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Nchọpụta na uru bara uru nke ngwaahịa eke nwere ike inye aka melite ndụ mmadụ.A na-eji kemịkalụ na-egbochi uto osisi eme ihe dị ka ahịhịa ahịhịa iji chịkwaa ahịhịa.N'ihi mkpa iji ụdị herbicides dị iche iche, ọ dị mkpa iji chọpụta ogige ndị nwere usoro ọhụrụ nke ime ihe.N'ime ọmụmụ ihe a, anyị chọpụtara akwụkwọ N-alkoxypyrrole compound, coumamonamide, sitere na Streptomyces werraensis MK493-CF1 wee guzobe usoro nhazi zuru oke.Site na nyocha ihe omume ndu, anyị chọpụtara na urs-monoamic acid bụ etiti sịntetịt nke urs-monoamide yana ike.ihe mgbochi ihe ọkụkụ.Na mgbakwunye, anyị emepụtala ụdị urbenonic acid dị iche iche, gụnyere urbenyloxy derivative (UDA), nke nwere ọrụ herbicidal dị elu na-enweghị emetụta uto nke mkpụrụ ndụ HeLa.Anyị chọpụtakwara na ihe ndị na-emepụta urmotonic acid na-akpaghasị microtubules osisi;na mgbakwunye, KAND na-emetụta actin filaments ma na-ebute ọnwụ cell;Mmetụta ndị a dị iche iche dị iche na nke ndị na-emechi microtubule a ma ama ma na-atụ aro usoro ọhụrụ nke ime ihe maka ursonic acid, nke na-anọchite anya uru dị mkpa na mmepe nke herbicides ọhụrụ.
Nchọpụta na ngwa bara uru nke ngwaahịa sitere n'okike na usoro ha bụ ụzọ isi melite ogo ndụ mmadụ.Metabolites nke abụọ na-emepụta site na microorganisms, osisi na ụmụ ahụhụ ebutela nnukwu ọganihu na nkà mmụta ọgwụ na ọrụ ugbo.Ọtụtụ ọgwụ nje na ọgwụ na-egbochi ọrịa leukemia sitere na ngwaahịa eke emepụtara.Ọzọkwa, ụdị dị iche icheọgwụ nje, fungicides na herbicides na-esi na ngwaahịa ndị a sitere n'okike maka iji na-arụ ọrụ ugbo.Karịsịa, ọgwụ mgbochi ahịhịa bụ ngwá ọrụ dị mkpa maka ịba ụba ihe ọkụkụ na ọrụ ugbo nke oge a, na-eji ụdị ogige dị iche iche eme ihe n'ahịa.Ọtụtụ usoro cellular na osisi, dị ka photosynthesis, amino acid metabolism, cell cell synthesis, regulation of mitosis, phytohormone signaling, or protein synthesis, ka a na-ewere dị ka ihe mgbaru ọsọ nke herbicides.Ngwakọta ndị na-egbochi ọrụ microtubule bụ otu klas nke ahịhịa ahịhịa na-emetụta uto osisi site na-emetụta ụkpụrụ mitotic2.
Microtubules bụ akụkụ nke cytoskeleton ma echekwara ya na sel eukaryotic.The tubulin heterodimer nwere α-tubulin na β-tubulin na-akpụ protofilaments microtubule linear, yana protofilaments 13 na-eme usoro cylindrical.Microtubules na-arụ ọtụtụ ọrụ na mkpụrụ ndụ ihe ọkụkụ, gụnyere ikpebi ụdị cell, nkewa sel, na njem intracellular3,4.Mkpụrụ ndụ osisi nwere microtubules n'okpuru interphase plasma membrane, na ihe ndị a na-akpọ cortical microtubules na-eche na-achịkwa nhazi nke cellulose microfibrils site na nhazi nke cellulose synthase complexes4,5.Cortical microtubules nke mgbọrọgwụ epidermal cell, dị na mpaghara nke ngwa ngwa elongation nke mgbọrọgwụ n'ọnụ, dị n'akụkụ, na cellulose microfibers na-eso ndị a microtubules na-amachi ntụziaka nke cell mgbasa, si otú na-akwalite anisotropic cell elongation.Ya mere, ọrụ microtubule nwere njikọ chiri anya na morphology osisi.Mgbanwe Amino acid na mkpụrụ ndụ ihe nketa na-eme ka tubulin na-eme ka skew nke cortical microtubule arrays na uto akụkụ aka ekpe ma ọ bụ aka nri na Arabidopsis 6,7.N'otu aka ahụ, ngbanwe dị na protein ndị metụtara microtubule nke na-achịkwa microtubule dynamics nwekwara ike ime ka mgbọrọgwụ gbagọrọ agbagọ8,9,10,11,12,13.Na mgbakwunye, ọgwụgwọ na microtubule na-akpaghasị ahịhịa ahịhịa dị ka disopyramide, nke a makwaara dị ka pretilachlor, na-ebutekwa mgbọrọgwụ oblique nke akụkụ aka ekpe14.Ihe data ndị a na-egosi na nhazi nke ọma nke ọrụ microtubule dị oke mkpa maka ikpebi ntụziaka nke uto osisi.
Achọpụtala ụdị ihe mgbochi microtubule dị iche iche, ọgwụ ndị a enyewokwa nnukwu onyinye na nyocha cytoskeletal, yana ọrụ ugbo na ọgwụ2.Karịsịa, oryzalin, ogige dinitroaniline, disopyramide, ogige ndị metụtara benzamide, na analogs ha nwere ike igbochi ọrụ microtubule ma si otú ahụ gbochie uto osisi.Ya mere, a na-eji ha eme ihe dị ka ahịhịa ahịhịa.Otú ọ dị, ebe ọ bụ na microtubules bụ akụkụ dị mkpa nke mkpụrụ ndụ osisi na anụmanụ, ọtụtụ ndị na-emechi microtubule bụ cytotoxic maka ụdị sel abụọ ahụ.Ya mere, n'agbanyeghị na ha ghọtara uru dị ka herbicides, a na-eji ọnụ ọgụgụ dị nta nke antimicrotubule eme ihe maka nzube bara uru.
