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ʻO ka ʻike ʻana a me ka hoʻohana pono ʻana i nā huahana kūlohelohe e hiki ke kōkua i ka hoʻomaikaʻi ʻana i ke ola kanaka. Hoʻohana nui ʻia nā kemika hoʻomake ulu mea kanu e like me nā herbicides e kāohi i nā nāhelehele. Ma muli o ka pono e hoʻohana i nā ʻano herbicides like ʻole, aia kahi pono e ʻike i nā hui me nā ʻano hana hou. Ma kēia haʻawina, ua ʻike mākou i kahi hui N -alkoxypyrrole hou, coumamonamide, mai Streptomyces werraensis MK493-CF1 a hoʻokumu i ke kaʻina hana synthesis piha. Ma o nā hoʻāʻo hana olaola, ua ʻike mākou ʻo ka urs-monoamic acid kahi waena synthetic o urs-monoamide a me kahi hiki.mea hoʻopaʻa ulu mea kanu. Eia kekahi, ua hoʻomohala mākou i nā ʻano derivatives urbenonic acid like ʻole, me ka urbenyloxy derivative (UDA), nona ka hana herbicidal kiʻekiʻe me ka ʻole o ka hoʻopilikia maikaʻi ʻole i ka ulu ʻana o nā cell HeLa. Ua ʻike pū mākou e hoʻopilikia nā derivatives urmotonic acid i nā microtubules mea kanu; eia kekahi, hoʻopilikia ʻo KAND i nā filaments actin a hoʻoulu i ka make o nā cell; ʻOkoʻa kēia mau hopena multifaceted mai nā mea i ʻike ʻia e nā mea hoʻopaʻa microtubule a hōʻike i kahi ʻano hana hou no ka ursonic acid, kahi e hōʻike ana i kahi pono koʻikoʻi i ka hoʻomohala ʻana i nā herbicides hou.
ʻO ka ʻike ʻana a me ka hoʻohana pono ʻana i nā huahana kūlohelohe pono a me kā lākou mau mea i loaʻa mai he ala ia e hoʻomaikaʻi ai i ke ʻano o ke ola kanaka. ʻO nā metabolites lua i hana ʻia e nā microorganisms, nā mea kanu a me nā pepeke ua alakaʻi i nā holomua nui i ka lāʻau lapaʻau a me ka mahiʻai. Ua hoʻomohala ʻia nā antibiotics he nui a me nā lāʻau anti-leukemia mai nā huahana kūlohelohe. Eia kekahi, nā ʻano like ʻole onā mea pepehi lāʻau make, ua unuhi ʻia nā fungicides a me nā herbicides mai kēia mau huahana kūlohelohe no ka hoʻohana ʻana i ka mahiʻai. ʻO ka mea nui, he mau mea hana koʻikoʻi nā herbicides kaohi mauʻu no ka hoʻonui ʻana i nā hua kanu i ka mahiʻai hou, a ua hoʻohana mua ʻia nā ʻano hui like ʻole i ke kālepa ʻana. Nui nā kaʻina hana cellular i nā mea kanu, e like me ka photosynthesis, ka metabolism amino acid, ka synthesis paia cell, ka hoʻoponopono ʻana o ka mitosis, ka hōʻailona phytohormone, a i ʻole ka synthesis protein, i manaʻo ʻia he mau pahuhopu maʻamau o nā herbicides. ʻO nā hui e kāohi ana i ka hana microtubule he papa maʻamau o nā herbicides e hoʻopilikia i ka ulu ʻana o nā mea kanu ma o ka hoʻopilikia ʻana i ka hoʻoponopono mitotic2.
ʻO nā Microtubules nā ʻāpana o ka cytoskeleton a ua mālama nui ʻia i loko o nā cell eukaryotic. ʻO ka tubulin heterodimer he α-tubulin a me β-tubulin e hana ana i nā protofilaments microtubule linear, me 13 mau protofilaments e hana ana i kahi ʻano cylindrical. He nui nā kuleana o nā Microtubules i loko o nā cell mea kanu, me ka hoʻoholo ʻana i ke ʻano o ke cell, ka mahele ʻana o ke cell, a me ka lawe ʻana o ka intracellular3,4. Loaʻa i nā cell mea kanu nā microtubules ma lalo o ka membrane plasma interphase, a ua manaʻo ʻia kēia mau microtubules cortical e kāohi i ka hoʻonohonoho ʻana o nā microfibrils cellulose ma o ka hoʻoponopono ʻana i nā complexes cellulose synthase4,5. Aia nā microtubules cortical o nā cell epidermal aʻa, aia ma ka ʻāpana o ka elongation wikiwiki o ka piko aʻa, ma ka ʻaoʻao, a hahai nā microfibers cellulose i kēia mau microtubules a kaupalena i ke kuhikuhi o ka hoʻonui ʻana o ke cell, no laila e hoʻolaha ana i ka elongation cell anisotropic. No laila, pili loa ka hana microtubule i ke ʻano o ka mea kanu. ʻO nā pani ʻana o ka waikawa amino i loko o nā genes e hoʻopili ana i ka tubulin ke kumu o ka skew o nā cortical microtubule arrays a me ka ulu ʻana ma ka ʻaoʻao hema a ʻākau paha ma Arabidopsis 6,7. Pēlā nō, ʻo nā mutations i loko o nā protein e pili ana i ka microtubule e hoʻoponopono ana i ka dynamics microtubule hiki ke alakaʻi i ka ulu ʻana o ke aʻa i hoʻololi ʻia8,9,10,11,12,13. Eia kekahi, ʻo ka mālama ʻana me nā herbicides e hoʻopilikia ana i ka microtubule e like me disopyramide, i ʻike ʻia hoʻi ʻo pretilachlor, ke kumu hoʻi i ka ulu ʻana o ke aʻa oblique ma ka ʻaoʻao hema14. Hōʻike kēia mau ʻikepili he mea koʻikoʻi ka hoʻoponopono pololei ʻana o ka hana microtubule no ka hoʻoholo ʻana i ke kuhikuhi o ka ulu ʻana o ka mea kanu.
Ua ʻike ʻia nā ʻano mea hoʻopaʻa microtubule like ʻole, a ua hāʻawi nui kēia mau lāʻau i ka noiʻi cytoskeletal, a me ka mahiʻai a me ka lāʻau lapaʻau2. Ma ke ʻano kūikawā, hiki i ka oryzalin, nā hui dinitroaniline, disopyramide, nā hui e pili ana i ka benzamide, a me kā lākou analogs ke kāohi i ka hana microtubule a pēlā e kāohi ai i ka ulu ʻana o nā mea kanu. No laila, hoʻohana nui ʻia lākou e like me nā herbicides. Eia nō naʻe, ʻoiai he ʻāpana koʻikoʻi nā microtubules o nā cell mea kanu a me nā holoholona, ʻo ka hapa nui o nā mea hoʻopaʻa microtubule he cytotoxic i nā ʻano cell ʻelua. No laila, ʻoiai ko lākou pono i ʻike ʻia he herbicides, hoʻohana ʻia kahi helu palena o nā mea antimicrotubule no nā kumu kūpono.
