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Kev tshawb pom thiab siv tau zoo ntawm cov khoom ntuj tuaj yeem pab txhim kho tib neeg lub neej. Nroj tsuag kev loj hlob inhibitory tshuaj yog dav siv los ua herbicides los tswj cov nroj tsuag. Vim qhov yuav tsum tau siv ntau hom tshuaj tua kab mob, nws yog qhov yuav tsum tau txheeb xyuas cov tebchaw nrog cov txheej txheem tshiab ntawm kev ua. Hauv txoj kev tshawb no, peb nrhiav tau ib qho tshiab N-alkoxypyrrole compound, coumamonamide, los ntawm Streptomyces werraensis MK493-CF1 thiab tsim kom tiav cov txheej txheem synthesis. Los ntawm kev soj ntsuam kev ua haujlwm lom neeg, peb pom tias urs-monoamic acid yog ib qho khoom nruab nrab ntawm urs-monoamide thiab muaj peev xwm.cog kev loj hlob inhibitor. Tsis tas li ntawd, peb tau tsim ntau yam urbenonic acid derivatives, suav nrog urbenyloxy derivative (UDA), uas muaj cov tshuaj tua kab mob siab uas tsis muaj kev cuam tshuam rau kev loj hlob ntawm HeLa hlwb. Peb kuj pom tias urmotonic acid derivatives cuam tshuam cov nroj tsuag microtubules; Tsis tas li ntawd, KAND cuam tshuam rau actin filaments thiab induces cell tuag; Cov teebmeem kev sib txawv no txawv ntawm cov neeg paub txog microtubule inhibitors thiab qhia txog kev ua haujlwm tshiab rau ursonic acid, uas sawv cev rau qhov txiaj ntsig tseem ceeb hauv kev tsim cov tshuaj tua kab tshiab.
Kev tshawb pom thiab kev siv tswv yim ntawm cov khoom muaj txiaj ntsig zoo thiab lawv cov derivatives yog ib txoj hauv kev txhim kho tib neeg lub neej zoo. Secondary metabolites tsim los ntawm cov kab mob, cov nroj tsuag thiab kab tau coj mus rau kev loj hlob ntawm cov tshuaj thiab kev ua liaj ua teb. Ntau cov tshuaj tua kab mob thiab tshuaj tiv thaiv kab mob qog ntshav qab zib tau tsim los ntawm cov khoom ntuj tsim. Tsis tas li ntawd, ntau homtshuaj tua kab, fungicides thiab herbicides yog muab rho tawm los ntawm cov khoom ntuj tsim los siv hauv kev ua liaj ua teb. Tshwj xeeb, cov tshuaj tswj cov nroj tsuag yog cov cuab yeej tseem ceeb rau kev nce qoob loo hauv kev ua liaj ua teb niaj hnub no, thiab ntau hom kev sib txuas tau siv los ua lag luam. Ntau cov txheej txheem ntawm tes hauv cov nroj tsuag, xws li photosynthesis, amino acid metabolism, cell phab ntsa synthesis, kev tswj ntawm mitosis, phytohormone signaling, los yog protein synthesis, yog suav hais tias yog lub hom phiaj ntawm cov tshuaj herbicides. Cov tshuaj uas inhibit microtubule muaj nuj nqi yog ib chav kawm ntawm cov tshuaj tua kab mob uas cuam tshuam rau cov nroj tsuag kev loj hlob los ntawm kev cuam tshuam mitotic regulation2.
Microtubules yog cov khoom ntawm cytoskeleton thiab tau txais kev txuag hauv cov hlwb eukaryotic. Tubulin heterodimer muaj α-tubulin thiab β-tubulin tsim cov kab microtubule protofilaments, nrog 13 protofilaments tsim cov qauv cylindrical. Microtubules ua si ntau lub luag haujlwm hauv cov nroj tsuag, suav nrog kev txiav txim siab cov duab ntawm tes, kev faib tawm ntawm tes, thiab kev thauj mus los hauv lub cev 3,4. Cov nroj tsuag muaj cov microtubules hauv qab ntawm interphase plasma membrane, thiab cov hu ua cortical microtubules tau xav tias yuav tswj lub koom haum ntawm cellulose microfibrils los ntawm kev tswj hwm ntawm cellulose synthase complexes4,5. Cortical microtubules ntawm cov hauv paus epidermal hlwb, tam sim no nyob rau hauv cheeb tsam ntawm ceev elongation ntawm lub hauv paus ntsis, yog nyob rau tom qab, thiab cellulose microfibers ua raws li cov microtubules thiab txwv cov kev taw qhia ntawm cell expansion, yog li txhawb anisotropic cell elongation. Yog li ntawd, microtubule muaj nuj nqi yog ze ze rau cov nroj tsuag morphology. Amino acid hloov nyob rau hauv cov noob encoding tubulin ua rau skew ntawm cortical microtubule arrays thiab sab laug-los yog sab xis txoj kev loj hlob nyob rau hauv Arabidopsis 6,7. Ib yam li ntawd, kev hloov pauv hauv microtubule-txuas nrog cov proteins uas tswj cov microtubule dynamics kuj tuaj yeem ua rau cov hauv paus hniav tsis zoo 8,9,10,11,12,13. Tsis tas li ntawd, kev kho mob nrog microtubule- cuam tshuam cov tshuaj tua kab mob xws li disopyramide, tseem hu ua pretilachlor, kuj ua rau sab laug-sab oblique cag loj hlob14. Cov ntaub ntawv no qhia tau hais tias cov kev cai ntawm microtubule muaj nuj nqi yog qhov tseem ceeb rau kev txiav txim siab txog kev loj hlob ntawm cov nroj tsuag.
Ntau hom microtubule inhibitors tau tshawb pom, thiab cov tshuaj no tau ua rau muaj txiaj ntsig zoo rau kev tshawb fawb cytoskeletal, nrog rau kev ua liaj ua teb thiab tshuaj2. Hauv particular, oryzalin, dinitroaniline tebchaw, disopyramide, benzamide-hais txog cov tebchaw, thiab lawv cov analogs tuaj yeem cuam tshuam microtubule muaj nuj nqi thiab yog li inhibit qhov kev loj hlob ntawm cov nroj tsuag. Yog li ntawd, lawv tau dav siv los ua herbicides. Txawm li cas los xij, txij li microtubules yog ib qho tseem ceeb ntawm cov nroj tsuag thiab tsiaj hlwb, feem ntau microtubule inhibitors yog cytotoxic rau ob hom cell. Yog li ntawd, txawm tias lawv cov khoom siv tau lees paub tias yog tshuaj tua kab mob, cov tshuaj tua kab mob tsawg tsawg yog siv rau cov laj thawj.
