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Kupeza ndi kugwiritsa ntchito bwino zinthu zachilengedwe kungathandize kusintha moyo wa anthu. Mankhwala oletsa kukula kwa zomera amagwiritsidwa ntchito kwambiri ngati mankhwala ophera udzu. Chifukwa cha kufunika kogwiritsa ntchito mitundu yosiyanasiyana ya mankhwala ophera udzu, pakufunika kuzindikira mankhwala omwe ali ndi njira zatsopano zogwirira ntchito. Mu kafukufukuyu, tapeza mankhwala atsopano a N-alkoxypyrrole, coumamonamide, ochokera ku Streptomyces werraensis MK493-CF1 ndipo takhazikitsa njira yonse yopangira. Kudzera mu kuyesa kwa zochita zamoyo, tapeza kuti urs-monoamic acid ndi mankhwala opangidwa ndi urs-monoamide komanso omwe ali ndi mphamvu.choletsa kukula kwa zomeraKuphatikiza apo, tapanga mitundu yosiyanasiyana ya urbenonic acid derivatives, kuphatikizapo urbenyloxy derivative (UDA), yomwe imakhala ndi mphamvu zambiri zowononga zomera popanda kusokoneza kukula kwa maselo a HeLa. Tapezanso kuti urmotonic acid derivatives imasokoneza microtubules za zomera; kuphatikiza apo, KAND imakhudza ulusi wa actin ndikuyambitsa kufa kwa maselo; Zotsatirazi zambiri zimasiyana ndi za zoletsa zodziwika bwino za microtubule ndipo zimasonyeza njira yatsopano yogwirira ntchito ya ursonic acid, yomwe imayimira phindu lofunika kwambiri pakupanga mankhwala atsopano ophera udzu.
Kupeza ndi kugwiritsa ntchito bwino zinthu zachilengedwe zothandiza ndi zochokera m'thupi ndi njira yowonjezerera moyo wa anthu. Zinthu zina zomwe zimapangidwa ndi tizilombo toyambitsa matenda, zomera ndi tizilombo zatsogolera ku kupita patsogolo kwakukulu mu zamankhwala ndi ulimi. Mankhwala ambiri opha maantibayotiki ndi mankhwala oletsa khansa ya m'magazi apangidwa kuchokera ku zinthu zachilengedwe. Kuphatikiza apo, mitundu yosiyanasiyana yamankhwala ophera tizilombo, mankhwala ophera udzu ndi mankhwala ophera udzu amachokera ku zinthu zachilengedwezi kuti agwiritsidwe ntchito mu ulimi. Makamaka, mankhwala ophera udzu ndi zida zofunika kwambiri pakuwonjezera zokolola mu ulimi wamakono, ndipo mitundu yosiyanasiyana ya mankhwala imagwiritsidwa ntchito kale m'malonda. Njira zingapo zamaselo m'zomera, monga photosynthesis, kagayidwe ka amino acid, kapangidwe ka makoma a maselo, kulamulira kwa mitosis, phytohormone signaling, kapena kupanga mapuloteni, zimaonedwa kuti ndi zolinga za herbicides. Mankhwala omwe amaletsa ntchito ya microtubule ndi gulu lofala la mankhwala ophera udzu omwe amakhudza kukula kwa zomera pokhudza malamulo a mitotic2.
Ma microtubules ndi zigawo za cytoskeleton ndipo amasungidwa kwambiri m'maselo a eukaryotic. Tubulin heterodimer imakhala ndi α-tubulin ndi β-tubulin zomwe zimapanga ma microtubule protofilaments olunjika, ndi ma protofilaments 13 omwe amapanga kapangidwe ka cylindrical. Ma microtubules amachita ntchito zingapo m'maselo a zomera, kuphatikizapo kudziwa mawonekedwe a maselo, kugawikana kwa maselo, ndi kunyamula kwa mkati mwa maselo3,4. Maselo a zomera amakhala ndi ma microtubules pansi pa nembanemba ya plasma ya interphase, ndipo ma microtubules otchedwa cortical awa akuganiziridwa kuti amayang'anira kapangidwe ka cellulose microfibrils kudzera mukuwongolera ma cellulose synthase complexes4,5. Ma microtubules a cortical a maselo a mizu ya epidermal, omwe amapezeka m'dera la kutalikitsa kwachangu kwa nsonga ya muzu, amapezeka mozungulira, ndipo ma microfibers a cellulose amatsatira ma microtubules awa ndikuchepetsa njira yokulira kwa maselo, motero amalimbikitsa kutalikitsa kwa maselo a anisotropic. Chifukwa chake, ntchito ya microtubule imagwirizana kwambiri ndi mawonekedwe a chomera. Kusinthidwa kwa amino acid m'majini omwe amalemba tubulin kumayambitsa kusokonekera kwa ma cortical microtubule arrays ndi kukula kwa kumanzere kapena kumanja mu Arabidopsis 6,7. Mofananamo, kusintha kwa mapuloteni okhudzana ndi microtubule omwe amawongolera mphamvu za microtubule kungayambitsenso kukula kwa mizu yolakwika8,9,10,11,12,13. Kuphatikiza apo, chithandizo ndi mankhwala ophera udzu omwe amasokoneza microtubule monga disopyramide, omwe amadziwikanso kuti pretilachlor, chimayambitsanso kukula kwa mizu yozungulira kumanzere14. Deta iyi ikuwonetsa kuti kuwongolera bwino ntchito ya microtubule ndikofunikira kwambiri podziwa komwe mbewu ikukula.
Mitundu yosiyanasiyana ya mankhwala oletsa tizilombo toyambitsa matenda yapezeka, ndipo mankhwala amenewa athandiza kwambiri pa kafukufuku wa cytoskeletal, komanso ulimi ndi mankhwala. Makamaka, oryzalin, mankhwala a dinitroaniline, disopyramide, mankhwala okhudzana ndi benzamide, ndi ma analogue awo amatha kuletsa ntchito ya tizilombo toyambitsa matenda ndipo motero amaletsa kukula kwa zomera. Chifukwa chake, amagwiritsidwa ntchito kwambiri ngati mankhwala ophera udzu. Komabe, popeza tizilombo toyambitsa matenda ndi gawo lofunika kwambiri la maselo a zomera ndi nyama, mankhwala ambiri oletsa tizilombo toyambitsa matenda ndi oopsa kwa mitundu yonse iwiri ya maselo. Chifukwa chake, ngakhale kuti amadziwika kuti ndi mankhwala ophera udzu, pali mankhwala ochepa oletsa tizilombo toyambitsa matenda omwe amagwiritsidwa ntchito pazifukwa zothandiza.