Streptomyces bụ genus nke ezinụlọ Streptomyces, nke gụnyere aerobic, gram-positive, bacteria filamentous na nke a maara nke ọma maka ikike ya ịmepụta ọtụtụ metabolites nke abụọ.Ya mere, a na-ewere ya dị ka otu n'ime ebe kachasị mkpa nke ngwaahịa okike ọhụrụ na-arụ ọrụ ndụ.N'ime ọmụmụ ihe ugbu a, anyị chọpụtara ihe ọhụrụ a na-akpọ coumamonamide, bụ nke dịpụrụ adịpụ na Streptomyces werraensis MK493-CF1 na S. werraensis ISP 5486. N'iji nyocha nke anya na nyocha zuru oke, a na-eji usoro nke coumamonamide mara ya na ọkpụkpụ N-alkoxypyrrole pụrụ iche. e kpebisiri ike.njikọ.Achọpụtara Ursmonic acid, ihe etiti sịntetik nke ursmonoamide na ihe ndị sitere na ya, na-egbochi uto na mpụta nke osisi Arabidopsis thaliana na-ewu ewu.N'ime ọmụmụ ihe omume-mmekọrịta ihe, anyị chọpụtara na ngwakọta nwere C9 gbanwere ka ọ bụrụ ursonic acid, nke a na-akpọ nonyloxy derivative nke ursonic acid (KAND), na-eme ka mmetụta mgbochi na uto na germination dịkwuo elu.N'ụzọ doro anya, onye na-egbochi ihe ọkụkụ nke achọpụtara ọhụrụ metụtakwara uto ụtaba na liverwort na ọ bụghị cytotoxic nye nje bacteria ma ọ bụ mkpụrụ ndụ HeLa.Ọzọkwa, ụfọdụ ụdị urmotonic acid na-ebute phenotype mgbọrọgwụ gbagọrọ agbagọ, na-egosi na usoro ndị a na-emetụta microtubules ozugbo ma ọ bụ na-apụtaghị ìhè.N'ikwekọ n'echiche a, ihe anyị chọpụtara banyere microtubules nke akpọrọ ma ọ bụ immunohistochemically ma ọ bụ na protein fluorescent na-egosi na ọgwụgwọ KAND na-ewepụ microtubules.Na mgbakwunye, ọgwụgwọ nke kumamotonic acid na-akpaghasị microfilaments actin.Ya mere, anyị achọpụtala ihe na-egbochi ihe ọkụkụ ọhụrụ nke usoro omume pụrụ iche gụnyere mbibi nke cytoskeleton.
Strain MK493-CF1 dịpụrụ adịpụ na ala dị na Shinagawa-ku, Tokyo.Strain MK493-CF1 kpụrụ mycelium stromal nwere alaka nke ọma.Ekpebiri usoro akụkụ nke 16S ribosomal RNA gene (1422 bp).Udiri a yiri S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: ụdị ụdị, 99.93%).Dabere na nsonaazụ a, e kpebisiri ike na eriri a nwere njikọ chiri anya na ụdị ụdị S. werraensis.Ya mere, anyị nwa oge kpọrọ ụdị a S. werraensis MK493-CF1.S. werraensis ISP 5486T na-emepụtakwa otu ogige bioactive.Ebe ọ bụ na ọ dị ntakịrị nyocha mbụ iji nweta ngwaahịa sitere na microorganism a, e mere nyocha nke kemịkal ọzọ.Mgbe cultivation nke S. werraensis MK493-CF1 na ọka bali ọkara site siri ike-ala gbaa ụka na 30 Celsius C maka 14 ụbọchị, na-ajụ e amịpụtara na 50% EtOH.Akpọkọrọ 60 ml nke ihe nlele iji nweta 59.5 mg nke mpụta crude.A na-edobe ihe mkpofu ahụ na-atụgharị oge HPLC iji nye N-methoxy-1H-pyrrole-2-carboxamide (1, aha ya bụ coumamonamide, 36.0 mg).Ngụkọta ego nke 1 bụ ihe dịka 60% nke mpụta crude.Ya mere, anyị kpebiri na-amụ n'ụzọ zuru ezu Njirimara nke kumamotoamide 1.
Coumamonamide 1 bụ ntụ ntụ amorphous na-acha ọcha na nnukwu spectrometry mass spectrometry (HRESIM) na-akwado C6H8N2O2 (Fig 1).A na-eji mpempe pyrrole nke C2 dochie anya δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH na 1H NMR ụdịdị: 4.5 Hz). , H-5) na δH 6.78 (1H, d, J = 2.5 Hz, H-6), na 13C NMR ụdịdị na-egosi ọnụnọ nke anọ sp2 carbon atom.Achọpụtara ọnụnọ nke otu amide na ọnọdụ C2 site na njikọ HMBC sitere na proton C-3 gaa na amide carbonyl carbon na δC 161.1.Na mgbakwunye, 1 H na 13 C NMR kacha elu na δH 4.10 (3H, S) na δC 68.3 na-egosi ọnụnọ nke otu N-methoxy na molekul.Ọ bụ ezie na ekpebibeghị ọnọdụ ziri ezi nke otu methoxy site na iji nyocha spectroscopic dị ka mmụba dị iche iche spectroscopy na nbibi nke nuklia (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide ghọrọ onyinye izizi mbụ.
Iji chọpụta nhazi ziri ezi nke 1, a na-eme nchịkọta mkpokọta (Fig 2a).Ọgwụgwọ nke 2-aminopyridine 2 dị na azụmaahịa nwere m-CPBA butere N-oxide 3 kwekọrọ na nha nha.Mgbe 2-aminoazidation nke 2 gasịrị, mmeghachi omume cyclocondensation kọwara Abramovich mere na benzene na 90 Celsius iji nweta 1-hydroxy-1H-pyrrole-2-carbonitrile 5 chọrọ na gram.Ọsọ ọsọ 60% (nkeji abụọ).15,16.Methylation na hydrolysis nke 4 wee nye 1-methoxy-1H-pyrrole-2-carboxylic acid (a na-akpọ "cumotonic acid", 6) na mkpụrụ dị mma (70%, nzọụkwụ abụọ).N'ikpeazụ, amidation via acid chloride intermediate 6 iji aqueous amonia nyere Kumamoto amide 1 na 98% mkpụrụ.All spectral data nke synthesized 1 yiri 1 dịpụrụ adịpụ, ya mere a na-ekpebi nhazi nke 1;
Nchịkọta na nyocha nke ọrụ ndu nke urbenamide na urbenic acid.(a) Ngụkọta mkpokọta Kumamoto amide.(b) Ụdị Arabidopsis Columbia (Col) osisi nke dị ụbọchị asaa ka a na-akụ na efere Murashige na Skoog (MS) nke nwere coumamonamide 6 ma ọ bụ coumamonamide 1 na mkpokọta egosiri.Ogwe ọnụ ọgụgụ = 1 cm.
Nke mbụ, anyị tụlere ihe omume ndu nke urbenamide na ndị nọ n'etiti ya maka ikike ha nwere imeziwanye ihe ọkụkụ.Anyị gbakwunyere ọkwa dị iche iche nke ursmonamide 1 ma ọ bụ ursmonic acid 6 na MS agar medium na omenala Arabidopsis thaliana seedlings na nke a.Nnwale ndị a gosipụtara na nnukwu mkpokọta (500 μM) nke 6 na-egbochi mgbọrọgwụ mgbọrọgwụ (Fig 2b).Ọzọ, anyị mepụtara usoro dị iche iche site n'ịgbanwe ọnọdụ N1 nke 6 ma rụọ ọrụ nhazi-mmekọrịta mmekọrịta na ha (a na-akọwa usoro nchịkọta analog na Ozi Nkwado (SI)).A na-akụ mkpụrụ osisi Arabidopsis n'obere ihe nwere 50 μM ursonic acid, wee tụọ ogologo mgbọrọgwụ.dị ka e gosiri na foto a.Dị ka e gosiri na 3a, b, na S1, coumamo acids nwere ogologo dị iche iche nke linear alkoxy chains (9, 10, 11, 12, and 13) ma ọ bụ nnukwu alkoxy chains (15, 16, na 17) na N1 ọnọdụ.Mwepụta ndị ahụ gosipụtara mgbochi dị ukwuu nke uto mgbọrọgwụ.Na mgbakwunye, anyị chọpụtara na ngwa nke 200 μM 10, 11, ma ọ bụ 17 gbochiri germination (Fig 3c na S2).