Streptomyces yog ib tug genus ntawm tsev neeg Streptomyces, uas muaj xws li aerobic, gram-positive, filamentous kab mob thiab yog dav paub rau nws muaj peev xwm los tsim ib tug ntau yam ntawm cov theem nrab metabolites. Yog li ntawd, nws raug suav hais tias yog ib qho tseem ceeb tshaj plaws ntawm cov khoom siv roj ntsha tshiab. Nyob rau hauv txoj kev tshawb no tam sim no, peb nrhiav tau ib tug tshiab compound hu ua coumamonamide, uas yog cais los ntawm Streptomyces werraensis MK493-CF1 thiab S. werraensis ISP 5486. Siv spectral tsom xam thiab tag nrho spectral tsom xam, cov qauv ntawm coumamonamide yog characterized thiab nws cov N-alkoxypyrrole skeleton tshwj xeeb. tau txiav txim. synthesis. Ursmonic acid, ib qho hluavtaws nruab nrab ntawm ursmonoamide thiab nws cov derivatives, tau pom tias inhibit qhov kev loj hlob thiab kev loj hlob ntawm cov qauv nrov Arabidopsis thaliana. Hauv kev tshawb fawb txog kev sib raug zoo-kev sib raug zoo, peb pom tias qhov sib xyaw nrog C9 tau hloov kho rau ursonic acid, hu ua nonyloxy derivative ntawm ursonic acid (KAND), ua rau muaj kev cuam tshuam zoo rau kev loj hlob thiab kev loj hlob. Tshwj xeeb tshaj yog, cov nroj tsuag loj hlob uas tau tshawb pom tshiab kuj cuam tshuam rau kev loj hlob ntawm cov luam yeeb thiab lub cev thiab tsis yog cytotoxic rau cov kab mob lossis HeLa hlwb. Ntxiv mus, qee cov urmotonic acid derivatives induce ib tug distorted paus phenotype, implying hais tias cov derivatives ncaj qha los yog indirectly cuam tshuam rau microtubules. Raws li lub tswv yim no, peb cov kev soj ntsuam ntawm microtubules sau los ntawm immunohistochemically los yog nrog fluorescent proteins qhia tias KAND kho depolymerizes microtubules. Tsis tas li ntawd, kev kho mob nrog kumamotonic acid derivatives cuam tshuam actin microfilaments. Yog li, peb tau tshawb pom ib qho tshiab cog kev loj hlob inhibitor uas nws cov txheej txheem tshwj xeeb ntawm kev ua cuam tshuam nrog kev puas tsuaj ntawm cytoskeleton.
Hom MK493-CF1 raug cais tawm ntawm cov av hauv Shinagawa-ku, Tokyo. Strain MK493-CF1 tsim tau zoo-branched stromal mycelium. Ib feem ntawm 16S ribosomal RNA gene (1422 bp) tau txiav txim siab. Cov kab mob no zoo ib yam li S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: typical strain, 99.93%). Raws li qhov tshwm sim no, nws tau txiav txim siab tias hom kab mob no muaj feem cuam tshuam nrog hom kab mob S. werraensis. Yog li ntawd, peb ib ntus hu ua tus kab mob no S. werraensis MK493-CF1. S. werraensis ISP 5486T kuj ua tau tib yam bioactive compounds. Txij li thaum muaj kev tshawb fawb me me kom tau txais cov khoom ntuj tsim los ntawm cov kab mob no, kev tshawb fawb tshuaj ntxiv tau ua tiav. Tom qab cog S. werraensis MK493-CF1 ntawm barley nruab nrab los ntawm solid-state fermentation ntawm 30 ° C rau 14 hnub, qhov nruab nrab tau muab rho tawm nrog 50% EtOH. 60 ml ntawm cov qauv tau qhuav kom tau 59.5 mg ntawm crude extract. Cov kua txiv hmab txiv ntoo tau raug rov qab rau theem HPLC muab N-methoxy-1H-pyrrole-2-carboxamide (1, hu ua coumamonamide, 36.0 mg). Tag nrho cov nyiaj ntawm 1 yog kwv yees li 60% ntawm cov khoom siv raw. Yog li ntawd, peb txiav txim siab los kawm hauv kev nthuav dav cov khoom ntawm kumamotoamide 1.
Coumamonamide 1 yog cov hmoov dawb amorphous thiab siab daws teeb meem loj spectrometry (HRESIMS) lees paub C6H8N2O2 (Fig. 1). C2-substituted pyrrole fragment ntawm no compound yog characterized by δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH hauv 1H NMR spectrum: 4.5 Hz , H-5) thiab δH 6.78 (1H, d, J = 2.5 Hz, H-6), thiab 13C NMR spectrum qhia pom muaj plaub sp2 carbon atoms. Lub xub ntiag ntawm ib pawg amide ntawm C2 txoj hauj lwm raug soj ntsuam los ntawm HMBC correlation los ntawm C-3 proton mus rau amide carbonyl carbon ntawm δC 161.1. Tsis tas li ntawd, 1 H thiab 13 C NMR peaks ntawm δH 4.10 (3H, S) thiab δC 68.3 qhia tias muaj N-methoxy pawg hauv cov molecule. Txawm hais tias qhov tseeb ntawm pawg methoxy tseem tsis tau txiav txim siab siv spectroscopic tsom xam xws li txhim kho qhov sib txawv spectroscopy thiab nuclear Overhauser abbreviation (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide tau los ua thawj tus neeg sib tw.
Txhawm rau txiav txim siab tus qauv raug ntawm 1, tag nrho cov synthesis tau ua tiav (Fig. 2a). Kev kho mob ntawm kev lag luam muaj 2-aminopyridine 2 nrog m-CPBA ua rau muaj qhov sib thooj N-oxide 3 nyob rau hauv kom muaj nuj nqis. Tom qab 2-aminoazidation ntawm 2, cov tshuaj tiv thaiv cyclocondensation piav qhia los ntawm Abramovich tau ua nyob rau hauv benzene ntawm 90 ° C kom tau qhov xav tau 1-hydroxy-1H-pyrrole-2-carbonitrile 5 hauv grams. Ceev 60% (ob theem). 15,16 ib. Methylation thiab hydrolysis ntawm 4 ces muab 1-methoxy-1H-pyrrole-2-carboxylic acid (lub sij hawm "cumotonic acid", 6) nyob rau hauv zoo yield (70%, ob kauj ruam). Thaum kawg, amidation ntawm acid chloride intermediate 6 siv aqueous ammonia muab Kumamoto amide 1 hauv 98% tawm los. Tag nrho cov ntaub ntawv spectral ntawm synthesized 1 zoo ib yam li cais 1, yog li cov qauv ntawm 1 tau txiav txim siab;
General synthesis thiab tsom xam ntawm kev ua haujlwm lom ntawm urbenamide thiab urbenic acid. (a) Tag nrho cov synthesis ntawm Kumamoto amide. (b) Xya-hnub-laus tsiaj qus-hom Arabidopsis Columbia (Col) cov noob tau cog rau ntawm Murashige thiab Skoog (MS) daim hlau uas muaj coumamonamide 6 los yog coumamonamide 1 ntawm qhov qhia tau ntau. Luas bar = 1 cm.