ʻO Streptomyces kahi ʻano o ka ʻohana Streptomyces, nona nā bacteria aerobic, gram-positive, filamentous a ua ʻike nui ʻia no kona hiki ke hana i nā ʻano metabolites lua. No laila, ua manaʻo ʻia ʻo ia kekahi o nā kumu nui loa o nā huahana kūlohelohe biologically active hou. Ma ke aʻo ʻana o kēia manawa, ua ʻike mākou i kahi hui hou i kapa ʻia ʻo coumamonamide, i hoʻokaʻawale ʻia mai Streptomyces werraensis MK493-CF1 a me S. werraensis ISP 5486. Ma ka hoʻohana ʻana i ka nānā ʻana i ka spectral a me ka nānā ʻana i ka spectral piha, ua wehewehe ʻia ke ʻano o ka coumamonamide a ua hoʻoholo ʻia kona iwi kū hoʻokahi N-alkoxypyrrole. synthesis. ʻO ka waikawa Ursmonic, kahi waena synthetic o ursmonoamide a me kāna mau derivatives, ua ʻike ʻia e kāohi i ka ulu ʻana a me ka germination o ka mea kanu hoʻohālike kaulana ʻo Arabidopsis thaliana. Ma kahi noiʻi pilina hana-ʻano, ua ʻike mākou he hui me C9 i hoʻololi ʻia i ka waikawa ursonic, i kapa ʻia ʻo nonyloxy derivative o ka waikawa ursonic (KAND), e hoʻonui nui i ka hopena inhibitory ma ka ulu ʻana a me ka germination. ʻO ka mea nui, ua hoʻopilikia pū ka mea hoʻopale ulu mea kanu i ʻike hou ʻia i ka ulu ʻana o ka paka a me ke akepaʻa a ʻaʻole ia he cytotoxic i nā bacteria a i ʻole nā pūnaewele HeLa. Eia kekahi, hoʻoulu kekahi mau derivatives urmotonic acid i kahi phenotype aʻa i hoʻololi ʻia, e hōʻike ana he pili pololei a ʻaʻole pololei paha kēia mau derivatives i nā microtubules. E kūlike me kēia manaʻo, ʻo kā mākou nānā ʻana i nā microtubules i kapa ʻia ʻo immunohistochemically a i ʻole me nā protein fluorescent e hōʻike ana e hoʻohemo ka mālama ʻana o KAND i nā microtubules. Eia kekahi, ua hoʻopau ka mālama ʻana me nā derivatives kumamotonic acid i nā microfilaments actin. No laila, ua ʻike mākou i kahi mea hoʻopale ulu mea kanu hou nona ke ʻano hana kūikawā e pili ana i ka luku ʻana i ka cytoskeleton.
Ua hoʻokaʻawale ʻia ke ʻano MK493-CF1 mai ka lepo ma Shinagawa-ku, Tokyo. Ua hoʻokumu ke ʻano MK493-CF1 i ka mycelium stromal lālā maikaʻi. Ua hoʻoholo ʻia ke kaʻina hapa o ka gene RNA ribosomal 16S (1422 bp). Ua like loa kēia ʻano me S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: ʻano maʻamau, 99.93%). Ma muli o kēia hopena, ua hoʻoholo ʻia ua pili loa kēia ʻano i ke ʻano o S. werraensis. No laila, ua kapa mua mākou i kēia ʻano S. werraensis MK493-CF1. Hoʻopuka pū ʻo S. werraensis ISP 5486T i nā pūhui bioactive like. No ka mea, ʻaʻohe noiʻi mua i ka loaʻa ʻana o nā huahana kūlohelohe mai kēia microorganism, ua hana ʻia nā noiʻi kemika hou aʻe. Ma hope o ka mahi ʻana o S. werraensis MK493-CF1 ma ka pā bale ma o ka fermentation solid-state ma 30°C no 14 mau lā, ua unuhi ʻia ka pā me 50% EtOH. Ua hoʻomaloʻo ʻia he 60 ml o ka laʻana e loaʻa ai he 59.5 mg o ka ʻaila maka. Ua hoʻokomo ʻia ka ʻaila maka i ka reverse phase HPLC e hāʻawi iā N-methoxy-1H-pyrrole-2-carboxamide (1, i kapa ʻia ʻo coumamonamide, 36.0 mg). ʻO ka nui o 1 ma kahi o 60% o ka ʻaila maka. No laila, ua hoʻoholo mākou e aʻo pono i nā waiwai o kumamotoamide 1.
He pauka amorphous keʻokeʻo ʻo Coumamonamide 1 a hōʻoia ka spectrometry mass resolution kiʻekiʻe (HRESIMS) iā C6H8N2O2 (Kiʻi 1). ʻO ka ʻāpana pyrrole i hoʻololi ʻia e C2 o kēia hui i hōʻailona ʻia e δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH ma ka spectrum 1H NMR: 4.5 Hz, H-5) a me δH 6.78 (1H, d, J = 2.5 Hz, H-6), a hōʻike ka spectrum 13C NMR i ke alo o ʻehā mau ʻātoma kalapona sp2. Ua loiloi ʻia ke alo o kahi hui amide ma ke kūlana C2 e ka pilina HMBC mai ka proton C-3 i ke kalapona amide carbonyl ma δC 161.1. Eia kekahi, ʻo nā piko 1 H a me 13 C NMR ma δH 4.10 (3H, S) a me δC 68.3 e hōʻike ana i ke alo o nā hui N-methoxy i loko o ka mole. ʻOiai ʻaʻole i hoʻoholo ʻia ke kūlana pololei o ka hui methoxy me ka hoʻohana ʻana i ka nānā spectroscopic e like me ka enhanced difference spectroscopy a me ka nuclear Overhauser abbreviation (NOEDF), ua lilo ʻo N-methoxy-1H-pyrrole-2-carboxamide i hui moho mua.
No ka hoʻoholo ʻana i ke ʻano kūpono o 1, ua hana ʻia kahi synthesis holoʻokoʻa (Kiʻi 2a). ʻO ka mālama ʻana o 2-aminopyridine 2 i loaʻa ma ke kālepa me ka m-CPBA i hopena i ka N-oxide 3 e like me ka hua quantitative. Ma hope o ka 2-aminoazidation o 2, ua hoʻokō ʻia ka hopena cyclocondensation i wehewehe ʻia e Abramovich ma benzene ma 90°C e loaʻa ai ka 1-hydroxy-1H-pyrrole-2-carbonitrile 5 i makemake ʻia ma grams. Speed 60% (ʻelua pae). 15,16. A laila hāʻawi ka Methylation a me ka hydrolysis o 4 i ka 1-methoxy-1H-pyrrole-2-carboxylic acid (i kapa ʻia ʻo "cumotonic acid", 6) i ka hua maikaʻi (70%, ʻelua mau ʻanuʻu). ʻO ka hope, ʻo ka amidation ma o ka acid chloride intermediate 6 me ka hoʻohana ʻana i ka aqueous ammonia i hāʻawi iā Kumamoto amide 1 i ka hua 98%. ʻO nā ʻikepili spectral āpau o synthesized 1 ua like me ka 1 i hoʻokaʻawale ʻia, no laila ua hoʻoholo ʻia ke ʻano o 1;
ʻO ka synthesis laulā a me ka nānā ʻana i ka hana olaola o ka urbenamide a me ka waikawa urbenic. (a) ʻO ka synthesis holoʻokoʻa o Kumamoto amide. (b) Ua ulu ʻia nā hua kanu Arabidopsis Columbia (Col) ʻano hihiu ʻehiku lā ma nā papa Murashige lāua ʻo Skoog (MS) e loaʻa ana ka coumamonamide 6 a i ʻole coumamonamide 1 ma nā ʻano i hōʻike ʻia. Ka pā unahi = 1 cm.