Streptomyces ndi mtundu wa banja la Streptomyces, lomwe limaphatikizapo mabakiteriya a aerobic, gram-positive, filamentous ndipo amadziwika kwambiri chifukwa cha kuthekera kwake kupanga mitundu yambiri ya metabolites yachiwiri. Chifukwa chake, imaonedwa kuti ndi imodzi mwa magwero ofunikira kwambiri a zinthu zatsopano zachilengedwe zomwe zimagwira ntchito m'thupi. Mu kafukufuku wapano, tapeza mankhwala atsopano otchedwa coumamonamide, omwe adachotsedwa ku Streptomyces werraensis MK493-CF1 ndi S. werraensis ISP 5486. Pogwiritsa ntchito kusanthula kwa spectral ndi kusanthula kwathunthu kwa spectral, kapangidwe ka coumamonamide kanadziwika ndipo mafupa ake apadera a N-alkoxypyrrole adadziwika. Ursmonic acid, yomwe ndi gawo lopangidwa la ursmonoamide ndi zotumphukira zake, idapezeka kuti ikuletsa kukula ndi kumera kwa chomera chodziwika bwino cha Arabidopsis thaliana. Mu kafukufuku wokhudzana ndi kapangidwe kake, tapeza kuti mankhwala okhala ndi C9 osinthidwa kukhala ursonic acid, otchedwa nonyloxy derivative of ursonic acid (KAND), amawonjezera kwambiri mphamvu yoletsa kukula ndi kumera. Chochititsa chidwi n'chakuti, choletsa kukula kwa zomera chomwe changopezeka kumenecho chinakhudzanso kukula kwa fodya ndi liverwort ndipo sichinali poizoni kwa mabakiteriya kapena maselo a HeLa. Komanso, zinthu zina zochokera ku urmotonic acid zimayambitsa mtundu wolakwika wa mizu, zomwe zikutanthauza kuti zinthu zochokera ku izi zimakhudza mwachindunji kapena mwanjira ina ma microtubules. Mogwirizana ndi lingaliro ili, zomwe tawona za ma microtubules omwe amalembedwa kuti ndi immunohistochemically kapena ndi mapuloteni a fluorescent zikusonyeza kuti chithandizo cha KAND chimachotsa ma microtubules. Kuphatikiza apo, chithandizo ndi kumamotonic acid derivatives chinasokoneza ma microfilaments a actin. Chifukwa chake, tapeza choletsa kukula kwa zomera chomwe njira yake yapadera yogwirira ntchito imaphatikizapo kuwononga cytoskeleton.
Mtundu wa MK493-CF1 unachotsedwa m'nthaka ku Shinagawa-ku, Tokyo. Mtundu wa MK493-CF1 unapanga mycelium ya nthambi yabwino. Gawo la jini la 16S ribosomal RNA (1422 bp) linapezeka. Mtundu uwu ndi wofanana kwambiri ndi S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: mtundu wamba, 99.93%). Kutengera ndi zotsatirazi, zinapezeka kuti mtundu uwu unali wogwirizana kwambiri ndi mtundu wa mtundu wa S. werraensis. Chifukwa chake, tinatcha mtundu uwu S. werraensis MK493-CF1 kwakanthawi. S. werraensis ISP 5486T imapanganso mankhwala omwewo a bioactive. Popeza panali kafukufuku wochepa woyambirira wopeza zinthu zachilengedwe kuchokera ku tizilombo toyambitsa matenda, kafukufuku wina wa mankhwala unachitika. Pambuyo polima S. werraensis MK493-CF1 pa barele medium pogwiritsa ntchito solid-state fermentation pa 30°C kwa masiku 14, medium inachotsedwa ndi 50% EtOH. 60 ml ya chitsanzo inaumitsidwa kuti ipeze 59.5 mg ya crude extract. crude extract inayikidwa mu reverse phase HPLC kuti ipereke N-methoxy-1H-pyrrole-2-carboxamide (1, yotchedwa coumamonamide, 36.0 mg). Chiwerengero chonse cha 1 ndi pafupifupi 60% ya crude extract. Chifukwa chake, tinaganiza zophunzira mwatsatanetsatane za kumamotoamide 1.
Coumamonamide 1 ndi ufa woyera wosasinthika komanso ma spectrometry amphamvu kwambiri (HRESIMS) amatsimikizira C6H8N2O2 (Chithunzi 1). Chidutswa cha pyrrole chosinthidwa ndi C2 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: 4.5 Hz, H-5) ndi δH 6.78 (1H, d, J = 2.5 Hz, H-6), ndipo 13C NMR spectrum ikuwonetsa kukhalapo kwa maatomu anayi a carbon sp2. Kupezeka kwa gulu la amide pamalo a C2 kunayesedwa ndi HMBC correlation kuchokera ku C-3 proton kupita ku amide carbonyl carbon pa δC 161.1. Kuphatikiza apo, 1 H ndi 13 C NMR peaks pa δH 4.10 (3H, S) ndi δC 68.3 zikusonyeza kukhalapo kwa magulu a N-methoxy mu molekyulu. Ngakhale malo oyenera a gulu la methoxy sanadziwikebe pogwiritsa ntchito kusanthula kwa spectroscopic monga enhanced difference spectroscopy ndi nuclear Overhauser abbreviation (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide inakhala chinthu choyamba chofunikira.
Kuti adziwe kapangidwe kolondola ka 1, kupangidwa konsekonse kunachitika (Chithunzi 2a). Kuchiza kwa 2-aminopyridine 2 yomwe imapezeka m'masitolo ndi m-CPBA kunapangitsa kuti N-oxide 3 yofanana ikhale yochuluka. Pambuyo pa 2-aminoazidation ya 2, cyclocondensation reaction yomwe Abramovich adafotokoza idachitika mu benzene pa 90°C kuti apeze 1-hydroxy-1H-pyrrole-2-carbonitrile 5 mu magalamu. Liwiro 60% (magawo awiri). 15,16. Methylation ndi hydrolysis ya 4 kenako idapereka 1-methoxy-1H-pyrrole-2-carboxylic acid (yotchedwa "cumotonic acid", 6) mu zokolola zabwino (70%, magawo awiri). Pomaliza, amidation kudzera mu acid chloride intermediate 6 pogwiritsa ntchito ammonia yamadzi inapatsa Kumamoto amide 1 mu zokolola 98%. Deta yonse ya spectral ya 1 yopangidwa inali yofanana ndi 1 yosiyana, kotero kapangidwe ka 1 kanatsimikiziridwa;
Kupanga ndi kusanthula kwa ntchito ya urbenamide ndi urbenic acid m'chilengedwe. (a) Kupanga konse kwa Kumamoto amide. (b) Mbeu zakuthengo zamtundu wa Arabidopsis Columbia (Col) za masiku asanu ndi awiri zinabzalidwa pa mbale za Murashige ndi Skoog (MS) zomwe zili ndi coumamonamide 6 kapena coumamonamide 1 pamlingo womwe wasonyezedwa. Chipilala cha sikelo = 1 cm.