Ua ntej, peb tau soj ntsuam cov dej num lom neeg ntawm urbenamide thiab nws cov intermediates rau lawv lub peev xwm los hloov cov nroj tsuag loj hlob. Peb ntxiv ntau qhov ntau ntawm ursmonamide 1 lossis ursmonic acid 6 rau MS agar nruab nrab thiab kab lis kev cai Arabidopsis thaliana seedlings ntawm qhov nruab nrab. Cov kev ntsuam xyuas no tau pom tias muaj cov concentrations siab (500 μM) ntawm 6 inhibited cag kev loj hlob (Fig. 2b). Tom ntej no, peb tau tsim ntau yam derivatives los ntawm kev hloov N1 txoj hauj lwm ntawm 6 thiab ua cov qauv-kev kawm kev sib raug zoo ntawm lawv (cov txheej txheem analogue synthesis tau piav qhia hauv Cov Lus Qhia Txhawb (SI)). Arabidopsis seedlings tau cog rau ntawm qhov nruab nrab uas muaj 50 μM ursonic acid derivatives, thiab cov cag ntev tau ntsuas. raws li nws pom hauv daim duab. Raws li pom hauv daim duab 3a, b, thiab S1, coumamo acids muaj qhov sib txawv ntawm cov kab alkoxy chains (9, 10, 11, 12, thiab 13) lossis loj alkoxy chains (15, 16, thiab 17) ntawm N1 txoj haujlwm. Cov derivatives pom qhov tseem ceeb inhibition ntawm kev loj hlob hauv paus. Ntxiv rau, peb pom tias daim ntawv thov ntawm 200 μM 10, 11, lossis 17 inhibited germination (Figs. 3c thiab S2).
Kawm txog cov qauv-kev sib raug zoo ntawm Kumamoto amide thiab lwm cov tebchaw. (a) Cov qauv thiab kev sib xyaw ua ke ntawm analogues. (b) Kev faib tawm ntawm cov cag ntev ntawm 7-hnub-laus seedlings zus ntawm MS nruab nrab nrog lossis tsis muaj 50 μM coumamonamide derivatives. Asterisks qhia qhov sib txawv tseem ceeb nrog kev kho mob sham (t test, p<0.05). n>18. Cov ntaub ntawv qhia tias txhais tau tias ± SD. nt txhais tau tias "tsis kuaj" vim tias ntau tshaj 50% ntawm cov noob tsis germinate. (c) Kev ua kom muaj nuj nqis ntawm germination tus nqi ntawm cov noob kho incubated rau 7 hnub nyob rau hauv MS nruab nrab nrog los yog tsis muaj 200 μM coumamonamide thiab lwm yam compounds. Asterisks qhia qhov sib txawv tseem ceeb nrog kev kho sham (chi-square test). n:96 ua.
Interestingly, qhov sib ntxiv ntawm alkyl sab chains ntev dua C9 txo cov kev ua haujlwm inhibitory, qhia tias kumamotoic acid-txog cov tebchaw xav tau cov saw hlau ntawm ib qho me me los nthuav qhia lawv cov kev ua haujlwm lom neeg.
Vim hais tias cov qauv-kev sib raug zoo kev txheeb xyuas pom tau tias C9 tau hloov kho rau ursonic acid thiab cov nonyloxy derivative ntawm ursonic acid (tom qab no hu ua KAND 11) yog cov nroj tsuag kev loj hlob zoo tshaj plaws inhibitor, peb tau ua ib qho kev qhia ntxaws ntxiv ntawm KAND 11. Kev kho mob ntawm Arabidopsis. nrog 50 μM KAND 11 yuav luag tag nrho tiv thaiv germination, whereas qis concentrations (40, 30, 20, los yog 10 μM) ntawm KAND 11 inhibited hauv paus kev loj hlob nyob rau hauv ib koob tshuaj raws li (Fig. 4a, b). Txhawm rau kuaj seb KAND 11 puas cuam tshuam rau hauv paus meristem, peb tau tshuaj xyuas cov hauv paus meristems stained nrog propidium iodide (PI) thiab ntsuas meristem cheeb tsam loj. Qhov luaj li cas ntawm meristem ntawm seedlings zus nyob rau hauv nruab nrab muaj 25 μM KAND-11 yog 151.1 ± 32.5 μm, thaum lub meristem ntawm seedlings zus nyob rau hauv ib tug tswj nruab nrab uas muaj DMSO yog 264.7 ± 30.8 μm (Fig. 4c, d) , uas qhia tias KAND-11 rov ua haujlwm ntawm tes. sib kis. Hauv paus meristem. Raws li qhov no, KAND 11 kev kho mob txo tus nqi ntawm cell division marker CDKB2;1p::CDKB2;1-GUS teeb liab nyob rau hauv lub hauv paus meristem (Fig. 4e) 17 . Cov txiaj ntsig no qhia tau tias KAND 11 inhibits kev loj hlob hauv paus los ntawm kev txo cov cell proliferation.
Kev tshuaj xyuas cov nyhuv inhibitory ntawm urbenonic acid derivatives (urbenyloxy derivatives) ntawm kev loj hlob. (a) 7-hnub qub qus-hom Col seedlings loj hlob rau ntawm MS daim hlau nrog cov qhia ntau npaum ntawm KAND 11. Scale bar = 1 cm. (b) Quantification ntawm cag ntev. Cov ntawv qhia qhov sib txawv tseem ceeb (Tukey HSD test, p<0.05). n>16. Cov ntaub ntawv qhia tias txhais tau tias ± SD. (c) Confocal microscopy ntawm propidium iodide-stained wild-type Col cov hauv paus qoob loo cog rau ntawm MS daim hlau nrog lossis tsis muaj 25 μM KAND 11. Cov kab ntawv dawb qhia cov hauv paus meristem. Scale bar = 100 µm. (d) Quantification ntawm cov hauv paus meristem loj (n = 10 txog 11). Kev ntsuas qhov sib txawv tau txiav txim siab siv t-test (p<0.05). Cov tuav sawv cev qhov nruab nrab meristem loj. (e) Qhov sib txawv cuam tshuam qhov sib txawv (DIC) microscopy ntawm lub hauv paus meristem uas muaj CDKB2 tsim; 1pro: CDKB2; 1-GUS stained thiab stained rau 5-hnub-laus cov noob cog rau ntawm MS daim hlau nrog lossis tsis muaj 25 µM KAND kev soj ntsuam.
Lub phytotoxicity ntawm KAND 11 tau sim ntxiv siv lwm cov nroj tsuag dicotyledonous, haus luam yeeb (Nicotiana tabacum), thiab cov av loj cog qauv kab mob, liverwort (Marchantia polymorpha). Raws li nyob rau hauv cov ntaub ntawv ntawm Arabidopsis, haus luam yeeb SR-1 seedlings zus nyob rau hauv nruab nrab muaj 25 μM KAND 11 ua rau luv luv keeb kwm (Fig. 5a). Tsis tas li ntawd, 40 ntawm 48 noob germinated rau ntawm daim hlau uas muaj 200 μM KAND 11, whereas tag nrho 48 noob germinated ntawm mock-kho tawm xov xwm, qhia tias ntau dua ntawm KAND yog qhov tseem ceeb (p<0.05; chi test -square) inhibited germination ntawm luam yeeb. (Fig. 5b). Tsis tas li ntawd, qhov concentration ntawm KAND 11 uas inhibited kab mob loj hlob nyob rau hauv liverwort zoo ib yam li cov concentration zoo nyob rau hauv Arabidopsis (Fig. 5c). Cov txiaj ntsig no qhia tau tias KAND 11 tuaj yeem cuam tshuam kev loj hlob ntawm ntau yam nroj tsuag. Tom qab ntawd peb tshawb xyuas qhov ua tau cytotoxicity ntawm dais monoamide-txog cov tebchaw hauv lwm yam kab mob, uas yog tib neeg HeLa hlwb thiab Escherichia coli strain DH5α, raws li cov neeg sawv cev ntawm cov tsiaj thiab cov kab mob siab dua, feem. Hauv kev soj ntsuam kev loj hlob ntawm tes, peb pom tias coumamonamide 1, coumamonamidic acid 6, thiab KAND 11 tsis cuam tshuam rau kev loj hlob ntawm HeLa lossis E. coli hlwb ntawm qhov ntau ntawm 100 μM (Fig. 5d, e).