ʻO ka mea mua, ua loiloi mākou i nā hana olaola o ka urbenamide a me kāna mau mea waena no ko lākou hiki ke hoʻololi i ka ulu ʻana o nā mea kanu. Ua hoʻohui mākou i nā ʻano like ʻole o ka ursmonamide 1 a i ʻole ka waikawa ursmonic 6 i ka MS agar medium a me nā mea kanu Arabidopsis thaliana i mahi ʻia ma kēia medium. Ua hōʻike kēia mau hoʻāʻo ʻana ua kāohi nā ʻano kiʻekiʻe (500 μM) o 6 i ka ulu ʻana o ke aʻa (Kiʻi 2b). A laila, ua hana mākou i nā derivatives like ʻole ma ke pani ʻana i ke kūlana N1 o 6 a ua hana i nā haʻawina pilina hana-hana ma luna o lākou (ua wehewehe ʻia ke kaʻina hana synthesis analogue ma ka ʻIke Kākoʻo (SI)). Ua ulu ʻia nā mea kanu Arabidopsis ma kahi medium e loaʻa ana he 50 μM ursonic acid derivatives, a ua ana ʻia ka lōʻihi o ke aʻa. e like me ka mea i hōʻike ʻia ma ke kiʻi. E like me ka mea i hōʻike ʻia ma nā Kiʻi 3a, b, a me S1, he ʻokoʻa ka lōʻihi o nā kaulahao alkoxy linear (9, 10, 11, 12, a me 13) a i ʻole nā kauoha alkoxy nui (15, 16, a me 17) ma ke kūlana N1. Ua hōʻike nā derivatives i ka pale nui ʻana i ka ulu ʻana o ke aʻa. Eia kekahi, ua ʻike mākou ua kāohi ka hoʻopili ʻana o 200 μM 10, 11, a i ʻole 17 i ka germination (Figs. 3c a me S2).
Ke aʻo ʻana i ka pilina hana-ʻano o Kumamoto amide a me nā hui pili. (a) ʻAno hoʻonohonoho a me ka synthesis o nā analogues. (b) Ka helu ʻana o ka lōʻihi o ke aʻa o nā hua kanu 7 lā i ulu ʻia ma ka MS medium me a i ʻole me ka ʻole o 50 μM coumamonamide derivatives. Hōʻike nā asterisks i nā ʻokoʻa koʻikoʻi me ka mālama sham (t test, p< 0.05). n>18. Hōʻike ʻia nā ʻikepili ma ke ʻano he awelika ± SD. ʻO ke ʻano o nt "ʻaʻole i hoʻāʻo ʻia" no ka mea ʻaʻole i ulu aʻe ma mua o 50% o nā hua. (c) Ka helu ʻana o ka nui o ka germination o nā hua i mālama ʻia i hoʻoulu ʻia no 7 mau lā ma ka MS medium me a ʻaʻohe paha 200 μM coumamonamide a me nā hui pili. Hōʻike nā asterisks i nā ʻokoʻa koʻikoʻi me ka mālama sham (hoʻāʻo chi-square). n=96.
ʻO ka mea hoihoi, ʻo ka hoʻohui ʻana o nā kaulahao ʻaoʻao alkyl i ʻoi aku ka lōʻihi ma mua o C9 i hōʻemi i ka hana inhibitory, e hōʻike ana he pono nā hui e pili ana i ka waikawa kumamotoic i nā kaulahao ʻaoʻao o kahi nui e hōʻike i kā lākou hana olaola.
Ma muli o ka hōʻike ʻana o ka pilina hana-hana ua hoʻololi ʻia ʻo C9 i ka waikawa ursonic a ʻo ka nonyloxy derivative o ka waikawa ursonic (i kapa ʻia ma hope ʻo KAND 11) ka mea hoʻopale ulu mea kanu maikaʻi loa, ua hana mākou i kahi ʻano kikoʻī o KAND 11. ʻO ka mālama ʻana iā Arabidopsis me 50 μM KAND 11 ua aneane pale loa i ka germination, ʻoiai ʻo nā ʻāpana haʻahaʻa (40, 30, 20, a i ʻole 10 μM) o KAND 11 i kāohi i ka ulu ʻana o ke aʻa ma ke ʻano hilinaʻi i ka lāʻau (Kiʻi 4a, b). No ka hoʻāʻo ʻana inā pili ʻo KAND 11 i ke ola o ka meristem aʻa, ua nānā mākou i nā meristem aʻa i hoʻoluʻu ʻia me ka propidium iodide (PI) a ua ana i ka nui o ka ʻāpana meristem. ʻO ka nui o ka meristem o nā mea kanu i ulu ʻia ma kahi medium e loaʻa ana he 25 μM KAND-11 he 151.1 ± 32.5 μm, ʻoiai ʻo ka nui o ka meristem o nā mea kanu i ulu ʻia ma kahi medium kaohi e loaʻa ana he DMSO he 264.7 ± 30.8 μm (Kiʻi 4c, d), e hōʻike ana e hoʻihoʻi ana ʻo KAND-11 i ka hana cellular. ka laha ʻana. Meristem aʻa. E kūlike me kēia, ua hoʻemi ka mālama ʻana o KAND 11 i ka nui o ka hōʻailona mahele cell CDKB2;1p::CDKB2;1-GUS i loko o ka meristem aʻa (Kiʻi 4e) 17. Hōʻike kēia mau hopena e kāohi ana ʻo KAND 11 i ka ulu ʻana o ke aʻa ma ka hōʻemi ʻana i ka hana hoʻonui cell.
Ka nānā ʻana i ka hopena pale o nā derivatives waikawa urbenonic (urbenyloxy derivatives) i ka ulu ʻana. (a) Nā hua kanu Col hihiu 7 lā i ulu ʻia ma nā papa MS me nā ʻano i hōʻike ʻia o KAND 11. Ka pā unahi = 1 cm. (b) Ka helu ʻana o ka lōʻihi o ke aʻa. Hōʻike nā leka i nā ʻokoʻa koʻikoʻi (hoʻāʻo Tukey HSD, p< 0.05). n>16. Hōʻike ʻia nā ʻikepili ma ke ʻano he awelika ± SD. (c) Microscopy confocal o nā aʻa Col wild-type i hoʻoluʻu ʻia i ka propidium iodide i ulu ʻia ma nā papa MS me a ʻaʻohe paha 25 μM KAND 11. Hōʻike nā pale keʻokeʻo i ka meristem aʻa. Pā unahi = 100 µm. (d) Ka helu ʻana o ka nui o ka meristem aʻa (n = 10 a 11). Ua hoʻoholo ʻia nā ʻokoʻa helu me ka hoʻohana ʻana i ka t-test (p< 0.05). Hōʻike nā kaola i ka nui o ka meristem awelika. (e) Microscopy hoʻohālikelike hoʻopilikia ʻokoʻa (DIC) o kahi meristem aʻa e loaʻa ana ka hana CDKB2; 1pro: CDKB2; 1-GUS i hoʻoluʻu ʻia a hoʻoluʻu ʻia ma nā hua kanu 5 lā i ulu ʻia ma nā papa MS me a i ʻole me ka ʻole o 25 µM KAND assay.