Choyamba, tinayesa ntchito za urbenamide ndi zinthu zake zapakati kuti tiwone momwe zimathandizira kukula kwa zomera. Tinawonjezera kuchuluka kwa ursmonamide 1 kapena ursmonic acid 6 ku MS agar medium ndi mbande za Arabidopsis thaliana zomwe zimalimidwa pa chomera ichi. Mayeso awa adawonetsa kuti kuchuluka kwakukulu (500 μM) kwa 6 kunaletsa kukula kwa mizu (Chithunzi 2b). Kenako, tinapanga zinthu zosiyanasiyana zotumphukira mwa kusintha malo a N1 a 6 ndikuchita maphunziro okhudza ubale wa kapangidwe kake (njira yopangira analog ikufotokozedwa mu Supporting Information (SI)). Mbeu za Arabidopsis zinalimidwa pa chomera chokhala ndi 50 μM ursonic acid derivatives, ndipo kutalika kwa mizu kunayesedwa. monga momwe zasonyezedwera pachithunzichi. Monga momwe zasonyezedwera mu Zithunzi 3a, b, ndi S1, ma coumamo acid ali ndi kutalika kosiyana kwa unyolo wa alkoxy wolunjika (9, 10, 11, 12, ndi 13) kapena unyolo waukulu wa alkoxy (15, 16, ndi 17) pamalo a N1. Zotumphukirazo zinawonetsa kuletsa kwakukulu kukula kwa mizu. Kuphatikiza apo, tinapeza kuti kugwiritsa ntchito 200 μM 10, 11, kapena 17 kunaletsa kumera (Zithunzi 3c ndi S2).
Kuphunzira za ubale wa kapangidwe ndi ntchito ya Kumamoto amide ndi mankhwala ena ofanana nawo. (a) Kapangidwe ndi kapangidwe ka ma analogues. (b) Kuyeza kutalika kwa mizu ya mbande za masiku 7 zomwe zimamera pa MS medium ndi kapena popanda 50 μM coumamonamide derivatives. Asterisks akuwonetsa kusiyana kwakukulu ndi chithandizo chabodza (t test, p< 0.05). n>18. Deta ikuwonetsedwa ngati avereji ± SD. nt imatanthauza "yosayesedwa" chifukwa mbewu zoposa 50% sizinamere. (c) Kuyeza kuchuluka kwa kumera kwa mbewu zomwe zathandizidwa zomwe zasungidwa kwa masiku 7 mu MS medium ndi kapena popanda 200 μM coumamonamide ndi mankhwala ena ofanana nawo. Asterisks akuwonetsa kusiyana kwakukulu ndi chithandizo cha sham (chi-square test). n=96.
Chochititsa chidwi n'chakuti, kuwonjezera ma chain a alkyl omwe ndi aatali kuposa C9 kunachepetsa ntchito yoletsa, zomwe zikusonyeza kuti mankhwala okhudzana ndi kumamotoic acid amafuna ma chain ambali a kukula kwina kuti awonetse ntchito yawo yachilengedwe.
Popeza kusanthula ubale wa kapangidwe ndi ntchito kunawonetsa kuti C9 inasinthidwa kukhala ursonic acid ndipo nonyloxy derivative ya ursonic acid (yomwe tsopano ikutchedwa KAND 11) inali yoletsa kukula kwa zomera kwambiri, tinachita kufotokoza mwatsatanetsatane za KAND 11. Chithandizo cha Arabidopsis ndi 50 μM KAND 11 chinaletsa kumera, pomwe kuchepa kwa KAND 11 kunaletsa kukula kwa mizu motengera mlingo (40, 30, 20, kapena 10 μM) (Chithunzi 4a, b). Kuti tiwone ngati KAND 11 imakhudza kulimba kwa mizu, tinayang'ana mizu ya meristems yokhala ndi propidium iodide (PI) ndikuyeza kukula kwa dera la meristem. Kukula kwa meristem ya mbande zomwe zinabzalidwa pa sing'anga yokhala ndi 25 μM KAND-11 kunali 151.1 ± 32.5 μm, pomwe kukula kwa meristem ya mbande zomwe zinabzalidwa pa sing'anga yolamulira yokhala ndi DMSO kunali 264.7 ± 30.8 μm (Chithunzi 4c, d), zomwe zikusonyeza kuti KAND-11 imabwezeretsa ntchito ya maselo. kufalikira. Root meristem. Mogwirizana ndi izi, chithandizo cha KAND 11 chinachepetsa kuchuluka kwa chizindikiro chogawa maselo CDKB2;1p::CDKB2;1-GUS mu root meristem (Chithunzi 4e) 17. Zotsatirazi zikusonyeza kuti KAND 11 imaletsa kukula kwa mizu pochepetsa kuchuluka kwa maselo.
Kusanthula kwa zotsatira zoletsa za urbenonic acid derivatives (urbenyloxy derivatives) pa kukula. (a) Mbeu za mtundu wa wild-type Col za masiku 7 zomwe zimamera pa mbale za MS zokhala ndi kuchuluka kwa KAND 11. Scale bar = 1 cm. (b) Kuyeza kutalika kwa mizu. Zilembo zimasonyeza kusiyana kwakukulu (Tukey HSD test, p.< 0.05). n>16. Deta ikuwonetsedwa ngati avereji ± SD. (c) Microscopy ya Confocal ya mizu ya mtundu wa Col ya propidium iodide yomwe imamera pa mbale za MS ndi kapena popanda 25 μM KAND 11. Mabulaketi oyera amasonyeza mizu ya meristem. Scale bar = 100 µm. (d) Kuyeza kukula kwa mizu ya meristem (n = 10 mpaka 11). Kusiyana kwa ziwerengero kunapezeka pogwiritsa ntchito t-test (p).< 0.05). Mipiringidzoyi ikuyimira kukula kwapakati pa meristem. (e) Microscopy ya differential interference contrast (DIC) ya root meristem yokhala ndi CDKB2 construct; 1pro: CDKB2; 1-GUS yokhala ndi utoto ndi utoto pa mbande za masiku 5 zomwe zimamera pa mbale za MS zokhala ndi kapena zopanda 25 µM KAND assay.
Kuopsa kwa KAND 11 kunayesedwanso pogwiritsa ntchito chomera china chotchedwa dicotyledonous, fodya (Nicotiana tabacum), ndi chomera chachikulu cha zomera, liverwort (Marchantia polymorpha). Monga momwe zinalili ndi Arabidopsis, mbande za fodya za SR-1 zomwe zinamera pa sing'anga yokhala ndi 25 μM KAND 11 zinapanga mizu yaifupi (Chithunzi 5a). Kuphatikiza apo, mbewu 40 mwa 48 zinamera pa mbale zomwe zili ndi 200 μM KAND 11, pomwe mbewu zonse 48 zinamera pa malo osungiramo zinthu, zomwe zikusonyeza kuti kuchuluka kwa KAND kunali kwakukulu (p).< 0.05; chi test -square) inaletsa kumera kwa fodya. (Chithunzi 5b). Kuphatikiza apo, kuchuluka kwa KAND 11 komwe kunaletsa kukula kwa mabakiteriya mu liverwort kunali kofanana ndi kuchuluka kogwira ntchito kwa Arabidopsis (Chithunzi 5c). Zotsatirazi zikusonyeza kuti KAND 11 ikhoza kuletsa kukula kwa zomera zosiyanasiyana. Kenako tinafufuza za poizoni wotheka wa mankhwala okhudzana ndi bear monoamide m'zamoyo zina, monga maselo a HeLa a anthu ndi Escherichia coli strain DH5α, monga oimira maselo apamwamba a nyama ndi mabakiteriya, motsatana. Mu mndandanda wa mayeso ochulukitsa maselo, tinaona kuti coumamonamide 1, coumamonamidic acid 6, ndi KAND 11 sizinakhudze kukula kwa maselo a HeLa kapena E. coli pa kuchuluka kwa 100 μM (Chithunzi 5d,e).