Kev loj hlob inhibition ntawm KAND 11 hauv cov kab mob uas tsis yog Arabidopsis. (a) Ob lub lis piam-laus-hom SR-1 cov nroj tsuag luam yeeb tau cog rau ntawm vertically positioned MS daim hlau uas muaj 25 μM KAND 11. (b) Ob-lub lim tiam-laus-hom SR-1 haus luam yeeb cov noob tau cog rau ntawm kab rov tav. MS daim hlau uas muaj 200 μM KAND 11. (c) Ob-lub lim tiam-laus qus-hom Tak-1 liverwort buds loj hlob rau ntawm Gamborg B5 daim hlau nrog rau qhov qhia tau hais tias muaj ntau ntawm KAND 11. Cov xub liab qhia cov kab mob uas tsis loj hlob nyob rau hauv ob lub lis piam incubation lub sij hawm. (d) Kev soj ntsuam kev loj hlob ntawm tes ntawm HeLa hlwb. Tus naj npawb ntawm cov hlwb siv tau raug ntsuas ntawm lub sijhawm tas sijhawm siv cov khoom siv ntawm tes suav 8 (Dojindo). Raws li kev tswj hwm, HeLa hlwb raug kho nrog 5 μg / ml actinomycin D (Act D), uas inhibits RNA polymerase transcription thiab ua rau cell tuag. Kev ntsuam xyuas tau ua hauv triplicate. (e) E. coli cell proliferation assay. E. coli kev loj hlob tau txheeb xyuas los ntawm kev ntsuas OD600. Raws li kev tswj hwm, cov hlwb raug kho nrog 50 μg / ml ampicillin (Amp), uas inhibits cov kab mob cell phab ntsa synthesis. Kev ntsuam xyuas tau ua hauv triplicate.
Txhawm rau txiav txim siab cov txheej txheem ntawm kev txiav txim ntawm cytotoxicity tshwm sim los ntawm uramide-txog cov tebchaw, peb rov ua dua urbenic acid derivatives nrog cov teebmeem cuam tshuam me ntsis. raws li nws pom hauv daim duab. Raws li pom nyob rau hauv daim duab 2b, 6a, yub cog rau ntawm agar daim hlau uas muaj siab concentrations (200 μM) ntawm urmotonic acid 6 ua luv luv thiab sab laug-kho cov hauv paus hniav (θ = - 23.7 ± 6.1), whereas ntawm seedlings zus nyob rau hauv kev tswj nruab nrab, lub seedlings tsim yuav luag ncaj cov hauv paus hniav (θ = - 3.8 ± 7.1). Qhov kev loj hlob ntawm oblique yog paub los ntawm kev ua haujlwm tsis zoo ntawm cortical microtubules14,18. Raws li qhov kev tshawb pom no, cov tshuaj microtubule-destabilizing tshuaj disopyramide thiab oryzalin induced zoo xws li cov cag tilting nyob rau hauv peb cov kev loj hlob (Figs. 2b, 6a). Nyob rau tib lub sijhawm, peb tau sim urmotonic acid derivatives thiab xaiv ob peb ntawm lawv uas, ntawm qee qhov concentrations, induced oblique cag loj hlob. Cov tshuaj 8, 9, thiab 15 tau hloov cov kev coj ntawm cov hauv paus hniav ntawm 75 μM, 50 μM, thiab 40 μM, raws li qhia tau hais tias cov tshuaj no tuaj yeem ua rau cov microtubules tsis muaj zog (Fig. 2b, 6a). Peb kuj tau sim qhov muaj zog tshaj plaws ursolic acid derivative, KAND 11, ntawm qhov qis dua (15 µM) thiab pom tias daim ntawv thov ntawm KAND 11 inhibited cag kev loj hlob thiab hais tias cov kev taw qhia ntawm cov hauv paus loj hlob tsis sib xws, txawm hais tias lawv tended nqes mus rau sab laug ( Daim duab C3). . Vim tias muaj ntau dua ntawm cov tshuaj microtubule-destabilizing qee zaum cuam tshuam cov nroj tsuag kev loj hlob ntau dua li ua rau hauv paus tilting, peb tom qab ntsuas qhov ua tau tias KAND 11 cuam tshuam rau microtubules los ntawm kev soj ntsuam cortical microtubules hauv cov hauv paus epidermal hlwb. Immunohistochemistry siv cov tshuaj tiv thaiv-β-tubulin cov tshuaj tiv thaiv kab mob hauv cov kab mob epidermal ntawm cov hauv paus cag kho nrog 25 μM KAND 11 pom qhov ploj ntawm yuav luag tag nrho cov cortical microtubules hauv cov hlwb epidermal hauv cheeb tsam elongation (Fig. 6b). Cov txiaj ntsig no qhia tau hais tias kumamotonic acid thiab nws cov derivatives ua ncaj qha lossis tsis ncaj rau ntawm microtubules los cuam tshuam rau lawv thiab tias cov tebchaw no yog cov tshiab microtubule inhibitors.
Ursonic acid thiab nws cov derivatives hloov cortical microtubules hauv Arabidopsis thaliana. (a) Lub kaum sab xis inclination ntsuas nyob rau hauv lub xub ntiag ntawm ntau yam urmotonic acid derivatives ntawm qhov qhia tau hais. Cov teebmeem ntawm ob lub tebchaw paub tias inhibit microtubules: disopyramide thiab oryzalin kuj tau txheeb xyuas. Lub inset qhia tus qauv siv los ntsuas lub hauv paus kev loj hlob lub kaum sab xis. Asterisks qhia qhov sib txawv tseem ceeb nrog kev kho sham (t test, p<0.05). n>19. Luas bar = 1 cm. (b) Cortical microtubules nyob rau hauv epidermal hlwb nyob rau hauv elongation cheeb tsam. Microtubules nyob rau hauv cov qus-hom Arabidopsis Col cov hauv paus hniav loj hlob ntawm MS daim hlau nrog lossis tsis muaj 25 μM KAND 11 tau pom los ntawm kev siv tshuaj tiv thaiv kab mob siv β-tubulin thawj cov tshuaj tiv thaiv thiab Alexa Fluor-conjugated Secondary antibodies. Luas bar = 10 µm. (c) Mitotic qauv ntawm microtubules hauv paus meristem. Microtubules tau pom los ntawm kev siv tshuaj tua kab mob immunohistochemical. Cov qauv mitotic, suav nrog thaj chaw prophase, spindles, thiab phragmoplasts, tau suav los ntawm cov duab confocal. Cov xub qhia cov qauv mitotic microtubule. Asterisks qhia qhov sib txawv tseem ceeb nrog kev kho mob sham (t test, p<0.05). n>9. Luas bar = 50 µm.