Ua hoʻāʻo hou ʻia ka phytotoxicity o KAND 11 me ka hoʻohana ʻana i kahi mea kanu dicotyledonous ʻē aʻe, ka paka (Nicotiana tabacum), a me kahi meaola hoʻohālike mea kanu ʻāina nui, ka liverwort (Marchantia polymorpha). E like me ke ʻano o Arabidopsis, ʻo nā mea kanu paka SR-1 i ulu ʻia ma ka medium e loaʻa ana he 25 μM KAND 11 i hua i nā aʻa pōkole (Kiʻi 5a). Eia kekahi, ua ulu aʻe he 40 o 48 mau hua ma nā papa e loaʻa ana he 200 μM KAND 11, ʻoiai ua ulu aʻe nā hua 48 a pau ma nā media hoʻopunipuni, e hōʻike ana he koʻikoʻi nā ʻano kiʻekiʻe o KAND (p< 0.05; hoʻāʻo chi -square) i kāohi i ka ulu ʻana o ka paka. (Kiʻi 5b). Eia kekahi, ʻo ka nui o KAND 11 i kāohi i ka ulu ʻana o ka bacteria i loko o ka liverwort ua like ia me ka nui kūpono ma Arabidopsis (Kiʻi 5c). Hōʻike kēia mau hopena hiki iā KAND 11 ke kāohi i ka ulu ʻana o nā ʻano mea kanu like ʻole. A laila ua noiʻi mākou i ka cytotoxicity hiki o nā hui e pili ana i ka bear monoamide i nā meaola ʻē aʻe, ʻo ia hoʻi nā cell HeLa kanaka a me ka Escherichia coli strain DH5α, ma ke ʻano he mau ʻelele o nā cell holoholona kiʻekiʻe a me nā cell bacteria, kēlā me kēia. Ma kahi moʻo o nā hoʻāʻo hoʻonui cell, ua ʻike mākou ʻaʻole i hoʻopilikia ka coumamonamide 1, coumamonamidic acid 6, a me KAND 11 i ka ulu ʻana o nā cell HeLa a i ʻole E. coli ma nā nui o 100 μM (Kiʻi 5d,e).
Ka pale ʻana i ka ulu ʻana o KAND 11 i nā meaola ʻaʻole Arabidopsis. (a) Ua ulu ʻia nā hua paka hihiu ʻano SR-1 ʻelua pule ma nā papa MS i hoʻonoho ʻia me 25 μM KAND 11. (b) Ua ulu ʻia nā hua paka hihiu ʻano SR-1 ʻelua pule ma nā papa MS i hoʻonoho ʻia me 200 μM KAND 11. (c) Nā ʻōpuʻu liverwort hihiu ʻano Tak-1 ʻelua pule i ulu ʻia ma nā papa Gamborg B5 me nā ʻano i hōʻike ʻia o KAND 11. Hōʻike nā pua ʻulaʻula i nā spores i oki i ka ulu ʻana i loko o ka manawa hoʻoulu ʻelua pule. (d) Ka hoʻāʻo hoʻonui ʻana o nā cell o nā cell HeLa. Ua ana ʻia ka helu o nā cell ola i nā manawa paʻa me ka hoʻohana ʻana i kahi pahu helu cell 8 (Dojindo). Ma ke ʻano he kaohi, ua mālama ʻia nā cell HeLa me 5 μg/ml actinomycin D (Act D), ka mea e pale ai i ka transcription RNA polymerase a hoʻoulu i ka make ʻana o nā cell. Ua hana ʻia nā loiloi ma triplicate. (e) Ka hoʻāʻo hoʻonui cell E. coli. Ua kālailai ʻia ka ulu ʻana o E. coli ma ke ana ʻana iā OD600. Ma ke ʻano he kaohi, ua mālama ʻia nā pūnaewele me 50 μg/ml ampicillin (Amp), ka mea e kāohi ai i ka hana ʻana o ka paia o nā pūnaewele bacteria. Ua hana ʻia nā loiloi ma ʻekolu mau hana.
No ka wehewehe ʻana i ke ʻano hana o ka cytotoxicity i hoʻokumu ʻia e nā hui e pili ana i ka uramide, ua nānā hou mākou i nā derivatives urbenic acid me nā hopena inhibitory waena. e like me ka mea i hōʻike ʻia ma ke kiʻi. E like me ka mea i hōʻike ʻia ma nā Kiʻi 2b, 6a, ʻo nā mea kanu i ulu ʻia ma nā papa agar e loaʻa ana nā kiʻekiʻe kiʻekiʻe (200 μM) o ka urmotonic acid 6 i hana i nā aʻa pōkole a me ka hema-curved (θ = – 23.7 ± 6.1), ʻoiai ʻo nā mea kanu i ulu ma ka medium control, ua hana nā mea kanu i nā aʻa kokoke pololei (θ = – 3.8 ± 7.1). ʻIke ʻia kēia ulu oblique hiʻohiʻona e hopena mai ka hana ʻole o nā microtubules cortical14,18. E kūlike me kēia ʻike, ʻo nā lāʻau microtubule-destabilizing disopyramide a me oryzalin i hoʻoulu i ke aʻa like ma lalo o kā mākou mau kūlana ulu (Figs. 2b, 6a). I ka manawa like, ua hoʻāʻo mākou i nā derivatives urmotonic acid a koho i kekahi o lākou, ma kekahi mau ʻano, i hoʻoulu i ka ulu ʻana o ke aʻa oblique. Ua hoʻololi nā hui 8, 9, a me 15 i ke kuhikuhi o ka ulu ʻana o ke aʻa ma 75 μM, 50 μM, a me 40 μM, kēlā me kēia, e hōʻike ana e hiki i kēia mau hui ke hoʻopau pono i nā microtubules (Kiʻi 2b, 6a). Ua hoʻāʻo pū mākou i ka derivative ursolic acid ikaika loa, ʻo KAND 11, ma kahi haʻahaʻa haʻahaʻa (15 µM) a ua ʻike ʻia ua kāohi ka hoʻopili ʻana o KAND 11 i ka ulu ʻana o ke aʻa a ʻaʻole like ke kuhikuhi o ka ulu ʻana o ke aʻa, ʻoiai ua ʻano like lākou i ka hema (Kiʻi C3). . No ka mea, ʻo nā kiʻekiʻe kiʻekiʻe o nā lāʻau lapaʻau microtubule-destabilizing i kekahi manawa e kāohi i ka ulu ʻana o ka mea kanu ma mua o ke kumu ʻana o ke aʻa tilting, ua loiloi mākou ma hope i ka hiki ke hoʻopilikia ʻo KAND 11 i nā microtubules ma ka nānā ʻana i nā microtubules cortical i nā pūnaewele epidermal aʻa. Ua hōʻike ka Immunohistochemistry e hoʻohana ana i nā antibodies anti-β-tubulin i nā pūnaewele epidermal o nā aʻa seedling i mālama ʻia me 25 μM KAND 11 i ka nalo ʻana o ka aneane o nā microtubules cortical i nā pūnaewele epidermal i ka elongation zone (Kiʻi 6b). Hōʻike kēia mau hopena e hana pololei a ʻaʻole pololei paha ka waikawa kumamotonic a me kāna mau mea i loaʻa ma nā microtubules e hoʻopilikia iā lākou a he mau mea hoʻopaʻa microtubule hou kēia mau hui.
