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Nchọpụta na ojiji bara uru nke ngwaahịa sitere n'okike nwere ike inyere aka melite ndụ mmadụ. A na-ejikarị kemịkalụ ndị na-egbochi uto osisi eme ihe dị ka ahịhịa ọgwụ iji chịkwaa ahịhịa. N'ihi mkpa ọ dị iji ụdị ahịhịa dị iche iche, ọ dị mkpa ịchọpụta ihe mejupụtara nwere usoro ọrụ ọhụrụ. N'ọmụmụ ihe a, anyị chọpụtara ihe mejupụtara N-alkoxypyrrole ọhụrụ, coumamonamide, sitere na Streptomyces werraensis MK493-CF1 ma guzobe usoro mmepụta zuru oke. Site na nnwale ọrụ bayoloji, anyị chọpụtara na urs-monoamic acid bụ ihe eji emepụta urs-monoamide na ihe nwere ike ime.ihe mgbochi uto osisiTinyere nke a, anyị emepụtala ọtụtụ ihe ndị sitere na urbenonic acid, gụnyere urbenyloxy derivative (UDA), nke nwere nnukwu ọrụ igbu ahịhịa na-enweghị mmetụta ọjọọ na uto nke mkpụrụ ndụ HeLa. Anyị chọpụtakwara na ihe ndị sitere na urmotonic acid na-emebi microtubules osisi; na mgbakwunye, KAND na-emetụta actin filaments ma na-akpata ọnwụ mkpụrụ ndụ; Mmetụta dị iche iche ndị a dị iche na nke ndị na-egbochi microtubule a ma ama ma na-atụ aro usoro ọhụrụ nke ọrụ maka ursonic acid, nke na-anọchite anya uru dị mkpa na mmepe nke ọgwụ ogwu ọhụrụ.
Nchọpụta na ojiji bara uru nke ngwaahịa sitere n'okike na ihe ndị sitere na ha bụ ụzọ isi melite ịdị mma nke ndụ mmadụ. Mkpụrụ ndụ ihe nketa nke microorganisms, osisi na ụmụ ahụhụ na-emepụta emeela ka ọganihu dị ukwuu na ọgwụ na ọrụ ugbo. E mepụtala ọtụtụ ọgwụ nje na ọgwụ mgbochi leukemia site na ngwaahịa eke. Na mgbakwunye, ụdị dị iche icheọgwụ ahụhụ, a na-esi na ngwaahịa ndị a sitere n'okike wepụta ọgwụ fungicides na ahịhịa ogwu maka ojiji ha n'ọrụ ugbo. Karịsịa, ọgwụ mgbochi ahịhịa bụ ngwaọrụ dị mkpa maka ịbawanye ihe ubi n'ọrụ ugbo nke oge a, a na-ejikwa ụdị ngwakọta dị iche iche n'ahịa. Ọtụtụ usoro sel dị na osisi, dị ka photosynthesis, amino acid metabolism, njikọ mgbidi sel, nhazi nke mitosis, phytohormone signaling, ma ọ bụ protein synthesis, bụ ihe a na-ewere dị ka ihe mgbaru ọsọ nke ahịhịa ọgwụ. Ihe ndị na-egbochi ọrụ microtubule bụ otu ụdị ahịhịa ọgwụ ndị a na-ahụkarị nke na-emetụta uto osisi site na imetụta iwu mitotic2.
Microtubules bụ akụkụ nke cytoskeleton ma echekwara ha nke ọma na mkpụrụ ndụ eukaryotic. Tubulin heterodimer nwere α-tubulin na β-tubulin na-emepụta protofilaments microtubule linear, yana protofilaments 13 na-emepụta usoro cylindrical. Microtubules na-arụ ọrụ dị iche iche na mkpụrụ ndụ osisi, gụnyere ịchọpụta ọdịdị mkpụrụ ndụ, nkewa mkpụrụ ndụ, na njem intracellular3,4. Mkpụrụ ndụ osisi nwere microtubules n'okpuru interphase plasma membrane, a na-echekwa na ihe a na-akpọ cortical microtubules na-achịkwa nhazi nke cellulose microfibrils site na nhazi nke cellulose synthase complexes4,5. Microtubules cortical nke mkpụrụ ndụ epidermal mgbọrọgwụ, dị na mpaghara nke ịgbatị ngwa ngwa nke nsọtụ mgbọrọgwụ, dị n'akụkụ, microfibers cellulose na-esokwa microtubules ndị a ma na-ebelata ntụziaka nke mgbasawanye mkpụrụ ndụ, si otú a na-akwalite ịgbatị mkpụrụ ndụ anisotropic. Ya mere, ọrụ microtubule nwere njikọ chiri anya na ọdịdị osisi. Mgbanwe amino acid na mkpụrụ ndụ ihe nketa nke na-agbanwe tubulin na-akpata mgbanwe nke usoro microtubule cortical na uto aka ekpe ma ọ bụ aka nri na Arabidopsis 6,7. N'otu aka ahụ, mgbanwe na protein ndị metụtara microtubule nke na-achịkwa usoro microtubule nwekwara ike ibute uto mgbọrọgwụ gbagọrọ agbagọ8,9,10,11,12,13. Na mgbakwunye, ọgwụgwọ site na iji ọgwụ ogwu na-emebi microtubule dị ka disopyramide, nke a makwaara dị ka pretilachlor, na-akpata uto mgbọrọgwụ oblique akụkụ aka ekpe14. Data ndị a na-egosi na nhazi ziri ezi nke ọrụ microtubule dị oke mkpa maka ịchọpụta ebe uto osisi si aga.
Achọpụtala ụdị dị iche iche nke ihe mgbochi microtubule, ọgwụ ndị a enyela aka dị ukwuu na nnyocha cytoskeletal, yana na ọrụ ugbo na ọgwụ2. Karịsịa, oryzalin, ngwakọta dinitroaniline, disopyramide, ihe ndị metụtara benzamide, na ihe ndị yiri ha nwere ike igbochi ọrụ microtubule ma si otú a gbochie uto osisi. Ya mere, a na-ejikarị ha eme ihe dị ka ihe ọkụkụ. Agbanyeghị, ebe ọ bụ na microtubules bụ ihe dị mkpa nke mkpụrụ osisi na anụmanụ, ọtụtụ ihe mgbochi microtubule na-egbu egbu nye ụdị mkpụrụ ndụ abụọ ahụ. Ya mere, n'agbanyeghị uru ha bara dị ka ihe ọkụkụ, a na-eji ọnụọgụgụ ole na ole nke ihe mgbochi microtubule eme ihe maka ebumnuche bara uru.
