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Kev tshawb pom thiab kev siv cov khoom ntuj tsim muaj txiaj ntsig zoo tuaj yeem pab txhim kho tib neeg lub neej. Cov tshuaj tua kab mob loj hlob ntawm cov nroj tsuag tau siv dav ua cov tshuaj tua kab los tswj cov nroj tsuag. Vim yog qhov xav tau siv ntau hom tshuaj tua kab, muaj qhov xav tau los txheeb xyuas cov tshuaj sib xyaw nrog cov txheej txheem tshiab ntawm kev ua haujlwm. Hauv kev tshawb fawb no, peb tau tshawb pom ib qho tshiab N -alkoxypyrrole compound, coumamonamide, los ntawm Streptomyces werraensis MK493-CF1 thiab tsim cov txheej txheem tsim ua tiav. Los ntawm kev ntsuam xyuas kev ua haujlwm ntawm cov kab mob, peb tau pom tias urs-monoamic acid yog ib qho tshuaj nruab nrab ntawm urs-monoamide thiab muaj peev xwmtshuaj tiv thaiv kev loj hlob ntawm cov nroj tsuagTsis tas li ntawd xwb, peb tau tsim ntau yam tshuaj urbenonic acid derivatives, suav nrog urbenyloxy derivative (UDA), uas muaj cov tshuaj tua kab mob siab yam tsis muaj kev cuam tshuam tsis zoo rau kev loj hlob ntawm HeLa hlwb. Peb kuj pom tias urmotonic acid derivatives cuam tshuam cov nroj tsuag microtubules; ntxiv rau, KAND cuam tshuam rau actin filaments thiab ua rau lub hlwb tuag; Cov teebmeem ntau yam no txawv ntawm cov tshuaj microtubule inhibitors paub thiab qhia txog cov txheej txheem tshiab ntawm kev ua haujlwm rau ursonic acid, uas sawv cev rau qhov zoo tseem ceeb hauv kev tsim cov tshuaj tua kab mob tshiab.
Kev tshawb pom thiab kev siv cov khoom ntuj tsim thiab lawv cov khoom siv yog ib txoj hauv kev los txhim kho lub neej zoo ntawm tib neeg. Cov metabolites theem ob uas tsim los ntawm cov kab mob me me, cov nroj tsuag thiab cov kab tau ua rau muaj kev vam meej loj hauv kev kho mob thiab kev ua liaj ua teb. Ntau yam tshuaj tua kab mob thiab tshuaj tiv thaiv kab mob leukemia tau tsim los ntawm cov khoom ntuj. Tsis tas li ntawd, ntau homtshuaj tua kab, cov tshuaj tua kab mob thiab cov tshuaj tua kab mob yog rho tawm los ntawm cov khoom ntuj no rau kev siv hauv kev ua liaj ua teb. Tshwj xeeb, cov tshuaj tua kab mob tswj cov nroj tsuag yog cov cuab yeej tseem ceeb rau kev ua kom cov qoob loo ntau ntxiv hauv kev ua liaj ua teb niaj hnub no, thiab ntau hom tshuaj sib xyaw twb tau siv rau kev lag luam lawm. Ntau cov txheej txheem cellular hauv cov nroj tsuag, xws li photosynthesis, amino acid metabolism, cell wall synthesis, kev tswj hwm ntawm mitosis, phytohormone signaling, lossis protein synthesis, yog suav tias yog cov hom phiaj ib txwm muaj ntawm cov tshuaj tua kab mob. Cov tshuaj uas inhibit microtubule ua haujlwm yog ib chav kawm ntawm cov tshuaj tua kab mob uas cuam tshuam rau kev loj hlob ntawm cov nroj tsuag los ntawm kev cuam tshuam rau mitotic kev tswj hwm2.
Cov Microtubules yog cov khoom ntawm cytoskeleton thiab tau khaws cia dav hauv cov hlwb eukaryotic. Lub tubulin heterodimer muaj α-tubulin thiab β-tubulin tsim cov kab linear microtubule protofilaments, nrog 13 protofilaments tsim cov qauv cylindrical. Microtubules ua ntau lub luag haujlwm hauv cov hlwb cog, suav nrog kev txiav txim siab cov duab ntawm cov hlwb, kev faib cov hlwb, thiab kev thauj mus los hauv hlwb3,4. Cov hlwb cog muaj cov microtubules hauv qab lub interphase plasma membrane, thiab cov no hu ua cortical microtubules tau xav tias yuav tswj hwm lub koom haum ntawm cellulose microfibrils los ntawm kev tswj hwm ntawm cellulose synthase complexes4,5. Cortical microtubules ntawm cov hlwb epidermal hauv paus, uas muaj nyob hauv thaj chaw ntawm kev nthuav dav sai ntawm cov hauv paus hniav, nyob rau sab nraud, thiab cellulose microfibers ua raws li cov microtubules no thiab txwv qhov kev taw qhia ntawm kev nthuav dav ntawm cov hlwb, yog li txhawb nqa anisotropic cell elongation. Yog li ntawd, microtubule ua haujlwm muaj feem cuam tshuam nrog cov qauv ntawm cov nroj tsuag. Cov amino acid hloov pauv hauv cov noob caj noob ces encoding tubulin ua rau muaj kev sib txawv ntawm cortical microtubule arrays thiab sab laug- lossis sab xis loj hlob hauv Arabidopsis 6,7. Ib yam li ntawd, kev hloov pauv hauv microtubule-cuam tshuam cov protein uas tswj hwm microtubule dynamics kuj tuaj yeem ua rau kev loj hlob ntawm cov hauv paus tsis zoo8,9,10,11,12,13. Tsis tas li ntawd, kev kho mob nrog microtubule-disrupting herbicides xws li disopyramide, tseem hu ua pretilachlor, kuj ua rau sab laug-sab laug oblique loj hlob14. Cov ntaub ntawv no qhia tau hais tias kev tswj hwm qhov tseeb ntawm microtubule ua haujlwm yog qhov tseem ceeb rau kev txiav txim siab qhov kev coj ntawm cov nroj tsuag loj hlob.
Muaj ntau hom tshuaj microtubule inhibitors tau raug tshawb pom, thiab cov tshuaj no tau ua rau muaj kev pab tseem ceeb rau kev tshawb fawb txog cytoskeletal, nrog rau kev ua liaj ua teb thiab tshuaj2. Tshwj xeeb, oryzalin, dinitroaniline compounds, disopyramide, benzamide-related compounds, thiab lawv cov analogs tuaj yeem inhibit microtubule function thiab yog li ntawd inhibit cog kev loj hlob. Yog li ntawd, lawv tau siv dav ua cov tshuaj tua kab. Txawm li cas los xij, txij li microtubules yog ib qho tseem ceeb ntawm cov nroj tsuag thiab tsiaj txhu, feem ntau microtubule inhibitors yog cytotoxic rau ob hom cell. Yog li ntawd, txawm hais tias lawv tau lees paub tias yog cov tshuaj tua kab, muaj tsawg tus neeg sawv cev antimicrotubule siv rau cov hom phiaj siv tau.
