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Kupezeka ndi kugwiritsa ntchito kopindulitsa kwa zinthu zachilengedwe kungathandize kusintha moyo wamunthu.Mankhwala oletsa kukula kwa zomera amagwiritsidwa ntchito kwambiri ngati mankhwala ophera udzu.Chifukwa cha kufunikira kogwiritsa ntchito mitundu yosiyanasiyana ya herbicides, pakufunika kuzindikira mankhwala omwe ali ndi njira zatsopano zogwirira ntchito.Mu phunziro ili, tapeza buku la N -alkoxypyrrole, coumamonamide, kuchokera ku Streptomyces werraensis MK493-CF1 ndikukhazikitsa njira yonse yophatikizira.Kupyolera mu kuyesa kwachilengedwe kwachilengedwe, tidapeza kuti urs-monoamic acid ndi gawo lapakati la urs-monoamide komanso kuthekera.choletsa kukula kwa zomera.Kuonjezera apo, tapanga mitundu yosiyanasiyana ya urbenonic acid, kuphatikizapo urbenyloxy derivative (UDA), yomwe imakhala ndi ntchito ya herbicidal yambiri popanda kusokoneza kukula kwa maselo a HeLa.Tinapezanso kuti zotumphukira za urmotonic acid zimasokoneza ma microtubules a zomera;kuonjezera apo, KAND imakhudza ma actin filaments ndipo imayambitsa kufa kwa selo;Zotsatira zambirizi zimasiyana ndi zomwe zimadziwika ndi ma microtubule inhibitors ndipo zimawonetsa njira yatsopano yochitira ursonic acid, yomwe imayimira mwayi wofunikira pakupanga mankhwala atsopano opha udzu.
Kupezeka ndi kugwiritsa ntchito kwachilengedwe kopindulitsa ndi zotuluka zake ndi njira yopititsira patsogolo moyo wamunthu.Ma metabolites achiwiri opangidwa ndi tizilombo tating'onoting'ono, zomera ndi tizilombo tapangitsa kuti pakhale chitukuko chachikulu chamankhwala ndi ulimi.Maantibayotiki ambiri ndi mankhwala oletsa khansa ya m'magazi apangidwa kuchokera kuzinthu zachilengedwe.Komanso, mitundu yosiyanasiyana yamankhwala ophera tizilombo, fungicides ndi herbicides amatengedwa kuchokera kuzinthu zachilengedwezi kuti azigwiritsa ntchito paulimi.Makamaka, mankhwala ophera udzu ndi zida zofunika zowonjezeretsa zokolola muulimi wamakono, ndipo mitundu yosiyanasiyana ya mankhwala imagwiritsidwa ntchito kale pamalonda.Njira zingapo zama cell m'zomera, monga photosynthesis, amino acid metabolism, cell wall synthesis, kuwongolera kwa mitosis, phytohormone signing, kapena protein synthesis, zimatengedwa ngati chandamale cha mankhwala ophera udzu.Mankhwala omwe amalepheretsa kugwira ntchito kwa microtubule ndi gulu lodziwika bwino la herbicides lomwe limakhudza kukula kwa mbewu pokhudza mitotic regulation2.
Ma microtubules ndi zigawo za cytoskeleton ndipo zimasungidwa kwambiri m'maselo a eukaryotic.Tubulin heterodimer imakhala ndi α-tubulin ndi β-tubulin yomwe imapanga ma microtubule protofilaments, ndi ma protofilaments 13 omwe amapanga mawonekedwe a cylindrical.Ma Microtubules amatenga maudindo angapo m'maselo a zomera, kuphatikizapo kudziwa mawonekedwe a maselo, kugawanika kwa maselo, ndi kayendedwe ka intracellular3,4.Maselo a zomera amakhala ndi ma microtubules pansi pa interphase plasma nembanemba, ndipo otchedwa cortical microtubules amaganiziridwa kuti amayang'anira bungwe la cellulose microfibrils kupyolera mu malamulo a cellulose synthase complexes4,5.Ma Cortical microtubules a muzu epidermal cell, omwe amapezeka m'dera la kufalikira mwachangu kwa nsonga ya muzu, amakhala mozungulira, ndipo ma cellulose microfiber amatsata ma microtubules ndikuchepetsa njira yakukulira kwa maselo, potero kulimbikitsa kukula kwa maselo a anisotropic.Choncho, ntchito ya microtubule imagwirizana kwambiri ndi morphology ya zomera.Ma amino acid olowa m'malo mwa jini ophatikizira tubulin amachititsa kusokonekera kwa ma cortical microtubule array ndi kukula kumanzere kapena kumanja ku Arabidopsis 6,7.Mofananamo, kusintha kwa mapuloteni opangidwa ndi ma microtubule omwe amayendetsa mphamvu ya microtubule kungayambitsenso kusokonezeka kwa mizu8,9,10,11,12,13.Kuonjezera apo, chithandizo ndi microtubule-kusokoneza herbicides monga disopyramide, yomwe imadziwikanso kuti pretilachlor, imayambitsanso kumanzere kwa mizu ya oblique kukula14.Deta iyi ikuwonetsa kuti kuwongolera bwino ntchito kwa ma microtubule ndikofunikira kuti mudziwe komwe mbewu ikukulira.
Mitundu yosiyanasiyana ya microtubule inhibitors yapezedwa, ndipo mankhwalawa athandizira kwambiri kafukufuku wa cytoskeletal, komanso ulimi ndi mankhwala2.Makamaka, oryzalin, dinitroaniline mankhwala, disopyramide, mankhwala okhudzana ndi benzamide, ndi ma analogi awo amatha kulepheretsa ntchito ya microtubule ndipo potero amalepheretsa kukula kwa zomera.Chifukwa chake, amagwiritsidwa ntchito kwambiri ngati mankhwala ophera udzu.Komabe, popeza ma microtubules ndi gawo lofunikira la maselo a zomera ndi zinyama, ma microtubule inhibitors ambiri ndi cytotoxic ku mitundu yonse ya maselo.Choncho, ngakhale kuti amagwiritsidwa ntchito ngati mankhwala a herbicides, mankhwala ochepa a antimicrotubule amagwiritsidwa ntchito pazinthu zothandiza.