Ọmụmụ nke nhazi-mmekọrịta ọrụ nke Kumamoto amide na ogige ndị metụtara ya.(a) Ọdịdị na nhazi atụmatụ nke analogues.(b) Nhazi ogologo mgbọrọgwụ nke mkpụrụ osisi dị ụbọchị asaa toro na MS ọkara nwere ma ọ bụ na-enweghị 50 μM coumamonamide.Asterisks na-egosi ọdịiche dị ukwuu na ọgwụgwọ sham (t test, p0.05).n>18. E gosipụtara data dị ka ± SD pụtara.nt pụtara "anaghị anwale" n'ihi na ihe karịrị 50% nke mkpụrụ osisi ahụ etolitebeghị.(c) Ọnụ ọgụgụ nke germination nke mkpụrụ osisi a na-agwọ agbakwunyere maka ụbọchị 7 na MS ọkara ma ọ bụ na-enweghị 200 μM coumamonamide na ogige ndị metụtara ya.Asterisks na-egosi ọdịiche dị ukwuu na ọgwụgwọ sham (nnwale chi-square).n=96.
N'ụzọ na-akpali mmasị, mgbakwunye nke agbụ akụkụ alkyl dị ogologo karịa C9 belatara ọrụ mgbochi, na-atụ aro na ogige ndị metụtara kumamotoic acid chọrọ agbụ akụkụ nke otu nha iji gosipụta ọrụ ndu ha.
N'ihi na nhazi-mmekọrịta mmekọrịta na-egosi na C9 gbanwere ka ọ bụrụ ursonic acid na nonyloxy derivative nke ursonic acid (nke a na-akpọ KAND 11) bụ ihe na-eme ka ihe ọkụkụ na-eto eto kacha dị irè, anyị duziri nkọwa zuru ezu nke KAND 11. Ọgwụgwọ Arabidopsis. na 50 μM KAND 11 fọrọ nke nta ka ọ bụrụ na ọ na-egbochi ịmalite ịmị mkpụrụ, ebe obere mkpokọta (40, 30, 20, ma ọ bụ 10 μM) nke KAND 11 gbochiri mgbọrọgwụ mgbọrọgwụ n'ụzọ dabere na dose (Fig. 4a, b).Iji nwalee ma KAND 11 ọ na-emetụta mgbọrọgwụ meristem viability, anyị nyochara mgbọrọgwụ meristems nwere propidium iodide (PI) wee tụọ nha mpaghara meristem.Ogo nke meristem nke mkpụrụ osisi toro na ọkara nwere 25 μM KAND-11 bụ 151.1 ± 32.5 μm, ebe nha nke meristem nke seedlings toro na usoro nchịkwa nwere DMSO bụ 264.7 ± 30.8 μm (Fig 4c, d) , nke na-egosi na KAND-11 na-eweghachi ọrụ cellular.na-agbasa.Mgbọrọgwụ Meristem.N'ikwekọ na nke a, ọgwụgwọ KAND 11 belatara ọnụ ọgụgụ nke akara nkewa cell CDKB2; 1p :: CDKB2; 1-GUS mgbaàmà na mgbọrọgwụ meristem (Fig 4e) 17.Nsonaazụ ndị a na-egosi na KAND 11 na-egbochi uto mgbọrọgwụ site n'ibelata ọrụ mmụba cell.
Nyocha nke mmetụta mgbochi nke urbenonic acid ewepụtara (urbenyloxy derivatives) na uto.(a) Ụdị Col seedlings nke dị ụbọchị 7 toro na efere MS nwere ihe ngosi egosipụtara nke KAND 11. Ogwe ọnụ ọgụgụ = 1 cm.(b) Nkọwa ogologo mgbọrọgwụ.Akwụkwọ ozi na-egosi nnukwu ọdịiche (Tukey HSD ule, p0.05).n>16. E gosipụtara data dị ka ± SD pụtara.(c) Confocal microscopy nke propidium iodide-ụbụrụ ụdị Col mgbọrọgwụ toro na efere MS nwere ma ọ bụ na-enweghị 25 μM KAND 11. Brackets ọcha na-egosi mgbọrọgwụ meristem.Ogwe ọnụ ọgụgụ = 100µm.(d) Ntụle nke mgbọrọgwụ meristem nha (n = 10 ruo 11).Ekpebiri ndịiche ọnụ ọgụgụ site na iji t-ule (p0.05).Ogwe ndị ahụ na-anọchi anya nha merisem nkezi.(e) Ntụgharị nnyonye anya dị iche (DIC) microscopy nke mgbọrọgwụ meristem nwere ihe nrụpụta CDKB2;1pro: CDKB2;1-GUS nwere ntụpọ na ntụpọ na mkpụrụ osisi dị ụbọchị 5 toro na efere MS nwere ma ọ bụ na-enweghị 25 µM KAND assay.
A nwalere phytotoxicity nke KAND 11 site na iji osisi dicotyledonous ọzọ, ụtaba (Nicotiana tabacum), na ụdị ụdị ihe ọkụkụ bụ isi ala, liverwort (Marchantia polymorpha).Dị ka ọ dị n'ihe banyere Arabidopsis, ụtaba SR-1 na-eto eto na ọkara nwere 25 μM KAND 11 mepụtara mgbọrọgwụ mkpụmkpụ (Fig 5a).Na mgbakwunye, 40 n'ime mkpụrụ 48 bilitere na efere nwere 200 μM KAND 11, ebe mkpụrụ 48 niile puru na mgbasa ozi a na-akwa emo, na-egosi na mkpokọta KAND dị elu dị ịrịba ama (p.<0.05;chi test -square) gbochiri germination nke ụtaba.(Fig 5b).Tụkwasị na nke ahụ, ntinye nke KAND 11 nke na-egbochi nje bacteria na liverwort yiri ntinye dị irè na Arabidopsis (Fig 5c).Nsonaazụ ndị a na-egosi na KAND 11 nwere ike igbochi uto nke osisi dị iche iche.Anyị wee nyochara cytotoxicity nke ihe jikọrọ anụ ọhịa bea monoamide n'ime ihe ndị ọzọ, ya bụ mkpụrụ ndụ HeLa mmadụ na Escherichia coli strain DH5a, dị ka ndị nnọchianya nke sel anụmanụ dị elu na nje nje, n'otu n'otu.N'ime usoro nyocha nke mmụba cell, anyị hụrụ na coumamonamide 1, coumamonamidic acid 6, na KAND 11 emetụtaghị uto nke mkpụrụ ndụ HeLa ma ọ bụ E. coli na ntinye nke 100 μM (Fig. 5d, e).