Streptomyces yog ib hom ntawm tsev neeg Streptomyces, uas suav nrog aerobic, gram-positive, filamentous bacteria thiab paub dav txog nws lub peev xwm los tsim ntau yam metabolites theem nrab. Yog li ntawd, nws suav hais tias yog ib qho ntawm cov chaw tseem ceeb tshaj plaws ntawm cov khoom tshiab biologically active. Hauv kev tshawb fawb tam sim no, peb tau tshawb pom ib qho tshuaj tshiab hu ua coumamonamide, uas tau rho tawm los ntawm Streptomyces werraensis MK493-CF1 thiab S. werraensis ISP 5486. Siv kev tshuaj xyuas spectral thiab kev tshuaj xyuas spectral tag nrho, cov qauv ntawm coumamonamide tau piav qhia thiab nws cov pob txha N-alkoxypyrrole tshwj xeeb tau txiav txim siab. synthesis. Ursmonic acid, ib qho tshuaj nruab nrab ntawm ursmonoamide thiab nws cov derivatives, tau pom tias inhibit kev loj hlob thiab germination ntawm cov qauv nrov Arabidopsis thaliana. Hauv kev tshawb fawb txog kev sib raug zoo ntawm cov qauv thiab kev ua ub no, peb pom tias ib qho tshuaj nrog C9 hloov kho rau ursonic acid, hu ua nonyloxy derivative ntawm ursonic acid (KAND), ua rau muaj kev cuam tshuam inhibitory rau kev loj hlob thiab germination. Qhov tseem ceeb, cov tshuaj tshiab uas nrhiav tau los ntawm cov nroj tsuag loj hlob kuj cuam tshuam rau kev loj hlob ntawm cov luam yeeb thiab cov nplooj siab thiab tsis yog cytotoxic rau cov kab mob lossis HeLa hlwb. Ntxiv mus, qee cov urmotonic acid derivatives ua rau muaj cov hauv paus hniav tsis zoo, uas qhia tias cov derivatives no ncaj qha lossis tsis ncaj qha cuam tshuam rau microtubules. Sib xws nrog lub tswv yim no, peb cov kev soj ntsuam ntawm microtubules uas muaj cim immunohistochemically lossis nrog cov protein fluorescent qhia tias KAND kev kho mob depolymerizes microtubules. Ntxiv rau, kev kho mob nrog kumamotonic acid derivatives cuam tshuam actin microfilaments. Yog li, peb tau tshawb pom ib qho tshuaj tshiab uas tiv thaiv kev loj hlob ntawm cov nroj tsuag uas nws txoj kev ua haujlwm tshwj xeeb cuam tshuam nrog kev rhuav tshem ntawm cytoskeleton.
Hom MK493-CF1 tau raug rho tawm ntawm cov av hauv Shinagawa-ku, Tokyo. Hom MK493-CF1 tau tsim cov mycelium stromal zoo. Cov kab ke ib nrab ntawm 16S ribosomal RNA gene (1422 bp) tau txiav txim siab. Hom no zoo ib yam li S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: hom ib txwm muaj, 99.93%). Raws li qhov tshwm sim no, nws tau txiav txim siab tias hom no muaj feem cuam tshuam nrog hom hom ntawm S. werraensis. Yog li ntawd, peb tau muab lub npe hom no rau S. werraensis MK493-CF1. S. werraensis ISP 5486T kuj tsim cov tshuaj bioactive zoo ib yam. Vim tias muaj kev tshawb fawb me ntsis thaum ntxov txog kev tau txais cov khoom ntuj tsim los ntawm cov kab mob me me no, kev tshawb fawb tshuaj ntxiv tau ua tiav. Tom qab cog S. werraensis MK493-CF1 rau ntawm cov khoom noj barley los ntawm kev fermentation khov kho ntawm 30 ° C rau 14 hnub, cov khoom noj tau rho tawm nrog 50% EtOH. 60 ml ntawm cov qauv tau qhuav kom tau txais 59.5 mg ntawm cov kua rho tawm nyoos. Cov kua rho tawm nyoos tau raug rau theem rov qab HPLC kom 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 kua rho tawm nyoos. Yog li ntawd, peb txiav txim siab los kawm txog cov khoom ntawm kumamotoamide 1.
Coumamonamide 1 yog hmoov dawb amorphous thiab high resolution mass spectrometry (HRESIMS) lees paub C6H8N2O2 (Daim Duab 1). C2-substituted pyrrole fragment ntawm cov compound no yog tus cwj pwm los ntawm 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 txog qhov muaj plaub sp2 carbon atoms. Qhov muaj ib pawg amide ntawm C2 txoj haujlwm tau soj ntsuam los ntawm HMBC correlation los ntawm C-3 proton mus rau amide carbonyl carbon ntawm δC 161.1. Ntxiv mus, 1 H thiab 13 C NMR cov ncov ntawm δH 4.10 (3H, S) thiab δC 68.3 qhia txog qhov muaj N-methoxy pawg hauv lub molecule. Txawm hais tias qhov chaw raug ntawm methoxy pawg tseem tsis tau txiav txim siab siv kev tshuaj xyuas spectroscopic xws li kev txhim kho qhov sib txawv spectroscopy thiab nuclear Overhauser abbreviation (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide tau dhau los ua thawj tus neeg sib tw compound.
Txhawm rau txiav txim siab qhov qauv tseeb ntawm 1, tag nrho cov synthesis tau ua tiav (Daim Duab 2a). Kev kho mob ntawm 2-aminopyridine 2 muaj nyob rau hauv kev lag luam nrog m-CPBA ua rau muaj N-oxide 3 sib xws hauv cov txiaj ntsig ntau. Tom qab 2-aminoazidation ntawm 2, qhov kev cuam tshuam cyclocondensation piav qhia los ntawm Abramovich tau ua tiav hauv benzene ntawm 90 ° C kom tau txais qhov xav tau 1-hydroxy-1H-pyrrole-2-carbonitrile 5 hauv grams. Ceev 60% (ob theem). 15,16. Methylation thiab hydrolysis ntawm 4 ces muab 1-methoxy-1H-pyrrole-2-carboxylic acid (hu ua "cumotonic acid", 6) hauv cov txiaj ntsig zoo (70%, ob kauj ruam). Thaum kawg, amidation ntawm acid chloride intermediate 6 siv aqueous ammonia muab Kumamoto amide 1 hauv 98% cov txiaj ntsig. 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;
Kev tsim thiab kev tshuaj xyuas dav dav ntawm cov haujlwm ntawm urbenamide thiab urbenic acid. (a) Tag nrho cov kev tsim ntawm Kumamoto amide. (b) Cov noob Arabidopsis Columbia (Col) uas muaj xya hnub tau loj hlob ntawm Murashige thiab Skoog (MS) cov phaj uas muaj coumamonamide 6 lossis coumamonamide 1 ntawm cov concentration qhia. Scale bar = 1 cm.