Streptomyces ndi mtundu wa banja la Streptomyces, lomwe limaphatikizapo mabakiteriya a aerobic, gram-positive, filamentous ndipo amadziwika kuti amatha kupanga ma metabolites ambiri achiwiri.Chifukwa chake, imatengedwa kuti ndi imodzi mwazinthu zofunika kwambiri zopangira zinthu zachilengedwe zatsopano.Pakafukufuku wamakono, tapeza gulu latsopano lotchedwa coumamonamide, lomwe linali lokhalokha ku Streptomyces werraensis MK493-CF1 ndi S. werraensis ISP 5486. Pogwiritsa ntchito kuwunika kowoneka bwino komanso kusanthula kwathunthu, mawonekedwe a coumamonamide anali odziwika komanso ma skeleton ake apadera a N-alkoxypyrrole. zinatsimikizika.kaphatikizidwe.Ursmonic acid, wopangidwa wapakatikati wa ursmoamide ndi zotuluka zake, adapezeka kuti amalepheretsa kukula ndi kumera kwa chomera chodziwika bwino cha Arabidopsis thaliana.Pakafukufuku wa ubale wamachitidwe, tidapeza kuti kaphatikizidwe ka C9 kosinthidwa kukhala ursonic acid, yotchedwa nonyloxy derivative of ursonic acid (KAND), imathandizira kwambiri kulepheretsa kukula ndi kumera.Makamaka, cholepheretsa kukula kwa zomera chomwe chinangopezeka kumene chinakhudzanso kukula kwa fodya ndi chiwindi ndipo sichinali cytotoxic kwa mabakiteriya kapena maselo a HeLa.Komanso, zotumphukira zina za urmotonic acid zimapangitsa kuti pakhale mizu yolakwika ya phenotype, kutanthauza kuti zotumphukira izi zimakhudza mwachindunji kapena molakwika ma microtubules.Mogwirizana ndi lingaliro ili, zomwe tikuwona za ma microtubules olembedwa ndi immunohistochemically kapena ndi mapuloteni a fulorosenti akuwonetsa kuti chithandizo cha KAND chimachotsa ma microtubules.Kuphatikiza apo, mankhwala opangidwa ndi kumamotonic acid amasokoneza ma microfilaments a actin.Chifukwa chake, tapeza choletsa chatsopano chakukula kwa zomera chomwe njira yake yapadera imaphatikizira kuwonongeka kwa cytoskeleton.
Strain MK493-CF1 idalekanitsidwa ndi dothi ku Shinagawa-ku, Tokyo.Strain MK493-CF1 idapanga stromal mycelium yanthambi yabwino.Kutsatira pang'ono kwa jini ya 16S ribosomal RNA (1422 bp) kudatsimikiziridwa.Vutoli ndi lofanana kwambiri ndi S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: kupsyinjika komweko, 99.93%).Malingana ndi zotsatirazi, zinatsimikiziridwa kuti vutoli linali logwirizana kwambiri ndi mtundu wa S. werraensis.Chifukwa chake, tinatcha mtundu uwu S. werraensis MK493-CF1.S. werraensis ISP 5486T imapanganso mankhwala omwewo a bioactive.Popeza panalibe kafukufuku woyambirira wopeza zinthu zachilengedwe kuchokera ku tizilombo tating'onoting'ono, kafukufuku winanso wamankhwala adachitika.Pambuyo pa kulima S. werraensis MK493-CF1 pa barele sing'anga ndi kuwira kolimba kwa boma pa 30 ° C kwa masiku 14, sing'angayo inatulutsidwa ndi 50% EtOH.60 ml ya zitsanzo anaumitsidwa kupeza 59.5 mg wa yaiwisi Tingafinye.Chotsitsacho chinasinthidwa ndi gawo la HPLC kuti apereke N-methoxy-1H-pyrrole-2-carboxamide (1, yotchedwa coumamonamide, 36.0 mg).Chiwerengero chonse cha 1 ndi pafupifupi 60% ya zotulutsa zopanda pake.Chifukwa chake, tidaganiza zophunzira mwatsatanetsatane za Kumamotoamide 1.
Coumamonamide 1 ndi ufa woyera wa amorphous ndi high resolution mass spectrometry (HRESIMS) imatsimikizira C6H8N2O2 (Mkuyu 1).Chidutswa cha C2-substituted pyrrole cha mankhwalawa chimadziwika ndi δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH mu 1H NMR spectrum 5Hz: 4. , H-5) ndi δH 6.78 (1H, d, J = 2.5 Hz, H-6), ndi sipekitiramu ya 13C NMR ikuwonetsa kukhalapo kwa ma atomu anayi a sp2 carbon.Kukhalapo kwa gulu la amide pa malo a C2 kunayesedwa ndi mgwirizano wa HMBC kuchokera ku C-3 proton kupita ku amide carbonyl carbon pa δC 161.1.Kuonjezera apo, 1 H ndi 13 C NMR pamwamba pa δH 4.10 (3H, S) ndi δC 68.3 amasonyeza kukhalapo kwa magulu a N-methoxy mu molekyulu.Ngakhale malo olondola a gulu la methoxy anali asanadziwikebe pogwiritsa ntchito kusanthula kwa spectroscopic monga kuwonjezereka kwa mawonekedwe a spectroscopy ndi chidule cha nyukiliya Overhauser (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide anakhala woyamba phungu pawiri.
Kudziwa dongosolo lolondola la 1, kuphatikizika kokwanira kunachitika (mkuyu 2a).Kuchiza kwa malonda a 2-aminopyridine 2 ndi m-CPBA kunapangitsa kuti N-oxide 3 yofananira ikhale ndi zokolola zambiri.Pambuyo pa 2-aminoazidation ya 2, cyclocondensation reaction yofotokozedwa ndi Abramovich idachitika mu benzene pa 90 ° C kuti ipeze 1-hydroxy-1H-pyrrole-2-carbonitrile 5 mu magalamu.Kuthamanga 60% (magawo awiri).15, 16.Methylation ndi hydrolysis ya 4 ndiye anapereka 1-methoxy-1H-pyrrole-2-carboxylic acid (otchedwa "cumotonic acid", 6) mu zokolola zabwino (70%, masitepe awiri).Pomaliza, amidation kudzera pa asidi chloride wapakatikati 6 kugwiritsa ntchito ammonia yamadzi ammonia kunapatsa Kumamoto amide 1 mu zokolola za 98%.Deta yonse ya spectral ya 1 yopangidwa inali yofanana ndi 1 yokha, kotero kuti mapangidwe a 1 adatsimikiziridwa;
General kaphatikizidwe ndi kusanthula kwachilengedwenso ntchito urbenamide ndi urbenic asidi.(a) Total synthesis ya Kumamoto amide.(b) Mbande zamasiku asanu ndi aŵiri zakuthengo za mtundu wa Arabidopsis Columbia (Col) zinabzalidwa pa mbale za Murashige ndi Skoog (MS) zomwe zili ndi coumamonamide 6 kapena coumamonamide 1 pazigawo zosonyezedwa.Kukula kwa bar = 1 cm.