Mgbochi uto nke KAND 11 n'ime ihe ndị na-abụghị Arabidopsis.(a) Osisi ụtaba SR-1 dị izu abụọ ka a na-akụ n'elu efere MS kwụ ọtọ nwere 25 μM KAND 11. Mpempe MS nwere 200 μM KAND 11. (c) Ụdị anụ ọhịa Tak-1 liverwort dị izu abụọ toro na efere Gamborg B5 na-egosi mkpokọta KAND 11. Àkụ na-acha uhie uhie na-egosi spores nke kwụsịrị ịmalite n'ime izu abụọ. oge.(d) Nnwale mmụba mkpụrụ ndụ nke mkpụrụ ndụ HeLa.A tụrụ ọnụọgụ mkpụrụ ndụ enwere ike n'oge oge a kapịrị ọnụ site na iji ngwa ọnụ ọgụgụ cell 8 (Dojindo).Dị ka njikwa, a na-emeso mkpụrụ ndụ HeLa na 5 μg/ml actinomycin D (Act D), nke na-egbochi nsụgharị RNA polymerase ma na-akpata ọnwụ cell.Emere nyocha n'ụzọ atọ.(e) E. coli cell proliferation assay.A tụlere uto E. coli site na nha OD600.Dị ka njikwa, a na-eji 50 μg / ml ampicillin (Amp) na-agwọ mkpụrụ ndụ, nke na-egbochi njikọ mgbidi cell.Emere nyocha n'ụzọ atọ.
Iji ghọta usoro ọrụ nke cytotoxicity kpatara site na ogige metụtara uramide, anyị jiri mmetụta mgbochi na-agafeghị oke nyochaa usoro urbenic acid.dị ka e gosiri na foto a.Dị ka e gosiri na ọnụ ọgụgụ 2b, 6a, mkpụrụ osisi toro na efere agar nke nwere ihe dị elu (200 μM) nke urmotonic acid 6 na-emepụta mgbọrọgwụ mkpụmkpụ na nke aka ekpe (θ = - 23.7 ± 6.1), ebe mkpụrụ osisi toro na nchịkwa nchịkwa, mkpụrụ osisi na-emepụta ihe fọrọ nke nta ka ọ bụrụ mgbọrọgwụ ogologo (θ = - 3.8 ± 7.1).Amara njirimara a na-eto eto na-esi na arụ ọrụ nke microtubules cortical14,18.N'ikwekọ na nchọpụta a, microtubule-destabilizing ọgwụ ọjọọ disopyramide na oryzalin mere ka mgbọrọgwụ gbanye mgbọrọgwụ n'okpuru ọnọdụ uto anyị (Figs 2b, 6a).N'otu oge ahụ, anyị nwalere usoro urmotonic acid wee họrọ ọtụtụ n'ime ha nke, na ụfọdụ mkpokọta, kpalitere mgbọrọgwụ mgbọrọgwụ oblique.Ngwakọta 8, 9, na 15 gbanwere ntụzịaka nke mgbọrọgwụ mgbọrọgwụ na 75 μM, 50 μM, na 40 μM, n'otu n'otu, na-egosi na ogige ndị a nwere ike imebi microtubules nke ọma (Fig 2b, 6a).Anyị nwalekwara ursolic acid kachasị ike, KAND 11, na ntinye dị ala (15 µM) wee chọpụta na ngwa nke KAND 11 gbochiri mgbọrọgwụ mgbọrọgwụ yana na ntụzịaka nke mgbọrọgwụ mgbọrọgwụ adịghị adabara, n'agbanyeghị na ha na-agbada n'aka ekpe. Foto C3)..N'ihi na ọnụ ọgụgụ dị elu nke microtubule-destabilizing ọgwụ mgbe ụfọdụ na-egbochi uto osisi kama ime ka mgbọrọgwụ gbanye mgbọrọgwụ, anyị mechara chọpụta na KAND 11 na-emetụta microtubules site na ịhụ microtubules cortical na sel epidermal mgbọrọgwụ.Immunohistochemistry na-eji mgbochi-β-tubulin antibodies na sel epidermal nke mgbọrọgwụ seedling na-emeso ya na 25 μM KAND 11 gosipụtara na-apụ n'anya nke fọrọ nke nta ka ọ bụrụ microtubules cortical niile na sel epidermal na mpaghara elongation (Fig 6b).Nsonaazụ ndị a na-egosi na kumamotonic acid na ihe ndị na-emepụta ya na-eme ozugbo ma ọ bụ na-apụtaghị ìhè na microtubules na-akpaghasị ha nakwa na ogige ndị a bụ ihe mgbochi microtubule ọhụrụ.
Ursonic acid na usoro ya na-agbanwe cortical microtubules na Arabidopsis thaliana.(a) Mgbọrọgwụ mgbọrọgwụ akụkụ atụnyere na ọnụnọ nke urmotonic acid dị iche iche na mkpokọta egosiri.A na-enyochakwa mmetụta nke ogige abụọ a maara na-egbochi microtubules: disopyramide na oryzalin.Ntinye ahụ na-egosi ọkọlọtọ ejiri tụọ akụkụ uto mgbọrọgwụ.Asterisks na-egosi ọdịiche dị ukwuu na ọgwụgwọ sham (t test, p0.05).n>19. Ogwe ọnụ ọgụgụ = 1 cm.(b) Microtubules cortical na sel epidermal na mpaghara elongation.Microtubules na ụdị Arabidopsis Col mgbọrọgwụ toro na efere MS nwere ma ọ bụ na-enweghị 25 μM KAND 11 ka a na-ahụ anya site na ntụpọ immunohistochemical site na iji β-tubulin pre antibodies na Alexa Fluor-conjugated secondary antibodies.Ogwe ọnụ ọgụgụ = 10µm.(c) Ọdịdị mitotic nke microtubules na mgbọrọgwụ meristem.A na-ahụ anya microtubules site na iji ntụpọ immunohistochemical.A na-agụta ihe owuwu mitotic, gụnyere mpaghara prophase, spindles, na phragmoplasts, site na onyonyo ndị na-atụgharị anya.Àkụ́ na-egosi nhazi microtubule mitotic.Asterisks na-egosi ọdịiche dị ukwuu na ọgwụgwọ sham (t test, p0.05).n>9. Ogwe ọnụ ọgụgụ = 50 µm.