Ua ntej, peb tau soj ntsuam cov haujlwm ntawm urbenamide thiab nws cov intermediates rau lawv lub peev xwm los hloov cov nroj tsuag loj hlob. Peb tau ntxiv ntau yam kev sib xyaw ntawm ursmonamide 1 lossis ursmonic acid 6 rau MS agar medium thiab cog cov noob Arabidopsis thaliana ntawm cov khoom no. Cov kev sim no qhia tau hais tias cov concentration siab (500 μM) ntawm 6 inhibited kev loj hlob ntawm cov hauv paus hniav (Daim duab 2b). Tom ntej no, peb tau tsim ntau yam derivatives los ntawm kev hloov qhov chaw N1 ntawm 6 thiab ua cov qauv-kev ua ub no kev tshawb fawb txog kev sib raug zoo ntawm lawv (cov txheej txheem analogue synthesis tau piav qhia hauv Cov Ntaub Ntawv Txhawb Nqa (SI)). Arabidopsis noob tau loj hlob ntawm cov khoom uas muaj 50 μM ursonic acid derivatives, thiab qhov ntev ntawm cov hauv paus hniav tau ntsuas. raws li nws tau pom ntawm daim duab. Raws li pom hauv Cov Duab 3a, b, thiab S1, coumamo acids muaj qhov ntev sib txawv ntawm cov saw hlau alkoxy linear (9, 10, 11, 12, thiab 13) lossis cov saw hlau alkoxy loj (15, 16, thiab 17) ntawm qhov chaw N1. Cov derivatives tau qhia txog kev txwv tsis pub cov hauv paus loj hlob. Tsis tas li ntawd xwb, peb pom tias kev siv 200 μM 10, 11, lossis 17 inhibited germination (Figs. 3c thiab S2).
Kev kawm txog kev sib raug zoo ntawm cov qauv thiab kev ua ub no ntawm Kumamoto amide thiab cov tshuaj sib xyaw. (a) Cov qauv thiab cov qauv tsim ntawm cov analogues. (b) Kev ntsuas qhov ntev ntawm cov hauv paus hniav ntawm cov noob 7-hnub uas loj hlob ntawm MS medium nrog lossis tsis muaj 50 μM coumamonamide derivatives. Cov cim hnub qub qhia txog qhov sib txawv tseem ceeb nrog kev kho mob sham (t xeem, p< 0.05). n>18. Cov ntaub ntawv tau qhia ua qhov nruab nrab ± SD. nt txhais tau tias "tsis tau sim" vim tias ntau dua 50% ntawm cov noob tsis tau tawg paj. (c) Kev ntsuas qhov nrawm ntawm kev tawg paj ntawm cov noob kho uas tau muab tso rau hauv MS medium rau 7 hnub nrog lossis tsis muaj 200 μM coumamonamide thiab cov tshuaj sib xyaw. Cov cim hnub qub qhia txog qhov sib txawv tseem ceeb nrog kev kho mob sham (chi-square test). n = 96.
Qhov nthuav yog, qhov ntxiv ntawm cov saw hlau alkyl ntev dua C9 txo qhov kev ua haujlwm inhibitory, qhia tias cov tshuaj sib xyaw ua ke kumamotoic acid xav tau cov saw hlau ntawm qee qhov loj me kom pom lawv cov haujlwm biological.
Vim tias kev tshuaj xyuas kev sib raug zoo ntawm cov qauv thiab kev ua ub no qhia tau hais tias C9 tau hloov kho rau ursonic acid thiab cov nonyloxy derivative ntawm ursonic acid (tom qab no hu ua KAND 11) yog qhov zoo tshaj plaws ntawm cov nroj tsuag loj hlob inhibitor, peb tau ua qhov kev piav qhia ntxiv ntawm KAND 11. Kev kho mob ntawm Arabidopsis nrog 50 μM KAND 11 yuav luag tiv thaiv kev tawg paj, thaum cov concentration qis dua (40, 30, 20, lossis 10 μM) ntawm KAND 11 inhibited kev loj hlob ntawm cov hauv paus hniav hauv txoj kev uas nyob ntawm koob tshuaj (Daim duab 4a, b). Txhawm rau sim seb KAND 11 puas cuam tshuam rau kev muaj sia nyob ntawm cov hauv paus hniav, peb tau tshuaj xyuas cov hauv paus hniav meristem uas muaj propidium iodide (PI) thiab ntsuas qhov loj me ntawm thaj chaw meristem. Qhov loj ntawm meristem ntawm cov noob cog qoob loo uas cog rau ntawm ib qho chaw nruab nrab uas muaj 25 μM KAND-11 yog 151.1 ± 32.5 μm, thaum qhov loj ntawm meristem ntawm cov noob cog qoob loo uas cog rau ntawm ib qho chaw nruab nrab uas muaj DMSO yog 264.7 ± 30.8 μm (Daim Duab 4c, d), uas qhia tau tias KAND-11 rov qab ua haujlwm ntawm tes. kev nthuav dav. Cov hauv paus meristem. Sib xws nrog qhov no, kev kho mob KAND 11 txo qhov ntau ntawm cov cim faib tes CDKB2; 1p:: CDKB2; 1-GUS teeb liab hauv cov hauv paus meristem (Daim Duab 4e) 17. Cov txiaj ntsig no qhia tau tias KAND 11 tiv thaiv kev loj hlob ntawm cov hauv paus los ntawm kev txo cov haujlwm ntawm tes.
Kev tshuaj xyuas ntawm cov nyhuv inhibitory ntawm urbenonic acid derivatives (urbenyloxy derivatives) ntawm kev loj hlob. (a) 7-hnub-laus qus-hom Col seedlings loj hlob ntawm MS phaj nrog cov concentration qhia ntawm KAND 11. Scale bar = 1 cm. (b) Kev ntsuas ntawm cov hauv paus ntev. Cov ntawv qhia txog qhov sib txawv tseem ceeb (Tukey HSD xeem, p< 0.05). n>16. Cov ntaub ntawv tau qhia ua qhov nruab nrab ± SD. (c) Confocal microscopy ntawm propidium iodide-stained wild-type Col cag uas loj hlob ntawm MS phaj nrog lossis tsis muaj 25 μM KAND 11. Cov ntawv cim dawb qhia txog cov cag meristem. Scale bar = 100 µm. (d) Kev ntsuas qhov loj ntawm cov cag meristem (n = 10 txog 11). Kev sib txawv ntawm cov lej tau txiav txim siab siv t-test (p<0.05). Cov kab sawv cev rau qhov nruab nrab meristem loj. (e) Differential interference contrast (DIC) microscopy ntawm cov hauv paus meristem uas muaj CDKB2 construct; 1pro: CDKB2; 1-GUS stained thiab stained rau ntawm 5-hnub-laus seedlings loj hlob ntawm MS phaj nrog lossis tsis muaj 25 µM KAND assay.