Streptomyces bụ ụdị nke ezinụlọ Streptomyces, nke gụnyere nje bacteria aerobic, gram-positive, na filamentous, a makwaara ya nke ọma maka ikike ya ịmepụta ọtụtụ metabolites nke abụọ. Ya mere, a na-ewere ya dị ka otu n'ime isi mmalite kachasị mkpa nke ngwaahịa okike ọhụrụ na-arụ ọrụ na bayoloji. N'ọmụmụ ihe a ugbu a, anyị chọpụtara ihe mejupụtara ọhụrụ a na-akpọ coumamonamide, nke e kewapụrụ na Streptomyces werraensis MK493-CF1 na S. werraensis ISP 5486. Site na iji nyocha spectral na nyocha spectral zuru oke, a kọwara nhazi nke coumamonamide ma chọpụta ọkpụkpụ N-alkoxypyrrole pụrụ iche ya. Achọpụtara na Ursmonic acid, etiti sịntetik nke ursmonoamide na ihe ndị sitere na ya, na-egbochi uto na ntolite nke osisi ihe nlereanya a ma ama bụ Arabidopsis thaliana. Na ọmụmụ mmekọrịta nhazi na ọrụ, anyị chọpụtara na ihe mejupụtara nwere C9 gbanwere ka ọ bụrụ ursonic acid, nke a na-akpọ nonyloxy derivative of ursonic acid (KAND), na-eme ka mmetụta mgbochi na uto na ntolite dịkwuo mma. Ihe pụrụ iche bụ na ihe mgbochi uto osisi a chọpụtara ọhụrụ metụtakwara uto nke ụtaba na liverwort, ọ bụghịkwa ihe na-egbu nje bacteria ma ọ bụ mkpụrụ ndụ HeLa. Ọzọkwa, ụfọdụ ihe sitere na urmotonic acid na-akpalite ọdịdị mgbọrọgwụ gbagọrọ agbagọ, na-egosi na ihe ndị a sitere na ya na-emetụta microtubules ozugbo ma ọ bụ n'ụzọ na-apụtaghị ìhè. Dịka echiche a si dị, ihe anyị hụrụ na microtubules nke ejiri immunohistochemical ma ọ bụ fluorescent protein mee gosiri na ọgwụgwọ KAND na-ewepụ microtubules. Na mgbakwunye, ọgwụgwọ na kumamotonic acid sitere na ya na-emebi microfilaments actin. Ya mere, anyị achọpụtala ihe mgbochi uto osisi ọhụrụ nke usoro ọrụ ya pụrụ iche gụnyere mbibi nke cytoskeleton.
E kewapụrụ ụdị MK493-CF1 site n'ala na Shinagawa-ku, Tokyo. Ụdị MK493-CF1 mepụtara mycelium stromal nke nwere alaka dị mma. A chọpụtara usoro akụkụ nke mkpụrụ ndụ ihe nketa RNA nke 16S (1422 bp). Ụdị a yiri nke S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: ụdị a na-ahụkarị, 99.93%). Dabere na nsonaazụ a, achọpụtara na ụdị a nwere njikọ chiri anya na ụdị ụdị S. werraensis. Ya mere, anyị kpọrọ ụdị a S. werraensis MK493-CF1 aha nwa oge. S. werraensis ISP 5486T na-emepụtakwa otu ihe ndị na-arụ ọrụ ndụ. Ebe ọ bụ na e nwebeghị nnyocha mbụ iji nweta ngwaahịa sitere n'okike site na obere nje a, e mere nnyocha kemịkalụ ọzọ. Mgbe a kụchara S. werraensis MK493-CF1 n'elu ọka bali site na ịgbaze ihe siri ike na 30°C ruo ụbọchị iri na anọ, e jiri 50% EtOH wepụta ihe ahụ. A kpọrọ nkụ 60 ml nke ihe nlele ahụ iji nweta 59.5 mg nke ihe e si na ya wepụta. E tinyere ihe e si na ya wepụta ihe ahụ n'okpuru HPLC nke na-agbanwe agbanwe iji nye N-methoxy-1H-pyrrole-2-carboxamide (1, nke a na-akpọ coumamonamide, 36.0 mg). Ngụkọta nke 1 bụ ihe dị ka 60% nke ihe e si na ya wepụta. Ya mere, anyị kpebiri ịmụ nke ọma ihe ndị dị na kumamotoamide 1.
Coumamonamide 1 bụ ntụ ntụ ọcha amorphous na spectrometry oke mkpebi (HRESIMS) na-akwado C6H8N2O2 (Foto 1). A na-eji 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 spectrum: 4.5 Hz, H-5) na δH 6.78 (1H, d, J = 2.5 Hz, H-6), na 13C NMR spectrum na-egosi ọnụnọ nke atọm carbon sp2 anọ. E jiri njikọ HMBC site na proton C-3 gaa na carbon carbon amide na δC 161.1 nyochaa ọnụnọ nke otu amide na ọnọdụ C2. Tinyere nke a, 1 H na 13 C NMR na-arị elu na δH 4.10 (3H, S) na δC 68.3 na-egosi ọnụnọ nke otu N-methoxy na molekul ahụ. Ọ bụ ezie na a chọpụtabeghị ọnọdụ ziri ezi nke otu methoxy site na iji nyocha spectroscopic dị ka enhanced difference spectroscopy na nuclear Overhauser abbreviation (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide ghọrọ ihe mbụ a ga-etinye n'ọrụ.
Iji chọpụta nhazi ziri ezi nke 1, e mere njikọta zuru oke (Foto 2a). Ọgwụgwọ nke 2-aminopyridine 2 dị n'ahịa na m-CPBA mere ka N-oxide 3 kwekọrọ na mmepụta ọnụọgụgụ. Mgbe 2-aminoazidation nke 2 gasịrị, emere mmeghachi omume cyclocondensation nke Abramovich kọwara na benzene na 90°C iji nweta 1-hydroxy-1H-pyrrole-2-carbonitrile 5 achọrọ na gram. Ọsọ 60% (usoro abụọ). 15,16. Methylation na hydrolysis nke 4 wee nye 1-methoxy-1H-pyrrole-2-carboxylic acid (akpọrọ "cumotonic acid", 6) na ezigbo mkpụrụ (70%, usoro abụọ). N'ikpeazụ, amidation site na acid chloride intermediate 6 site na iji ammonia mmiri nyere Kumamoto amide 1 na 98% mkpụrụ. Data spectral niile nke synthesized 1 yiri nke dịpụrụ adịpụ 1, yabụ achọpụtara nhazi nke 1;
Nchịkọta na nyocha izugbe nke ọrụ ndu nke urbenamide na urbenic acid. (a) Nchikota zuru oke nke Kumamoto amide. (b) A kụrụ mkpụrụ Arabidopsis Columbia (Col) nke ụdị ọhịa ụbọchị asaa na efere Murashige na Skoog (MS) nke nwere coumamonamide 6 ma ọ bụ coumamonamide 1 na oke egosiri. Ogwe nha = 1 cm.