Kuletsa kukula kwa KAND 11 m'zamoyo zomwe si Arabidopsis. (a) Mbeu za fodya za mtundu wa SR-1 zakuthengo za milungu iwiri zinabzalidwa pa mbale za MS zokhazikika molunjika zomwe zili ndi 25 μM KAND 11. (b) Mbeu za fodya za mtundu wa SR-1 zakuthengo za milungu iwiri zinabzalidwa pa mbale za MS zokhazikika molunjika zomwe zili ndi 200 μM KAND 11. (c) Masamba a mtundu wa Tak-1 a mtundu wa kuthengo a milungu iwiri omwe anabzalidwa pa mbale za Gamborg B5 ndi kuchuluka kwa KAND 11 komwe kunawonetsedwa. Mivi yofiira imasonyeza spores zomwe zinasiya kukula mkati mwa nthawi yoberekera ya milungu iwiri. (d) Kuyesa kuchuluka kwa maselo a HeLa. Chiwerengero cha maselo okhazikika chinayesedwa nthawi yokhazikika pogwiritsa ntchito chida chowerengera maselo 8 (Dojindo). Monga chowongolera, maselo a HeLa anachiritsidwa ndi 5 μg/ml actinomycin D (Act D), yomwe imaletsa RNA polymerase transcription ndikuyambitsa kufa kwa maselo. Kusanthula kunachitika katatu. (e) Kuyesa kuchuluka kwa maselo a E. coli. Kukula kwa E. coli kunawunikidwa poyesa OD600. Monga njira yowongolera, maselo anapatsidwa mankhwala a 50 μg/ml ampicillin (Amp), omwe amaletsa kupanga makoma a maselo a bakiteriya. Kusanthula kunachitika katatu.
Kuti tidziwe momwe ntchito ya cytotoxicity imagwirira ntchito chifukwa cha mankhwala okhudzana ndi uramide, tinasanthulanso zinthu zochokera ku urbenic acid ndi zotsatira zoletsa pang'ono. Monga momwe zasonyezedwera pachithunzichi. Monga momwe zasonyezedwera pa Zithunzi 2b, 6a, mbande zomwe zimamera pa mbale za agar zomwe zimakhala ndi kuchuluka kwakukulu (200 μM) kwa urmotonic acid 6 zinapanga mizu yaifupi komanso yokhota kumanzere (θ = – 23.7 ± 6.1), pomwe mbande zomwe zimamera pa malo olamulira, mbandezo zinapanga mizu yowongoka (θ = – 3.8 ± 7.1). Kukula kwapadera kumeneku kumadziwika kuti kumachitika chifukwa cha kusagwira ntchito bwino kwa ma microtubules14,18. Mogwirizana ndi zomwe zapezekazi, mankhwala oletsa kukhazikika kwa ma microtubule disopyramide ndi oryzalin adayambitsa kupendekeka kwa mizu yofanana pansi pa mikhalidwe yathu yokulira (Zithunzi 2b, 6a). Nthawi yomweyo, tinayesa zinthu zochokera ku urmotonic acid ndipo tinasankha zingapo mwa izo zomwe, pamlingo wina, zidayambitsa kukula kwa mizu yokhota. Ma compounds 8, 9, ndi 15 adasintha momwe mizu imakulira pa 75 μM, 50 μM, ndi 40 μM, motsatana, zomwe zikusonyeza kuti mankhwala awa amatha kusokoneza bwino ma microtubules (Chithunzi 2b, 6a). Tinayesanso mankhwala amphamvu kwambiri a ursolic acid, KAND 11, pamlingo wochepa (15 µM) ndipo tinapeza kuti kugwiritsa ntchito KAND 11 kunaletsa kukula kwa mizu ndipo kuti njira yokulira kwa mizu sinali yofanana, ngakhale kuti nthawi zambiri ankatsetsereka kumanzere (Chithunzi C3). . Chifukwa kuchuluka kwa mankhwala ochepetsa mphamvu ya microtubule nthawi zina kumaletsa kukula kwa zomera m'malo moyambitsa kupendekeka kwa mizu, pambuyo pake tinayesa kuthekera kwakuti KAND 11 imakhudza ma microtubules powona ma cortical microtubules m'maselo a mizu ya epidermal. Immunohistochemistry pogwiritsa ntchito ma antibodies a anti-β-tubulin m'maselo a epidermal a mizu ya mbande yomwe yathandizidwa ndi 25 μM KAND 11 inawonetsa kutha kwa pafupifupi ma cortical microtubules onse m'maselo a epidermal m'dera lotalikira (Chithunzi 6b). Zotsatirazi zikusonyeza kuti kumamotonic acid ndi zinthu zochokera mkati mwake zimagwira ntchito mwachindunji kapena mwanjira ina pa ma microtubules kuti zisokoneze ndipo mankhwala awa ndi oletsa atsopano a microtubule.
Ursonic acid ndi zotumphukira zake zimasintha ma cortical microtubules mu Arabidopsis thaliana. (a) Ngodya yopendekera kwa mizu yoyesedwa pamaso pa mitundu yosiyanasiyana ya urmotonic acid derivatives pamlingo wosonyezedwa. Zotsatira za mankhwala awiri odziwika kuti amaletsa ma microtubules: disopyramide ndi oryzalin zinawunikidwanso. Chithunzi chamkati chikuwonetsa muyezo womwe umagwiritsidwa ntchito poyesa ngodya ya kukula kwa mizu. Ma asterisks akuwonetsa kusiyana kwakukulu ndi chithandizo chabodza (t test, p< 0.05). n>19. Chipilala chopingasa = 1 cm. (b) Ma microtubules a cortical m'maselo a epidermal m'dera lotalikira. Ma microtubules mu mizu ya Arabidopsis Col yamtundu wachilengedwe yomwe imakula pa mbale za MS zokhala ndi kapena zopanda 25 μM KAND 11 zinawonetsedwa ndi utoto wa immunohistochemical pogwiritsa ntchito ma antibodies a β-tubulin primary ndi ma antibodies a Alexa Fluor-conjugated secondary. Chipilala chopingasa = 10 µm. (c) Kapangidwe ka mitotic ka ma microtubules mu root meristem. Ma microtubules anawonetsedwa pogwiritsa ntchito utoto wa immunohistochemical. Kapangidwe ka mitotic, kuphatikiza ma prophase zones, spindles, ndi phragmoplasts, zinawerengedwa kuchokera ku zithunzi za confocal. Mivi imasonyeza kapangidwe ka mitotic microtubule. Asterisks imasonyeza kusiyana kwakukulu ndi chithandizo chachinyengo (t test, p< 0.05). n>9. Chipilala cha sikelo = 50 µm.