Choyamba, tidawunika zochitika zachilengedwe za urbenamide ndi zapakati zake kuti athe kusintha kukula kwa mbewu.Tidawonjeza mitundu yosiyanasiyana ya ursmonamide 1 kapena ursmonic acid 6 ku MS agar medium ndi mbande za Arabidopsis thaliana panjira iyi.Mayeserowa adawonetsa kuti kuchuluka kwakukulu (500 μM) kwa 6 kumalepheretsa kukula kwa mizu (mkuyu 2b).Kenako, tidapanga zotumphukira zosiyanasiyana polowa m'malo mwa N1 malo 6 ndikuchita maphunziro a ubale-zochitika (kaphatikizidwe ka analogi akufotokozedwa mu Supporting Information (SI)).Mbande za Arabidopsis zidakulitsidwa pa sing'anga yokhala ndi 50 μM zotumphukira za ursonic acid, ndipo kutalika kwa mizu kumayesedwa.monga zikuwonetsedwa pachithunzichi.Monga momwe tawonetsera mu Zithunzi 3a, b, ndi S1, ma coumamo acid ali ndi utali wosiyana wa unyolo wa alkoxy (9, 10, 11, 12, ndi 13) kapena unyolo waukulu wa alkoxy (15, 16, ndi 17) pa malo a N1.Zotulutsa zinawonetsa kulepheretsa kwakukulu kwa mizu.Komanso, tinapeza kuti ntchito 200 μM 10, 11, kapena 17 inhibited kumera (mkuyu. 3c ndi S2).
Kuphunzira za ubale-ntchito ya Kumamoto amide ndi mankhwala okhudzana nawo.(a) Kapangidwe ndi kaphatikizidwe ka ma analogues.(b) Kuyeza kutalika kwa mizu ya mbande zamasiku 7 zomwe zabzalidwa pa MS medium kapena popanda 50 μM zotumphukira za coumamonamide.Nyenyezi zimawonetsa kusiyana kwakukulu ndi chithandizo chabodza (t test, p<0.05]n >18. Deta imawonetsedwa ngati ± SD.nt amatanthauza "sanayesedwe" chifukwa mbewu zopitilira 50% sizinamere.(c) Kuchulukitsidwa kwa kameredwe ka mbeu zothiriridwa zosungidwa kwa masiku 7 mu MS medium ndi kapena popanda 200 μM coumamonamide ndi mankhwala ogwirizana nawo.Nyenyezi zimasonyeza kusiyana kwakukulu ndi chithandizo cha sham (chi-square test).n = 96.
Chochititsa chidwi n'chakuti, kuwonjezera kwa maunyolo a alkyl aatali kuposa C9 kunachepetsa ntchito yolepheretsa, kutanthauza kuti mankhwala okhudzana ndi kumamotoic acid amafuna maunyolo am'mbali a kukula kwake kuti awonetsere zochitika zawo zamoyo.
Chifukwa kusanthula kwa ubale-ntchito kunawonetsa kuti C9 idasinthidwa kukhala ursonic acid komanso chotumphukira cha nonyloxy cha ursonic acid (pambuyo pake chomwe chimatchedwa KAND 11) chinali choletsa kukula kwa zomera, tinachita zambiri za KAND 11. Chithandizo cha Arabidopsis ndi 50 μM KAND 11 pafupifupi kulepheretsa kumera kwathunthu, pomwe ndende zotsika (40, 30, 20, kapena 10 μM) za KAND 11 zimalepheretsa kukula kwa mizu m'njira yodalira mlingo (mkuyu 4a, b).Kuti tiwone ngati KAND 11 imakhudza mayendedwe a mizu, tidapenda mizu yokhala ndi propidium iodide (PI) ndikuyesa kukula kwa dera la meristem.Kukula kwa meristem ya mbande wakula pa sing'anga munali 25 μM KAND-11 anali 151.1 ± 32.5 μm, pamene kukula kwa meristem wa mbande anakulira pa ulamuliro sing'anga munali DMSO anali 264.7 ± 30,8 μm (mkuyu. 4c, d) , zomwe zikuwonetsa kuti KAND-11 imabwezeretsa ntchito zama cell.kufalikira.Root meristem.Mogwirizana ndi izi, chithandizo cha KAND 11 chinachepetsa chiwerengero cha ma cell division marker CDKB2; 1p :: CDKB2; 1-GUS chizindikiro mu mizu meristem (Mkuyu 4e) 17.Zotsatira izi zikuwonetsa kuti KAND 11 imalepheretsa kukula kwa mizu pochepetsa ntchito yochulukitsa maselo.
Kuwunika kwa inhibitory zotsatira za urbenonic acid zotumphukira (zotengera za urbenyloxy) pakukula.(a) mbande zakuthengo zamasiku 7 zakutchire za Col zomwe zabzalidwa m'mbale za MS zomwe zikuwonetsa kuti KAND 11. Sikero = 1 cm.(b) Chiwerengero cha kutalika kwa mizu.Malembo amawonetsa kusiyana kwakukulu (mayeso a Tukey HSD, p<0.05]n >16. Deta imawonetsedwa ngati ± SD.(c) Confocal microscopy ya mtundu wa propidium iodide wothimbirira wamtundu wa Col mizu yomwe imamera pamasamba a MS okhala kapena opanda 25 μM KAND 11. Mabulaketi oyera amawonetsa mizu meristem.Scale bar = 100 µm.(d) Kuwerengera kwa kukula kwa mizu ya meristem (n = 10 mpaka 11).Kusiyana kwa ziwerengero kunatsimikiziridwa pogwiritsa ntchito t-test (p<0.05]Mipiringidzo imayimira kukula kwa meristem.(e) Differential interference difference (DIC) microscopy ya root meristem yomwe ili ndi CDKB2 construct;1pro: CDKB2;1-GUS wothimbirira komanso wothimbirira pa mbande zamasiku asanu zakubadwa zomwe zabzalidwa pa mbale za MS zoyesera kapena popanda 25 µM KAND.
Phytotoxicity ya KAND 11 inayesedwanso pogwiritsa ntchito chomera china cha dicotyledonous, fodya (Nicotiana tabacum), ndi zamoyo zazikulu zamtundu wa nthaka, liverwort (Marchantia polymorpha).Monga mu nkhani ya Arabidopsis, fodya SR-1 mbande wamkulu pa sing'anga munali 25 μM KAND 11 opangidwa wamfupi mizu (mkuyu. 5a).Kuphatikiza apo, mbewu 40 mwa 48 zidamera m'mbale zomwe zili ndi 200 μM KAND 11, pomwe mbewu zonse 48 zidamera pazankhani zoseketsa, kuwonetsa kuti kuchuluka kwa KAND kunali kofunikira (p.<0.05;chi test -square) amalepheretsa kumera kwa fodya.(Mku. 5b).Kuonjezera apo, chiwerengero cha KAND 11 chomwe chinalepheretsa kukula kwa bakiteriya mu chiwindi cha chiwindi chinali chofanana ndi ndende yothandiza mu Arabidopsis (mkuyu 5c).Zotsatirazi zikuwonetsa kuti KAND 11 imatha kulepheretsa kukula kwa mbewu zosiyanasiyana.Kenako tinafufuza za cytotoxicity yotheka ya mankhwala okhudzana ndi chimbalangondo cha monoamide mu zamoyo zina, zomwe ndi maselo a HeLa aumunthu ndi Escherichia coli strain DH5α, monga oimira maselo apamwamba a nyama ndi mabakiteriya, motsatira.M'mayesero owonjezereka a maselo, tinawona kuti coumamonamide 1, coumamonamidic acid 6, ndi KAND 11 sizinakhudze kukula kwa maselo a HeLa kapena E. coli pamagulu a 100 μM (Mkuyu 5d, e).