Qhov phytotoxicity ntawm KAND 11 tau sim ntxiv siv lwm cov nroj tsuag dicotyledonous, luam yeeb (Nicotiana tabacum), thiab ib qho tseem ceeb ntawm cov nroj tsuag qauv av, liverwort (Marchantia polymorpha). Ib yam li Arabidopsis, luam yeeb SR-1 cov noob cog rau ntawm cov khoom nruab nrab uas muaj 25 μM KAND 11 tsim cov hauv paus luv dua (Daim duab 5a). Tsis tas li ntawd, 40 ntawm 48 noob tau tawg rau ntawm cov phaj uas muaj 200 μM KAND 11, thaum tag nrho 48 noob tau tawg rau ntawm cov khoom nruab nrab kho, qhia tias cov concentration ntawm KAND ntau dua yog qhov tseem ceeb (p<0.05; chi test -square) inhibited qhov germination ntawm cov luam yeeb. (Daim duab 5b). Ntxiv rau, qhov concentration ntawm KAND 11 uas inhibited cov kab mob loj hlob hauv liverwort zoo ib yam li qhov concentration zoo hauv Arabidopsis (Daim duab 5c). Cov txiaj ntsig no qhia tau tias KAND 11 tuaj yeem inhibit qhov kev loj hlob ntawm ntau yam nroj tsuag. Tom qab ntawd peb tshawb xyuas qhov ua tau cytotoxicity ntawm dais monoamide-cuam tshuam cov tshuaj hauv lwm yam kab mob, uas yog tib neeg HeLa hlwb thiab Escherichia coli hom DH5α, ua tus sawv cev ntawm cov tsiaj txhu thiab cov kab mob hlwb siab dua, raws li. Hauv cov koob ntawm cov cell proliferation assays, peb tau 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 concentration ntawm 100 μM (Daim duab 5d, e).
Kev txwv tsis pub loj hlob ntawm KAND 11 hauv cov kab mob uas tsis yog Arabidopsis. (a) Cov noob luam yeeb SR-1 qus uas muaj ob lub lis piam tau cog rau ntawm cov phaj MS uas muaj 25 μM KAND 11. (b) Cov noob luam yeeb SR-1 qus uas muaj ob lub lis piam tau cog rau ntawm cov phaj MS uas muaj 200 μM KAND 11. (c) Cov noob Tak-1 liverwort qus uas muaj ob lub lis piam tau cog rau ntawm cov phaj Gambog B5 nrog cov ntsiab lus ntawm KAND 11. Cov xub liab qhia txog cov noob uas tsis loj hlob hauv ob lub lis piam. (d) Kev ntsuas kev loj hlob ntawm cov hlwb HeLa. Tus naj npawb ntawm cov hlwb uas muaj sia nyob tau ntsuas ntawm lub sijhawm teem tseg siv cov khoom siv suav cov hlwb 8 (Dojindo). Ua tus tswj, cov hlwb HeLa tau kho nrog 5 μg/ml actinomycin D (Act D), uas tiv thaiv RNA polymerase transcription thiab ua rau cov hlwb tuag. Kev tshuaj xyuas tau ua tiav hauv triplicate. (e) Kev ntsuas kev loj hlob ntawm cov hlwb E. coli. Kev loj hlob ntawm E. coli tau raug soj ntsuam los ntawm kev ntsuas OD600. Ua ib qho kev tswj hwm, cov hlwb tau kho nrog 50 μg / ml ampicillin (Amp), uas tiv thaiv cov kab mob cell phab ntsa synthesis. Kev soj ntsuam tau ua tiav hauv triplicate.
Txhawm rau txiav txim siab txog cov txheej txheem ntawm kev ua haujlwm ntawm cytotoxicity los ntawm cov tshuaj uramide, peb tau rov tshuaj xyuas cov urbenic acid derivatives nrog cov teebmeem inhibitory nruab nrab. raws li nws tau pom ntawm daim duab. Raws li pom hauv Daim Duab 2b, 6a, cov noob cog qoob loo ntawm cov phaj agar uas muaj cov concentration siab (200 μM) ntawm urmotonic acid 6 tsim cov hauv paus luv luv thiab sab laug-curved (θ = - 23.7 ± 6.1), thaum cov noob cog qoob loo ntawm cov khoom tswj hwm, cov noob cog qoob loo tsim cov hauv paus ncaj nraim (θ = - 3.8 ± 7.1). Qhov kev loj hlob oblique no paub tias yog los ntawm kev ua haujlwm tsis zoo ntawm cortical microtubules14,18. Sib xws nrog qhov kev tshawb pom no, cov tshuaj microtubule-destabilizing disopyramide thiab oryzalin ua rau cov hauv paus zoo sib xws tilting nyob rau hauv peb cov xwm txheej kev loj hlob (Figs. 2b, 6a). Tib lub sijhawm, peb tau sim urmotonic acid derivatives thiab xaiv ntau ntawm lawv uas, ntawm qee qhov concentration, ua rau cov hauv paus oblique loj hlob. Cov tshuaj sib xyaw 8, 9, thiab 15 tau hloov qhov kev coj ntawm kev loj hlob ntawm cov hauv paus hniav ntawm 75 μM, 50 μM, thiab 40 μM, raws li, qhia tias cov tshuaj sib xyaw no tuaj yeem ua rau cov microtubules tsis ruaj khov (Daim duab 2b, 6a). Peb kuj tau sim cov tshuaj ursolic acid derivative uas muaj zog tshaj plaws, KAND 11, ntawm qhov concentration qis dua (15 µM) thiab pom tias kev siv KAND 11 inhibits kev loj hlob ntawm cov hauv paus hniav thiab tias qhov kev coj ntawm kev loj hlob ntawm cov hauv paus hniav tsis sib xws, txawm hais tias lawv nyiam nqes mus rau sab laug (Daim duab C3). . Vim tias cov tshuaj microtubule-destabilizing ntau dua qee zaum inhibits kev loj hlob ntawm cov nroj tsuag es tsis yog ua rau cov hauv paus hniav tilting, tom qab ntawd peb tau soj ntsuam qhov ua tau tias KAND 11 cuam tshuam rau microtubules los ntawm kev soj ntsuam cov cortical microtubules hauv cov hlwb epidermal hauv paus hniav. Immunohistochemistry siv cov tshuaj tiv thaiv anti-β-tubulin hauv cov hlwb epidermal ntawm cov hauv paus hniav seedling kho nrog 25 μM KAND 11 qhia txog kev ploj ntawm yuav luag txhua lub cortical microtubules hauv cov hlwb epidermal hauv thaj chaw elongation (Daim duab 6b). Cov txiaj ntsig no qhia tau hais tias kumamotonic acid thiab nws cov derivatives ua haujlwm ncaj qha lossis tsis ncaj qha rau microtubules los cuam tshuam lawv thiab tias cov tshuaj no yog cov tshuaj microtubule inhibitors tshiab.