Nke mbụ, anyị lere anya n'ọrụ ndu nke urbenamide na ihe ndị dị n'ime ya maka ikike ha nwere ịgbanwe uto osisi. Anyị tinyere ọtụtụ ursmonamide 1 ma ọ bụ ursmonic acid 6 na mkpụrụ osisi agar nke MS na nke a na-akọ na Arabidopsis thaliana na usoro a. Nnyocha ndị a gosiri na oke njupụta (500 μM) nke 6 gbochiri uto mgbọrọgwụ (Fig. 2b). Na-esote, anyị mepụtara ọtụtụ ihe ndị sitere na ya site n'ịnọchi ọnọdụ N1 nke 6 ma mee ọmụmụ ihe gbasara nhazi na ọrụ na ha (a kọwara usoro njikọta analog na Ozi Nkwado (SI)). A kụrụ mkpụrụ osisi Arabidopsis na usoro nwere ihe ndị sitere na ursonic acid 50 μM, a tụkwara ogologo mgbọrọgwụ. dịka egosiri na foto a. Dịka egosiri na Fig 3a, b, na S1, coumamo acids nwere ogologo dị iche iche nke agbụ alkoxy linear (9, 10, 11, 12, na 13) ma ọ bụ nnukwu agbụ alkoxy (15, 16, na 17) na ọnọdụ N1. Ihe ndị e si na ya pụta gosiri nnukwu mgbochi nke uto mgbọrọgwụ. Na mgbakwunye, anyị chọpụtara na itinye 200 μM 10, 11, ma ọ bụ 17 gbochiri mkpụrụ (Fig. 3c na S2).
Ọmụmụ ihe gbasara mmekọrịta nhazi na ọrụ nke Kumamoto amide na ihe ndị metụtara ya. (a) Atụmatụ nhazi na njikọta nke analogues. (b) Ngụkọta ogologo mgbọrọgwụ nke mkpụrụ osisi dị ụbọchị 7 a kụrụ na MS nwere ma ọ bụ na-enweghị 50 μM coumamonamide sitere na ya. Akara kpakpando na-egosi nnukwu ọdịiche na ọgwụgwọ aghụghọ (ule t, p<0.05). n>18. E gosiri data dị ka nkezi ± SD. nt pụtara "anaghị anwale" n'ihi na ihe karịrị 50% nke mkpụrụ ndị ahụ emeghị ka ha pụta ìhè. (c) Ngụkọta ọnụego mkpụrụ ndị a gwọrọ agwọ nke e tinyere n'ime mkpụrụ MS ruo ụbọchị 7 ma ọ bụ na-enweghị 200 μM coumamonamide na ihe ndị metụtara ya. Akara kpakpando na-egosi nnukwu ọdịiche dị na ọgwụgwọ sham (ule chi-square). n=96.
N'ụzọ na-adọrọ mmasị, mgbakwunye nke agbụ akụkụ alkyl karịa C9 belatara ọrụ mgbochi ahụ, na-egosi na ngwakọta ndị metụtara kumamotoic acid chọrọ agbụ akụkụ nke nha ụfọdụ iji gosipụta ọrụ bayoloji ha.
Ebe ọ bụ na nyocha mmekọrịta nhazi na ọrụ gosiri na agbanwere C9 ka ọ bụrụ ursonic acid na ihe sitere na nonyloxy nke ursonic acid (nke a na-akpọ KAND 11 ma emechaa) bụ ihe kacha mma na-egbochi uto osisi, anyị mere nkọwa zuru ezu nke KAND 11. Ọgwụgwọ nke Arabidopsis na 50 μM KAND 11 fọrọ nke nta ka ọ gbochie ntolite kpamkpam, ebe obere njupụta (40, 30, 20, ma ọ bụ 10 μM) nke KAND 11 gbochiri uto mgbọrọgwụ n'ụzọ dabere na dose (Fig. 4a, b). Iji nwalee ma KAND 11 na-emetụta ndụ mgbọrọgwụ nke meristem, anyị lere anya na mpụta mgbọrọgwụ nke propidium iodide (PI) kpuchiri ma tụọ nha mpaghara meristem. Nha nke mkpụrụ osisi a kụrụ n'elu ihe nwere 25 μM KAND-11 bụ 151.1 ± 32.5 μm, ebe nha nke mkpụrụ osisi a kụrụ n'elu ihe njikwa nwere DMSO bụ 264.7 ± 30.8 μm (Foto 4c, d), nke na-egosi na KAND-11 na-eweghachi ọrụ mkpụrụ ndụ. na-agbasa. Mgbọrọgwụ meristem. N'ikwekọ na nke a, ọgwụgwọ KAND 11 belatara ọnụọgụ nke akara nkewa mkpụrụ ndụ CDKB2;1p::CDKB2;1-GUS na mgbọrọgwụ meristem (Foto 4e) 17. Nsonaazụ ndị a na-egosi na KAND 11 na-egbochi uto mgbọrọgwụ site na ibelata ọrụ mmụba mkpụrụ ndụ.
Nyocha nke mmetụta mgbochi nke ihe ndị sitere na urbenonic acid (urbenyloxy derivatives) na uto. (a) Mkpụrụ osisi Col dị ụbọchị asaa nke a kụrụ n'elu efere MS nwere oke KAND 11 egosiri. Ogwe nha = 1 cm. (b) Ọnụọgụ ogologo mgbọrọgwụ. Mkpụrụ akwụkwọ na-egosi nnukwu ọdịiche (ule Tukey HSD, p<0.05). n>16. E gosiri data dị ka nkezi ± SD. (c) Nyocha confocal nke propidium iodide-wild-type Col roots tolitere na efere MS nwere ma ọ bụ na-enweghị 25 μM KAND 11. Brackets ọcha na-egosi mgbọrọgwụ meristem. Ogwe nha = 100 µm. (d) Ọnụọgụ nke nha mgbọrọgwụ meristem (n = 10 ruo 11). E ji t-test chọpụta ọdịiche ọnụọgụgụ site na iji t-test (p< 0.05). Ogwe ndị ahụ na-anọchite anya nha nkezi nke meristem. (e) Nyocha ọdịiche dị iche iche (DIC) nke meristem mgbọrọgwụ nwere ihe owuwu CDKB2; 1pro: CDKB2; 1-GUS nke e tinyere agba ma tee agba na mkpụrụ osisi dị ụbọchị ise a kụrụ na efere MS ma ọ bụ na-enweghị 25 µM KAND.
A nwalekwara ihe ndị na-egbu egbu nke KAND 11 site na iji osisi dicotyledonous ọzọ, ụtaba (Nicotiana tabacum), na ihe dị ndụ dị mkpa n'ala, liverwort (Marchantia polymorpha). Dịka ọ dị n'ihe gbasara Arabidopsis, mkpụrụ osisi ụtaba SR-1 a kụrụ n'elu ihe nwere 25 μM KAND 11 mepụtara mgbọrọgwụ dị mkpụmkpụ (Fig. 5a). Na mgbakwunye, mkpụrụ 40 n'ime 48 gbapụrụ n'elu efere nwere 200 μM KAND 11, ebe mkpụrụ 48 niile gbapụrụ n'elu ihe ndị a na-agwọ agwọ, nke na-egosi na oke KAND dị elu dị oke mkpa (p<0.05; chi test -square) gbochiri uto nke ụtaba. (Foto 5b). Na mgbakwunye, njupụta nke KAND 11 nke gbochiri uto nje bacteria na liverwort yiri njupụta dị irè na Arabidopsis (Foto 5c). Nsonaazụ ndị a na-egosi na KAND 11 nwere ike igbochi uto nke ụdị osisi dị iche iche. Anyị mechara nyochaa ike cytotoxicity nke ihe ndị metụtara bea monoamide na ihe ndị ọzọ dị ndụ, ya bụ mkpụrụ ndụ HeLa mmadụ na ụdị Escherichia coli DH5α, dị ka ndị nnọchi anya mkpụrụ ndụ anụmanụ na nje bacteria dị elu, n'otu n'otu. Na usoro nyocha mmụba mkpụrụ ndụ, anyị hụrụ na coumamonamide 1, coumamonamidic acid 6, na KAND 11 emetụtaghị uto nke mkpụrụ ndụ HeLa ma ọ bụ E. coli na njupụta nke 100 μM (Foto 5d,e).