Ngakhale Ursa ili ndi mphamvu yosokoneza ntchito ya microtubule, njira yake yogwirira ntchito ikuyembekezeka kukhala yosiyana ndi mankhwala wamba a microtubule depolymerizing. Mwachitsanzo, kuchuluka kwa mankhwala a microtubule depolymerizing monga disopyramide ndi oryzalin kumapangitsa kuti maselo a epidermal afalikire kwambiri, pomwe KAND 11 siitero. Kuphatikiza apo, kugwiritsa ntchito pamodzi kwa KAND 11 ndi disopyramide kunapangitsa kuti mizu ikule bwino chifukwa cha disopyramide ndipo kuletsa kukula kwa KAND 11 kunawonedwa (Chithunzi S4). Tinafufuzanso momwe disopyramide 1-1 (phs1-1) imayankhira ku KAND 11. phs1-1 ili ndi kusintha kwa tubulin kinase point komwe sikuli kovomerezeka ndipo imapanga mizu yayifupi ikathandizidwa ndi disopyramide9,20. Mbeu za phs1-1 mutant zomwe zimamera pa agar medium zomwe zili ndi KAND 11 zinali ndi mizu yayifupi yofanana ndi yomwe imamera pa disopyramid (chithunzi S5).
Kuphatikiza apo, tinaona mapangidwe a mitotic microtubule, monga prophase zones, spindles, ndi phragmoplasts, mu mizu ya mbande zomwe zapatsidwa KAND 11. Mogwirizana ndi zomwe zawonedwa pa CDKB2;1p::CDKB2;1-GUS, kuchepa kwakukulu kwa chiwerengero cha mitotic microtubules kunawonedwa (Chithunzi .6c).
Kuti tizindikire kuchuluka kwa poizoni wa KAND 11 pa maselo ang'onoang'ono, tinachiza maselo oimitsidwa a BY-2 ndi KAND 11 ndipo tinaona momwe amayankhira. Choyamba tinawonjezera KAND 11 ku maselo a BY-2 omwe amawonetsa TagRFP-TUA6, yomwe imalemba ma microtubules mopepuka, kuti tiwone momwe KAND 11 imakhudzira ma microtubules a cortical. Kuchuluka kwa ma microtubule a cortical kunayesedwa pogwiritsa ntchito kusanthula kwa chithunzi, komwe kunayesa kuchuluka kwa ma pixel a cytoskeletal pakati pa ma pixel a cytoplasmic. Zotsatira za mayeso zinawonetsa kuti pambuyo pa chithandizo ndi 50 μM kapena 100 μM KAND 11 kwa ola limodzi, kuchuluka kunachepa kwambiri kufika pa 0.94 ± 0.74% kapena 0.23 ± 0.28%, motsatana, pomwe kuchuluka kwa maselo omwe amathandizidwa ndi DMSO, kunali 1.61 ± 0.34% (Chithunzi 7a). Zotsatirazi zikugwirizana ndi zomwe zapezeka mu Arabidopsis kuti chithandizo cha KAND 11 chimayambitsa kuchotsedwa kwa ma microtubules a cortical (Chithunzi 6b). Tinayang'ananso mzere wa BY-2 wokhala ndi ma filaments a actin olembedwa ndi GFP-ABD pambuyo pochiza ndi kuchuluka komweko kwa KAND 11 ndipo tinaona kuti chithandizo cha KAND 11 chinasokoneza ma filaments a actin. Chithandizo ndi 50 μM kapena 100 μM KAND 11 kwa ola limodzi chinachepetsa kwambiri kuchuluka kwa actin filament kufika pa 1.20 ± 0.62% kapena 0.61 ± 0.26%, motsatana, pomwe kuchuluka kwa maselo omwe amachiritsidwa ndi DMSO kunali 1.69 ± 0.51% (Chithunzi 2). 7b). Zotsatirazi zimasiyana ndi zotsatira za propyzamide, zomwe sizikhudza ma filaments a actin, ndi latrunculin B, actin depolymerizer yomwe sikhudza ma microtubules (Chithunzi SI S6). Kuphatikiza apo, chithandizo ndi coumamonamide 1, coumamonamide acid 6, kapena KAND 11 sichinakhudze ma microtubules m'maselo a HeLa (Chithunzi cha SI S7). Chifukwa chake, njira yogwirira ntchito ya KAND 11 imakhulupirira kuti ndi yosiyana ndi ya ma cytoskeleton disruptors odziwika bwino. Kuphatikiza apo, kuyang'ana kwathu kwa microscopic kwa maselo a BY-2 omwe adachiritsidwa ndi KAND 11 kunawonetsa kuyambika kwa kufa kwa maselo panthawi ya chithandizo cha KAND 11 ndipo kunawonetsa kuti kuchuluka kwa maselo akufa a Evans omwe adapakidwa buluu sikunakulire kwambiri pambuyo pa mphindi 30 za chithandizo cha KAND 11, pomwe pambuyo pa mphindi 90 za chithandizo ndi 50 μM kapena 100 μM KAND, chiwerengero cha maselo akufa chinawonjezeka kufika pa 43.7% kapena 80.1%, motsatana (Chithunzi 7c). Ponseponse, deta iyi ikuwonetsa kuti ursolic acid yochokera ku KAND 11 ndi choletsa cytoskeletal cha zomera chomwe sichinadziwike kale.
KAND imakhudza ma microtubules a cortical, actin filaments, ndi kuthekera kwa maselo a BY-2 a fodya. (a) Kuwona ma microtubules a cortical m'maselo a BY-2 pamaso pa TagRFP-TUA6. Maselo a BY-2 omwe adathandizidwa ndi KAND 11 (50 μM kapena 100 μM) kapena DMSO adawunikidwa pogwiritsa ntchito microscopy ya confocal. Kuchuluka kwa ma microtubule a cortical kunawerengedwa kuchokera ku ma micrograph a maselo 25 odziyimira pawokha. Zilembo zimasonyeza kusiyana kwakukulu (Tukey HSD test, p.< 0.05). Scale bar = 10 µm. (b) Cortical actin filaments mu maselo a BY-2 omwe akuwonetsedwa pamaso pa GFP-ABD2. Maselo a BY-2 omwe adathandizidwa ndi KAND 11 (50 μM kapena 100 μM) kapena DMSO adawunikidwa pogwiritsa ntchito confocal microscopy. Kuchuluka kwa cortical actin filaments kudawerengedwa kuchokera ku micrographs ya maselo 25 odziyimira pawokha. Zilembo zimasonyeza kusiyana kwakukulu (Tukey HSD test, p.< 0.05). Scale bar = 10 µm. (c) Kuwona maselo a BY-2 akufa pogwiritsa ntchito Evans blue staining. Maselo a BY-2 omwe adathandizidwa ndi KAND 11 (50 μM kapena 100 μM) kapena DMSO adawunikidwa pogwiritsa ntchito bright-field microscopy. n=3. Scale bar = 100 µm.