Ursonic acid thiab nws cov derivatives hloov cortical microtubules hauv Arabidopsis thaliana. (a) Lub kaum sab xis ntawm cov hauv paus hniav ntsuas nyob rau hauv qhov muaj ntau yam urmotonic acid derivatives ntawm cov concentration qhia. Cov teebmeem ntawm ob lub tebchaw paub tias inhibit microtubules: disopyramide thiab oryzalin kuj tau soj ntsuam. Daim duab qhia txog tus qauv siv los ntsuas lub kaum sab xis ntawm cov hauv paus hniav. Cov cim asterisks qhia txog qhov sib txawv tseem ceeb nrog kev kho mob sham (t xeem, p< 0.05). n>19. Scale bar = 1 cm. (b) Cortical microtubules hauv epidermal hlwb hauv thaj chaw elongation. Microtubules hauv cov qus-hom Arabidopsis Col cag loj hlob ntawm MS phaj nrog lossis tsis muaj 25 μM KAND 11 tau pom los ntawm immunohistochemical staining siv β-tubulin thawj cov tshuaj tiv thaiv thiab Alexa Fluor-conjugated secondary antibodies. Scale bar = 10 µm. (c) Mitotic qauv ntawm microtubules hauv cov hauv paus meristem. Microtubules tau pom los ntawm kev siv immunohistochemical staining. Mitotic qauv, suav nrog prophase zones, spindles, thiab phragmoplasts, tau suav los ntawm confocal duab. Xub qhia txog mitotic microtubule qauv. Asterisks qhia txog qhov sib txawv tseem ceeb nrog kev kho mob sham (t xeem, p< 0.05). n>9. Qhov ntsuas ntsuas = 50 µm.
Txawm hais tias Ursa muaj peev xwm cuam tshuam kev ua haujlwm ntawm microtubule, nws txoj kev ua haujlwm yuav txawv ntawm cov neeg sawv cev microtubule depolymerizing. Piv txwv li, cov tshuaj microtubule depolymerizing ntau dua xws li disopyramide thiab oryzalin ua rau muaj kev nthuav dav anisotropic ntawm cov hlwb epidermal, thaum KAND 11 tsis ua. Tsis tas li ntawd, kev siv KAND 11 thiab disopyramide ua rau muaj kev sib xyaw ua ke ntawm disopyramide-induced root growth response thiab KAND 11-induced growth inhibition tau pom (Daim Duab S4). Peb kuj tau tshuaj xyuas cov lus teb ntawm hypersensitive disopyramide 1-1 (phs1-1) mutant rau KAND 11. phs1-1 muaj qhov tsis yog canonical tubulin kinase point mutation thiab tsim cov hauv paus luv dua thaum kho nrog disopyramide9,20. phs1-1 mutant seedlings cog rau ntawm agar medium uas muaj KAND 11 muaj cov hauv paus luv dua zoo ib yam li cov cog rau ntawm disopyramid (duab S5).
Ntxiv rau, peb tau pom cov qauv microtubule mitotic, xws li prophase zones, spindles, thiab phragmoplasts, hauv cov hauv paus hniav ntawm cov noob cog uas kho nrog KAND 11. Sib xws nrog cov kev soj ntsuam rau CDKB2;1p::CDKB2;1-GUS, qhov txo qis tseem ceeb ntawm cov lej ntawm mitotic microtubules tau pom (Daim duab .6c).
Yuav kom paub txog qhov cytotoxicity ntawm KAND 11 ntawm qhov kev daws teeb meem subcellular, peb tau kho cov hlwb haus luam yeeb BY-2 nrog KAND 11 thiab saib lawv cov lus teb. Peb thawj zaug ntxiv KAND 11 rau BY-2 hlwb qhia TagRFP-TUA6, uas fluorescently labels microtubules, los ntsuas qhov cuam tshuam ntawm KAND 11 rau cortical microtubules. Cortical microtubule density tau soj ntsuam siv kev tshuaj xyuas duab, uas ntsuas qhov feem pua ntawm cytoskeletal pixels ntawm cytoplasmic pixels. Cov txiaj ntsig assay qhia tau tias tom qab kho nrog 50 μM lossis 100 μM KAND 11 rau 1 teev, qhov ceev tau txo qis mus rau 0.94 ± 0.74% lossis 0.23 ± 0.28%, feem, thaum qhov ceev ntawm cov hlwb kho nrog DMSO, yog 1.61 ± 0.34% (Daim duab 7a). Cov txiaj ntsig no yog sib xws nrog qhov kev soj ntsuam hauv Arabidopsis tias KAND 11 kev kho mob ua rau depolymerization ntawm cortical microtubules (Daim duab 6b). Peb kuj tau tshuaj xyuas kab BY-2 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 teev txo qis actin filament ceev rau 1.20 ± 0.62% lossis 0.61 ± 0.26%, feem, thaum qhov ceev hauv DMSO-kho hlwb yog 1.69 ± 0.51% (Daim duab 2). 7b). Cov txiaj ntsig no sib txawv nrog cov teebmeem ntawm propyzamide, uas tsis cuam tshuam rau actin filaments, thiab latrunculin B, ib qho actin depolymerizer uas tsis cuam tshuam rau microtubules (SI Daim duab S6). Ntxiv mus, kev kho mob nrog coumamonamide 1, coumamonamide acid 6, lossis KAND 11 tsis cuam tshuam rau microtubules hauv HeLa hlwb (SI Daim Duab S7). Yog li, lub tshuab ua haujlwm ntawm KAND 11 ntseeg tias txawv ntawm cov paub cytoskeleton disruptors. Ntxiv mus, peb qhov kev soj ntsuam microscopic ntawm BY-2 hlwb kho nrog KAND 11 tau qhia txog qhov pib ntawm kev tuag ntawm tes thaum lub sijhawm KAND 11 kho thiab qhia tias qhov feem pua ntawm Evans xiav-stained tuag hlwb tsis nce ntxiv tom qab 30 feeb ntawm KAND 11 kho, thaum tom qab 90 feeb ntawm kev kho mob nrog 50 μM lossis 100 μM KAND, tus lej ntawm cov hlwb tuag nce mus txog 43.7% lossis 80.1%, feem (Daim Duab 7c). Thaum muab ua ke, cov ntaub ntawv no qhia tau hais tias cov tshuaj ursolic acid derivative KAND 11 tshiab yog cov nroj tsuag-tshwj xeeb cytoskeletal inhibitor nrog lub tshuab ua haujlwm tsis paub yav dhau los.
KAND cuam tshuam rau cortical microtubules, actin filaments, thiab kev muaj sia nyob ntawm cov hlwb BY-2. (a) Kev pom cov cortical microtubules hauv BY-2 hlwb nyob rau hauv qhov muaj 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 tau suav los ntawm micrographs ntawm 25 lub hlwb ywj pheej. Cov ntawv qhia txog qhov sib txawv tseem ceeb (Tukey HSD xeem, p<0.05). Scale bar = 10 µm. (b) Cortical actin filaments hauv BY-2 hlwb pom tau tias muaj 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 tau suav los ntawm micrographs ntawm 25 lub hlwb ywj pheej. Cov ntawv qhia txog qhov sib txawv tseem ceeb (Tukey HSD xeem, p<0.05). Scale bar = 10 µm. (c) Kev soj ntsuam ntawm cov hlwb BY-2 tuag los ntawm Evans xiav staining. BY-2 hlwb kho nrog KAND 11 (50 μM lossis 100 μM) lossis DMSO tau kuaj xyuas los ntawm lub tshuab microscopy ci ntsa iab. n = 3. Scale bar = 100 µm.