Txawm hais tias Ursa muaj peev xwm cuam tshuam microtubule kev ua haujlwm, nws cov txheej txheem ntawm kev ua haujlwm yuav tsum txawv ntawm cov tshuaj microtubule depolymerizing. Piv txwv li, ntau dua ntawm microtubule depolymerizing cov neeg ua haujlwm xws li disopyramide thiab oryzalin induce anisotropic expansion ntawm epidermal cells, whereas KAND 11 tsis. Tsis tas li ntawd, kev sib koom ua ke ntawm KAND 11 thiab disopyramide ua rau muaj kev sib koom ua ke ntawm disopyramide-induced hauv paus kev loj hlob teb thiab KAND 11-induced kev loj hlob inhibition tau pom (Fig. S4). Peb kuj tau soj ntsuam cov lus teb ntawm hypersensitive disopyramide 1-1 (phs1-1) mutant rau KAND 11. phs1-1 muaj qhov tsis yog-canonical tubulin kinase point kev hloov pauv thiab ua rau cov hauv paus hniav luv dua thaum kho nrog disopyramide9,20. phs1-1 mutant seedlings cog rau ntawm agar nruab nrab uas muaj KAND 11 muaj cov hauv paus hniav luv zoo ib yam li cov cog ntawm disopyramid (daim duab S5).
Tsis tas li ntawd, peb tau pom cov qauv mitotic microtubule, xws li thaj chaw prophase, spindles, thiab phragmoplasts, nyob rau hauv lub hauv paus meristem ntawm seedlings kho nrog KAND 11. Raws li cov kev soj ntsuam rau CDKB2; 1p:: CDKB2; 1-GUS, ib tug tseem ceeb txo nyob rau hauv pom tus naj npawb ntawm mitotic microtubules (Fig. .6c).
Txhawm rau ua tus yam ntxwv ntawm cytotoxicity ntawm KAND 11 ntawm kev daws teeb meem hauv subcellular, peb tau kho cov kab mob BY-2 ncua kev kawm nrog KAND 11 thiab soj ntsuam lawv cov lus teb. Peb thawj zaug ntxiv KAND 11 rau BY-2 hlwb qhia TagRFP-TUA6, uas fluorescently sau cov microtubules, los soj ntsuam cov nyhuv ntawm KAND 11 ntawm cortical microtubules. Cortical microtubule ntom ntom tau soj ntsuam siv cov duab tsom, uas ntsuas qhov feem pua ntawm cytoskeletal pixels ntawm cytoplasmic pixels. Cov txiaj ntsig kev soj ntsuam tau pom tias tom qab kev kho mob nrog 50 μM lossis 100 μM KAND 11 rau 1 teev, qhov ceev tau txo qis rau 0.94 ± 0.74% lossis 0.23 ± 0.28%, feem, thaum qhov ceev ntawm cov hlwb kho nrog DMSO , 61 ± 1 . % (Fig. 7a). Cov txiaj ntsig no zoo ib yam nrog kev soj ntsuam hauv Arabidopsis tias KAND 11 kev kho mob induces depolymerization ntawm cortical microtubules (Fig. 6b). Peb kuj tau tshuaj xyuas BY-2 kab nrog GFP-ABD-labeled actin filaments tom qab kev kho mob nrog tib qhov concentration ntawm KAND 11 thiab pom tias KAND 11 kev kho mob cuam tshuam cov actin filaments. Kev kho mob nrog 50 μM lossis 100 μM KAND 11 rau 1 h txo qis actin filament ceev mus rau 1.20 ± 0.62% lossis 0.61 ± 0.26%, feem, qhov ceev hauv DMSO-kho hlwb yog 1.69% 0.69% (2. 7 b). Cov txiaj ntsig no sib piv nrog cov teebmeem ntawm propyzamide, uas tsis cuam tshuam rau actin filaments, thiab latrunculin B, actin depolymerizer uas tsis cuam tshuam rau microtubules (SI Figure S6). Tsis tas li ntawd, kev kho mob nrog coumamonamide 1, coumamonamide acid 6, lossis KAND 11 tsis cuam tshuam rau microtubules hauv HeLa hlwb (SI Figure S7). Yog li, cov txheej txheem ntawm kev ua ntawm KAND 11 ntseeg tau tias txawv ntawm cov neeg paub txog cytoskeleton cuam tshuam. Tsis tas li ntawd, peb qhov kev soj ntsuam microscopic ntawm BY-2 hlwb kho nrog KAND 11 tau nthuav tawm qhov pib ntawm kev tuag ntawm tes thaum kho KAND 11 thiab pom tias qhov feem pua ntawm Evans xiav-stained cov hlwb tuag tsis tau nce ntxiv tom qab 30 min ntawm KAND 11 kev kho mob, whereas tom qab 90 feeb ntawm kev kho mob nrog 50 μM lossis 100 μM KAND, cov hlwb tuag tau nce mus rau 43.7% lossis 80.1%, feem (Fig. 7c). Ua ke, cov ntaub ntawv no qhia tau hais tias cov tshiab ursolic acid derivative KAND 11 yog cov nroj tsuag tshwj xeeb cytoskeletal inhibitor nrog rau yav dhau los tsis paub txog kev ua haujlwm.
KAND cuam tshuam rau cortical microtubules, actin filaments, thiab muaj peev xwm ntawm kev haus luam yeeb BY-2 hlwb. (a) Visualization ntawm cortical microtubules nyob rau hauv BY-2 hlwb nyob rau hauv lub xub ntiag ntawm TagRFP-TUA6. BY-2 hlwb kho nrog KAND 11 (50 μM lossis 100 μM) lossis DMSO tau kuaj xyuas los ntawm confocal microscopy. Cortical microtubule density yog xam los ntawm micrographs ntawm 25 lub hlwb ywj pheej. Cov ntawv qhia qhov sib txawv tseem ceeb (Tukey HSD test, p<0.05). Luas bar = 10 µm. (b) Cortical actin filaments nyob rau hauv BY-2 hlwb pom nyob rau hauv lub xub ntiag ntawm GFP-ABD2. BY-2 hlwb kho nrog KAND 11 (50 μM lossis 100 μM) lossis DMSO tau kuaj xyuas los ntawm confocal microscopy. Qhov ceev ntawm cortical actin filaments yog xam los ntawm micrographs ntawm 25 lub hlwb ywj pheej. Cov ntawv qhia qhov sib txawv tseem ceeb (Tukey HSD test, p<0.05). Luas bar = 10 µm. (c) Kev soj ntsuam ntawm cov hlwb tuag BY-2 los ntawm Evans xiav staining. BY-2 hlwb tau kho nrog KAND 11 (50 μM lossis 100 μM) lossis DMSO tau kuaj xyuas los ntawm lub teeb pom kev zoo. n=3. Scale bar = 100 µm.
