li ne li lengoa ka kamoreng ea ho hōla 'me palo ea lipeo tse melang e ile ea baloa ka mor'a matsatsi a 14.Bakeng sa tlhahlobo ea thibelo ea ho hōla ha sebete, li-embryo tse 9 ho tloha poleiting e 'ngoe le e' ngoe li ne li kenngoa ka har'a agar medium e nang le likhahla tse bontšitsoeng tsa KAND kapa DMSO 'me li kenngoa ka kamoreng ea kholo ka matsatsi a 14.
Sebelisa lipeo tse silafalitsoeng ka 5 mg/ml propidium iodide (PI) ho bona ka mahlo a kelello mokhatlo o hlophisitsoeng.Lipontšo tsa PI li ile tsa bonoa ka microscope ea fluorescence ho sebelisoa microscope ea TCS SPE confocal laser scanning (Leica Microsystems).
Histochemical staining of roots with β-glucuronidase (GUS) e ile ea etsoa ho ea ka protocol e hlalositsoeng ke Malami le Benfey36.Lipeo li ne li tsitsitsoe ka 90% ea acetone ka bosiu bo le bong, e silafalitsoeng ka 0.5 mg / ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid ka GUS buffer bakeng sa hora ea 1 ebe e kenngoa ka tharollo ea hydrated chloraldehyde.(8 g chloral hydrate, 2 ml ea metsi le 1 ml glycerol) 'me e hlokomeloe ka tšitiso e fapaneng ea phapang ea microscopy ho sebelisa maekorosekopo ea Axio Imager M1 (Carl Zeiss).
Li-angles tsa metso li ne li lekanngoa ka lipeo tse nang le matsatsi a 7 tse hōlileng ka lipoleiti tse behiloeng holimo.Lekanya angle ea motso ho tloha lehlakoreng la vector ea khoheli joalokaha ho hlalositsoe mohatong oa 6.
Ho hlophisoa ha li-microtubules tsa cortical ho ile ha hlokomeloa joalokaha ho hlalositsoe, ka liphetoho tse nyenyane ho protocol 37.Anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) le Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) e ne e sebelisoa e le li-antibodies tsa mantlha le tsa bobeli ho 1: 1000 le 1: 100 dilutions, ka ho latellana.Litšoantšo tsa Fluorescence li ile tsa fumanoa ho sebelisoa maekorosekopo ea TCS SPE confocal laser scanning (Leica Microsystems).Fumana litšoantšo tsa Z-stack 'me u thehe likhakanyo tse matla ho latela litaelo tsa moetsi.
Teko ea ho ata ha lisele tsa HeLa e entsoe ho sebelisoa Cell Counting Kit 8 (Dojindo) ho latela litaelo tsa moetsi.
Khōlo ea E. coli DH5α e ile ea hlahlobjoa ka ho lekanya palo ea lisele tsa setso ho sebelisa spectrophotometer ho 600 nm (OD600).
Mokhatlo oa Cytoskeletal ka liseleng tsa transgenic BY-2 o ile oa bonoa ho sebelisoa microscope ea fluorescence e nang le mochine oa CSU-X1 confocal scanning (Yokogawa) le khamera ea sCMOS (Zyla, Andor Technology).Cytoskeletal density e ile ea hlahlojoa ka tlhahlobo ea litšoantšo, e ileng ea lekanya liperesente tsa lipikselse tsa cytoskeletal har'a lipikselse tsa cytoplasmic litšoantšong tsa confocal ho sebelisa software ea ImageJ joalo ka ha ho hlalositsoe38,39.
Ho lemoha lefu la sele ka har'a lisele tsa BY-2, aliquot ea ho emisoa ha sele e ne e kenngoa ka 0.05% Evans blue ka metsotso e 10 mocheso oa kamore.Khethollo ea Evans e putsoa ea lisele tse shoeleng e ipapisitse le extrusion ea dae e tsoang liseleng tse sebetsang ka lesela le sa sebetseng la plasma40.Lisele tse silafetseng li ile tsa bonoa ho sebelisoa microscope ea tšimo e khanyang (BX53, Olympus).
Lisele tsa HeLa li ne li lengoa ka DMEM e tlatsitsoeng ka 10% FBS ka incubator e nang le mongobo ho 37 ° C le 5% CO2.Lisele li ne li tšoaroa ka 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng / ml colcemid (Gibco), kapa 100 ng / ml Nocodmaze (Sigma) bakeng sa 6 h ho 37 ° C.Lisele li ne li tsitsitsoe ka MetOH bakeng sa 10 min ebe ka acetate bakeng sa 5 min mocheso oa kamore.Lisele tse tsitsitseng li ne li kenngoa ka β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) e hloekisitsoeng ka 0.5% BSA / PBS bakeng sa lihora tsa 2, e hlatsuoa ka makhetlo a 3 ka TBST, ebe e kenngoa ka Alexa Fluor antibody ea pōli.4881 hora.- Mouse IgG (Thermo Fisher Scientific: A11001) le 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) e hlapolotsoeng ka 0.5% BSA/PBS.Ka mor'a ho hlatsoa ka TBST ka makhetlo a mararo, lisele tse nang le litšila li ile tsa hlokomeloa ka microscope e fetotsoeng ea Nikon Eclipse Ti-E.Litšoantšo li nkiloe ka k'hamera e pholileng ea Hamamatsu ORCA-R2 CCD ho sebelisa software ea MetaMorph (Molecular Devices).
Nako ea poso: Jun-17-2024