Kupezeka ndi kugwiritsa ntchito zinthu zatsopano zachilengedwe kwapangitsa kuti pakhale kupita patsogolo kwakukulu m'mbali zosiyanasiyana za moyo wa anthu, kuphatikizapo mankhwala ndi ulimi. Kafukufuku wakale wachitika kuti apeze mankhwala othandiza kuchokera ku zinthu zachilengedwe. Makamaka, actinomycetes amadziwika kuti ndi othandiza ngati maantibayotiki oletsa tizilombo toyambitsa matenda chifukwa cha kuthekera kwawo kupanga zinthu zina monga avermectin, mankhwala otsogolera a ivermectin ndi bleomycin ndi zotumphukira zake, zomwe zimagwiritsidwa ntchito ngati mankhwala oletsa khansa21,22. Momwemonso, mitundu yosiyanasiyana ya mankhwala ophera udzu yapezeka kuchokera ku actinomycetes, ena mwa iwo agwiritsidwa ntchito kale m'malonda1,23. Chifukwa chake, kusanthula kwa metabolites a actinomycete kuti tisiyanitse zinthu zachilengedwe zomwe zili ndi ntchito zofunika zamoyo kumaonedwa kuti ndi njira yothandiza. Mu kafukufukuyu, tinapeza mankhwala atsopano, coumamonamide, ochokera ku S. werraensis ndipo tinapanga bwino. Ursonic acid ndi mankhwala opangidwa a urbenamide ndi zotumphukira zake. Imatha kuyambitsa kupindika kwa mizu, kuwonetsa mphamvu yapakati mpaka yamphamvu yopha udzu, komanso kuwononga microtubules za zomera mwachindunji kapena mwanjira ina. Komabe, njira yogwirira ntchito ya urmotonic acid ikhoza kusiyana ndi njira yogwiritsira ntchito microtubule inhibitors yomwe ilipo, popeza KAND 11 imasokonezanso ulusi wa actin ndikuyambitsa kufa kwa maselo, zomwe zikusonyeza njira yowongolera yomwe urmotonic acid ndi zotumphukira zake zimakhudzira mitundu yosiyanasiyana ya ma cytoskeletal.
Kufotokozera mwatsatanetsatane za urbenonic acid kudzakuthandizani kumvetsetsa bwino momwe urbenonic acid imagwirira ntchito. Makamaka, cholinga chotsatira ndikuwunika momwe ursonic acid imagwirira ntchito ku microtubules zochepa kuti mudziwe ngati ursonic acid ndi zotumphukira zake zimagwira ntchito mwachindunji pa microtubules ndikuzichotsa, kapena ngati zochita zawo zimapangitsa kuti microtubules zisamayende bwino. Kuphatikiza apo, ngati microtubules si chandamale mwachindunji, kuzindikira malo omwe ursonic acid imagwirira ntchito komanso zolinga za molekyulu pa maselo a zomera kudzakuthandizani kumvetsetsa bwino makhalidwe a mankhwala okhudzana ndi izi komanso njira zomwe zingatheke zowonjezera mphamvu yophera udzu. Kuyesa kwathu kwa bioactivity kunavumbula mphamvu yapadera ya cytotoxic ya ursonic acid pakukula kwa zomera monga Arabidopsis thaliana, fodya ndi liverwort, pomwe maselo a E. coli kapena HeLa sanakhudzidwe. Kuchepa kapena kusaphatikizika kwa poizoni ku maselo a nyama ndi ubwino wa ursonic acid derivatives ngati apangidwa ngati mankhwala ophera udzu kuti agwiritsidwe ntchito m'minda yotseguka yaulimi. Inde, popeza microtubules ndi kapangidwe kofala mu eukaryotes, kuletsa kwawo kusankha m'zomera ndikofunikira kwambiri pa mankhwala ophera udzu. Mwachitsanzo, propyzamide, mankhwala ochotsera ma polymer omwe amalumikizana mwachindunji ndi tubulin ndikuletsa polymerization, amagwiritsidwa ntchito ngati mankhwala ophera tizilombo chifukwa cha poizoni wochepa ku maselo a nyama. Mosiyana ndi disopyramide, benzamides ofanana nawo ali ndi ma specifics osiyanasiyana. Kuphatikiza pa ma microtubules a zomera, RH-4032 kapena benzoxamide imaletsanso ma microtubules a maselo a nyama kapena oomycetes, motsatana, ndipo zalilamide imagwiritsidwa ntchito ngati fungicide chifukwa cha poizoni wochepa wa phyto25,26,27. Chimbalangondo chomwe changopezeka kumene ndi ma derivatives ake chikuwonetsa kuopsa kosankha zomera, koma ndikofunikira kudziwa kuti kusintha kwina kungasinthe specific yawo, zomwe zingapereke zowonjezera zotsutsana ndi bowa kapena oomycetes.
Makhalidwe apadera a urbenonic acid ndi zotumphukira zake ndi othandiza pakukula kwawo ngati mankhwala ophera udzu komanso kugwiritsa ntchito ngati zida zofufuzira. Kufunika kwa cytoskeleton pakulamulira mawonekedwe a maselo a chomera kumadziwika kwambiri. Kafukufuku wakale wasonyeza kuti zomera zapanga njira zovuta zokonzera ma microtubule a cortical powongolera ma microtubule dynamics kuti ziwongolere bwino morphogenesis. Mamolekyu ambiri omwe amayang'anira ntchito ya microtubule apezeka, ndipo kafukufuku wofanana nawo akadali kupitilira3,4,28. Kumvetsetsa kwathu kwamakono kwa ma microtubule dynamics m'maselo a zomera sikukufotokoza bwino momwe ma cortical microtubule structure amagwirira ntchito. Mwachitsanzo, ngakhale kuti disopyramide ndi oryzalin zonse zimatha kuchotsa polymerization ya ma microtubule, disopyramide imayambitsa kusokonekera kwa mizu pomwe oryzalin ili ndi zotsatira zochepa. Kuphatikiza apo, kusintha kwa tubulin, komwe kumakhazikitsa ma microtubule, kumayambitsanso dextrorotation mu mizu, pomwe paclitaxel, yomwe imakhazikitsanso ma microtubule dynamics, sichoncho. Chifukwa chake, kuphunzira ndikuzindikira zolinga za ma molekyulu a ursolic acid kuyenera kupereka chidziwitso chatsopano pakulamulira ma cortical microtubule a zomera. Mofananamo, kufananiza mtsogolo kwa mankhwala omwe angathandize kukulitsa kukula kolakwika, monga disopyramide, ndi mankhwala osagwira ntchito bwino, monga oryzalin kapena kumamotoric acid, kudzapereka zizindikiro za momwe kukula kolakwika kumachitikira.