Kev tshawb pom thiab kev siv cov khoom tshiab ntuj tsim tau ua rau muaj kev nce qib tseem ceeb 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 cov khoom siv los ntawm cov peev txheej ntuj. Tshwj xeeb, actinomycetes paub tias muaj txiaj ntsig zoo li cov tshuaj tua kab mob rau nematodes vim lawv muaj peev xwm tsim ntau yam metabolites theem nrab xws li avermectin, cov khoom ua los ntawm ivermectin thiab bleomycin thiab nws cov derivatives, siv tshuaj ua tus neeg sawv cev anticancer21,22. Ib yam li ntawd, ntau yam tshuaj tua kab mob tau pom los ntawm actinomycetes, qee qhov twb tau siv rau kev lag luam1,23. Yog li ntawd, kev tshuaj xyuas ntawm actinomycete metabolites los cais cov khoom ntuj nrog cov haujlwm xav tau yog suav tias yog ib qho tswv yim zoo. Hauv kev tshawb fawb no, peb tau pom ib qho tshuaj tshiab, coumamonamide, los ntawm S. werraensis thiab ua tiav synthesized nws. Ursonic acid yog ib qho tshuaj nruab nrab ntawm urbenamide thiab nws cov derivatives. Nws tuaj yeem ua rau cov hauv paus hniav curling, qhia txog kev ua haujlwm tshuaj tua kab mob nruab nrab mus rau muaj zog, thiab ncaj qha lossis tsis ncaj qha ua rau cov nroj tsuag microtubules puas tsuaj. Txawm li cas los xij, cov txheej txheem ntawm kev ua haujlwm ntawm urmotonic acid yuav txawv ntawm cov tshuaj microtubule inhibitors uas twb muaj lawm, vim KAND 11 kuj cuam tshuam cov actin filaments thiab ua rau lub hlwb tuag, qhia txog cov txheej txheem tswj hwm uas urmotonic acid thiab nws cov derivatives cuam tshuam rau ntau yam qauv cytoskeletal.
Kev piav qhia ntxiv txog urbenonic acid yuav pab kom nkag siab zoo dua txog cov txheej txheem ntawm kev ua haujlwm ntawm urbenonic acid. Tshwj xeeb, lub hom phiaj tom ntej yog los soj ntsuam qhov muaj peev xwm ntawm ursonic acid los khi rau cov microtubules txo qis kom txiav txim siab seb ursonic acid thiab nws cov derivatives ua haujlwm ncaj qha rau microtubules thiab depolymerize lawv, lossis seb lawv qhov kev ua haujlwm ua rau microtubule destabilization. Tsis tas li ntawd, nyob rau hauv rooj plaub uas microtubules tsis yog lub hom phiaj ncaj qha, kev txheeb xyuas qhov chaw ntawm kev ua haujlwm thiab cov hom phiaj molecular ntawm ursonic acid ntawm cov nroj tsuag hlwb yuav pab nkag siab ntxiv txog cov khoom ntawm cov tshuaj sib txuas thiab cov hau kev los txhim kho kev ua haujlwm ntawm tshuaj ntsuab. Peb qhov kev sim bioactivity tau qhia txog lub peev xwm cytotoxic tshwj xeeb ntawm ursonic acid ntawm kev loj hlob ntawm cov nroj tsuag xws li Arabidopsis thaliana, haus luam yeeb thiab liverwort, thaum tsis yog E. coli lossis HeLa hlwb raug cuam tshuam. Me ntsis lossis tsis muaj tshuaj lom rau cov tsiaj hlwb yog qhov zoo ntawm ursonic acid derivatives yog tias lawv tau tsim los ua tshuaj ntsuab rau kev siv hauv cov teb ua liaj ua teb qhib. Tseeb tiag, txij li microtubules yog cov qauv sib xws hauv eukaryotes, lawv cov kev xaiv inhibition hauv cov nroj tsuag yog qhov tseem ceeb rau cov tshuaj ntsuab. Piv txwv li, propyzamide, ib tug microtubule depolymerizing agent uas ncaj qha khi rau tubulin thiab inhibits polymerization, yog siv ua tshuaj tua kab vim nws tsis muaj tshuaj lom rau cov tsiaj txhu cell24. Nyob rau hauv sib piv rau disopyramide, cov benzamides cuam tshuam muaj cov hom phiaj tshwj xeeb sib txawv. Ntxiv rau cov nroj tsuag microtubules, RH-4032 lossis benzoxamide kuj inhibits microtubules ntawm cov tsiaj txhu cell lossis oomycetes, feem, thiab zalilamide yog siv ua fungicide vim nws qis phytotoxicity25,26,27. Tus dais tshiab nrhiav tau thiab nws cov derivatives qhia txog kev xaiv cytotoxicity tawm tsam cov nroj tsuag, tab sis nws tsim nyog sau cia tias kev hloov kho ntxiv tuaj yeem hloov pauv lawv lub hom phiaj tshwj xeeb, tej zaum yuav muab cov derivatives ntxiv rau kev tswj hwm cov kab mob pathogenic lossis oomycetes.
Cov khoom tshwj xeeb ntawm urbenonic acid thiab nws cov derivatives yog qhov muaj txiaj ntsig zoo rau lawv txoj kev loj hlob ua cov tshuaj tua kab thiab siv ua cov cuab yeej tshawb fawb. Qhov tseem ceeb ntawm cytoskeleton hauv kev tswj cov duab ntawm cov nroj tsuag tau lees paub dav. Cov kev tshawb fawb ua ntej tau qhia tias cov nroj tsuag tau hloov pauv cov txheej txheem nyuaj ntawm cortical microtubule lub koom haum los ntawm kev tswj hwm microtubule dynamics kom tswj tau morphogenesis. Muaj ntau cov molecules uas muaj lub luag haujlwm rau kev tswj hwm ntawm microtubule kev ua ub no tau txheeb xyuas, thiab kev tshawb fawb cuam tshuam tseem tab tom ua mus3,4,28. Peb qhov kev nkag siab tam sim no ntawm microtubule dynamics hauv cov nroj tsuag cell tsis piav qhia tag nrho cov txheej txheem 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 hauv paus thaum oryzalin muaj qhov cuam tshuam me me. Ntxiv mus, kev hloov pauv hauv tubulin, uas ua rau microtubules ruaj khov, kuj ua rau dextrorotation hauv cov hauv paus, thaum paclitaxel, uas kuj ua rau microtubule dynamics ruaj khov, tsis ua. Yog li ntawd, kev kawm thiab txheeb xyuas cov hom phiaj molecular ntawm ursolic acid yuav tsum muab cov kev nkag siab tshiab rau kev tswj hwm cov microtubules cortical ntawm cov nroj tsuag. 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 muaj txiaj ntsig zoo, xws li oryzalin lossis kumamotoric acid, yuav muab cov lus qhia txog kev loj hlob tsis zoo li cas.