Kev tshawb pom thiab kev siv cov khoom siv ntuj tshiab tau ua rau muaj kev vam meej hauv ntau yam ntawm tib neeg lub neej, suav nrog tshuaj thiab kev ua liaj ua teb. Kev tshawb fawb keeb kwm tau ua tiav kom tau txais txiaj ntsig zoo los ntawm cov peev txheej ntuj. Tshwj xeeb, actinomycetes paub tias muaj txiaj ntsig zoo li cov tshuaj tua kab mob rau cov nematodes vim lawv muaj peev xwm tsim ntau yam metabolites xws li avermectin, cov hmoov lead compound ntawm ivermectin thiab bleomycin thiab nws cov derivatives, siv tshuaj raws li tus neeg sawv cev anticancer21,22. Ib yam li ntawd, ntau yam tshuaj ntsuab tshuaj ntsuab tau pom los ntawm actinomycetes, qee qhov twb tau siv ua lag luam 1,23. Yog li ntawd, kev tsom xam ntawm actinomycete metabolites cais cov khoom ntuj tsim nrog kev ua haujlwm lom neeg yog suav tias yog lub tswv yim zoo. Nyob rau hauv txoj kev tshawb no, peb nrhiav tau ib tug tshiab compound, coumamonamide, los ntawm S. werraensis thiab ntse synthesized nws. Ursonic acid yog ib qho hluavtaws nruab nrab ntawm urbenamide thiab nws cov derivatives. Nws tuaj yeem ua rau tus yam ntxwv hauv paus curling, nthuav tawm nruab nrab mus rau muaj zog tshuaj tua kab mob, thiab ncaj qha lossis ncaj qha ua rau cov nroj tsuag microtubules. Txawm li cas los xij, cov txheej txheem ntawm kev ua ntawm urmotonic acid yuav txawv ntawm cov microtubule inhibitors uas twb muaj lawm, txij li KAND 11 kuj cuam tshuam cov actin filaments thiab ua rau cov cell tuag, qhia txog kev tswj hwm txoj cai uas urmotonic acid thiab nws cov derivatives cuam tshuam ntau yam ntawm cov qauv cytoskeletal. .
Cov ncauj lus kom ntxaws ntxiv ntawm urbenonic acid yuav pab kom nkag siab zoo dua cov txheej txheem ntawm kev ua ntawm urbenonic acid. Tshwj xeeb, lub hom phiaj tom ntej yog los ntsuas lub peev xwm ntawm ursonic acid los khi kom txo cov microtubules los txiav txim seb ursonic acid thiab nws cov derivatives ua ncaj qha rau microtubules thiab depolymerize lawv, los yog seb lawv qhov kev txiav txim ua rau microtubule destabilization. Tsis tas li ntawd, nyob rau hauv rooj plaub uas microtubules tsis yog lub hom phiaj ncaj qha, txheeb xyuas qhov chaw ntawm kev ua thiab molecular lub hom phiaj ntawm ursonic acid ntawm cov nroj tsuag cog yuav pab kom nkag siab ntxiv txog cov khoom ntawm cov khoom sib txuas thiab muaj peev xwm los txhim kho kev ua haujlwm ntawm herbicidal. Peb qhov kev ntsuam xyuas bioactivity tau qhia txog qhov tshwj xeeb cytotoxic peev xwm ntawm ursonic acid ntawm kev loj hlob ntawm cov nroj tsuag xws li Arabidopsis thaliana, haus luam yeeb thiab liverwort, thaum tsis muaj E. coli lossis HeLa hlwb raug cuam tshuam. Me me los yog tsis muaj tshuaj lom rau cov tsiaj hlwb yog qhov zoo ntawm ursonic acid derivatives yog tias lawv tau tsim los ua tshuaj tua kab mob rau siv hauv kev ua liaj ua teb qhib. Tseeb tiag, txij li microtubules yog cov qauv tsim hauv eukaryotes, lawv cov kev xaiv inhibition hauv cov nroj tsuag yog qhov yuav tsum tau ua rau cov tshuaj tua kab mob. Piv txwv li, propyzamide, microtubule depolymerizing tus neeg sawv cev uas ncaj qha khi rau tubulin thiab inhibits polymerization, yog siv los ua tshuaj tua kab mob vim nws tsis tshua muaj toxicity rau tsiaj hlwb24. Nyob rau hauv sib piv rau disopyramide, ntsig txog benzamides muaj cov hom phiaj sib txawv. Ntxiv rau cov nroj tsuag microtubules, RH-4032 los yog benzoxamide kuj inhibits microtubules ntawm cov tsiaj hlwb los yog oomycetes, raws li, thiab zalilamide yog siv raws li ib tug fungicide vim nws tsis tshua muaj phytotoxicity25,26,27. Tus dais uas nyuam qhuav pom thiab nws cov derivatives muaj kev xaiv cytotoxicity tawm tsam cov nroj tsuag, tab sis nws tsim nyog sau cia tias kev hloov kho ntxiv tuaj yeem hloov kho lawv lub hom phiaj tshwj xeeb, muaj peev xwm muab cov txiaj ntsig ntxiv rau kev tswj cov kab mob fungi lossis oomycetes.
Cov khoom tshwj xeeb ntawm urbenonic acid thiab nws cov derivatives muaj txiaj ntsig zoo rau lawv txoj kev loj hlob raws li cov tshuaj tua kab mob thiab siv los ua cov cuab yeej tshawb fawb. Qhov tseem ceeb ntawm cytoskeleton hauv kev tswj cov nroj tsuag cell zoo yog dav lees paub. Cov kev tshawb fawb yav dhau los tau pom tias cov nroj tsuag tau hloov kho cov txheej txheem nyuaj ntawm cortical microtubule lub koom haum los ntawm kev tswj cov microtubule dynamics kom tswj morphogenesis. Ib tug loj tus naj npawb ntawm cov molecules lub luag hauj lwm rau cov kev cai ntawm microtubule kev ua ub no tau raug txheeb xyuas, thiab lwm yam kev tshawb fawb tseem tab tom ua 3,4,28. Peb qhov kev nkag siab tam sim no ntawm microtubule dynamics nyob rau hauv cov nroj tsuag hlwb tsis tau piav qhia tag nrho cov mechanisms ntawm cortical microtubule lub koom haum. Piv txwv li, txawm hais tias ob qho tib si disopyramide thiab oryzalin tuaj yeem depolymerize microtubules, disopyramide ua rau muaj kev cuam tshuam loj heev thaum oryzalin muaj cov nyhuv me me. Tsis tas li ntawd, kev hloov pauv hauv tubulin, uas ruaj khov microtubules, kuj ua rau dextrorotation hauv cov hauv paus hniav, hos paclitaxel, uas kuj stabilizes microtubule dynamics, tsis. Yog li, kev kawm thiab txheeb xyuas cov hom phiaj molecular ntawm ursolic acid yuav tsum muab cov kev nkag siab tshiab rau hauv kev tswj hwm ntawm cov nroj tsuag cortical microtubules. Ib yam li ntawd, kev sib piv yav tom ntej ntawm cov tshuaj uas muaj txiaj ntsig zoo hauv kev txhawb nqa kev loj hlob tsis zoo, xws li disopyramide, thiab cov tshuaj tsis zoo, xws li oryzalin lossis kumamotoric acid, yuav muab cov lus qhia txog kev loj hlob cuam tshuam li cas.