Kuletsa kukula kwa KAND 11 mu zamoyo zomwe si Arabidopsis.A Mbalame za MS zomwe zili ndi 200 μM KAND 11. ( c) Masamba amtundu wa Tak-1 wamtundu wa Tak-1 wa nyama zakutchire wa masabata awiri omwe amakula pa mbale za Gamborg B5 zomwe zimasonyeza KAND 11. Mivi yofiira imasonyeza spores zomwe zinasiya kukula mkati mwa masabata awiri nthawi.(d) Kuyesa kuchuluka kwa ma cell a ma cell a HeLa.Chiwerengero cha maselo otheka chinayesedwa pakapita nthawi pogwiritsa ntchito zida zowerengera ma cell 8 (Dojindo).Monga kuwongolera, ma cell a HeLa adathandizidwa ndi 5 μg/ml actinomycin D (Act D), yomwe imaletsa kulembedwa kwa RNA polymerase ndikuyambitsa kufa kwa cell.Kusanthula kunachitika katatu.(e) E. coli cell proliferation assay.Kukula kwa E. koli kunawunikidwa poyesa OD600.Monga kuwongolera, ma cell adathandizidwa ndi 50 μg/ml ampicillin (Amp), yomwe imalepheretsa kaphatikizidwe ka khoma la bakiteriya.Kusanthula kunachitika katatu.
Kuti tidziwe momwe cytotoxicity imagwirira ntchito chifukwa cha mankhwala okhudzana ndi uramide, tidasanthulanso zotumphukira za urbenic acid zomwe zimalepheretsa pang'ono.monga zikuwonetsedwa pachithunzichi.Monga momwe zikusonyezedwera mu Zithunzi 2b, 6a, mbande zomwe zabzalidwa pa mbale za agar zomwe zimakhala ndi madzi ambiri (200 μM) za urmotonic acid 6 zimatulutsa mizu yaifupi komanso yokhotakhota kumanzere (θ = - 23.7 ± 6.1), pamene mbande zomwe zabzalidwa pazitsulo zowongolera, mbande zotulutsa pafupifupi mizu yowongoka (θ = - 3.8 ± 7.1).Kukula kowoneka bwino kumeneku kumadziwika chifukwa cha kusagwira ntchito kwa ma cortical microtubules14,18.Mogwirizana ndi izi, microtubule-destabilizing mankhwala disopyramide ndi oryzalin anachititsa muzu ofanana tilting pansi pa kukula kwathu mikhalidwe (Figs. 2b, 6a).Panthawi imodzimodziyo, tinayesa zotumphukira za urmotonic acid ndikusankha zingapo zomwe, panthawi zina, zidapangitsa kuti mizu ya oblique ikule.Mankhwala 8, 9, ndi 15 anasintha njira ya kukula kwa mizu pa 75 μM, 50 μM, ndi 40 μM, motero, kusonyeza kuti mankhwalawa amatha kusokoneza ma microtubules (mkuyu 2b, 6a).Tidayesanso chotengera champhamvu kwambiri cha ursolic acid, KAND 11, pamlingo wocheperako (15 µM) ndipo tidapeza kuti kugwiritsa ntchito KAND 11 kumalepheretsa mizu kukula komanso komwe komwe mizu sikuli kofanana, ngakhale imakonda kutsetsereka kumanzere. Chithunzi C3)..Chifukwa kuchuluka kwa mankhwala owononga ma microtubule nthawi zina kumalepheretsa kukula kwa mbewu m'malo mopangitsa kuti mizu ipendekeke, tidawonanso kuthekera kwakuti KAND 11 imakhudza ma microtubules poyang'ana ma cortical microtubules m'ma cell epidermal cell.Immunohistochemistry pogwiritsa ntchito ma anti-β-tubulin antibodies m'maselo a epidermal a mizu ya mbande yothandizidwa ndi 25 μM KAND 11 inasonyeza kutha kwa pafupifupi ma microtubules onse a cortical m'maselo a epidermal m'dera la elongation (mkuyu 6b).Zotsatirazi zikuwonetsa kuti kumamotonic acid ndi zotumphukira zake amachita mwachindunji kapena mosalunjika pa ma microtubules kuti awasokoneze komanso kuti mankhwalawa ndi ma microtubule inhibitors.
Ursonic acid ndi zotuluka zake zimasintha ma cortical microtubules mu Arabidopsis thaliana.(a) Kupendekera kwa mizu kumayezedwa pamaso pa zotumphukira zosiyanasiyana za urmotonic acid pazomwe zawonetsedwa.Zotsatira za mankhwala awiri omwe amadziwika kuti amaletsa ma microtubules: disopyramide ndi oryzalin adafufuzidwanso.Tsambali likuwonetsa mulingo womwe umagwiritsidwa ntchito kuyeza kukula kwa mizu.Nyenyezi zimawonetsa kusiyana kwakukulu ndi chithandizo chabodza (t test, p<0.05]n >19. Mlingo wa bar = 1 cm.(b) Ma cortical microtubules m'maselo a epidermal mu malo otalikirapo.Ma Microtubules mumtundu wamtundu wa Arabidopsis Col mizu yomwe idakula pamasamba a MS omwe ali ndi 25 μM KAND 11 kapena opanda 25 μM KAND 11 adawonetsedwa ndi immunohistochemical staining pogwiritsa ntchito β-tubulin primary antibodies ndi Alexa Fluor-conjugated secondary antibodies.Scale bar = 10 µm.(c) Mitotic kapangidwe ka microtubules muzu meristem.Ma Microtubules adawonetsedwa pogwiritsa ntchito ma immunohistochemical staining.Zomangamanga za Mitotic, kuphatikiza ma prophase zones, spindles, ndi phragmoplasts, zidawerengedwa kuchokera pazithunzi zolumikizana.Mivi imawonetsa mapangidwe a mitotic microtubule.Nyenyezi zimawonetsa kusiyana kwakukulu ndi chithandizo chabodza (t test, p<0.05]n >9. Scale bar = 50 µm.