Hoʻololi ka waikawa Ursonic a me kāna mau mea i loaʻa i nā microtubules cortical ma Arabidopsis thaliana. (a) Ke kihi o ke aʻa i ana ʻia i ke alo o nā mea like ʻole o ka waikawa urmotonic ma nā ʻano i hōʻike ʻia. Ua kālailai ʻia nā hopena o nā hui ʻelua i ʻike ʻia e kāohi i nā microtubules: disopyramide a me oryzalin. Hōʻike ka inset i ke kūlana i hoʻohana ʻia e ana i ke kihi ulu aʻa. Hōʻike nā asterisks i nā ʻokoʻa koʻikoʻi me ka mālama sham (t test, p< 0.05). n>19. Ka pā unahi = 1 kenimika. (b) Nā microtubules cortical i loko o nā pūnaewele epidermal ma ka ʻāpana elongation. Ua ʻike ʻia nā Microtubules i nā aʻa Arabidopsis Col hihiu i ulu ʻia ma nā papa MS me a ʻaʻohe paha 25 μM KAND 11 e ka hoʻoluʻu immunohistochemical me ka hoʻohana ʻana i nā antibodies mua β-tubulin a me nā antibodies lua Alexa Fluor-conjugated. Ka pā unahi = 10 µm. (c) ʻAno mitotic o nā microtubules i loko o ke aʻa meristem. Ua ʻike ʻia nā Microtubules me ka hoʻohana ʻana i ka hoʻoluʻu immunohistochemical. Ua helu ʻia nā ʻano mitotic, me nā ʻāpana prophase, nā spindles, a me nā phragmoplasts, mai nā kiʻi confocal. Hōʻike nā pua i nā ʻano microtubule mitotic. Hōʻike nā asterisks i nā ʻokoʻa koʻikoʻi me ka mālama sham (t test, p< 0.05). n>9. Pā unahi = 50 µm.
ʻOiai ʻo Ursa ka hiki ke hoʻopilikia i ka hana microtubule, manaʻo ʻia e ʻokoʻa kona ʻano hana mai nā mea hana depolymerizing microtubule maʻamau. No ka laʻana, ʻo nā ʻāpana kiʻekiʻe o nā mea hana depolymerizing microtubule e like me ka disopyramide a me ka oryzalin e hoʻoulu ai i ka hoʻonui anisotropic o nā cell epidermal, ʻoiai ʻaʻole ʻo KAND 11. Eia kekahi, ʻo ka hoʻopili pū ʻana o KAND 11 a me ka disopyramide i hopena i ka pane ulu aʻa i hoʻokomo ʻia e ka disopyramide a ua ʻike ʻia ka pale ʻana i ka ulu ʻana o KAND 11 (Kiʻi S4). Ua kālailai pū mākou i ka pane o ka mutant hypersensitive disopyramide 1-1 (phs1-1) i KAND 11. Loaʻa iā phs1-1 kahi mutation tubulin kinase point non-canonical a hana i nā aʻa pōkole ke mālama ʻia me disopyramide9,20. ʻO nā hua kanu mutant phs1-1 i ulu ʻia ma ka medium agar e loaʻa ana iā KAND 11 he mau aʻa pōkole e like me nā mea i ulu ʻia ma ka disopyramid (kiʻi S5).
Eia kekahi, ua ʻike mākou i nā ʻano microtubule mitotic, e like me nā prophase zones, spindles, a me nā phragmoplasts, i loko o ke aʻa meristem o nā mea kanu i mālama ʻia me KAND 11. E like me nā nānā ʻana no CDKB2;1p::CDKB2;1-GUS, ua ʻike ʻia kahi emi nui o ka helu o nā microtubules mitotic (Kiʻi .6c).
No ke ʻano o ka cytotoxicity o KAND 11 ma ka hoʻonā subcellular, ua mālama mākou i nā cell suspension paka BY-2 me KAND 11 a nānā i kā lākou pane. Ua hoʻohui mua mākou iā KAND 11 i nā cell BY-2 e hōʻike ana iā TagRFP-TUA6, ka mea e lepili fluorescently i nā microtubules, e loiloi i ka hopena o KAND 11 ma nā microtubules cortical. Ua loiloi ʻia ka nui o ka microtubule cortical me ka hoʻohana ʻana i ka nānā ʻana i ke kiʻi, kahi i helu ai i ka pakeneka o nā pixels cytoskeletal ma waena o nā pixels cytoplasmic. Ua hōʻike nā hopena assay ma hope o ka mālama ʻana me 50 μM a i ʻole 100 μM KAND 11 no 1 hola, ua emi nui ka nui i 0.94 ± 0.74% a i ʻole 0.23 ± 0.28%, kēlā me kēia, ʻoiai ʻo ka nui o nā cell i mālama ʻia me DMSO, ua like ia me 1.61 ± 0.34% (Kiʻi 7a). Kūlike kēia mau hopena me ka nānā ʻana ma Arabidopsis e hoʻoulu ana ka mālama ʻana o KAND 11 i ka depolymerization o nā microtubules cortical (Kiʻi 6b). Ua nānā pū mākou i ka laina BY-2 me nā filament actin i hōʻailona ʻia e GFP-ABD ma hope o ka mālama ʻana me ka nui like o KAND 11 a ua ʻike ʻia ua hoʻopilikia ka mālama ʻana o KAND 11 i nā filament actin. ʻO ka mālama ʻana me 50 μM a i ʻole 100 μM KAND 11 no 1 h ua hōʻemi nui i ka nui o ka filament actin i 1.20 ± 0.62% a i ʻole 0.61 ± 0.26%, kēlā me kēia, ʻoiai ʻo ka nui i loko o nā cell i mālama ʻia me DMSO he 1.69 ± 0.51% (Kiʻi 2). 7b). Kūʻē kēia mau hopena me nā hopena o propyzamide, ʻaʻole ia e hoʻopilikia i nā filament actin, a me latrunculin B, kahi depolymerizer actin ʻaʻole ia e hoʻopilikia i nā microtubules (SI Figure S6). Eia kekahi, ʻo ka mālama ʻana me coumamonamide 1, coumamonamide acid 6, a i ʻole KAND 11 ʻaʻole ia i hoʻopilikia i nā microtubules i loko o nā cell HeLa (SI Figure S7). No laila, ua manaʻo ʻia he ʻokoʻa ke ʻano hana o KAND 11 mai nā mea hoʻopilikia cytoskeleton i ʻike ʻia. Eia kekahi, ua hōʻike kā mākou nānā ʻana i nā cell BY-2 i mālama ʻia me KAND 11 i ka hoʻomaka ʻana o ka make ʻana o nā cell i ka wā o ka mālama ʻana iā KAND 11 a ua hōʻike ʻia ʻaʻole i piʻi nui ka hapa o nā cell make i hoʻoluʻu ʻia e Evans ma hope o 30 min o ka mālama ʻana iā KAND 11, ʻoiai ma hope o 90 mau minuke o ka mālama ʻana me 50 μM a i ʻole 100 μM KAND, ua piʻi ka helu o nā cell make i 43.7% a i ʻole 80.1%, kēlā me kēia (Kiʻi 7c). I ka hui pū ʻia ʻana, hōʻike kēia ʻikepili ʻo ka ursolic acid derivative KAND 11 hou he mea hoʻopaʻa cytoskeletal kikoʻī i nā mea kanu me kahi ʻano hana i ʻike ʻole ʻia ma mua.