Ọ bụ ezie na Ursa nwere ikike ịkpaghasị ọrụ microtubule, a na-atụ anya na usoro ya ga-adị iche na ihe ndị na-emepụta microtubule depolymerizing.Dịka ọmụmaatụ, ọnụ ọgụgụ dị elu nke microtubule depolymerizing agents dị ka disopyramide na oryzalin na-ebute mgbasawanye anisotropic nke mkpụrụ ndụ epidermal, ebe KAND 11 adịghị.Tụkwasị na nke ahụ, ntinye akwụkwọ nke KAND 11 na disopyramide mere ka a na-eme ka mmeghachi omume mgbọrọgwụ na-akpata disopyramide na-akpata na KAND 11 na-eme ka ihe mgbochi na-eto eto hụrụ (Fig. S4).Anyị nyochakwara nzaghachi nke hypersensitive disopyramide 1-1 (phs1-1) mutant na KAND 11. phs1-1 nwere mgbanwe na-abụghị nke tubulin kinase ma na-emepụta mgbọrọgwụ dị mkpụmkpụ mgbe a na-emeso ya na disopyramide9,20.phs1-1 mutant seedlings toro na agar ọkara nwere KAND 11 nwere mgbọrọgwụ dị mkpụmkpụ dịka ndị toro na disopyramid (fig. S5).
Na mgbakwunye, anyị hụrụ akụkụ microtubule mitotic, dị ka mpaghara prophase, spindles, na phragmoplasts, na mgbọrọgwụ meristem nke mkpụrụ osisi a na-emeso ya na KAND 11. N'ikwekọ na nleba anya maka CDKB2; 1p :: CDKB2; 1-GUS, mbelata dị ukwuu na A hụrụ ọnụ ọgụgụ nke microtubules mitotic (Fig. 6c).
Iji mara cytotoxicity nke KAND 11 na mkpebi subcellular, anyị na-eji KAND 11 gwọọ ụtaba BY-2 mkpụrụ ndụ nkwusioru ma hụ nzaghachi ha.Anyị buru ụzọ gbakwunye KAND 11 na sel BY-2 na-ekwupụta TagRFP-TUA6, nke na-akpọ microtubules na-egbuke egbuke, iji chọpụta mmetụta nke KAND 11 na microtubules cortical.A tụlere njupụta cortical microtubule site na iji nyocha onyonyo, nke gbapụtara pasentị pikselụ cytoskeletal n'etiti pikselụ cytoplasmic.Nsonaazụ nyocha gosipụtara na mgbe ọgwụgwọ na 50 μM ma ọ bụ 100 μM KAND 11 maka awa 1, njupụta ahụ belatara nke ukwuu ruo 0.94 ± 0.74% ma ọ bụ 0.23 ± 0.28%, n'otu n'otu, ebe njupụta nke mkpụrụ ndụ na-emeso DMSO, ruru 1.634 ± % (Fig. 7a).Nsonaazụ ndị a kwekọrọ na nleba anya na Arabidopsis na ọgwụgwọ KAND 11 na-ebute depolymerization nke microtubules cortical (Fig 6b).Anyị nyochakwara ahịrị BY-2 na GFP-ABD-labeled actin filaments mgbe a gwọchara ya na otu ntinye nke KAND 11 wee hụ na ọgwụgwọ KAND 11 mebiri actin filaments.Ọgwụgwọ na 50 μM ma ọ bụ 100 μM KAND 11 maka 1 h budata njupụta actin filament njupụta na 1.20 ± 0.62% ma ọ bụ 0.61 ± 0.26%, n'otu n'otu, ebe njupụta dị na DMSO-mere cell bụ 1.69 ± 0.51% (Fig).nke 7b).Nsonaazụ ndị a dị iche na mmetụta nke propyzamide, nke na-adịghị emetụta actin filaments, na latrunculin B, actin depolymerizer nke na-adịghị emetụta microtubules (SI Figure S6).Na mgbakwunye, ọgwụgwọ na coumamonamide 1, coumamonamide acid 6, ma ọ bụ KAND 11 emetụtaghị microtubules na sel HeLa (SI Figure S7).Ya mere, a kwenyere na usoro ọrụ nke KAND 11 dị iche na nke ndị na-akpaghasị cytoskeleton ama ama.Tụkwasị na nke ahụ, nyocha anyị nke microscopic nke mkpụrụ ndụ BY-2 nke a na-emeso KAND 11 gosipụtara mmalite nke ọnwụ cell n'oge ọgwụgwọ KAND 11 wee gosi na ọnụ ọgụgụ nke Evans sel nwụrụ anwụ na-acha anụnụ anụnụ amụbaghị nke ọma mgbe 30 min nke KAND 11 gachara, ma. mgbe 90 nkeji ọgwụgwọ na 50 μM ma ọ bụ 100 μM KAND, ọnụ ọgụgụ nke mkpụrụ ndụ nwụrụ anwụ mụbara ruo 43.7% ma ọ bụ 80.1%, n'otu n'otu (Fig 7c).N'ịchịkọta ọnụ, data ndị a na-egosi na akwụkwọ akụkọ ursolic acid na-emepụta KAND 11 bụ ihe na-egbochi cytoskeletal kpọmkwem ihe ọkụkụ nwere usoro a na-amaghị na mbụ.
KAND na-emetụta microtubules cortical, actin filaments, na ike nke ụtaba BY-2.(a) Nleba anya nke microtubules cortical na sel BY-2 n'ihu TagRFP-TUA6.A na-enyocha mkpụrụ ndụ BY-2 na KAND 11 (50 μM ma ọ bụ 100 μM) ma ọ bụ DMSO site na nyocha nke confocal.A gbakọrọ njupụta cortical microtubule site na micrographs nke sel 25 nọọrọ onwe ha.Akwụkwọ ozi na-egosi nnukwu ọdịiche (Tukey HSD ule, p0.05).Ogwe ọnụ ọgụgụ = 10µm.(b) Cortical actin filaments na sel BY-2 na-ahụ anya n'ihu GFP-ABD2.A na-enyocha mkpụrụ ndụ BY-2 na KAND 11 (50 μM ma ọ bụ 100 μM) ma ọ bụ DMSO site na nyocha nke confocal.A gbakọrọ njupụta nke cortical actin filaments site na micrographs nke sel 25 nọọrọ onwe ha.Akwụkwọ ozi na-egosi nnukwu ọdịiche (Tukey HSD ule, p0.05).Ogwe ọnụ ọgụgụ = 10µm.(c) Nleba anya nke mkpụrụ ndụ BY-2 nwụrụ anwụ site na Evans blue staining.A na-enyocha mkpụrụ ndụ BY-2 na KAND 11 (50 μM ma ọ bụ 100 μM) ma ọ bụ DMSO site na microscopy na-egbuke egbuke.n=3.Ogwe ọnụ ọgụgụ = 100µm.