Ngakhale Ursa amatha kusokoneza ntchito ya microtubule, njira yake yochitira zinthu ikuyembekezeka kukhala yosiyana ndi ma microtubule depolymerizing agents.Mwachitsanzo, kuchuluka kwa ma microtubule depolymerizing agents monga disopyramide ndi oryzalin kumapangitsa kuti maselo a epidermal achuluke, pamene KAND 11 sichitero.Kuonjezera apo, kugwiritsa ntchito mgwirizano wa KAND 11 ndi disopyramide kunapangitsa kuti disopyramide-induced root growth response ndi KAND 11-induced kukula inhibition inhibition (Fig. S4).Tinasanthulanso kuyankha kwa hypersensitive disopyramide 1-1 (phs1-1) mutant ku KAND 11. phs1-1 ili ndi kusintha kosasinthika kwa tubulin kinase point ndipo imapanga mizu yaifupi ikachitidwa ndi disopyramide9,20.phs1-1 mbande zosasinthika zomwe zidabzalidwa pa sing'anga ya agar yomwe ili ndi KAND 11 inali ndi mizu yaifupi yofanana ndi yomwe imamera pa disopyramid (mkuyu. S5).
Kuphatikiza apo, tidawona mawonekedwe a mitotic microtubule, monga ma prophase zones, spindles, ndi phragmoplasts, muzu wa mbande zomwe zimathandizidwa ndi KAND 11. Mogwirizana ndi zomwe CDKB2; 1p:: CDKB2; chiwerengero cha mitotic microtubules chinawonedwa (mkuyu .6c).
Kuti tiwonetsere cytotoxicity ya KAND 11 pakusankha kwapang'onopang'ono, tidachiritsa ma cell oyimitsidwa a fodya BY-2 ndi KAND 11 ndikuwona kuyankha kwawo.Poyamba tidawonjezera KAND 11 ku ma BY-2 ma cell a TagRFP-TUA6, omwe amalemba ma microtubules, kuti awone momwe KAND 11 imakhudzira ma cortical microtubules.Cortical microtubule density idawunikidwa pogwiritsa ntchito kusanthula kwazithunzi, komwe kumawerengera kuchuluka kwa ma cytoskeletal pixels pakati pa ma cytoplasmic pixels.Zotsatira zoyesa zasonyeza kuti pambuyo pa chithandizo ndi 50 μM kapena 100 μM KAND 11 kwa ola la 1, kachulukidwe kameneka kanatsika kwambiri mpaka 0.94 ± 0.74% kapena 0.23 ± 0.28%, motero, pamene kuchuluka kwa maselo omwe amachitidwa ndi DMSO, kunakwana 10.63 ± 1. % (mkuyu 7a).Zotsatira izi zimagwirizana ndi kuwonetsetsa kwa Arabidopsis kuti chithandizo cha KAND 11 chimapangitsa depolymerization ya cortical microtubules (mkuyu 6b).Tinayang'ananso mzere wa BY-2 ndi GFP-ABD-otchedwa actin filaments pambuyo pa chithandizo chofanana ndi KAND 11 ndikuwona kuti chithandizo cha KAND 11 chinasokoneza ma actin filaments.Kuchiza ndi 50 μM kapena 100 μM KAND 11 kwa 1 h kunachepetsa kwambiri kachulukidwe ka actin filament ku 1.20 ± 0.62% kapena 0.61 ± 0.26%, motsatana, pomwe kachulukidwe m'maselo opangidwa ndi DMSO anali 1.69 ± 0.51% (mkuyu 0.51).7b).Zotsatirazi zimasiyana ndi zotsatira za propyzamide, zomwe sizimakhudza actin filaments, ndi latrunculin B, actin depolymerizer yomwe imakhudza ma microtubules (SI Chithunzi S6).Kuonjezera apo, chithandizo cha coumamonamide 1, coumamonamide acid 6, kapena KAND 11 sichinakhudze ma microtubules m'maselo a HeLa (SI Chithunzi S7).Chifukwa chake, njira yogwirira ntchito ya KAND 11 imakhulupirira kuti ndi yosiyana ndi ya osokoneza cytoskeleton odziwika.Kuonjezera apo, kuyang'ana kwathu kochepa kwambiri kwa maselo a BY-2 omwe amathandizidwa ndi KAND 11 kunavumbulutsa kuyambika kwa imfa ya selo panthawi ya chithandizo cha KAND 11 ndikuwonetsa kuti chiwerengero cha maselo akufa a buluu a Evans sichinachuluke kwambiri pambuyo pa 30 min ya chithandizo cha KAND 11, pamene. pambuyo 90 mphindi mankhwala ndi 50 μM kapena 100 μM KAND, chiwerengero cha maselo akufa chinawonjezeka 43,7% kapena 80,1%, motero (mkuyu. 7c).Kuphatikizidwa, izi zikuwonetsa kuti buku la ursolic acid lochokera ku KAND 11 ndi chomera chodziwikiratu cha cytoskeletal inhibitor chokhala ndi kachitidwe kosadziwika kale.
KAND imakhudza ma cortical microtubules, actin filaments, komanso mphamvu ya ma cell a fodya BY-2.(a) Kuwona ma cortical microtubules m'maselo a BY-2 pamaso pa TagRFP-TUA6.Maselo a BY-2 omwe amathandizidwa ndi KAND 11 (50 μM kapena 100 μM) kapena DMSO adawunikidwa ndi ma microscopy a confocal.Cortical microtubule density idawerengedwa kuchokera ku ma micrographs a 25 odziyimira pawokha.Malembo amawonetsa kusiyana kwakukulu (mayeso a Tukey HSD, p<0.05]Scale bar = 10 µm.(b) Ma Cortical actin filaments m'maselo a BY-2 omwe amawonekera pamaso pa GFP-ABD2.Maselo a BY-2 omwe amathandizidwa ndi KAND 11 (50 μM kapena 100 μM) kapena DMSO adawunikidwa ndi ma microscopy a confocal.Kuchulukana kwa ma cortical actin filaments kunawerengedwa kuchokera ku ma micrograph a ma cell 25 odziyimira pawokha.Malembo amawonetsa kusiyana kwakukulu (mayeso a Tukey HSD, p<0.05]Scale bar = 10 µm.(c) Kuwona kwa ma cell a BY-2 akufa ndi Evans buluu.Maselo a BY-2 omwe amathandizidwa ndi KAND 11 (50 μM kapena 100 μM) kapena DMSO adawunikidwa ndi microscope yowala.n=3.Scale bar = 100 µm.