Kumbali ina, kukonzedwanso kwa ma cytoskeletal okhudzana ndi chitetezo ndi njira ina yofotokozera kuopsa kwa ursonic acid. Kufalikira kwa tizilombo toyambitsa matenda kapena kulowetsedwa kwa elicitor m'maselo a zomera nthawi zina kumayambitsa kuwonongeka kwa cytoskeleton ndi kufa kwa maselo pambuyo pake29. Mwachitsanzo, cryptoxanthin yochokera ku oomycete yanenedwa kuti imasokoneza ma microtubules ndi ma filaments a actin maselo asanayambe kufa, mofanana ndi zomwe zimachitika ndi chithandizo cha KAND30,31. Kufanana pakati pa mayankho a chitetezo ndi mayankho a maselo omwe amayambitsidwa ndi ursonic acid kwatipangitsa kuganiza kuti zimayambitsa njira zodziwika bwino za maselo, ngakhale kuti zotsatira za ursonic acid mwachangu komanso mwamphamvu kuposa cryptoxanthin zikuwonekera. Komabe, kafukufuku wasonyeza kuti kusokonezeka kwa ma filaments a actin kumalimbikitsa kufa kwa maselo mwadzidzidzi, komwe sikuti nthawi zonse kumayenderana ndi kusokonezeka kwa ma microtubule29. Kuphatikiza apo, zikuwonekerabe ngati tizilombo toyambitsa matenda kapena elicitor zimayambitsa kukula kwa mizu yolakwika, monga momwe ursonic acid derivatives imachitira. Chifukwa chake, chidziwitso cha mamolekyulu cholumikiza mayankho a chitetezo ndi cytoskeleton ndi vuto lokongola lomwe liyenera kuthetsedwa. Mwa kugwiritsa ntchito kupezeka kwa mankhwala otsika a molekyulu okhudzana ndi ursonic acid, komanso mitundu yosiyanasiyana ya zinthu zomwe zimakhala ndi mphamvu zosiyanasiyana, zingapereke mwayi wolunjika ku njira zosadziwika za maselo.
Ponseponse, kupeza ndi kugwiritsa ntchito mankhwala atsopano omwe amasintha mphamvu za microtubule kudzapereka njira zamphamvu zothetsera njira zovuta zamamolekyu zomwe zimapangitsa kuti pakhale mawonekedwe a maselo a zomera. Pachifukwa ichi, compound urmotonic acid yomwe yapangidwa posachedwapa, yomwe imakhudza microtubules ndi actin filaments ndikuyambitsa kufa kwa maselo, ingapereke mwayi wopeza kulumikizana pakati pa kulamulira microtubule ndi njira zina izi. Chifukwa chake, kusanthula kwa mankhwala ndi zamoyo pogwiritsa ntchito urbenonic acid kudzatithandiza kumvetsetsa njira zowongolera mamolekyu zomwe zimalamulira cytoskeleton ya zomera.
Ikani botolo la S. werraensis MK493-CF1 mu botolo la Erlenmeyer la 500 mL losakanizidwa lomwe lili ndi 110 mL ya mbewu 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) chimanga chochotsera (KOGOSTCH Co., Ltd., Japan), 0.2% (w/v) (NH4)2SO4 ndi 0.2% CaCO3 m'madzi oyeretsedwa. (pH 7.4 musanayeretsedwe). Mbewu zobzalidwa zinayikidwa mu chogwedeza chozungulira (180 rpm) pa 27°C kwa masiku awiri. Kulima kopangidwa pogwiritsa ntchito solid state fermentation. Kubzala mbewu (7 ml) kunasamutsidwa mu botolo la 500 ml K-1 lomwe linali ndi 40 g ya malo opangira zinthu okhala ndi 15 g ya barele wokakamizidwa (MUSO Co., Ltd., Japan) ndi 25 g ya madzi oyeretsedwa (pH sinasinthidwe musanayeretsedwe). Kuwiritsa kunachitidwa pa 30°C mumdima kwa masiku 14. Zinthu zowiritsa zinachotsedwa ndi 40 ml/botolo la EtOH ndipo zinayikidwa mu centrifuge (1500 g, 4°C, mphindi 10). Supernatant yokulira (60 ml) inachotsedwa ndi chisakanizo cha 10% MeOH/EtOAc. Gawo lachilengedwe linasanduka nthunzi pansi pa kupanikizika kochepa kuti lipeze zotsalira (59.5 mg), zomwe zinayikidwa mu HPLC ndi gradient elution (mphindi 0–10: 90%) pa reverse phase column (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × kutalika 250 mm) H2O/CH3CN, mphindi 10–35: 90% H2O/CH3CN mpaka 70% H2O/CH3CN (gradient), mphindi 35–45: 90% H2O/EtOH, mphindi 45–155: 90% H2O/EtOH mpaka 100% EtOH (gradient (gradient), mphindi 155–200: 100% EtOH) pa flow rate ya 1.5 ml/min, coumamonamide (1, 36.0 mg) inapezeka ngati ufa woyera wosasinthika.
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 wowerengedwa: 141.0659, mtengo woyesedwa: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
Mbewu za ku Columbia (Col-0) zinapezeka ku Arabidopsis Biological Resource Center (ABRC) ndi chilolezo choti zigwiritsidwe ntchito pa kafukufuku. Mbewu za Col-0 zinafalitsidwa ndikusungidwa pansi pa mikhalidwe yathu ya labotale ndipo zinagwiritsidwa ntchito ngati zomera zakuthengo za Arabidopsis. Mbewu za Arabidopsis zinaphikidwa pamwamba ndikulimidwa mu Murashige ndi Skoog medium yamphamvu 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 mtundu wa SR-1 zinaperekedwa ndi T. Hashimoto (Nara Institute of Science and Technology) ndipo zinagwiritsidwa ntchito ngati zomera za fodya zakuthengo. Mbewu za fodya zinayeretsedwa pamwamba ndi kuviikidwa m'madzi osawilitsidwa kwa masiku atatu kuti zipititse patsogolo kumera, kenako zinayikidwa mu yankho la mphamvu ya theka lomwe lili ndi 2% sucrose, 0.05% (w/v) MES, ndi 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige. ndi Skoog medium) yokhala ndi pH 5.7 ndipo zinayikidwa pa 23°C pansi pa kuwala kosalekeza.
Mankhwala a Strain Tak-1 adaperekedwa ndi T. Kohchi (Yunivesite ya Kyoto) ndipo adagwiritsidwa ntchito ngati gawo loyesera la kafukufuku wa liverwort. Gemma idapezeka kuchokera ku zomera zobzalidwa mopanda kuwiritsa kenako idayikidwa pa Gamborg B5 medium (Fujifilm Wako Pure Chemical) yokhala ndi 1% sucrose ndi 0.3% gellan chingamu ndikuyikidwa pa 23°C pansi pa kuwala kosalekeza.
Ma cell a fodya a BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2) adaperekedwa ndi S. Hasezawa (University of Tokyo). Ma cell a BY-2 adachepetsedwa ka 95-fold mu Linsmeier yosinthidwa ndi Skoog medium ndipo adawonjezeredwa sabata iliyonse ndi 2,4-dichlorophenoxyacetic acid 32. Kuyimitsidwa kwa maselo kunasakanizidwa pa rotary shaker pa 130 rpm pa 27°C mumdima. Tsukani maselo ndi voliyumu yoposa 10 ya medium yatsopano ndikuyimitsidwanso mu medium yomweyo. Mizere ya maselo a BY-2 transgenic yomwe ikuwonetsa bwino chizindikiro cha microtubule TagRFP-TUA6 kapena chizindikiro cha actin filament GFP-ABD2 pansi pa cauliflower mosaic virus 35S promoter idapangidwa monga momwe tafotokozera33,34,35. Mizere ya maselo iyi ikhoza kusungidwa ndikugwirizanitsidwa pogwiritsa ntchito njira zofanana ndi zomwe zidagwiritsidwa ntchito pa mzere woyambirira wa maselo a BY-2.