Mgbochi uto nke KAND 11 n'ime ihe ndị na-abụghị Arabidopsis. (a) A kụrụ mkpụrụ osisi ụtaba SR-1 nke dị izu abụọ n'elu efere MS nke dị n'usoro nke nwere 25 μM KAND 11. (b) A kụrụ mkpụrụ osisi ụtaba SR-1 nke dị izu abụọ n'elu efere MS nke dị n'usoro nke nwere 200 μM KAND 11. (c) Mkpụrụ osisi liverwort Tak-1 nke dị izu abụọ nke toro na efere Gamborg B5 nke nwere oke KAND 11 egosiri. Akụ uhie na-egosi spores kwụsịrị itolite n'ime oge incubation izu abụọ. (d) Nnyocha mmụba mkpụrụ ndụ nke mkpụrụ ndụ HeLa. A tụrụ ọnụọgụgụ mkpụrụ ndụ ndị dị ndụ na oge a kapịrị ọnụ site na iji ngwa ọnụọgụgụ mkpụrụ ndụ 8 (Dojindo). Dịka njikwa, a gwọrọ mkpụrụ ndụ HeLa na 5 μg/ml actinomycin D (Act D), nke na-egbochi ntụgharị RNA polymerase ma na-akpata ọnwụ mkpụrụ ndụ. Emere nyocha na atọ. (e) Nnyocha mmụba mkpụrụ ndụ E. coli. A tụlere uto E. coli site n'ịtụ OD600. Dịka njikwa, e ji 50 μg/ml ampicillin (Amp) gwọọ mkpụrụ ndụ, nke na-egbochi mmepụta mgbidi mkpụrụ ndụ nje. E mere nyocha ahụ n'ụzọ atọ.
Iji ghọta usoro nke ihe ndị metụtara uramide na-akpata site na cytotoxicity, anyị nyochaghachiri ihe ndị sitere na urbenic acid nwere mmetụta mgbochi dị ala. dịka egosiri na foto a. Dịka egosiri na Foto 2b, 6a, mkpụrụ osisi a kụrụ na efere agar nwere nnukwu mkpokọta (200 μM) nke urmotonic acid 6 mepụtara mgbọrọgwụ dị mkpụmkpụ na nke gbagọrọ agbagọ n'aka ekpe (θ = - 23.7 ± 6.1), ebe nke mkpụrụ osisi a kụrụ na usoro njikwa, mkpụrụ osisi ahụ mepụtara mgbọrọgwụ kwụ ọtọ (θ = - 3.8 ± 7.1). A maara na uto oblique a bụ nke sitere na nsogbu nke microtubules cortical14,18. Dịka nchọpụta a si dị, ọgwụ microtubule na-emebi emebi disopyramide na oryzalin kpatara mgbanwe mgbọrọgwụ yiri nke ahụ n'okpuru ọnọdụ uto anyị (Fig. 2b, 6a). N'otu oge ahụ, anyị nwalere ihe ndị sitere na urmotonic acid ma họrọ ọtụtụ n'ime ha nke, na njupụta ụfọdụ, kpatara uto mgbọrọgwụ oblique. Ngwakọta 8, 9, na 15 gbanwere ụzọ uto mgbọrọgwụ si aga na 75 μM, 50 μM, na 40 μM, n'otu n'otu, na-egosi na ihe ndị a nwere ike imebi microtubules nke ọma (Fig. 2b, 6a). Anyị nwalekwara ihe kachasị ike nke ursolic acid derivative, KAND 11, na obere njupụta (15 µM) wee chọpụta na itinye KAND 11 na-egbochi uto mgbọrọgwụ nakwa na ntụziaka nke uto mgbọrọgwụ adịghị mma, ọ bụ ezie na ha na-agbada n'aka ekpe (Foto C3). . N'ihi na oke njupụta nke ọgwụ microtubule na-egbochi uto osisi mgbe ụfọdụ kama ịkpata ntụgharị mgbọrọgwụ, anyị mechara nyochaa ohere nke KAND 11 na-emetụta microtubules site n'ile anya na microtubules cortical na mkpụrụ ndụ epidermal mgbọrọgwụ. Immunohistochemistry site na iji anti-β-tubulin antibodies na mkpụrụ ndụ epidermal nke mgbọrọgwụ seedling ejiri 25 μM KAND 11 gwọọ gosiri mfu nke ihe fọrọ nke nta ka ọ bụrụ microtubules cortical niile na mkpụrụ ndụ epidermal na mpaghara elongation (Fig. 6b). Nsonaazụ ndị a na-egosi na kumamotonic acid na ihe ndị sitere na ya na-arụ ọrụ kpọmkwem ma ọ bụ na-apụtaghị ìhè na microtubules iji mebie ha, nakwa na ihe ndị a bụ ihe mgbochi microtubule ọhụrụ.
Ursonic acid na ihe ndị sitere na ya na-agbanwe obere akụkụ cortical dị na Arabidopsis thaliana. (a) A tụrụ akụkụ nhịgharị mgbọrọgwụ n'ihu ọtụtụ ihe sitere na urmotonic acid na oke egosiri. A tụlekwara mmetụta nke ihe abụọ a maara na-egbochi obere akụkụ: disopyramide na oryzalin. Ihe dị n'ime ya na-egosi ọkọlọtọ e ji atụ akụkụ uto mgbọrọgwụ. Akara kpakpando na-egosi nnukwu ọdịiche na ọgwụgwọ sham (t test, p<0.05). n>19. Ogwe nha = 1 cm. (b) Microtubules cortical na mkpụrụ ndụ epidermal na mpaghara ogologo. E jiri ma ọ bụ na-enweghị 25 μM KAND 11 hụ microtubules na mbadamba MS nwere ma ọ bụ na-enweghị ya site na iji immunohistochemical staining site na iji β-tubulin primary antibodies na Alexa Fluor-conjugated secondary antibodies. Ogwe nha = 10 µm. (c) Nhazi mitotic nke microtubules na mgbọrọgwụ meristem. E jiri immunohistochemical staining hụ microtubules. A gụrụ usoro mitotic, gụnyere mpaghara prophase, spindles, na phragmoplasts, site na onyonyo confocal. Akụ na-egosi nhazi mitotic microtubule. Akara kpakpando na-egosi nnukwu ọdịiche na ọgwụgwọ sham (ule t, p<0.05). n>9. Ogwe nha = 50 µm.