Kupezeka ndi kugwiritsa ntchito zinthu zatsopano zachilengedwe kwadzetsa kupita patsogolo kwakukulu m'mbali zosiyanasiyana za moyo wa anthu, kuphatikiza zamankhwala ndi ulimi.Kafukufuku wakale wachitika kuti apeze mankhwala othandiza kuchokera kuzinthu zachilengedwe.Makamaka, actinomycetes amadziwika kuti ndi othandiza monga antiparasitic maantibayotiki a nematodes chifukwa cha kuthekera kwawo kupanga metabolites yachiwiri yachiwiri monga avermectin, gulu lotsogolera la ivermectin ndi bleomycin ndi zotumphukira zake, zomwe zimagwiritsidwa ntchito ngati mankhwala ngati anticancer agent21,22.Momwemonso, mankhwala osiyanasiyana a herbicidal apezeka kuchokera ku actinomycetes, ena omwe amagwiritsidwa ntchito kale pamalonda1,23.Chifukwa chake, kusanthula kwa metabolites ya actinomycete kudzipatula kwachilengedwe ndi zinthu zomwe zimafunidwa kwachilengedwe kumawonedwa ngati njira yabwino.Mu phunziro ili, tinapeza gulu latsopano, coumamonamide, kuchokera ku S. werraensis ndipo tinapanga bwino.Ursonic acid ndi wapakatikati wa urbenamide ndi zotumphukira zake.Zitha kuyambitsa kupindika kwa mizu, kuwonetsa zolimbitsa thupi za herbicidal, ndikuwononga mwachindunji kapena mwanjira ina ma microtubules.Komabe, makina a urmotonic acid amatha kusiyana ndi omwe alipo, popeza KAND 11 imasokonezanso ma actin filaments ndipo imayambitsa kufa kwa maselo, kutanthauza njira yoyendetsera yomwe urmotonic acid ndi zotumphukira zake zimakhudza mitundu yambiri ya cytoskeletal..
Kufotokozera mwatsatanetsatane kwa urbenonic acid kudzakuthandizani kumvetsetsa bwino momwe urbenonic acid imagwirira ntchito.Makamaka, cholinga chotsatira ndicho kuyesa mphamvu ya ursonic acid kumangiriza kuchepetsedwa kwa ma microtubules kuti adziwe ngati ursonic acid ndi zotumphukira zake zimagwira ntchito mwachindunji pa ma microtubules ndikuwachotsa, kapena ngati zochita zawo zimabweretsa kuwonongeka kwa microtubule.Komanso, pamene microtubules si chandamale chandamale, kuzindikira malo zochita ndi maselo mipherezero ursonic asidi pa zomera zomera zingathandize kumvetsa katundu wa mankhwala okhudzana ndi njira zotheka kusintha herbicidal ntchito.Kufufuza kwathu kwa bioactivity kunavumbula mphamvu yapadera ya cytotoxic ya ursonic acid pakukula kwa zomera monga Arabidopsis thaliana, fodya ndi chiwindi, pamene maselo a E. coli kapena HeLa sanakhudzidwe.Kawopsedwe kakang'ono kapena kulibe ku maselo a nyama ndi mwayi wa zotumphukira za ursonic acid ngati atapangidwa ngati mankhwala ophera udzu kuti agwiritsidwe ntchito m'minda yotseguka yaulimi.Zowonadi, popeza ma microtubules ndizomwe zimachitika mu eukaryotes, kuletsa kwawo kumera ndikofunikira kwambiri pamankhwala ophera udzu.Mwachitsanzo, propyzamide, microtubule depolymerizing agent yomwe imamangiriza mwachindunji ku tubulin ndikuletsa polymerization, imagwiritsidwa ntchito ngati mankhwala ophera udzu chifukwa cha kuchepa kwake kwa kawopsedwe ku maselo a nyama24.Mosiyana ndi disopyramid, benzamides ogwirizana ali ndi zolinga zosiyana.Kuphatikiza pa ma microtubules a zomera, RH-4032 kapena benzoxamide imalepheretsanso ma microtubules a maselo a nyama kapena oomycetes, motero, ndipo zalilamide amagwiritsidwa ntchito ngati fungicide chifukwa cha kuchepa kwake kwa phytotoxicity25,26,27.Chimbalangondo chomwe changopezedwa kumene ndi zotuluka zake zimawonetsa cytotoxicity yosankha motsutsana ndi zomera, koma ndikofunikira kudziwa kuti kusinthidwa kwina kungathe kusintha zomwe akufuna, zomwe zitha kupereka zina zowonjezera zowongolera bowa kapena oomycetes.
Makhalidwe apadera a urbenonic acid ndi zotuluka zake ndizothandiza pakukula kwawo monga mankhwala ophera udzu ndikugwiritsa ntchito ngati zida zofufuzira.Kufunika kwa cytoskeleton pakuwongolera mawonekedwe a cell cell kumadziwika kwambiri.Kafukufuku wakale wasonyeza kuti zomera zasintha njira zovuta za bungwe la cortical microtubule mwa kulamulira mphamvu za microtubule kuti zithetse bwino morphogenesis.Mamolekyu ambiri omwe ali ndi udindo woyang'anira ntchito ya microtubule adziwika, ndipo kafukufuku wokhudzana ndi izi akupitirirabe3,4,28.Kumvetsetsa kwathu kwaposachedwa kwamphamvu kwa ma microtubule m'maselo a zomera sikumalongosola bwino njira za bungwe la cortical microtubule.Mwachitsanzo, ngakhale onse a disopyramide ndi oryzalin amatha kutulutsa ma microtubules, disopyramide imayambitsa kupotoza kwa mizu pomwe oryzalin imakhala ndi zotsatira zochepa.Komanso, kusintha kwa tubulin, komwe kumapangitsa kuti ma microtubules akhazikike, kumayambitsanso dextrorotation mumizu, pamene paclitaxel, yomwe imakhazikitsanso mphamvu za microtubule, sizitero.Chifukwa chake, kuphunzira ndikuzindikira zolinga za mamolekyulu a ursolic acid kuyenera kupereka zidziwitso zatsopano pakuwongolera ma cortical microtubules.Momwemonso, kufananitsa kwamtsogolo kwa mankhwala omwe amathandiza kulimbikitsa kukula kolakwika, monga disopyramide, ndi mankhwala osagwira ntchito, monga oryzalin kapena kumamotoric acid, adzapereka zidziwitso za momwe kukula kolakwika kumachitikira.
Kumbali ina, kukonzanso zokhudzana ndi chitetezo cha cytoskeletal ndi njira ina yofotokozera za cytotoxicity ya ursonic acid.Kupatsirana kwa tizilombo toyambitsa matenda kapena kulowetsedwa kwa chotulutsa m'maselo a zomera nthawi zina kumayambitsa kuwonongeka kwa cytoskeleton ndi kufa kwa cell29.Mwachitsanzo, cryptoxanthin yochokera ku oomycete yanenedwa kuti imasokoneza ma microtubules ndi actin filaments isanayambe kufa kwa maselo a fodya, mofanana ndi zomwe zimachitika ndi chithandizo cha KAND30,31.Kufanana pakati pa mayankho a chitetezo ndi mayankho a ma cell omwe amapangidwa ndi ursonic acid adatipangitsa kuti tiganize kuti zimayambitsa njira zodziwika bwino zama cell, ngakhale kuti mphamvu ya ursonic acid imawonekera mwachangu komanso mwamphamvu kuposa cryptoxanthin.Komabe, kafukufuku wasonyeza kuti kusokonezeka kwa ma actin filaments kumalimbikitsa kufa kwa maselo, komwe sikumakhala limodzi ndi kusokonezeka kwa microtubule29.Kuphatikiza apo, zikuyenera kuwonedwa ngati tizilombo toyambitsa matenda kapena chotulutsa chimayambitsa kusokonezeka kwa mizu, monga momwe zotumphukira za ursonic acid zimachitira.Chifukwa chake, chidziwitso cha mamolekyu cholumikiza mayankho achitetezo ndi cytoskeleton ndivuto lokongola lomwe liyenera kuthetsedwa.Pogwiritsa ntchito kukhalapo kwa mamolekyu otsika kwambiri okhudzana ndi ursonic acid, komanso zotumphukira zosiyanasiyana zomwe zimakhala ndi mphamvu zosiyanasiyana, zimatha kupereka mwayi wolunjika kumayendedwe osadziwika a ma cell.