Maselo a HeLa adakulitsidwa mu Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) yowonjezera 10% fetal bovine serum, 1.2 U/ml penicillin, ndi 1.2 μg/ml streptomycin mu incubator ya 37°C yokhala ndi 5% CO2.
Mayesero onse omwe afotokozedwa m'bukuli adachitika motsatira malamulo ndi malangizo a chitetezo cha chilengedwe aku Japan.
Ma compounds anasungunuka mu dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) ngati njira zosungiramo mbewu ndipo anachepetsedwa mu MS medium ya Arabidopsis ndi fodya kapena Gamborg B5 medium ya liverwort. Pa kuyesa koletsa kukula kwa mizu, mbewu zoposa 10 pa mbale iliyonse zinafesedwa pa agar medium yokhala ndi mankhwala omwe asonyezedwa kapena DMSO. Mbewu zinaikidwa mu chipinda chokulirapo kwa masiku 7. Mbewu zinajambulidwa ndipo kutalika kwa mizu kunayesedwa. Pa kuyesa kumera kwa Arabidopsis, mbewu 48 pa mbale iliyonse zinafesedwa pa agar medium yokhala ndi 200 μM compound kapena DMSO. Mbewu za Arabidopsis zinafesedwa mu chipinda chokulirapo ndipo chiwerengero cha mbewu zomwe zinamera chinawerengedwa patatha masiku 7 zitamera (dag). Pa kuyesa kumera kwa fodya, mbewu 24 pa mbale iliyonse zinafesedwa pa agar medium yokhala ndi 200 μM KAND kapena DMSO. Mbewu za fodya zinafesedwa mu chipinda chokulirapo ndipo chiwerengero cha mbewu zomwe zinamera chinawerengedwa patatha masiku 14. Pa mayeso oletsa kukula kwa liverwort, mazira 9 ochokera pa mbale iliyonse adayikidwa pa agar medium yokhala ndi kuchuluka kwa KAND kapena DMSO komwe kumawonetsedwa ndipo adayikidwa mu chipinda chokulirapo kwa masiku 14.
Gwiritsani ntchito mbande zopakidwa utoto wa 5 mg/ml wa propidium iodide (PI) kuti muwone bwino momwe mizu ya meristem imagwirira ntchito. Zizindikiro za PI zinawonedwa pogwiritsa ntchito maikulosikopu ya fluorescence pogwiritsa ntchito maikulosikopu ya TCS SPE confocal laser scanning (Leica Microsystems).
Kupaka utoto wa histochemical pa mizu ndi β-glucuronidase (GUS) kunachitika motsatira njira yomwe Malami ndi Benfey36 adafotokoza. Mbande zinakhazikika mu 90% acetone usiku wonse, zopaka utoto wa 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid mu GUS buffer kwa ola limodzi ndipo zinayikidwa mu hydrated chloraldehyde solution. (8 g chloral hydrate, 2 ml madzi ndi 1 ml glycerol) ndipo zinawonedwa ndi differential interference contrast microscopy pogwiritsa ntchito Axio Imager M1 microscope (Carl Zeiss).
Makona a mizu adayesedwa pa mbande za masiku 7 zomwe zidabzalidwa pa mbale zoyimitsidwa. Yesani ngodya ya mizu kuchokera mbali ya vekitala yokoka monga tafotokozera mu gawo 6.
Kapangidwe ka ma cortical microtubules kadawonedwa monga momwe tafotokozera, ndi kusintha pang'ono kwa protocol 37. Anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) ndi Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) zidagwiritsidwa ntchito ngati ma antibodies oyambira ndi achiwiri pa 1:1000 ndi 1:100 dilutions, motsatana. Zithunzi za Fluorescence zidapezeka pogwiritsa ntchito TCS SPE confocal laser scanning microscope (Leica Microsystems). Pezani zithunzi za Z-stack ndikupanga ma projection amphamvu kwambiri malinga ndi malangizo a wopanga.
Kuyesa kuchuluka kwa maselo a HeLa kunachitika pogwiritsa ntchito Cell Counting Kit 8 (Dojindo) motsatira malangizo a wopanga.
Kukula kwa E. coli DH5α kunafufuzidwa poyesa kuchuluka kwa maselo m'chilengedwe pogwiritsa ntchito spectrophotometer pa 600 nm (OD600).
Kulinganiza kwa ma cytoskeletal m'maselo a BY-2 opangidwa ndi transgenic kunawonedwa pogwiritsa ntchito maikulosikopu ya fluorescence yokhala ndi chipangizo chojambulira cha CSU-X1 confocal (Yokogawa) ndi kamera ya sCMOS (Zyla, Andor Technology). Kuchuluka kwa ma cytoskeletal kunayesedwa pogwiritsa ntchito kusanthula kwa zithunzi, komwe kunayesa kuchuluka kwa ma cytoskeletal pixels pakati pa ma cytoplasmic pixels m'zithunzi za confocal pogwiritsa ntchito pulogalamu ya ImageJ monga momwe tafotokozera38,39.
Kuti azindikire kufa kwa maselo mu maselo a BY-2, gawo limodzi la selo loyimitsidwa linayikidwa ndi 0.05% Evans blue kwa mphindi 10 kutentha kwa chipinda. Kupaka utoto wabuluu wa Evans wa maselo akufa kumadalira kutulutsa utoto kuchokera ku maselo okhazikika ndi nembanemba ya plasma yosatha40. Maselo opaka utoto adawonedwa pogwiritsa ntchito maikulosikopu yowala (BX53, Olympus).
Maselo a HeLa adakulitsidwa mu DMEM owonjezeredwa ndi 10% FBS mu incubator yonyowa pa 37°C ndi 5% CO2. Maselo adachiritsidwa ndi 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), kapena 100 ng/ml Nocodmaze (Sigma) kwa maola 6 pa 37°C. Maselo adakhazikika ndi MetOH kwa mphindi 10 kenako ndi acetate kwa mphindi 5 kutentha kwa chipinda. Maselo okhazikika adakhazikika ndi β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) yochepetsedwa mu 0.5% BSA/PBS kwa maola awiri, kutsukidwa katatu ndi TBST, kenako kukhazikika ndi Alexa Fluor goat antibody. 488 Ola limodzi. – Mbewa IgG (Thermo Fisher Scientific: A11001) ndi 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) yochepetsedwa mu 0.5% BSA/PBS. Pambuyo potsuka ndi TBST katatu, maselo opaka utoto adawonedwa pa maikulosikopu yozungulira ya Nikon Eclipse Ti-E. Zithunzi zidajambulidwa ndi kamera yoziziritsidwa ya Hamamatsu ORCA-R2 CCD pogwiritsa ntchito pulogalamu ya MetaMorph (Molecular Devices).
Nthawi yotumizira: Juni-17-2024