Ntawm qhov tod tes, kev hloov pauv cytoskeletal uas cuam tshuam nrog kev tiv thaiv yog lwm qhov ua tau los piav qhia txog cytotoxicity ntawm ursonic acid. Kev kis tus kab mob lossis kev qhia txog elicitor rau hauv cov hlwb cog qee zaum ua rau kev puas tsuaj ntawm cytoskeleton thiab tom qab ntawd cell tuag29. Piv txwv li, oomycete-derived cryptoxanthin tau tshaj tawm tias cuam tshuam microtubules thiab actin filaments ua ntej kev tuag ntawm cov hlwb haus luam yeeb, 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 cellular los ntawm ursonic acid ua rau peb xav tias lawv ua rau cov txheej txheem cellular, txawm hais tias qhov cuam tshuam sai dua thiab muaj zog ntawm ursonic acid dua li cryptoxanthin yog qhov pom tseeb. Txawm li cas los xij, kev tshawb fawb tau qhia tias kev cuam tshuam ntawm actin filaments txhawb kev tuag ntawm tes, uas tsis yog ib txwm nrog microtubule cuam tshuam29. Tsis tas li ntawd, nws tseem yuav tsum tau pom seb tus kab mob lossis elicitor ua rau kev loj hlob ntawm cov hauv paus hniav tsis zoo, zoo li ursonic acid derivatives ua. Yog li, kev paub txog molecular txuas cov lus teb tiv thaiv thiab cytoskeleton yog qhov teeb meem zoo uas yuav tsum tau hais. Los ntawm kev siv qhov muaj cov tshuaj sib xyaw ua ke uas muaj cov molecular hnyav tsawg uas cuam tshuam nrog ursonic acid, nrog rau ntau yam derivatives nrog ntau yam zog, lawv yuav muab sijhawm los tsom mus rau cov txheej txheem cellular tsis paub.
Thaum muab ua ke, kev tshawb pom thiab kev siv cov tshuaj tshiab uas hloov kho microtubule dynamics yuav muab cov txheej txheem muaj zog los daws cov txheej txheem molecular nyuaj uas yog lub hauv paus ntawm kev txiav txim siab cov duab ntawm cov cell. Hauv cov ntsiab lus no, cov tshuaj urmotonic acid uas nyuam qhuav tsim, uas cuam tshuam rau microtubules thiab actin filaments thiab ua rau cov cell tuag, yuav muab sijhawm los txiav txim siab qhov kev sib txuas ntawm kev tswj hwm microtubule thiab lwm cov txheej txheem no. Yog li, kev tshuaj xyuas tshuaj lom neeg thiab kev siv tshuaj lom neeg siv urbenonic acid yuav pab peb nkag siab txog cov txheej txheem tswj hwm molecular uas tswj cov cytoskeleton ntawm cov nroj tsuag.
Txhaj tshuaj S. werraensis MK493-CF1 rau hauv lub raj mis Erlenmeyer 500 mL uas muaj 110 mL ntawm cov noob nruab nrab uas muaj 2% (w/v) galactose, 2% (w/v) Essence paste, 1% (w/v) Bacto composition. -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 incubated rau ntawm lub rotary shaker (180 rpm) ntawm 27 ° C rau 2 hnub. Kev cog qoob loo ntawm kev fermentation khov kho. Cov noob kab lis kev cai (7 ml) tau raug xa mus rau hauv lub raj mis 500 ml K-1 uas muaj 40 g ntawm cov khoom tsim tawm uas muaj 15 g ntawm cov barley nias (MUSO Co., Ltd., Nyiv) thiab 25 g ntawm dej deionized (pH tsis tau kho ua ntej sterilization). ). Kev fermentation tau ua tiav ntawm 30 ° C hauv qhov tsaus ntuj rau 14 hnub. Cov khoom siv fermentation tau rho tawm nrog 40 ml / lub raj mis EtOH thiab centrifuged (1500 g, 4 ° C, 10 feeb). Cov kab lis kev cai supernatant (60 ml) tau rho tawm nrog kev sib xyaw ntawm 10% MeOH / EtOAc. Cov txheej organic tau evaporated nyob rau hauv lub siab txo kom tau txais cov residue (59.5 mg), uas tau raug rau HPLC nrog gradient elution (0–10 feeb: 90%) ntawm ib kem theem rov qab (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × ntev 250 mm) 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 txog 100% EtOH (gradient (gradient), 155–200 feeb: 100% EtOH) ntawm tus nqi ntws ntawm 1.5 ml/min, coumamonamide (1, 36.0 mg) tau raug rho tawm ua 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]+ tus nqi xam: 141.0659, tus nqi ntsuas: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
Cov noob Columbia (Col-0) tau txais los ntawm Arabidopsis Biological Resource Center (ABRC) nrog kev tso cai rau kev tshawb fawb siv. Cov noob Col-0 tau nthuav dav thiab tswj hwm nyob rau hauv peb cov xwm txheej hauv chav kuaj thiab siv ua cov nroj tsuag Arabidopsis qus. Cov noob Arabidopsis tau raug tua kab mob ntawm qhov chaw thiab cog rau hauv 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 and Technology).
Cov noob ntawm hom SR-1 tau muab los ntawm T. Hashimoto (Nara Institute of Science and Technology) thiab siv ua cov nroj tsuag luam yeeb qus. Cov noob luam yeeb tau raug ntxuav kom huv thiab muab tso rau hauv dej huv rau peb hmos kom txhawb kev tawg paj, tom qab ntawd muab tso rau hauv cov tshuaj ib nrab uas muaj 2% sucrose, 0.05% (w/v) MES, thiab 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige. thiab Skoog medium) nrog pH 5.7 thiab muab tso rau ntawm 23°C hauv qab lub teeb tas li.
Hom kab mob Tak-1 tau muab los ntawm T. Kohchi (Kyoto University) thiab tau siv ua chav kuaj sim rau kev kawm txog liverwort. Gemma tau txais los ntawm cov nroj tsuag uas tau sterilized thiab tom qab ntawd plated rau ntawm Gamborgic B5 medium (Fujifilm Wako Pure Chemical) uas muaj 1% sucrose thiab 0.3% gellan gum thiab incubated ntawm 23 ° C nyob rau hauv lub teeb tas mus li.
Cov hlwb luam yeeb BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) tau muab los ntawm S. Hasezawa (University of Tokyo). Cov hlwb BY-2 tau diluted 95-fold hauv Linsmeier thiab Skoog nruab nrab hloov kho thiab ntxiv txhua lub lim tiam nrog 2,4-dichlorophenoxyacetic acid 32. Cov cell ncua tau sib xyaw rau ntawm lub shaker rotary ntawm 130 rpm ntawm 27 ° C hauv qhov tsaus ntuj. Ntxuav cov hlwb nrog 10 npaug ntawm qhov ntim ntawm cov khoom nruab nrab tshiab thiab rov ua dua hauv tib qho nruab nrab. BY-2 transgenic cell kab ruaj khov qhia txog microtubule marker TagRFP-TUA6 lossis actin filament marker GFP-ABD2 nyob rau hauv cauliflower mosaic virus 35S promoter tau tsim raws li tau piav qhia 33,34,35. Cov kab cell no tuaj yeem tswj hwm thiab sib dhos siv cov txheej txheem zoo ib yam li cov uas siv rau kab BY-2 cell thawj.