Kuphatikizidwa pamodzi, kutulukira ndi kugwiritsa ntchito mankhwala atsopano omwe amawongolera ma microtubule dynamics adzapereka njira zamphamvu zothetsera makina ovuta a maselo omwe amachititsa kuti maselo apangidwe apangidwe.M'nkhaniyi, posachedwapa urmotonic acid yomwe imapangidwa posachedwapa, yomwe imakhudza ma microtubules ndi actin filaments ndipo imayambitsa kufa kwa maselo, ikhoza kupereka mpata wofotokozera kugwirizana pakati pa microtubule control ndi njira zina izi.Chifukwa chake, kusanthula kwamankhwala ndi kwachilengedwe pogwiritsa ntchito urbenonic acid kudzatithandiza kumvetsetsa njira zoyendetsera ma cell zomwe zimawongolera chomera cha cytoskeleton.
Inoculate S. werraensis MK493-CF1 mu botolo la Erlenmeyer lodabwitsa la 500 mL lomwe lili ndi 110 mL ya sing'anga yambewu yokhala ndi 2% (w/v) galactose, 2% (w/v) Essence paste, 1% (w/v) Bacto composition .-soyton (Thermo Fisher Scientific, Inc.), 0.5% (w / v) chotsitsa cha chimanga (KOGOSTCH Co., Ltd., Japan), 0.2% (w / v) (NH4) 2SO4 ndi 0.2% CaCO3 m'madzi a deionized.(pH 7.4 musanatseke cholera).Zikhalidwe za mbewuzo zidakulungidwa pa shaker yozungulira (180 rpm) pa 27 ° C kwa masiku awiri.Kulima pogwiritsa ntchito fermentation yolimba.Chikhalidwe cha mbeu (7 ml) chinasamutsidwa mu botolo la 500 ml K-1 lomwe linali ndi 40 g ya sing'anga yopangira yomwe imakhala ndi 15 g ya balere woponderezedwa (MUSO Co., Ltd., Japan) ndi 25 g wa madzi osungunuka (pH osasinthidwa musanatseke chotchinga).).Kutentha kunachitika pa 30 ° C mumdima kwa masiku 14.Zinthu zowotchera zidatengedwa ndi 40 ml/botolo EtOH ndi centrifuged (1500 g, 4°C, 10 min).Chikhalidwe cha supernatant (60 ml) chinachotsedwa ndi 10% MeOH/EtOAc.organic wosanjikiza anasanduka nthunzi pansi pa kupanikizika kochepa kuti apeze zotsalira (59.5 mg), zomwe zinaperekedwa kwa HPLC ndi gradient elution (0-10 mphindi: 90%) pa reverse gawo ndime (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × kutalika 250 mm) H2O/CH3CN, 10–35 mphindi: 90% H2O/CH3CN mpaka 70% H2O/CH3CN (gradient), 35–45 mphindi: 90% H2O/EtOH, 45–155 mphindi: 90% H2O / EtOH ku 100% EtOH (gradient (gradient), 155-200 min: 100% EtOH) pamtunda wothamanga wa 1.5 ml / min, coumamonamide (1, 36.0 mg) inalekanitsidwa ngati ufa woyera wa 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]+ mtengo wowerengetsera: 141.0659, mtengo woyezedwa: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
Mbewu za Columbia (Col-0) zinapezedwa ku Arabidopsis Biological Resource Center (ABRC) ndi chilolezo chogwiritsa ntchito kafukufuku.Mbeu za Col-0 zidafalitsidwa ndikusungidwa m'malo athu a labotale ndipo zimagwiritsidwa ntchito ngati zomera zakutchire za Arabidopsis.Mbeu za Arabidopsis zinali pamwamba zosawilitsidwa ndi kulimidwa mu theka la mphamvu Murashige ndi Skoog sing'anga yokhala ndi 2% sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino) ethanesulfonic acid (MES) (Fujifilm Wako Pure Chemical ).) ndi 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, pa 23 °C ndi kuwala kosalekeza.Mbewu za phs1-1 mutant zinaperekedwa ndi T. Hashimoto (Nara Institute of Science and Technology).
Mbewu za SR-1 zinaperekedwa ndi T. Hashimoto (Nara Institute of Science and Technology) ndipo zimagwiritsidwa ntchito ngati zomera zamtundu wa fodya.Mbeu za fodya zidathiridwa pamwamba ndikunyowetsedwa m'madzi osabala kwa mausiku atatu kuti zimere, kenako zimayikidwa mumtsuko wamphamvu-hafu wokhala ndi 2% sucrose, 0.05% (w/v) MES, ndi 0.8% gellan chingamu (Fujifilm Wako Pure Chemical) Murashige.ndi Skoog medium) yokhala ndi pH 5.7 ndipo imakulungidwa pa 23°C pansi pa kuwala kosalekeza.
Strain Tak-1 idaperekedwa ndi T. Kohchi (Yunivesite ya Kyoto) ndipo idagwiritsidwa ntchito ngati gawo loyeserera la kafukufuku wa liverwort.Gemma inapezedwa kuchokera ku zomera zosautsa zolimidwa ndikuzikutidwa pa sing'anga ya Gamborg B5 (Fujifilm Wako Pure Chemical) yomwe ili ndi 1% sucrose ndi 0.3% ya chingamu ya gellan ndikumakulitsidwa pa 23 ° C pansi pa kuwala kosalekeza.
Maselo a Fodya BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) anaperekedwa ndi S. Hasezawa (University of Tokyo).Maselo a BY-2 adachepetsedwa 95-fold in modified Linsmeier ndi Skoog medium ndi kuwonjezeredwa mlungu uliwonse ndi 2,4-dichlorophenoxyacetic acid 32.Kuyimitsidwa kwa selo kunasakanizidwa pa shaker yozungulira pa 130 rpm pa 27 ° C mumdima.Sambani maselo ndi 10 kuwirikiza kuchuluka kwa sing'anga yatsopano ndikuyimitsanso chimodzimodzi.BY-2 transgenic cell mizere yofotokozera mokhazikika cholembera cha microtubule TagRFP-TUA6 kapena cholembera cha actin filament GFP-ABD2 pansi pa cholimbikitsa cha cauliflower mosaic virus 35S adapangidwa monga momwe amafotokozera33,34,35.Mizere yama cellwa imatha kusamalidwa ndikuyanjanitsidwa pogwiritsa ntchito njira zofananira ndi zomwe zimagwiritsidwa ntchito pama cell a BY-2 oyambirira.