Nchọpụta na itinye ngwaahịa ọhụrụ sitere n'okike ebutela ọganihu dị ukwuu n'akụkụ dị iche iche nke ndụ mmadụ, gụnyere ọgwụ na ọrụ ugbo.Emeela nyocha akụkọ ihe mere eme iji nweta ogige bara uru sitere na ihe ndị sitere n'okike.Karịsịa, a maara actinomycetes bara uru dị ka ọgwụ nje antiparasitic maka nematodes n'ihi ikike ha nwere ịmepụta metabolites dị iche iche dị ka avermectin, ihe nchịkọta nke ivermectin na bleomycin na ihe ndị na-emepụta ya, na-eji ọgwụ eme ihe dị ka onye na-agwọ ọrịa cancer21,22.N'otu aka ahụ, a chọpụtala ogige herbicidal dị iche iche site na actinomycetes, ụfọdụ n'ime ha ejirila ahịa1,23.Ya mere, a na-ewere nyocha nke actinomycete metabolites iji kewapụ ngwaahịa sitere n'okike na ihe omume ndụ chọrọ dị ka atụmatụ dị irè.N'ime ọmụmụ ihe a, anyị chọpụtara ogige ọhụrụ, coumamonamide, sitere na S. werraensis ma mejupụta ya nke ọma.Ursonic acid bụ etiti sịntetik nke urbenamide na ihe ndị sitere na ya.Ọ nwere ike ịkpata mgbanaka mgbọrọgwụ ejiri mara, gosipụta ọrụ herbicidal siri ike na-agafeghị oke, yana mebie microtubules osisi ozugbo ma ọ bụ na-apụtaghị ìhè.Otú ọ dị, usoro nke urmotonic acid nwere ike ịdị iche na nke ndị na-emechi microtubule dị ugbu a, ebe ọ bụ na KAND 11 na-akpaghasịkwa actin filaments ma na-akpata ọnwụ cell, na-atụ aro usoro nhazi nke urmotonic acid na ihe ndị na-emepụta ya na-emetụta ọtụtụ ụdị cytoskeletal..
Nkọwa nkọwapụta nke urbenonic acid ga-enyere aka ịghọta nke ọma usoro ọrụ nke urbenonic acid.Karịsịa, ihe mgbaru ọsọ na-esote bụ iji nyochaa ikike nke ursonic acid na-ejikọta na microtubules dị ntakịrị iji chọpụta ma ursonic acid na ihe ndị na-emepụta ya na-eme ihe ozugbo na microtubules na depolymerize ha, ma ọ bụ na omume ha na-eme ka microtubule destabilization.Na mgbakwunye, n'ọnọdụ ebe microtubules na-abụghị ebumnuche kpọmkwem, ịchọpụta ebe a na-eme ihe na ebumnuche molekụla nke ursonic acid na mkpụrụ ndụ osisi ga-enyere aka ịghọtakwu njirimara nke ogige ndị metụtara ya na ụzọ ndị nwere ike isi melite ọrụ herbicidal.Nyocha bioactivity anyị gosipụtara ikike cytotoxic pụrụ iche nke ursonic acid na uto nke osisi dị ka Arabidopsis thaliana, ụtaba na liverwort, ebe emetụtaghị mkpụrụ ndụ E. coli ma ọ bụ HeLa.Obere ma ọ bụ enweghị nsi nye mkpụrụ ndụ anụmanụ bụ uru nke ursonic acid ewepụtara ma ọ bụrụ na emepụtara ha dị ka ahịhịa ahịhịa maka iji n'ubi ugbo mepere emepe.N'ezie, ebe ọ bụ na microtubules bụ ihe a na-ahụkarị na eukaryotes, nkwụsị ha na-ahọrọ na osisi bụ isi ihe a chọrọ maka herbicides.Dịka ọmụmaatụ, propyzamide, microtubule depolymerizing agent nke na-ejikọta ya na tubulin ozugbo ma na-egbochi polymerization, na-eji dị ka ahịhịa ahịhịa n'ihi obere nsí ya na mkpụrụ ndụ anụmanụ24.N'adịghị ka disopyramide, benzamides metụtara nwere nkọwa ebumnuche dị iche iche.Na mgbakwunye na microtubules osisi, RH-4032 ma ọ bụ benzoxamide na-egbochikwa microtubules nke mkpụrụ ndụ anụmanụ ma ọ bụ oomycetes, n'otu n'otu, na zalilamide na-eji dị ka fungicides n'ihi obere phytotoxicity25,26,27.Anụ ọhịa bea ahụ achọpụtara ọhụrụ na ihe ndị na-emepụta ya na-egosipụta cytotoxicity nke ahọpụtara megide osisi, mana ọ dị mma ịmara na mgbanwe ndị ọzọ nwere ike ịgbanwe nkọwapụta ebumnuche ha, nwere ike inye ihe ndị ọzọ maka njikwa fungi pathogenic ma ọ bụ oomycetes.
Njirimara pụrụ iche nke urbenonic acid na ihe ndị na-emepụta ya bara uru maka mmepe ha dị ka ahịhịa na-eji dị ka ngwá ọrụ nyocha.A ghọtara mkpa cytoskeleton dị n'ịchịkwa ụdị mkpụrụ ndụ ihe ọkụkụ.Nnyocha ndị gara aga egosila na osisi ewepụtala usoro mgbagwoju anya nke cortical microtubule nzukọ site na ịchịkwa microtubule dynamics iji chịkwaa morphogenesis nke ọma.Achọpụtala ọnụ ọgụgụ dị ukwuu nke ụmụ irighiri ihe maka nhazi nke ọrụ microtubule, nchọpụta metụtara ya ka na-aga n'ihu3,4,28.Nghọta anyị ugbu a banyere microtubule dynamics na mkpụrụ ndụ osisi anaghị akọwa n'ụzọ zuru ezu usoro nke cortical microtubule nzukọ.Dịka ọmụmaatụ, ọ bụ ezie na ma disopyramide na oryzalin nwere ike imebi microtubules, disopyramide na-akpata mgbagha mgbọrọgwụ siri ike ebe oryzalin nwere mmetụta dị nro.Ọzọkwa, mmụgharị na tubulin, nke na-eme ka microtubules kwụsie ike, na-ebutekwa dextrorotation na mgbọrọgwụ, ebe paclitaxel, nke na-emekwa ka microtubule dynamics, adịghị eme.Ya mere, ịmụ na ịchọpụta ebumnuche molekụla nke ursolic acid kwesịrị inye nghọta ọhụrụ na nhazi nke microtubules cortical osisi.N'otu aka ahụ, ntụnyere kemịkalụ n'ọdịnihu nke dị irè n'ịkwalite uto gbagọọ agbagọ, dị ka disopyramide, na kemịkalụ na-adịchaghị irè, dị ka oryzalin ma ọ bụ kumamotoric acid, ga-enye ihe ngosi maka ka uto gbagọọ agbagọ si eme.