Cov hlwb HeLa tau cog qoob loo hauv Dulbecco's modified Eagle's medium (DMEM) (Life 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 uas tau piav qhia hauv daim ntawv sau no tau ua raws li cov cai thiab cov lus qhia txog kev nyab xeeb ntawm Nyiv Pooj.
Cov tshuaj tau yaj rau hauv dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) ua cov tshuaj thiab diluted hauv MS medium rau Arabidopsis thiab haus luam yeeb lossis Gambog B5 medium rau liverwort. Rau qhov kev ntsuam xyuas kev loj hlob ntawm cov hauv paus hniav, ntau tshaj 10 noob ib lub phaj tau cog rau ntawm agar medium uas muaj cov tshuaj qhia lossis DMSO. Cov noob tau incubated hauv chav loj hlob rau 7 hnub. Cov noob tau thaij duab thiab ntsuas qhov ntev ntawm cov hauv paus hniav. Rau Arabidopsis germination assay, 48 noob ib lub phaj tau cog rau ntawm agar medium uas muaj 200 μM compound lossis DMSO. Arabidopsis noob tau cog rau hauv chav loj hlob thiab tus lej ntawm cov noob germinated tau suav 7 hnub tom qab germination (dag). Rau qhov kev ntsuam xyuas kev loj hlob ntawm cov luam yeeb, 24 noob ib lub phaj tau cog rau ntawm agar medium uas muaj 200 μM KAND lossis DMSO. Cov noob luam yeeb tau cog rau hauv chav loj hlob thiab tus lej ntawm cov noob germinated tau suav tom qab 14 hnub. Rau qhov kev ntsuam xyuas kev loj hlob ntawm cov kab mob siab, 9 lub qe menyuam los ntawm txhua lub phaj tau muab tso rau ntawm agar medium uas muaj cov concentration ntawm KAND lossis DMSO thiab muab tso rau hauv chav loj hlob rau 14 hnub.
Siv cov noob uas muaj 5 mg/ml propidium iodide (PI) uas pleev xim rau kom pom tau qhov sib txawv ntawm cov cag. Cov cim PI tau pom los ntawm kev siv lub tshuab tsom iav fluorescence siv lub tshuab tsom iav TCS SPE confocal laser scanning microscope (Leica Microsystems).
Kev siv β-glucuronidase (GUS) los pleev xim rau cov cag raws li Malami thiab Benfey36 tau piav qhia. Cov noob tau muab tso rau hauv 90% acetone ib hmos, pleev xim rau nrog 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid hauv GUS buffer rau 1 teev thiab muab tso rau hauv cov kua chloraldehyde hydrated. (8 g chloral hydrate, 2 ml dej thiab 1 ml glycerol) thiab pom los ntawm kev siv lub tshuab tsom iav sib txawv siv lub tshuab tsom iav Axio Imager M1 (Carl Zeiss).
Cov kaum ntawm cov hauv paus hniav raug ntsuas rau ntawm cov noob uas muaj 7 hnub uas cog rau ntawm cov phaj uas muab tso rau ntsug. Ntsuas lub kaum ntawm cov hauv paus hniav los ntawm qhov kev taw qhia ntawm lub zog gravity raws li tau piav qhia hauv kauj ruam 6.
Qhov kev npaj ntawm cortical microtubules tau pom raws li tau piav qhia, nrog rau kev hloov kho me me rau cov txheej txheem 37. Anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) thiab Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) tau siv ua cov tshuaj tiv thaiv thawj zaug thiab theem ob ntawm 1: 1000 thiab 1: 100 dilutions, feem. Cov duab fluorescence tau txais los ntawm kev siv lub tshuab microscope TCS SPE confocal laser scanning (Leica Microsystems). Tau txais cov duab Z-stack thiab tsim cov duab siab tshaj plaws raws li cov lus qhia ntawm cov chaw tsim khoom.
Kev ntsuam xyuas HeLa cell proliferation tau ua tiav los ntawm kev siv Cell Counting Kit 8 (Dojindo) raws li cov lus qhia ntawm cov chaw tsim khoom.
Kev loj hlob ntawm E. coli DH5α tau raug soj ntsuam los ntawm kev ntsuas qhov ceev ntawm cov cell hauv kev cog qoob loo siv lub spectrophotometer ntawm 600 nm (OD600).
Kev teeb tsa Cytoskeletal hauv cov hlwb transgenic BY-2 tau pom siv lub tshuab microscope fluorescence uas muaj lub cuab yeej CSU-X1 confocal scanning (Yokogawa) thiab lub koob yees duab sCMOS (Zyla, Andor Technology). Cytoskeletal density tau soj ntsuam los ntawm kev tshuaj xyuas duab, uas ntsuas qhov feem pua ntawm cov cytoskeletal pixels ntawm cov cytoplasmic pixels hauv cov duab confocal siv ImageJ software raws li tau piav qhia 38,39.
Yuav kom pom cov cell tuag hauv BY-2 cell, ib qho aliquot ntawm cov cell suspension tau incubated nrog 0.05% Evans xiav rau 10 feeb ntawm chav tsev kub. Selective Evans xiav staining ntawm cov cell tuag nyob ntawm extrusion ntawm cov xim los ntawm cov cell ciaj sia los ntawm cov plasma membrane intact40. Cov cell stained tau pom siv lub tshuab microscope ci ntsa iab (BX53, Olympus).
Cov hlwb HeLa tau loj hlob hauv DMEM ntxiv nrog 10% FBS hauv lub tshuab ua kom noo noo ntawm 37 ° C thiab 5% CO2. Cov hlwb tau kho nrog 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng / ml colcemid (Gibco), lossis 100 ng / ml Nocodmaze (Sigma) rau 6 teev ntawm 37 ° C. Cov hlwb tau kho nrog MetOH rau 10 feeb thiab tom qab ntawd nrog acetate rau 5 feeb ntawm chav tsev kub. Cov hlwb kho tau incubated nrog β-tubulin thawj antibody (1D4A4, Proteintech: 66240-1) diluted hauv 0.5% BSA / PBS rau 2 teev, ntxuav 3 zaug nrog TBST, thiab tom qab ntawd incubated nrog Alexa Fluor tshis antibody. 488 1 teev. – Nas 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, cov hlwb stained tau pom ntawm Nikon Eclipse Ti-E inverted microscope. Cov duab tau raug ntes nrog lub koob yees duab Hamamatsu ORCA-R2 CCD txias siv MetaMorph software (Molecular Devices).
Lub sijhawm tshaj tawm: Lub Rau Hli-17-2024