Ma cell a HeLa adapangidwa mu Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) yowonjezeredwa ndi 10% ya fetal bovine serum, 1.2 U/ml penicillin, ndi 1.2 μg/ml streptomycin mu chofungatira cha 37°C chokhala ndi 5% CO2.
Zoyeserera zonse zomwe zafotokozedwa m'bukhuli zidachitika motsatira malamulo ndi malangizo aku Japan oteteza zachilengedwe.
Ma Compounds adasungunuka mu dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) ngati mayankho a stock ndikuchepetsedwa mu MS sing'anga ya Arabidopsis ndi fodya kapena Gamborg B5 sing'anga ya liverwort.Poyesa kuletsa kukula kwa mizu, mbewu zopitilira 10 pa mbale zimafesedwa pa sing'anga ya agar yomwe ili ndi mankhwala owonetsedwa kapena DMSO.Mbewuzo zidakwiriridwa m'chipinda chokulirapo kwa masiku 7.Mbewuzo zinajambulidwa ndipo utali wa mizu ankapimidwa.Poyesa kumera kwa Arabidopsis, mbewu 48 pa mbale zinafesedwa pa agar medium yomwe ili ndi 200 μM kapena DMSO.Mbewu za Arabidopsis zidakulitsidwa m'chipinda chokulirapo ndipo kuchuluka kwa mbande zomwe zidamera zidawerengedwa patatha masiku 7 kumera (dag).Poyesa kumera kwa fodya, mbewu 24 pa mbale iliyonse zidafesedwa pa sing'anga ya agar yomwe ili ndi 200 μM KAND kapena DMSO.Mbewu za fodya zimabzalidwa m'chipinda chokulirapo ndipo chiwerengero cha mbande zomwe zidamera zidawerengedwa pakadutsa masiku 14.Pachiyeso choletsa kukula kwa chiwindi, mazira 9 kuchokera m'mbale iliyonse adakutidwa pa sing'anga ya agar yomwe ili ndi kuchuluka kwa KAND kapena DMSO ndikuyikidwa m'chipinda chokulirapo kwa masiku 14.
Gwiritsani ntchito mbande zothimbirira ndi 5 mg/ml ya propidium iodide (PI) kuti muone momwe mizu ikuyendera.Zizindikiro za PI zidawonedwa ndi ma microscopy a fluorescence pogwiritsa ntchito maikolofoni ya TCS SPE confocal laser scanning (Leica Microsystems).
Kudetsa kwa histochemical kwa mizu ndi β-glucuronidase (GUS) kunachitidwa molingana ndi ndondomeko yofotokozedwa ndi Malami ndi Benfey36.Mbande inakhazikitsidwa mu 90% acetone usiku wonse, yodetsedwa ndi 0.5 mg / ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid mu GUS buffer kwa ola la 1 ndikuyika mu hydrated chloraldehyde solution.(8 g chloral hydrate, 2 ml madzi ndi 1 ml glycerol) ndipo amawonedwa ndi kusokoneza kusiyanitsa maikulosikopu pogwiritsa ntchito maikulosikopu ya Axio Imager M1 (Carl Zeiss).
Makona amizu adayezedwa pa mbande zamasiku 7 zakubadwa zomwe zidabzalidwa pambale zoyikidwa molunjika.Yezerani ngodya ya muzu kuchokera kolowera komwe kuli vekitala yokoka monga momwe tafotokozera mu sitepe 6.
Kukonzekera kwa ma cortical microtubules kunawonedwa monga momwe tafotokozera, ndi zosintha zazing'ono za protocol 37.Anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) ndi Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) anagwiritsidwa ntchito ngati ma antibodies oyambirira ndi achiwiri pa 1: 1000 ndi 1: 100 dilutions, motsatira.Zithunzi za fluorescence zinapezedwa pogwiritsa ntchito maikulosikopu ya TCS SPE confocal laser scanning (Leica Microsystems).Pezani zithunzi za Z-stack ndikupanga zowonera mwamphamvu molingana ndi malangizo a wopanga.
HeLa cell proliferation assay idachitidwa pogwiritsa ntchito Cell Counting Kit 8 (Dojindo) molingana ndi malangizo a wopanga.
Kukula kwa E. coli DH5α kunayesedwa poyesa kuchuluka kwa maselo mu chikhalidwe pogwiritsa ntchito spectrophotometer pa 600 nm (OD600).
Bungwe la Cytoskeletal m'maselo a transgenic BY-2 adawonedwa pogwiritsa ntchito microscope ya fluorescence yokhala ndi CSU-X1 confocal scanning device (Yokogawa) ndi kamera ya sCMOS (Zyla, Andor Technology).Kuchuluka kwa cytoskeletal density kudawunikidwa ndi kusanthula kwazithunzi, komwe kunawerengera kuchuluka kwa ma cytoskeletal pixels pakati pa ma cytoplasmic pixels muzithunzi zolumikizana pogwiritsa ntchito pulogalamu ya ImageJ monga momwe akufotokozedwera38,39.
Kuti muwone kufa kwa cell m'maselo a BY-2, aliquot ya kuyimitsidwa kwa cell idalumikizidwa ndi 0.05% Evans buluu kwa mphindi 10 kutentha.Kusankha kwa Evans buluu kwa maselo akufa kumadalira kutulutsa utoto kuchokera ku maselo otheka ndi plasma membrane40.Maselo oipitsidwa adawonedwa pogwiritsa ntchito microscope yowala (BX53, Olympus).
Maselo a HeLa anakula mu DMEM yowonjezeredwa ndi 10% FBS mu chofungatira cha humidified pa 37 ° C ndi 5% CO2.Maselo amathandizidwa ndi 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), kapena 100 ng/ml Nocodmaze (Sigma) kwa 6 h pa 37 ° C.Maselo adakonzedwa ndi MetOH kwa 10 min ndiyeno ndi acetate kwa 5 min kutentha.Maselo osasunthika adakulungidwa ndi β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) yosungunuka mu 0.5% BSA / PBS kwa maola 2, kutsukidwa nthawi za 3 ndi TBST, kenaka kulowetsedwa ndi Alexa Fluor mbuzi antibody.4881 ora.- Mouse IgG (Thermo Fisher Scientific: A11001) ndi 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) yochepetsedwa mu 0.5% BSA/PBS.Pambuyo kutsuka ndi TBST katatu, maselo odetsedwa adawonedwa pa microscope yopindika ya Nikon Eclipse Ti-E.Zithunzi zinajambulidwa ndi kamera yoziziritsa ya Hamamatsu ORCA-R2 CCD pogwiritsa ntchito pulogalamu ya MetaMorph (Molecular Devices).
Nthawi yotumiza: Jun-17-2024