N'aka nke ọzọ, nhazigharị cytoskeletal metụtara nchekwa bụ ohere ọzọ iji kọwaa cytotoxicity nke ursonic acid.Mbufe ọrịa nje ma ọ bụ iwebata elicitor n'ime mkpụrụ ndụ osisi mgbe ụfọdụ na-ebute mbibi nke cytoskeleton na mkpụrụ ndụ na-esote ọnwụ29.Dịka ọmụmaatụ, a kọwawo cryptoxanthin nke sitere na oomycete na-akpaghasị microtubules na actin filaments tupu ọnwụ ụtaba anwụọ, dịka ihe na-eme na ọgwụgwọ KAND30,31.Myirịta dị n'etiti nzaghachi nchebe na nzaghachi cellular nke ursonic acid kpatara mere ka anyị chee na ha na-ebute usoro cellular nkịtị, ọ bụ ezie na mmetụta ngwa ngwa na ike siri ike nke ursonic acid karịa cryptoxanthin pụtara ìhè.Otú ọ dị, ọmụmụ egosila na nkwụsị nke actin filaments na-akwalite ọnwụ cell na-enweghị onwe ya, nke na-adịghị ejikọta ya na microtubule disruption29.Tụkwasị na nke a, a ka ga-ahụ ma pathogen ma ọ bụ elicitor na-eme ka mgbọrọgwụ gbagọrọ agbagọ, dị ka ursonic acid na-eme.Ya mere, ihe omuma nke molikula na-ejikọta nzaghachi agbachitere na cytoskeleton bụ nsogbu mara mma nke a ga-edozi.Site n'iji ọnụnọ nke ogige dị ala dị ala metụtara ursonic acid, yana ọtụtụ usoro nwere ike dị iche iche, ha nwere ike inye ohere iji gbado usoro cellular amabeghị.
N'ịkọkọta ọnụ, nchọpụta na ntinye nke ogige ọhụrụ nke na-emezigharị microtubule dynamics ga-enye ụzọ dị ike iji dozie usoro mgbagwoju anya dị mgbagwoju anya na-adabere na mkpebi nke mkpụrụ ndụ osisi.N'okwu a, urmotonic acid na-emepụta n'oge na-adịbeghị anya, nke na-emetụta microtubules na actin filaments ma na-ebute ọnwụ cell, nwere ike inye ohere iji chọpụta njikọ dị n'etiti njikwa microtubule na usoro ndị ọzọ.Ya mere, nyocha nke kemịkalụ na nke ndu site na iji urbenonic acid ga-enyere anyị aka ịghọta usoro nhazi nke molikula na-achịkwa cytoskeleton osisi.
Inoculate S. werraensis MK493-CF1 n'ime 500 mL gbagwojuru anya Erlenmeyer flask nwere 110 mL nke mkpụrụ ọkara nke nwere 2% (w / v) galactose, 2% (w / v) Essence mado, 1% ( w / v) Bacto mejupụtara .-soyton (Thermo Fisher Scientific, Inc.), 0.5% (w / v) ọka wepụ (KOGOSTCH Co., Ltd., Japan), 0.2% (w / v) (NH4) 2SO4 na 0.2% CaCO3 na mmiri deionized.(pH 7.4 tupu sterilization).A na-etinye omenala mkpụrụ osisi na rotary shaker (180 rpm) na 27 Celsius C maka ụbọchị 2.Mmepụta cultivation site siri ike steeti gbaa ụka.A na-ebufe omenala mkpụrụ (7 ml) n'ime 500 ml K-1 flask nwere 40 g nke mmepụta ihe mejupụtara 15 g nke ọka bali (MUSO Co., Ltd., Japan) na 25 g nke mmiri deionized (pH adịghị edozi ya). tupu sterilization).).A na-eme nri na 30 Celsius C n'ọchịchịrị maka ụbọchị 14.Ejiri 40 ml / karama EtOH wepụta ihe ịgba ụka na centrifuged (1500 g, 4°C, 10 min).E ji ngwakọta nke 10% MeOH/EtOAc wepụta omenaala dị elu (60 ml).A na-ekpochapụ oyi akwa organic n'okpuru nrụgide dị ntakịrị iji nweta ihe fọdụrụ (59.5 mg), bụ nke edoro HPLC na gradient elution (0-10 nkeji: 90%) na kọlụm ntụgharị (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID). 10 mm × ogologo 250 mm) H2O/CH3CN, 10-35 nkeji: 90% H2O/CH3CN ruo 70% H2O/CH3CN (gradient), 35-45 nkeji: 90% H2O/EtOH, 45-155 nkeji: 90% H2O / EtOH na 100% EtOH (gradient (gradient), 155-200 min: 100% EtOH) na ọnụọgụ nke 1.5 ml / min, coumamonamide (1, 36.0 mg) dịpụrụ adịpụ dị ka ntụ ntụ amorphous ọcha.
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]+ uru gbakọrọ: 141.0659, uru tụrụ: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593-153 cm
Enwetara mkpụrụ osisi Columbia (Col-0) site na Arabidopsis Biological Resource Center (ABRC) na ikike maka iji nyocha.A na-agbasa mkpụrụ osisi Col-0 ma dokwa ya n'okpuru ọnọdụ ụlọ nyocha anyị ma jiri ya dị ka osisi Arabidopsis anụ ọhịa.A na-ekpochasị mkpụrụ osisi Arabidopsis n'elu wee zụlite ya na ọkara ike Murashige na Skoog nwere 2% sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2- (4-morpholino) ethanesulfonic acid (MES) (Fujifilm Wako Pure Chemical). ).) na 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, na 23 Celsius C na ọkụ mgbe niile.Ọ bụ T. Hashimoto (Nara Institute of Science and Technology) nyere mkpụrụ nke phs1-1 mutant.
Mkpụrụ nke nje SR-1 bụ T. Hashimoto (Nara Institute of Science and Technology) nyere ma jiri ya dị ka osisi ụtaba anụ ọhịa.A na-ekpocha mkpụrụ osisi ụtaba n'elu ma tinye ya na mmiri na-adịghị mma maka abalị atọ iji kwalite germination, wee tinye ya na ngwọta nke ọkara nke nwere 2% sucrose, 0.05% (w / v) MES, na 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murshige.na Skoog ọkara) nwere pH 5.7 ma tinye ya na 23 Celsius C n'okpuru ọkụ mgbe niile.
T. Kohchi (Mahadum Kyoto) nyere Strain Tak-1 ma jiri ya mee ihe dị ka ọkọlọtọ nnwale maka ọmụmụ imeju.A nwetara Gemma site na osisi ndị a na-eme ọgwụ mgbochi ma tinyezie ya na Gamborg B5 medium (Fujifilm Wako Pure Chemical) nwere 1% sucrose na 0.3% gellan gum ma tinye ya na 23 Celsius C n'okpuru ọkụ na-aga n'ihu.
Ụta ụtaba BY-2 cell (Nicotiana tabacum L. cv. Bright Yellow 2) bụ S. Hasezawa (Mahadum Tokyo) nyere.A na-agbanye mkpụrụ ndụ BY-2 95-fold na mgbanwe Linsmeier na Skoog na-agbakwunye kwa izu na 2,4-dichlorophenoxyacetic acid 32.Agwakọtara nkwusioru cell na rotary shaker na 130 rpm na 27 Celsius C n'ọchịchịrị.Na-asa mkpụrụ ndụ na 10 ugboro olu nke ọhụrụ ọkara na resupend na otu ọkara.BY-2 transgenic cell ahịrị na-egosipụta nke ọma microtubule marker TagRFP-TUA6 ma ọ bụ actin filament markker GFP-ABD2 n'okpuru kọlịflawa mosaic virus 35S onye na-akwalite dị ka akọwara33,34,35.Enwere ike idowe ma mekọrịta ahịrị sel ndị a site na iji usoro yiri nke ejiri maka ahịrị sel BY-2 izizi.