Hoʻopilikia ʻo KAND i nā microtubules cortical, nā filaments actin, a me ke ola o nā pūnaewele BY-2 paka. (a) ʻIke ʻia o nā microtubules cortical i loko o nā pūnaewele BY-2 i ke alo o TagRFP-TUA6. Ua nānā ʻia nā pūnaewele BY-2 i mālama ʻia me KAND 11 (50 μM a i ʻole 100 μM) a i ʻole DMSO e ka microscopy confocal. Ua helu ʻia ka nui o ka microtubule cortical mai nā micrographs o 25 mau pūnaewele kūʻokoʻa. Hōʻike nā leka i nā ʻokoʻa koʻikoʻi (hoʻāʻo Tukey HSD, p< 0.05). Pā unahi = 10 µm. (b) Nā filament actin cortical i loko o nā pūnaewele BY-2 i ʻike ʻia i ke alo o GFP-ABD2. Ua nānā ʻia nā pūnaewele BY-2 i mālama ʻia me KAND 11 (50 μM a i ʻole 100 μM) a i ʻole DMSO e ka microscopy confocal. Ua helu ʻia ka nui o nā filament actin cortical mai nā micrographs o 25 mau pūnaewele kūʻokoʻa. Hōʻike nā leka i nā ʻokoʻa koʻikoʻi (hoʻāʻo Tukey HSD, p< 0.05). Ka pā unahi = 10 µm. (c) Nānā ʻana i nā pūnaewele BY-2 make e ka pena polū Evans. Ua nānā ʻia nā pūnaewele BY-2 i mālama ʻia me KAND 11 (50 μM a i ʻole 100 μM) a i ʻole DMSO e ka microscopy ʻālohilohi. n=3. Ka pā unahi = 100 µm.
ʻO ka ʻike ʻana a me ka hoʻohana ʻana i nā huahana kūlohelohe hou ua alakaʻi i nā holomua koʻikoʻi i nā ʻano like ʻole o ke ola kanaka, me ka lāʻau lapaʻau a me ka mahiʻai. Ua hana ʻia nā noiʻi mōʻaukala e loaʻa ai nā hui pono mai nā kumuwaiwai kūlohelohe. Ma ke ʻano kūikawā, ua ʻike ʻia nā actinomycetes he mea pono e like me nā antibiotics antiparasitic no nā nematodes ma muli o ko lākou hiki ke hana i nā metabolites lua like ʻole e like me avermectin, ka hui alakaʻi o ivermectin a me bleomycin a me kāna mau derivatives, i hoʻohana ʻia i ka lāʻau lapaʻau ma ke ʻano he mea anticancer21,22. Pēlā nō, ua ʻike ʻia nā ʻano hui herbicidal like ʻole mai actinomycetes, ua hoʻohana mua ʻia kekahi o ia mau mea i ke kālepa1,23. No laila, ʻo ka nānā ʻana i nā metabolites actinomycete e hoʻokaʻawale i nā huahana kūlohelohe me nā hana olaola i makemake ʻia ua manaʻo ʻia he hoʻolālā kūpono. Ma kēia haʻawina, ua ʻike mākou i kahi hui hou, coumamonamide, mai S. werraensis a ua synthesize maikaʻi ʻia. ʻO ka waikawa Ursonic kahi waena synthetic o urbenamide a me kāna mau derivatives. Hiki iā ia ke hana i ke ʻano o ke aʻa curling, hōʻike i ka hana herbicidal waena a ikaika, a hōʻino pololei a ʻaʻole pololei paha i nā microtubules mea kanu. Eia nō naʻe, ʻokoʻa paha ke ʻano hana o ka waikawa urmotonic mai ko nā mea hoʻopaʻa microtubule e kū nei, ʻoiai ʻo KAND 11 ke hoʻopilikia nei i nā filaments actin a ke kumu o ka make ʻana o nā cell, e hōʻike ana i kahi ʻano hana hoʻoponopono e hiki ai i ka waikawa urmotonic a me kāna mau mea i loaʻa ke hoʻohuli i kahi ākea o nā ʻano cytoskeletal. .
ʻO ka wehewehe kikoʻī hou aku o ka waikawa urbenonic e kōkua i ka hoʻomaopopo maikaʻi ʻana i ke ʻano o ka hana o ka waikawa urbenonic. ʻO ka pahuhopu aʻe, ʻo ia ka loiloi i ka hiki o ka waikawa ursonic ke hoʻopaʻa i nā microtubules i hoʻemi ʻia e hoʻoholo ai inā hana pololei ka waikawa ursonic a me kāna mau derivatives ma nā microtubules a hoʻohemo iā lākou, a i ʻole inā hopena kā lākou hana i ka destabilization microtubule. Eia kekahi, i ka hihia kahi ʻaʻole he pahuhopu pololei nā microtubules, ʻo ka ʻike ʻana i kahi o ka hana a me nā pahuhopu molekala o ka waikawa ursonic ma nā cell mea kanu e kōkua i ka hoʻomaopopo hou ʻana i nā waiwai o nā hui pili a me nā ala hiki ke hoʻomaikaʻi i ka hana herbicidal. Ua hōʻike kā mākou hoʻāʻo bioactivity i ka hiki cytotoxic kū hoʻokahi o ka waikawa ursonic ma ka ulu ʻana o nā mea kanu e like me Arabidopsis thaliana, paka a me ka liverwort, ʻoiai ʻaʻole i hoʻopilikia ʻia nā cell E. coli a me HeLa. ʻO ka liʻiliʻi a ʻaʻohe paha o ka ʻawaʻawa i nā cell holoholona he pono ia o nā derivatives waikawa ursonic inā hoʻomohala ʻia lākou ma ke ʻano herbicides no ka hoʻohana ʻana i nā mahina mahiʻai ākea. ʻOiaʻiʻo, ʻoiai he mau ʻano maʻamau nā microtubules i nā eukaryotes, ʻo kā lākou kāohi koho i nā mea kanu he koi nui ia no nā herbicides. No ka laʻana, ʻo propyzamide, kahi mea hoʻokaʻawale microtubule e hoʻopaʻa pololei ana i ka tubulin a kāohi i ka polymerization, hoʻohana ʻia ma ke ʻano he herbicide ma muli o kona haʻahaʻa haʻahaʻa i nā cell holoholona24. I ka hoʻohālikelike ʻana me ka disopyramide, loaʻa i nā benzamides pili nā kikoʻī kikoʻī like ʻole. Ma waho aʻe o nā microtubules mea kanu, ʻo RH-4032 a i ʻole benzoxamide ke kāohi pū i nā microtubules o nā cell holoholona a i ʻole oomycetes, kēlā me kēia, a hoʻohana ʻia ka zalilamide ma ke ʻano he fungicide ma muli o kona phytotoxicity haʻahaʻa25,26,27. Hōʻike ka bea i ʻike hou ʻia a me kāna mau derivatives i ka cytotoxicity koho e kūʻē i nā mea kanu, akā he mea kūpono ke hoʻomaopopo ʻana e hiki i nā hoʻololi hou aʻe ke hoʻololi i kā lākou kikoʻī kikoʻī, e hāʻawi ana i nā derivatives hou aʻe no ka kaohi ʻana i nā fungi pathogenic a i ʻole oomycetes.