Ọ bụ ezie na Ursa nwere ike imebi ọrụ microtubule, a na-atụ anya na usoro ọrụ ya ga-adị iche na ihe ndị na-eme ka microtubule depolymerizing dị iche. Dịka ọmụmaatụ, oke ọnụọgụ nke ihe ndị na-eme ka microtubule depolymerizing dị ka disopyramide na oryzalin na-akpali mmụba anisotropic nke mkpụrụ ndụ epidermal, ebe KAND 11 anaghị eme ya. Na mgbakwunye, itinye KAND 11 na disopyramide n'otu oge butere mmeghachi omume uto mgbọrọgwụ nke disopyramide kpatara na a hụrụ mgbochi uto nke KAND 11 (Foto S4). Anyị nyochakwara mmeghachi omume nke disopyramide 1-1 (phs1-1) nke nwere mmetụta na KAND 11. phs1-1 nwere mgbanwe isi tubulin kinase na-abụghị canonical ma na-emepụta mgbọrọgwụ dị mkpụmkpụ mgbe ejiri disopyramide9,20 gwọọ ya. Mkpụrụ osisi mutant phs1-1 nke a kụrụ na agar medium nwere KAND 11 nwere mgbọrọgwụ dị mkpụmkpụ yiri nke a kụrụ na disopyramid (Foto S5).
Na mgbakwunye, anyị hụrụ usoro mitotic microtubule, dị ka mpaghara prophase, spindles, na phragmoplasts, na mgbọrọgwụ nke mkpụrụ osisi e ji KAND 11 gwọọ. Dịka ihe ndị a chọpụtara maka CDKB2;1p::CDKB2;1-GUS si dị, a hụrụ mbelata dị ukwuu na ọnụọgụgụ mitotic microtubules (Foto .6c).
Iji mara etu KAND 11 si emetụta mkpụrụ ndụ ihe nketa, anyị jiri KAND 11 gwọọ mkpụrụ ndụ ihe nketa BY-2 ma hụ mmeghachi omume ha. Anyị buru ụzọ tinye KAND 11 na mkpụrụ ndụ BY-2 nke na-egosipụta TagRFP-TUA6, nke na-akpọ microtubules nke ọma, iji chọpụta mmetụta nke KAND 11 na microtubules cortical. E jiri nyocha onyonyo nyochaa njupụta microtubule cortical, nke gosipụtara pasent nke pikselụ cytoskeletal n'etiti pikselụ cytoplasmic. Nsonaazụ nnwale ahụ gosiri na mgbe ejiri 50 μM ma ọ bụ 100 μM KAND 11 gwọọ ya ruo otu awa, 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 ejiri DMSO gwọọ, ruru 1.61 ± 0.34% (Fig. 7a). Nsonaazụ ndị a kwekọrọ na nchọpụta dị na Arabidopsis na ọgwụgwọ KAND 11 na-akpali mwepụ nke obere akpa ume cortical (Foto 6b). Anyị lerekwa akara BY-2 na eriri actin nke GFP-ABD mara mgbe ọgwụgwọ ahụ gasịrị na otu mkpokọta KAND 11 ma hụ na ọgwụgwọ KAND 11 mebiri eriri actin. Ọgwụgwọ na 50 μM ma ọ bụ 100 μM KAND 11 maka awa 1 belatara njupụta eriri actin nke ukwuu ruo 1.20 ± 0.62% ma ọ bụ 0.61 ± 0.26%, n'otu n'otu, ebe njupụta dị na mkpụrụ ndụ DMSO gwọọ bụ 1.69 ± 0.51% (Foto 2). 7b). Nsonaazụ ndị a dị iche na mmetụta nke propyzamide, nke na-emetụtaghị eriri actin, na latrunculin B, onye na-egbochi actin nke na-emetụtaghị obere akpa ume (SI Foto S6). Tinyere nke ahụ, ọgwụgwọ coumamonamide 1, coumamonamide acid 6, ma ọ bụ KAND 11 emetụtaghị obere tubules dị na mkpụrụ ndụ HeLa (SI Figure S7). Ya mere, a kwenyere na usoro ọrụ nke KAND 11 dị iche na nke ndị na-emebi cytoskeleton a maara. Na mgbakwunye, nleba anya anyị nke mkpụrụ ndụ BY-2 ejiri KAND 11 gwọọ gosiri mmalite nke ọnwụ mkpụrụ ndụ n'oge ọgwụgwọ KAND 11 ma gosi na oke nke mkpụrụ ndụ nwụrụ anwụ nwere ntụpọ acha anụnụ anụnụ Evans amụbaghị nke ukwuu mgbe nkeji 30 nke ọgwụgwọ KAND 11 gasịrị, ebe mgbe nkeji 90 nke ọgwụgwọ na 50 μM ma ọ bụ 100 μM KAND gasịrị, ọnụọgụ nke mkpụrụ ndụ nwụrụ anwụ mụbara ruo 43.7% ma ọ bụ 80.1%, n'otu n'otu (Fig. 7c). A na-ejikọta data ndị a ọnụ, data ndị a na-egosi na ursolic acid derivative KAND 11 bụ ihe na-egbochi cytoskeletal nke osisi nwere usoro ọrụ a na-amaghị na mbụ.
KAND na-emetụta obere akpa ume, eriri actin, na ike nke mkpụrụ ndụ ụtaba BY-2. (a) Nlereanya nke obere akpa ume na mkpụrụ ndụ BY-2 n'ihu TagRFP-TUA6. E jiri obere akpa ume na-enyocha mkpụrụ ndụ BY-2 ejiri KAND 11 (50 μM ma ọ bụ 100 μM) ma ọ bụ DMSO gwọọ. E jiri obere akpa ume nke mkpụrụ ndụ 25 nọọrọ onwe ha gbakọọ njupụta obere akpa ume (Tukey HSD test, p< 0.05). Ogwe nha = 10 µm. (b) E jiri ihe onyonyo hụta eriri actin cortical na mkpụrụ ndụ BY-2 n'ihu GFP-ABD2. E jiri ihe nyocha confocal microscopy nyochaa mkpụrụ ndụ BY-2 ejiri KAND 11 (50 μM ma ọ bụ 100 μM) ma ọ bụ DMSO gwọọ ha. E jiri obere ihe nyocha nke mkpụrụ ndụ 25 nọọrọ onwe ha gbakọọ njupụta nke eriri actin cortical. Mkpụrụ akwụkwọ na-egosi nnukwu ọdịiche (ule Tukey HSD, p< 0.05). Ogwe nha = 10 µm. (c) Nleba anya nke mkpụrụ ndụ BY-2 nwụrụ anwụ site na iji Evans blue staining. E jiri igwe onyonyo bright-field nyochaa mkpụrụ ndụ BAND-2 ejiri KAND 11 (50 μM ma ọ bụ 100 μM) ma ọ bụ DMSO gwọọ ha. n=3. Ogwe nha = 100 µm.