Azụlitere mkpụrụ ndụ HeLa na Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) agbakwunyere na serum bovine nwa ebu n'afọ 10%, penicillin 1.2 U/ml, na 1.2 μg/ml streptomycin na 37°C incubator nwere 5% CO2.
Emere nnwale niile akọwara n'ihe odide a dabere na ụkpụrụ na ụkpụrụ biosafety Japanese.
A gbazere ngwakọta na dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) dị ka ngwaahịa ngwaahịa na diluted na MS medium maka Arabidopsis na ụtaba ma ọ bụ Gamborg B5 ọkara maka liverwort.Maka nyocha ihe mgbochi mgbochi mgbọrọgwụ, a na-akụ ihe karịrị mkpụrụ 10 n'otu efere na agar media nwere ogige egosipụtara ma ọ bụ DMSO.A na-etinye mkpụrụ n'ime ụlọ na-eto eto maka ụbọchị 7.A na-ese foto osisi ndị ahụ ma tụọ ogologo mgbọrọgwụ.Maka nyocha mkpụrụ nke Arabidopsis, a na-akụ mkpụrụ iri anọ na asatọ n'otu efere na agar media nwere 200 μM compound ma ọ bụ DMSO.A na-akụ mkpụrụ Arabidopsis n'ime ụlọ na-eto eto na ọnụ ọgụgụ nke mkpụrụ osisi germinated na-agụta ụbọchị 7 ka germination (dag).Maka nyocha mkpụrụ osisi ụtaba, a na-akụ mkpụrụ iri abụọ na anọ n'otu efere na agar media nwere 200 μM KAND ma ọ bụ DMSO.A na-akụ mkpụrụ ụtaba n'ime ụlọ na-eto eto, a na-agụkwa ọnụ ọgụgụ nke mkpụrụ osisi germinated mgbe ụbọchị iri na anọ gachara.Maka nyocha mgbochi mmụba nke liverwort, embrayo 9 sitere na efere nke ọ bụla ka etinyere na agar media nke nwere mkpokọta KAND ma ọ bụ DMSO egosipụtara ma tinye ya n'ime ụlọ uto maka ụbọchị iri na anọ.
Jiri mkpụrụ osisi nwere ntụpọ 5 mg/ml propidium iodide (PI) iji were anya nke uche hụ nhazi mgbọrọgwụ meristem.Ahụrụ akara PI site na microscopy fluorescence site na iji microscope TCS SPE confocal laser scanning (Leica Microsystems).
A na-eme ntụpọ histochemical nke mgbọrọgwụ ya na β-glucuronidase (GUS) dịka usoro nke Malami na Benfey36 kọwara.A na-edozi mkpụrụ osisi na 90% acetone n'otu ntabi anya, na 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid na GUS buffer maka 1 hour ma tinye ya na ngwọta chloraldehyde hydrated.(8 g chloral hydrate, 2 ml mmiri na 1 ml glycerol) wee hụ site na nnyonye anya dị iche iche na-eji microscope Axio Imager M1 (Carl Zeiss).
A tụrụ akụkụ mgbọrọgwụ n'osisi ndị dị ụbọchị 7 toro na efere ndị edobere na kwụ ọtọ.Tulee akụkụ nke mgbọrọgwụ site na ntụzịaka nke vector ike ndọda dịka akọwara na nzọụkwụ 6.
A na-ahụ nhazi nke microtubules cortical dị ka akọwara ya, na obere mgbanwe na protocol 37.Anti-β-tubulin mgbochi (KMX-1, Merk Millipore: MAB3408) na Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) ka ejiri mee ihe dị ka ọgwụ mgbochi isi na nke abụọ na 1: 1000 na 1: 100 dilutions, n'otu n'otu.Ejiri TCS SPE confocal laser scanning microscope (Leica Microsystems).Nweta onyogho Z-stack wee mepụta ntule kachasi ike dịka ntuziaka onye nrụpụta siri dị.
A na-eme nyocha mmụba cell HeLa site na iji Kit Counting Kit 8 (Dojindo) dịka ntuziaka onye nrụpụta siri dị.
A na-enyocha uto nke E. coli DH5a site na iji spectrophotometer na 600 nm (OD600).
A na-ahụ nzukọ cytoskeletal na sel transgenic BY-2 site na iji microscope fluorescence nke nwere CSU-X1 confocal scanning device (Yokogawa) na igwefoto sCMOS (Zyla, Andor Technology).A na-enyocha njupụta nke cytoskeletal site na nyocha onyonyo, bụ nke gụpụtara pasentị nke pikselụ cytoskeletal n'etiti pikselụ cytoplasmic na foto confocal site na iji ngwa ngwa ImageJ dị ka akọwara38,39.
Iji chọpụta ọnwụ cell n'ime mkpụrụ ndụ BY-2, a na-eji 0.05% Evans acha anụnụ anụnụ tinye aliquot nke nkwusioru cell maka nkeji iri na okpomọkụ.Nhọrọ nke Evans na-acha anụnụ anụnụ nke mkpụrụ ndụ nwụrụ anwụ na-adabere na mwepu nke agba sitere na mkpụrụ ndụ nwere ike ime site na membrane plasma na-emebibeghị40.A na-ahụ mkpụrụ ndụ ntụpọ site na iji microscope na-egbuke egbuke (BX53, Olympus).
A na-eto mkpụrụ ndụ HeLa na DMEM gbakwunyere na 10% FBS na incubator humidified na 37°C na 5% CO2.A na-agwọ mkpụrụ ndụ na 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), ma ọ bụ 100 ng/ml Nocodmaze (Sigma) maka 6 h na 37 ° C.A na-edozi cell na MetOH maka nkeji 10 wee jiri acetate maka 5 min na okpomọkụ ụlọ.A na-eji β-tubulin primary antibody tinye sel ndị a kapachara (1D4A4, Proteintech: 66240-1) na 0.5% BSA/PBS maka awa 2, were TBST sachaa ugboro 3, wee tinye ya na mgbochi ewu ewu Alexa Fluor.4881 elekere.- Mouse IgG (Thermo Fisher Scientific: A11001) na 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) gbazere na 0.5% BSA/PBS.Mgbe ejiri TBST sachaa ugboro atọ, a hụrụ mkpụrụ ndụ nwere ntụpọ na microscope nke Nikon Eclipse Ti-E.Ejiri igwefoto CCD Hamamatsu ORCA-R2 jụrụ oyi wee weghara onyonyo site na iji ngwa MetaMorph (Ngwaọrụ Molecular).
Oge nzipu: Jun-17-2024