He mea pono nā waiwai kūikawā o ka waikawa urbenonic a me kāna mau mea i loaʻa no ko lākou hoʻomohala ʻana ma ke ʻano herbicides a hoʻohana ʻia ma ke ʻano he mau mea hana noiʻi. Ua ʻike nui ʻia ke koʻikoʻi o ka cytoskeleton i ka hoʻomalu ʻana i ke ʻano o nā cell mea kanu. Ua hōʻike nā haʻawina mua ua hoʻomohala nā mea kanu i nā ʻano hana paʻakikī o ka hoʻonohonoho microtubule cortical ma o ka hoʻomalu ʻana i ka dynamics microtubule e hoʻomalu pono i ka morphogenesis. Ua ʻike ʻia kahi helu nui o nā molekala kuleana no ka hoʻoponopono ʻana i ka hana microtubule, a ke hoʻomau nei ka noiʻi pili3,4,28. ʻAʻole wehewehe piha kā mākou ʻike o kēia manawa i ka dynamics microtubule i loko o nā cell mea kanu i nā ʻano hana o ka hoʻonohonoho microtubule cortical. No ka laʻana, ʻoiai hiki i ka disopyramide a me ka oryzalin ke depolymerize i nā microtubules, hoʻoulu ka disopyramide i ka distortion aʻa koʻikoʻi ʻoiai he hopena iki ka oryzalin. Eia kekahi, ʻo nā mutations i loko o ka tubulin, ka mea e hoʻopaʻa ai i nā microtubules, ke kumu pū kekahi i ka dextrorotation i loko o nā aʻa, ʻoiai ʻo ka paclitaxel, ka mea e hoʻopaʻa ai i ka dynamics microtubule, ʻaʻole. No laila, ʻo ke aʻo ʻana a me ka ʻike ʻana i nā pahuhopu molekala o ka waikawa ursolic e hāʻawi i nā ʻike hou i ka hoʻoponopono ʻana i nā microtubules cortical mea kanu. Pēlā nō, ʻo nā hoʻohālikelike ʻana i nā kemika i ka wā e hiki mai ana e kūpono i ka hoʻoulu ʻana i ka ulu ʻana i hoʻopilikia ʻia, e like me ka disopyramide, a me nā kemika i emi iki ka maikaʻi, e like me ka oryzalin a i ʻole ka kumamotoric acid, e hāʻawi i nā hōʻailona i ke ʻano o ka ulu ʻana i hoʻopilikia ʻia.
Ma ka ʻaoʻao ʻē aʻe, ʻo nā hoʻonohonoho hou ʻana o ka cytoskeletal e pili ana i ka pale ʻana kekahi mea hiki ke wehewehe i ka cytotoxicity o ka waikawa ursonic. ʻO ka maʻi ʻana o kahi pathogen a i ʻole ka hoʻokomo ʻana o kahi elicitor i loko o nā cell mea kanu i kekahi manawa ke kumu o ka luku ʻia ʻana o ka cytoskeleton a me ka make ʻana o ka cell ma hope29. No ka laʻana, ua hōʻike ʻia ka cryptoxanthin i loaʻa mai oomycete e hoʻopilikia i nā microtubules a me nā filaments actin ma mua o ka make ʻana o nā cell paka, e like me ka mea e hana ʻia me ka mālama ʻana o KAND30,31. ʻO nā ʻano like ma waena o nā pane pale a me nā pane cellular i hoʻokumu ʻia e ka waikawa ursonic i alakaʻi iā mākou e kuhiakau e hoʻoulu lākou i nā kaʻina hana cellular maʻamau, ʻoiai he hopena wikiwiki a ikaika hoʻi o ka waikawa ursonic ma mua o ka cryptoxanthin. Eia nō naʻe, ua hōʻike nā haʻawina e hoʻolaha ka hoʻohaunaele ʻana o nā filaments actin i ka make cell spontaneous, ʻaʻole i hele mau ʻia e ka hoʻohaunaele microtubule29. Eia kekahi, ke kali nei e ʻike ʻia inā paha ʻo ka pathogen a i ʻole ka elicitor ke kumu o ka ulu ʻana o ke aʻa i hoʻopilikia ʻia, e like me ka hana a nā derivatives acid ursonic. No laila, ʻo ka ʻike molecular e hoʻopili ana i nā pane pale a me ka cytoskeleton he pilikia hoihoi ia e hoʻoponopono ʻia. Ma ka hoʻohana ʻana i ke alo o nā pūhui kaumaha molekala haʻahaʻa e pili ana i ka waikawa ursonic, a me kahi ʻano o nā derivatives me nā mana like ʻole, hiki iā lākou ke hāʻawi i nā manawa kūpono e hoʻopaʻa i nā ʻano cellular i ʻike ʻole ʻia.
I ka hui pū ʻia ʻana, ʻo ka ʻike ʻana a me ka hoʻohana ʻana i nā hui hou e hoʻololi i ka dynamics microtubule e hāʻawi i nā ʻano hana ikaika e hoʻoponopono ai i nā ʻano hana molekala paʻakikī e pili ana i ka hoʻoholo ʻana i ke ʻano o nā cell mea kanu. Ma kēia ʻano, ʻo ka hui urmotonic acid i hoʻomohala hou ʻia, ka mea e hoʻopilikia ai i nā microtubules a me nā filaments actin a hoʻoulu i ka make ʻana o nā cell, hiki ke hāʻawi i kahi manawa kūpono e wehewehe ai i ka pilina ma waena o ka mana microtubule a me kēia mau ʻano hana ʻē aʻe. No laila, ʻo ka loiloi kemika a me ka biological e hoʻohana ana i ka urbenonic acid e kōkua iā mākou e hoʻomaopopo i nā ʻano hana hoʻoponopono molekala e kaohi ana i ka cytoskeleton mea kanu.