Nchọpụta na itinye ngwaahịa ọhụrụ sitere n'okike n'ọrụ emeela ka e nwee ọganihu dị ukwuu n'akụkụ dị iche iche nke ndụ mmadụ, gụnyere ọgwụ na ọrụ ugbo. Emeela nnyocha akụkọ ihe mere eme iji nweta ihe ndị bara uru site na akụrụngwa sitere n'okike. Karịsịa, a maara na actinomycetes bara uru dị ka ọgwụ mgbochi nje maka nematodes n'ihi ikike ha nwere imepụta ọtụtụ metabolites nke abụọ dị ka avermectin, ihe ndu nke ivermectin na bleomycin na ihe ndị sitere na ya, nke a na-eji ọgwụ eme ihe dị ka ihe na-egbochi kansa21,22. N'otu aka ahụ, achọpụtala ọtụtụ ihe ndị na-egbu ahịhịa site na actinomycetes, ụfọdụ n'ime ha ejirila azụmaahịa1,23. Ya mere, a na-ewere nyocha nke actinomycete metabolites iji kewapụ ngwaahịa sitere n'okike na ihe ndị dị ndụ achọrọ dị ka atụmatụ dị irè. N'ọmụmụ ihe a, anyị chọpụtara ihe ọhụrụ, coumamonamide, site na S. werraensis ma mepụta ya nke ọma. Ursonic acid bụ ihe e ji eme ihe nke urbenamide na ihe ndị sitere na ya. Ọ nwere ike ibute mgbagọ mgbọrọgwụ njirimara, gosipụta ọrụ igbu egbu ahịhịa siri ike ruo na nke na-apụtaghị ìhè, ma mebie microtubules osisi ozugbo ma ọ bụ n'ụzọ na-apụtaghị ìhè. Agbanyeghị, usoro ọrụ nke urmotonic acid nwere ike ịdị iche na nke ndị na-egbochi microtubule dị adị, ebe ọ bụ na KAND 11 na-akpaghasịkwa eriri actin ma na-akpata ọnwụ mkpụrụ ndụ, na-atụ aro usoro nhazi nke urmotonic acid na ihe ndị sitere na ya na-emetụta ọtụtụ usoro cytoskeletal.
Nkọwa zuru ezu nke urbenonic acid ga-enyere aka ịghọta usoro ọrụ nke urbenonic acid. Karịsịa, ihe mgbaru ọsọ ọzọ bụ inyocha ikike ursonic acid iji jikọta na obere tubules iji chọpụta ma ursonic acid na ihe ndị sitere na ya na-arụ ọrụ ozugbo na obere tubules ma depolymerize ha, ma ọ bụ ma ihe ha na-eme na-ebute mmebi microtubule. Na mgbakwunye, n'ọnọdụ ebe obere tubules abụghị ihe mgbaru ọsọ kpọmkwem, ịchọpụta ebe ihe na ihe mgbaru ọsọ molekul nke ursonic acid dị na mkpụrụ ndụ osisi ga-enyere aka ịghọtakwu ihe onwunwe nke ihe ndị metụtara ya na ụzọ ndị enwere ike isi melite ọrụ igbu egbu. Nnyocha bioactivity anyị kpughere ikike pụrụ iche nke ursonic acid na uto nke osisi dịka Arabidopsis thaliana, ụtaba na liverwort, ebe ọ dịghị mkpụrụ ndụ E. coli ma ọ bụ HeLa emetụtara. Nsí na-adịghị mma na mkpụrụ ndụ anụmanụ bụ uru nke ihe ndị sitere na ursonic acid ma ọ bụrụ na emepụtara ha dị ka ihe ọkụkụ maka ojiji n'ubi ugbo mepere emepe. N'ezie, ebe ọ bụ na microtubules bụ ihe owuwu a na-ahụkarị na eukaryotes, mgbochi nhọrọ ha na osisi bụ ihe dị mkpa maka ọgwụ ahịhịa. Dịka ọmụmaatụ, a na-eji propyzamide, ihe na-eme ka microtubule depolymerizing nke na-ejikọ kpọmkwem na tubulin ma na-egbochi polymerization, dị ka ọgwụ ahịhịa n'ihi obere nsị ya na mkpụrụ ndụ anụmanụ24. N'adịghị ka disopyramide, benzamides ndị yiri ya nwere ihe dị iche iche a na-elekwasị anya. 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, a na-ejikwa zalilamide dị ka ọgwụ fungicide n'ihi obere phytotoxicity ya25,26,27. Bea a chọtara ọhụrụ na ihe ndị sitere na ya na-egosipụta cytotoxicity nhọrọ megide osisi, mana ọ dị mkpa ịmara na mgbanwe ndị ọzọ nwere ike ịgbanwe nkọwa ha, nwere ike inye ihe mgbakwunye maka njikwa fungi ma ọ bụ oomycetes na-akpata ọrịa.
Njirimara pụrụ iche nke urbenonic acid na ihe ndị sitere na ya bara uru maka mmepe ha dị ka ihe ọkụkụ na ojiji dị ka ngwaọrụ nyocha. A maara mkpa cytoskeleton dị n'ịchịkwa ọdịdị mkpụrụ ndụ osisi nke ọma. Ọmụmụ ihe ndị gara aga egosila na osisi emepụtala usoro dị mgbagwoju anya nke nhazi microtubule cortical site na ijikwa ike microtubule iji chịkwaa morphogenesis nke ọma. Achọpụtala ọtụtụ molekul na-ahụ maka nhazi ọrụ microtubule, nchọpụta ndị metụtara ya ka na-aga n'ihu3,4,28. Nghọta anyị ugbu a banyere ike microtubule na mkpụrụ ndụ osisi anaghị akọwa usoro nke nhazi microtubule cortical nke ọma. Dịka ọmụmaatụ, ọ bụ ezie na disopyramide na oryzalin nwere ike iwepụ microtubules, disopyramide na-akpata nnukwu mgbagwoju anya mgbọrọgwụ ebe oryzalin nwere mmetụta dịtụ obere. Ọzọkwa, mgbanwe na tubulin, nke na-eme ka microtubules kwụsie ike, na-akpatakwa dextrorotation na mgbọrọgwụ, ebe paclitaxel, nke na-emekwa ka ike microtubule kwụsie ike, anaghị eme. Ya mere, ịmụ na ịchọpụta ihe mgbaru ọsọ molekul nke ursolic acid kwesịrị inye nghọta ọhụrụ na nhazi nke microtubules cortical osisi. N'otu aka ahụ, ntụnyere kemịkalụ ndị dị irè n'ịkwalite uto gbagọrọ agbagọ, dịka disopyramide, na kemịkalụ ndị na-adịghị arụ ọrụ nke ọma, dịka oryzalin ma ọ bụ kumamotoric acid, ga-enye ihe ngosi maka otu uto gbagọrọ agbagọ si eme.