Ntawm qhov tod tes, kev tiv thaiv ntsig txog cytoskeletal rearrangements yog lwm qhov ua tau los piav qhia txog cytotoxicity ntawm ursonic acid. Kev kis tus kab mob los yog kev taw qhia ntawm ib qho elicitor rau hauv cov nroj tsuag qee zaum ua rau kev puas tsuaj ntawm cytoskeleton thiab cov cell tuag tom ntej. Piv txwv li, oomycete-derived cryptoxanthin tau tshaj tawm los cuam tshuam microtubules thiab actin filaments ua ntej haus luam yeeb cell tuag, zoo ib yam li qhov tshwm sim nrog KAND kev kho mob30,31. Qhov zoo sib xws ntawm kev tiv thaiv cov lus teb thiab cov lus teb ntawm tes raug ntxias los ntawm ursonic acid ua rau peb xav tias lawv ua rau cov txheej txheem ntawm tes, txawm hais tias muaj kev cuam tshuam sai thiab muaj zog ntawm ursonic acid dua li cryptoxanthin yog qhov tseeb. Txawm li cas los xij, kev tshawb fawb tau pom tias kev cuam tshuam ntawm actin filaments txhawb kev tuag ntawm tes, uas tsis yog ib txwm nrog microtubule cuam tshuam. Tsis tas li ntawd, nws tseem yuav tsum tau pom seb puas yog cov kab mob lossis elicitor ua rau cov hauv paus hniav tsis zoo, raws li ursonic acid derivatives ua. Yog li, kev paub txog molecular txuas cov lus teb tiv thaiv thiab cytoskeleton yog qhov teeb meem txaus nyiam yuav tsum tau daws. Los ntawm kev siv lub xub ntiag ntawm cov khoom hnyav molecular uas tsis tshua muaj feem cuam tshuam nrog ursonic acid, nrog rau ntau yam ntawm cov khoom siv sib txawv nrog cov potencies sib txawv, lawv tuaj yeem muab sijhawm rau cov phiaj xwm tsis paub txog cellular mechanisms.
Ua ke, kev tshawb pom thiab kev siv cov khoom sib txuas tshiab uas hloov kho microtubule dynamics yuav muab txoj hauv kev muaj zog los hais txog cov txheej txheem molecular nyuaj hauv qab cog cog cell zoo li kev txiav txim siab. Nyob rau hauv cov ntsiab lus no, qhov tsis ntev los no tsim compound urmotonic acid, uas cuam tshuam rau microtubules thiab actin filaments thiab induces cell tuag, tej zaum yuav muab ib lub sij hawm rau decipher kev sib txuas ntawm microtubule tswj thiab lwm yam mechanisms. Yog li, tshuaj lom neeg thiab tshuaj lom neeg kev tshuaj xyuas siv urbenonic acid yuav pab peb nkag siab txog cov txheej txheem tswj hwm molecular uas tswj cov nroj tsuag cytoskeleton.
Inoculate S. werraensis MK493-CF1 rau hauv 500 mL baffled Erlenmeyer fwj uas muaj 110 mL ntawm cov noob nruab nrab muaj 2% (w/v) galactose, 2% (w/v) Essence muab tshuaj txhuam, 1% (w/v) Bacto muaj pes tsawg leeg . -soyton (Thermo Fisher Scientific, Inc.), 0.5% (w/v) pob kws extract (KOGOSTCH Co., Ltd., Nyiv), 0.2% (w/v) (NH4) 2SO4 thiab 0.2% CaCO3 hauv dej deionized. (pH 7.4 ua ntej sterilization). Cov noob kab lis kev cai tau muab tso rau ntawm lub tshuab nqus dej (180 rpm) ntawm 27 ° C rau 2 hnub. Kev cog qoob loo ntau lawm los ntawm lub xeev fermentation. Cov noob kab lis kev cai (7 ml) tau hloov mus rau hauv 500 ml K-1 lub khob uas muaj 40 g ntawm cov khoom nruab nrab uas muaj 15 g ntawm cov barley pressed (MUSO Co., Ltd., Nyiv) thiab 25 g ntawm deionized dej (pH tsis kho. ua ntej sterilization). ). Fermentation yog nqa tawm ntawm 30 ° C hauv qhov tsaus ntuj rau 14 hnub. Cov khoom fermentation tau muab rho tawm nrog 40 ml / lub raj mis EtOH thiab centrifuged (1500 g, 4 ° C, 10 min). Cov kab lis kev cai supernatant (60 ml) tau muab rho tawm nrog kev sib xyaw ntawm 10% MeOH / EtOAc. Cov txheej txheem organic tau evaporated nyob rau hauv lub siab txo kom tau ib qho residue (59.5 mg), uas tau raug HPLC nrog gradient elution (0-10 feeb: 90%) ntawm cov theem rov qab (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 hli × ntev 250 hli) H2O / CH3CN, 10-35 feeb: 90% H2O/CH3CN txog 70% H2O/CH3CN (gradient), 35–45 feeb: 90% H2O/EtOH, 45–155 feeb: 90% H2O/EtOH rau 100% EtOH (gradient (gradient), 155–200 min: 100 % EtOH) ntawm tus nqi ntws ntawm 1.5 ml / min, coumamonamide (1, 36.0 mg) raug cais raws li cov hmoov dawb 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]+ xam tus nqi: 141.0659, ntsuas tus nqi: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603–1593, 1537 cm
Columbia noob (Col-0) tau txais los ntawm Arabidopsis Biological Resource Center (ABRC) nrog kev tso cai rau kev tshawb fawb siv. Col-0 noob tau propagated thiab khaws cia nyob rau hauv peb lub chaw kuaj mob thiab siv raws li qus-hom Arabidopsis nroj tsuag. Arabidopsis noob tau nto sterilized thiab coj mus rau hauv ib nrab-lub zog Murashige thiab Skoog nruab nrab uas muaj 2% sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino) ethanesulfonic acid (MES) ( Fujifilm Wako Pure Chemical) ). ) thiab 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, ntawm 23 ° C thiab lub teeb tas li. Cov noob ntawm phs1-1 mutant tau muab los ntawm T. Hashimoto (Nara Institute of Science thiab Technology).
Cov noob ntawm hom SR-1 tau muab los ntawm T. Hashimoto (Nara Institute of Science thiab Technology) thiab siv los ua cov nroj tsuag tsiaj qus. Cov noob luam yeeb tau nto sterilized thiab tsau rau hauv dej tsis muaj menyuam rau peb hmo los txhawb kev loj hlob, ces muab tso rau hauv ib nrab-lub zog tov uas muaj 2% sucrose, 0.05% (w/v) MES, thiab 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige. thiab Skoog nruab nrab) nrog pH 5.7 thiab incubated ntawm 23 ° C nyob rau hauv lub teeb tas li.
Strain Tak-1 tau muab los ntawm T. Kohchi (Kyoto University) thiab tau siv los ua tus qauv sim chav rau kev kawm txog kev siv lub cev. Gemma tau txais los ntawm cov nroj tsuag uas tsis muaj kab mob thiab tom qab ntawd plated rau Gamborg B5 nruab nrab (Fujifilm Wako Pure Chemical) uas muaj 1% sucrose thiab 0.3% gellan gum thiab incubated ntawm 23 ° C nyob rau hauv lub teeb nruam.
Tobacco BY-2 hlwb (Nicotiana tabacum L. cv. Bright Yellow 2) tau muab los ntawm S. Hasezawa (University of Tokyo). BY-2 hlwb tau diluted 95-fold hauv kev hloov kho Linsmeier thiab Skoog nruab nrab thiab ntxiv txhua lub lim tiam nrog 2,4-dichlorophenoxyacetic acid 32 . Lub cell ncua kev kawm ntawv tau muab sib xyaw rau ntawm ib lub shaker teb ntawm 130 rpm ntawm 27 ° C hauv qhov tsaus ntuj. Ntxuav cov hlwb nrog 10 npaug ntawm qhov ntim ntawm qhov nruab nrab tshiab thiab rov ua dua hauv tib qhov nruab nrab. BY-2 transgenic cell kab stably qhia lub microtubule marker TagRFP-TUA6 los yog cov actin filament marker GFP-ABD2 nyob rau hauv lub cauliflower mosaic virus 35S txhawb tau tsim raws li piav qhia 33,34,35. Cov kab xovtooj ntawm no tuaj yeem khaws cia thiab sib dhos siv cov txheej txheem zoo ib yam li cov uas siv rau thawj BY-2 kab ntawm tes.