N'aka nke ọzọ, nhazi cytoskeletal metụtara nchekwa bụ ihe ọzọ nwere ike ịkọwa cytotoxicity nke ursonic acid. Ọrịa nke nje bacteria ma ọ bụ itinye ihe na-akpalite n'ime mkpụrụ ndụ osisi na-akpata mbibi nke cytoskeleton na ọnwụ mkpụrụ ndụ na-esote29. Dịka ọmụmaatụ, akọpụtala na cryptoxanthin sitere na oomycete na-akpaghasị microtubules na actin filaments tupu ọnwụ mkpụrụ ndụ ụtaba, yiri ihe na-eme na ọgwụgwọ KAND30,31. Myirịta dị n'etiti nzaghachi nchekwa na nzaghachi mkpụrụ ndụ nke ursonic acid kpatara mere ka anyị chee na ha na-akpali usoro mkpụrụ ndụ nkịtị, ọ bụ ezie na mmetụta dị ngwa na ike nke ursonic acid karịa cryptoxanthin pụtara ìhè. Agbanyeghị, ọmụmụ ihe egosila na mmebi nke actin filaments na-akwalite ọnwụ mkpụrụ ndụ na-enweghị ihe ọ bụla, nke anaghị eso mgbe niile na nsogbu microtubule29. Na mgbakwunye, a ka ga-ahụ ma nje bacteria ma ọ bụ ihe na-akpalite na-akpata uto mgbọrọgwụ gbagọrọ agbagọ, dịka ihe ndị sitere na ursonic acid si eme. Ya mere, ihe ọmụma molekul nke na-ejikọ nzaghachi nchebe na cytoskeleton bụ nsogbu mara mma a ga-edozi. Site n'iji ọnụnọ nke obere ihe ndị dị arọ molekul metụtara ursonic acid, yana ọtụtụ ihe ndị sitere na ya nwere ike dị iche iche, ha nwere ike inye ohere iji lekwasị anya na usoro sel ndị a na-amaghị.
Ijikọta ya, nchọpụta na itinye ihe ndị ọhụrụ e ji eme ihe nke na-agbanwe usoro microtubule ga-enye ụzọ dị ike iji dozie usoro molekul dị mgbagwoju anya nke na-adabere na mkpebi ọdịdị mkpụrụ ndụ osisi. N'ọnọdụ a, urmotonic acid e mepụtara n'oge na-adịbeghị anya, nke na-emetụta microtubules na actin filaments ma na-akpata ọnwụ mkpụrụ ndụ, nwere ike inye ohere ịchọpụta njikọ dị n'etiti njikwa microtubule na usoro ndị ọzọ a. Ya mere, nyocha kemịkalụ na bayọlọji site na iji urbenonic acid ga-enyere anyị aka ịghọta usoro iwu molekul nke na-achịkwa cytoskeleton osisi.
Tinye ọgwụ S. werraensis MK493-CF1 n'ime karama Erlenmeyer 500 mL nke nwere 110 mL nke mkpụrụ osisi 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) ihe e si n'ọka wepụta (KOGOSTCH Co., Ltd., Japan), 0.2% (w/v) (NH4)2SO4 na 0.2% CaCO3 n'ime mmiri e si n'ọkpụkpụ gbapụta. (pH 7.4 tupu a mịchaa ya). E tinyere mkpụrụ osisi ndị ahụ n'ime ihe e ji atụ ntụ ntụ (180 rpm) na 27°C ruo ụbọchị abụọ. Ịkụ mkpụrụ site na iji usoro ịgba mmiri siri ike. E tinyere mkpụrụ osisi ahụ (7 ml) n'ime karama K-1 500 ml nke nwere 40 g nke ihe eji emepụta ihe nke gụnyere 15 g nke ọka bali a mịrị amị (MUSO Co., Ltd., Japan) na 25 g nke mmiri e tinyere ion (pH anaghị agbanwe tupu a mịchaa ya). E mere ịgbakọ ya na 30°C n'ọchịchịrị ruo ụbọchị 14. E jiri 40 ml/karama EtOH wepụta ihe a na-agbakọ ihe ahụ ma tinye ya na centrifuge (1500 g, 4°C, nkeji 10). E jiri ngwakọta nke 10% MeOH/EtOAc wepụta ihe na-agbakọ ihe (60 ml). A gbapụrụ oyi akwa organic ahụ n'okpuru nrụgide dị ala iji nweta ihe fọdụrụ (59.5 mg), nke e tinyere HPLC na mkpụcha gradient (nkeji 0–10: 90%) na kọlụm nkebi azụ (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × ogologo 250 mm) H2O/CH3CN, nkeji 10–35: 90% H2O/CH3CN ruo 70% H2O/CH3CN (gradient), nkeji 35–45: 90% H2O/EtOH, nkeji 45–155: 90% H2O/EtOH ruo 100% EtOH (gradient (gradient), nkeji 155–200: 100% EtOH) na ọnụego mmiri nke 1.5 ml/min, e kewapụrụ coumamonamide (1, 36.0 mg) dị ka ntụ ntụ ọcha 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]+ uru agbakọrọ: 141.0659, uru a tụrụ: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
E si n'ụlọ ọrụ Arabidopsis Biological Resource Center (ABRC) nweta mkpụrụ Columbia (Col-0) ma nye ikike maka ojiji nyocha. A na-agbasa ma na-elekọta mkpụrụ Col-0 n'okpuru ọnọdụ ụlọ nyocha anyị ma jiri ya dị ka osisi Arabidopsis nke ụdị ọhịa. E tinyere mkpụrụ Arabidopsis n'elu ma kụọ ya n'ime ọkara ike Murashige na Skoog nke 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 °C na ìhè na-adịgide adịgide. T. Hashimoto (Nara Institute of Science and Technology) nyere mkpụrụ nke phs1-1 mutant.
T. Hashimoto (Nara Institute of Science and Technology) nyere mkpụrụ nke ụdị SR-1 ma jiri ya mee ihe dị ka osisi ụtaba ọhịa. E tinyere mkpụrụ ụtaba n'elu ma tinye ya na mmiri dị ọcha ruo abalị atọ iji mee ka ọ pụta ìhè, wee tinye ya na mmiri ọkara ike nke nwere 2% sucrose, 0.05% (w/v) MES, na 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige. na Skoog medium) nwere pH 5.7 ma tinye ya na 23°C n'okpuru ìhè na-adịgide adịgide.
T. Kohchi (Mahadum Kyoto) nyere Strain Tak-1, e jikwa ya mee ihe dị ka ihe nnwale ọkọlọtọ maka ọmụmụ ihe liverwort. E si n'osisi ndị a kụrụ ahịhịa nweta Gemma wee tee ya na Gamborg B5 medium (Fujifilm Wako Pure Chemical) nke nwere 1% sucrose na 0.3% gellan gum ma tinye ya na okpomọkụ 23°C n'okpuru ìhè na-aga n'ihu.
S. Hasezawa (Mahadum Tokyo) nyere mkpụrụ ndụ ụtaba BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2). A gwakọtara mkpụrụ ndụ BY-2 ugboro 95 na Linsmeier na Skoog gbanwere agbanwe ma tinye 2,4-dichlorophenoxyacetic acid 32 kwa izu. A gwakọtara nkwụsịtụ mkpụrụ ndụ ahụ na rotary shaker na 130 rpm na 27°C n'ọchịchịrị. Sachaa mkpụrụ ndụ ahụ ugboro 10 nke olu ọhụrụ wee weghachite ya n'otu etiti ahụ. E mepụtara ahịrị mkpụrụ ndụ BY-2 transgenic nke na-egosipụta akara microtubule TagRFP-TUA6 nke ọma ma ọ bụ akara actin filament GFP-ABD2 n'okpuru cauliflower mosaic virus 35S promoter dịka akọwara33,34,35. Enwere ike idobe ma mekọrịta ahịrị mkpụrụ ndụ ndị a site na iji usoro ndị yiri nke ejiri maka ahịrị mkpụrụ ndụ BY-2 mbụ.
E tinyere mkpụrụ ndụ HeLa n'ime ihe e ji eme ka anụ ahụ dị ndụ nke Dulbecco gbanwere (DMEM) (Life Technologies) nke e tinyere 10% serum anụ ehi nwa ebu n'afọ, penicillin 1.2 U/ml, na 1.2 μg/ml streptomycin n'ime ihe na-eme ka anụ ahụ dị ọkụ nke 37°C nke nwere 5% CO2.
Emere nnwale niile a kọwara n'akwụkwọ a dịka iwu na ntuziaka nchekwa bio nke ndị Japan si dị.
A gbazere ngwakọta ndị ahụ na dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) dị ka ihe ngwọta ngwaahịa ma gwakọta ya na MS medium maka Arabidopsis na ụtaba ma ọ bụ Gamborg B5 medium maka liverwort. Maka nnwale mgbochi uto mgbọrọgwụ, a kụrụ ihe karịrị mkpụrụ iri kwa efere na agar medium nwere ihe ndị a tụrụ aro ma ọ bụ DMSO. A kpọchiri mkpụrụ n'ime ụlọ uto ruo ụbọchị 7. A sere foto mkpụrụ ndị ahụ ma tụọ ogologo mgbọrọgwụ ha. Maka nnwale germination Arabidopsis, a kụrụ mkpụrụ 48 kwa efere na agar medium nwere 200 μM compound ma ọ bụ DMSO. A kụrụ mkpụrụ Arabidopsis n'ime ụlọ uto ma gụọ ọnụọgụ nke mkpụrụ ndị a kụrụ etolite ụbọchị 7 mgbe ha chara (dag). Maka nnwale germination ụtaba, a kụrụ mkpụrụ 24 kwa efere na agar medium nwere 200 μM KAND ma ọ bụ DMSO. A kụrụ mkpụrụ ụtaba n'ime ụlọ uto ma gụọ ọnụọgụ nke mkpụrụ ndị a kụrụ etolite mgbe ụbọchị 14 gachara. Maka nnwale mgbochi uto nke imeju, e tinyere akwa embrayo itoolu site na efere ọ bụla n'elu ihe e ji agar mee nke nwere oke KAND ma ọ bụ DMSO gosiri ma tinye ha n'ime ụlọ uto ruo ụbọchị iri na anọ.
Jiri mkpụrụ osisi e ji 5 mg/ml propidium iodide (PI) mee ka ha dị nro iji hụ nhazi mgbọrọgwụ nke meristem. E ji igwe onyonyo fluorescence mee ihe site na iji igwe onyonyo nyocha laser confocal TCS SPE (Leica Microsystems).
E mere β-glucuronidase (GUS) ka mgbọrọgwụ ndị ahụ dị ọcha dịka usoro Malami na Benfey36 si kọwaa si dị. E tinyere mkpụrụ osisi n'ime 90% acetone n'otu abalị, tinye 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid n'ime ihe nchekwa GUS ruo otu awa ma tinye ha n'ime mmiri chloraldehyde hydrated. (8 g chloral hydrate, 2 ml mmiri na 1 ml glycerol) ma hụ ha site na iji microscope Axio Imager M1 (Carl Zeiss) na-enyocha ha site na differential nnyonye anya.
A tụrụ akụkụ mgbọrọgwụ n'elu mkpụrụ osisi dị ụbọchị asaa a kụrụ n'elu efere ndị a kwụ ọtọ. Tụọ akụkụ mgbọrọgwụ ahụ site na ntụziaka nke vektọ ike ndọda dịka akọwara na nzọụkwụ nke isii.
A hụrụ nhazi nke obere akpa ume dị n'ime cortical dịka akọwara ya, yana obere mgbanwe na protocol 37. Ejiri antibody anti-β-tubulin (KMX-1, Merk Millipore: MAB3408) na Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) mee ihe dị ka ọgwụ mgbochi mbụ na nke abụọ na 1:1000 na 1:100 dilutions, n'otu n'otu. E ji igwe nyocha laser confocal TCS SPE (Leica Microsystems) nweta onyonyo fluorescence. Nweta onyonyo Z-stack ma mepụta atụmatụ ike kachasị dịka ntuziaka onye nrụpụta si dị.
E jiri ngwa 8 (Dojindo) mee nnwale mmụba nke mkpụrụ ndụ HeLa dịka ntuziaka nke onye nrụpụta si dị.
A nyochachara uto nke E. coli DH5α site na iji igwe spectrophotometer tụọ njupụta sel na ọdịbendị site na iji igwe spectrophotometer na 600 nm (OD600).
A hụrụ nhazi cytoskeletal na mkpụrụ ndụ BY-2 transgenic site na iji igwe microscope fluorescence nke nwere ngwaọrụ nyocha confocal CSU-X1 (Yokogawa) na igwefoto sCMOS (Zyla, Andor Technology). E jiri nyocha onyonyo nyochaa njupụta cytoskeletal, nke gosipụtara pasent nke cytoskeletal pixels n'etiti pikselụ cytoplasmic na onyonyo confocal site na iji ngwanrọ ImageJ dịka akọwara38,39.
Iji chọpụta ọnwụ mkpụrụ ndụ na mkpụrụ ndụ BY-2, e tinyere 0.05% Evans blue n'ime otu akụkụ nke ihe mkpuchi mkpụrụ ndụ ahụ ruo nkeji iri n'ọnọdụ okpomọkụ ụlọ. Nchacha mkpụrụ ndụ nwụrụ anwụ nke Evans blue dabere na mpụta nke agba sitere na mkpụrụ ndụ ndị dị ndụ site na akpụkpọ ahụ plasma na-adịghị agwụ agwụ40. A hụrụ mkpụrụ ndụ ndị nwere ntụpọ site na iji igwe microscope na-egbuke egbuke (BX53, Olympus).
A zụlitere mkpụrụ ndụ HeLa na DMEM tinyere 10% FBS n'ime ihe na-eme ka mmiri dị n'ime ya na 37°C na 5% CO2. E jiri 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), ma ọ bụ 100 ng/ml Nocodmaze (Sigma) gwọọ mkpụrụ ndụ ahụ ruo awa isii na 37°C. E jiri MetOH dozie mkpụrụ ndụ ahụ ruo nkeji iri wee jiri acetate mee ihe ruo nkeji ise n'ọnọdụ okpomọkụ ụlọ. E tinyere β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) gwakọtara mkpụrụ ndụ ndị a kapịrị ọnụ na 0.5% BSA/PBS ruo awa abụọ, saa ya ugboro atọ na TBST, wee jiri ọgwụ mgbochi ewu Alexa Fluor mee ihe. 488 otu awa. – Òké IgG (Thermo Fisher Scientific: A11001) na 15 ng/ml 4′, 6-diamidino-2-phenylindole (DAPI) agwakọtara na 0.5% BSA/PBS. Mgbe ejiri TBST saa ya ugboro atọ, a hụrụ mkpụrụ ndụ ndị nwere ntụpọ na mikroskopu Nikon Eclipse Ti-E nke a tụgharịrị agbanwe. E ji kamera Hamamatsu ORCA-R2 CCD oyi see foto site na iji ngwanrọ MetaMorph (Ngwaọrụ Molecular).
Oge ozi: Jun-17-2024