HeLa hlwb raug coj los ntawm Dulbecco's modified Eagle's nruab nrab (DMEM) (Lub Neej Technologies) ntxiv nrog 10% fetal bovine serum, 1.2 U / ml penicillin, thiab 1.2 μg / ml streptomycin hauv 37 ° C incubator nrog 5% CO2.
Txhua qhov kev sim piav qhia hauv phau ntawv no tau ua raws li Japanese biosafety cov cai thiab cov lus qhia.
Cov tshuaj tau yaj nyob rau hauv dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) raws li cov kev daws teeb meem thiab diluted hauv MS nruab nrab rau Arabidopsis thiab haus luam yeeb lossis Gamborg B5 nruab nrab rau liverwort. Rau cov hauv paus kev loj hlob inhibition assay, ntau tshaj 10 noob rau ib phaj tau sown on agar medium uas muaj cov qhia tebchaw los yog DMSO. Noob yog incubated nyob rau hauv ib tug loj hlob chamber rau 7 hnub. Cov noob tau yees duab thiab qhov ntev ntawm cov hauv paus hniav raug ntsuas. Rau Arabidopsis germination assay, 48 noob rau ib phaj tau sown on agar medium uas muaj 200 μM compound los yog DMSO. Arabidopsis noob tau zus nyob rau hauv ib tug loj hlob chamber thiab tus naj npawb ntawm germinated seedlings raug suav 7 hnub tom qab germination (dag). Rau kev soj ntsuam kev haus luam yeeb germination, 24 noob ib phaj tau sown rau ntawm agar nruab nrab uas muaj 200 μM KAND lossis DMSO. Cov noob luam yeeb tau cog rau hauv chav loj hlob thiab cov noob qoob loo raug suav tom qab 14 hnub. Rau qhov kev ntsuam xyuas kev loj hlob ntawm lub siab, 9 embryos los ntawm txhua lub phaj tau muab tso rau ntawm agar nruab nrab uas muaj cov ntsiab lus ntawm KAND lossis DMSO thiab incubated hauv chav loj hlob rau 14 hnub.
Siv cov noob stained nrog 5 mg / ml propidium iodide (PI) kom pom cov hauv paus meristem lub koom haum. PI cov cim tau pom los ntawm fluorescence microscopy siv TCS SPE confocal laser scanning microscope (Leica Microsystems).
Histochemical staining ntawm cov hauv paus hniav nrog β-glucuronidase (GUS) tau ua raws li cov txheej txheem piav qhia los ntawm Malami thiab Benfey36. Cov noob tau tsau rau hauv 90% acetone thaum hmo ntuj, stained nrog 0.5 mg / ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid hauv GUS tsis rau 1 teev thiab muab tso rau hauv cov tshuaj chloraldehyde hydrated. (8 g chloral hydrate, 2 ml dej thiab 1 ml glycerol) thiab pom los ntawm kev cuam tshuam sib txawv ntawm qhov sib txawv ntawm lub tshuab siv Axio Imager M1 microscope (Carl Zeiss).
Hauv paus cov ces kaum tau ntsuas ntawm 7-hnub-laus cov noob qoob loo loj hlob ntawm cov phiaj xwm vertically. Ntsuas lub kaum sab xis ntawm lub hauv paus los ntawm kev taw qhia ntawm lub ntiajteb txawj nqus vector raws li tau piav qhia hauv qib 6.
Kev npaj ntawm cortical microtubules tau pom raws li tau piav qhia, nrog kev hloov kho me me rau txoj cai 37 . Anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) thiab Alexa Fluor 488-conjugated anti-nas IgG (Thermo Fisher Scientific: A32723) tau siv los ua thawj thiab theem nrab cov tshuaj tiv thaiv ntawm 1:1000 thiab 1:100 dilutions, raws. Cov duab fluorescence tau txais los ntawm TCS SPE confocal laser scanning microscope (Leica Microsystems). Tau Z-pawg dluab thiab tsim qhov kev siv ntau tshaj plaws raws li cov chaw tsim khoom cov lus qhia.
HeLa cell proliferation assay tau ua tiav siv Cell Counting Kit 8 (Dojindo) raws li cov chaw tsim khoom cov lus qhia.
Kev loj hlob ntawm E. coli DH5α tau soj ntsuam los ntawm kev ntsuas cell ceev hauv kab lis kev cai siv lub spectrophotometer ntawm 600 nm (OD600).
Cytoskeletal lub koom haum nyob rau hauv transgenic BY-2 hlwb tau pom siv lub fluorescence microscope nruab nrog CSU-X1 confocal scanning device (Yokogawa) thiab sCMOS koob yees duab (Zyla, Andor Technology). Cytoskeletal ntom tau soj ntsuam los ntawm cov duab tsom xam, uas ntsuas qhov feem pua ntawm cytoskeletal pixels ntawm cytoplasmic pixels hauv cov duab confocal siv ImageJ software raws li tau piav qhia 38,39.
Txhawm rau txheeb xyuas cov cell tuag hauv BY-2 hlwb, ib qho aliquot ntawm lub cell ncua kev kawm tau incubated nrog 0.05% Evans xiav rau 10 feeb ntawm chav tsev kub. Selective Evans xiav staining ntawm cov hlwb tuag nyob ntawm extrusion ntawm cov dye los ntawm cov hlwb siv tau los ntawm cov ntshav plasma membrane40. Stained cells raug soj ntsuam siv lub teeb pom kev zoo (BX53, Olympus).
HeLa hlwb tau loj hlob nyob rau hauv DMEM ntxiv nrog 10% FBS nyob rau hauv ib tug humidified incubator ntawm 37 ° C thiab 5% CO2. Cells tau kho nrog 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng / ml colcemid (Gibco), los yog 100 ng / ml Nocodmaze (Sigma) rau 6 h ntawm 37 ° C. Cells raug kho nrog MetOH rau 10 min thiab tom qab ntawd nrog acetate rau 5 min ntawm chav tsev kub. Tsau hlwb raug incubated nrog β-tubulin thawj antibody (1D4A4, Proteintech: 66240-1) diluted nyob rau hauv 0.5% BSA / PBS rau 2 teev, ntxuav 3 zaug nrog TBST, thiab ces incubated nrog Alexa Fluor tshis antibody. 4881h ua. Mouse IgG (Thermo Fisher Scientific: A11001) thiab 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) diluted hauv 0.5% BSA/PBS. Tom qab ntxuav nrog TBST peb zaug, stained hlwb raug soj ntsuam ntawm Nikon Eclipse Ti-E inverted microscope. Cov duab tau raug ntes nrog lub koob yees duab txias Hamamatsu ORCA-R2 CCD siv MetaMorph software (Molecular Devices).
Post lub sij hawm: Jun-17-2024