E hoʻokomo iā S. werraensis MK493-CF1 i loko o kahi ʻōmole Erlenmeyer baffled 500 mL e loaʻa ana he 110 mL o ka medium hua i haku ʻia me 2% (w/v) galactose, 2% (w/v) Essence paste, 1% (w/v) Bacto composition. -soyton (Thermo Fisher Scientific, Inc.), 0.5% (w/v) corn extract (KOGOSTCH Co., Ltd., Iapana), 0.2% (w/v) (NH4)2SO4 a me 0.2% CaCO3 i loko o ka wai deionized. (pH 7.4 ma mua o ka sterilization). Ua hoʻoulu ʻia nā moʻomeheu hua ma kahi rotary shaker (180 rpm) ma 27°C no 2 mau lā. Hoʻoulu hana ma o ka fermentation solid state. Ua hoʻoili ʻia ka moʻomeheu hua (7 ml) i loko o kahi ʻōmole K-1 500 ml e loaʻa ana he 40 g o ka mea hana i loko o 15 g o ka bale i kaomi ʻia (MUSO Co., Ltd., Iapana) a me 25 g o ka wai deionized (ʻaʻole i hoʻoponopono ʻia ka pH ma mua o ka sterilization). Ua hana ʻia ka fermentation ma 30°C i ka pouli no 14 mau lā. Ua unuhi ʻia ka mea fermentation me 40 ml/ʻōmole EtOH a ua centrifuged ʻia (1500 g, 4°C, 10 min). Ua unuhi ʻia ka supernatant moʻomeheu (60 ml) me ka hui ʻana o 10% MeOH/EtOAc. Ua hoʻoheheʻe ʻia ka papa organik ma lalo o ke kaomi i hoʻemi ʻia e loaʻa ai ke koena (59.5 mg), kahi i hoʻokomo ʻia i ka HPLC me ka gradient elution (0-10 mau minuke: 90%) ma kahi kolamu pae hoʻohuli (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × lōʻihi 250 mm) H2O/CH3CN, 10-35 mau minuke: 90% H2O/CH3CN a i 70% H2O/CH3CN (gradient), 35-45 mau minuke: 90% H2O/EtOH, 45-155 mau minuke: 90% H2O/EtOH a i 100% EtOH (gradient (gradient), 155-200 min: 100% EtOH) ma kahi kahe o 1.5 ml/min, ua hoʻokaʻawale ʻia ka coumamonamide (1, 36.0 mg) ma ke ʻano he pauka amorphous keʻokeʻo.
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]+ waiwai i helu ʻia: 141.0659, waiwai i ana ʻia: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
Ua loaʻa nā hua Columbia (Col-0) mai ke Arabidopsis Biological Resource Center (ABRC) me ka ʻae no ka hoʻohana noiʻi. Ua hoʻolaha ʻia a mālama ʻia nā hua Col-0 ma lalo o kā mākou mau kūlana hoʻokolohua a hoʻohana ʻia e like me nā mea kanu Arabidopsis ʻano hihiu. Ua hoʻomaʻemaʻe ʻia nā hua Arabidopsis ma ka ʻili a ua mahi ʻia i loko o ka hapalua ikaika o Murashige a me Skoog medium e loaʻa ana he 2% sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino)ethanesulfonic acid (MES) (Fujifilm Wako Pure Chemical). ) a me 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, ma 23 °C a me ka mālamalama mau. Ua hāʻawi ʻia nā hua o ka mutant phs1-1 e T. Hashimoto (Nara Institute of Science and Technology).
Ua hāʻawi ʻia nā hua o ke ʻano SR-1 e T. Hashimoto (Nara Institute of Science and Technology) a hoʻohana ʻia e like me nā mea kanu paka hihiu. Ua hoʻomaʻemaʻe ʻia nā hua paka ma ka ʻili a hoʻomoʻa ʻia i loko o ka wai sterile no ʻekolu pō e hoʻoulu ai i ka germination, a laila waiho ʻia i loko o kahi hopena hapalua ikaika e loaʻa ana he 2% sucrose, 0.05% (w/v) MES, a me 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige. a me Skoog medium) me ka pH 5.7 a hoʻomoʻa ʻia ma 23°C ma lalo o ka mālamalama mau.
Ua hāʻawi ʻia ke ʻano Tak-1 e T. Kohchi (Kyoto University) a ua hoʻohana ʻia ma ke ʻano he ʻāpana hoʻokolohua maʻamau no ke aʻo ʻana i ka liverwort. Ua loaʻa ʻo Gemma mai nā mea kanu i hoʻomaʻemaʻe ʻia a laila ua kau ʻia ma luna o ka media Gamborg B5 (Fujifilm Wako Pure Chemical) nona ka 1% sucrose a me 0.3% gellan gum a ua hoʻomoʻa ʻia ma 23°C ma lalo o ka mālamalama mau.
Ua hāʻawi ʻia nā pūnaewele BY-2 paka (Nicotiana tabacum L. cv. Bright Yellow 2) e S. Hasezawa (Ke Kulanui o Tokyo). Ua hoʻoheheʻe ʻia nā pūnaewele BY-2 he 95-fold i loko o ka Linsmeier a me Skoog medium i hoʻololi ʻia a hoʻohui ʻia i kēlā me kēia pule me ka 2,4-dichlorophenoxyacetic acid 32. Ua hui ʻia ka hoʻokuʻu ʻana o ke kelepona ma kahi shaker rotary ma 130 rpm ma 27°C i ka pouli. E holoi i nā kelepona me 10 mau manawa o ka nui o ka medium hou a hoʻokuʻu hou i loko o ka medium like. Ua hoʻokumu ʻia nā laina kelepona transgenic BY-2 e hōʻike paʻa ana i ka māka microtubule TagRFP-TUA6 a i ʻole ka māka filament actin GFP-ABD2 ma lalo o ka mea hoʻolaha cauliflower mosaic virus 35S e like me ka mea i wehewehe ʻia 33,34,35. Hiki ke mālama ʻia a hoʻonohonoho ʻia kēia mau laina kelepona me ka hoʻohana ʻana i nā kaʻina hana like me nā mea i hoʻohana ʻia no ka laina kelepona BY-2 mua.
Ua hoʻoulu ʻia nā pūnaewele HeLa i loko o ka Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) i hoʻohui ʻia me 10% fetal bovine serum, 1.2 U/ml penicillin, a me 1.2 μg/ml streptomycin i loko o kahi incubator 37°C me 5% CO2.
Ua hana ʻia nā hoʻokolohua āpau i wehewehe ʻia ma kēia palapala e like me nā lula a me nā alakaʻi biosafety Kepani.
Ua hoʻoheheʻe ʻia nā hui i loko o ka dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) ma ke ʻano he mau hopena waihona a ua hoʻoheheʻe ʻia i loko o ka MS medium no Arabidopsis a me ka paka a i ʻole ka Gamborg B5 medium no ka liverwort. No ka hoʻāʻo pale ulu aʻa, ua lūlū ʻia ma mua o 10 mau hua ma kēlā me kēia pā ma ka medium agar e loaʻa ana nā hui i hōʻike ʻia a i ʻole DMSO. Ua hoʻoulu ʻia nā hua i loko o kahi keʻena ulu no 7 mau lā. Ua kiʻi ʻia nā mea kanu a ua ana ʻia ka lōʻihi o nā aʻa. No ka hoʻāʻo germination Arabidopsis, ua lūlū ʻia he 48 mau hua ma kēlā me kēia pā ma ka medium agar e loaʻa ana he 200 μM compound a i ʻole DMSO. Ua ulu ʻia nā hua Arabidopsis i loko o kahi keʻena ulu a ua helu ʻia ka helu o nā mea kanu i ulu ʻia i 7 mau lā ma hope o ka germination (dag). No ka hoʻāʻo germination paka, ua lūlū ʻia he 24 mau hua ma kēlā me kēia pā ma ka medium agar e loaʻa ana he 200 μM KAND a i ʻole DMSO. Ua ulu ʻia nā hua paka i loko o kahi keʻena ulu a ua helu ʻia ka helu o nā mea kanu i ulu ʻia ma hope o 14 mau lā. No ka hoʻāʻo kāohi ulu o ke akepaʻa, ua kau ʻia he 9 mau embryos mai kēlā me kēia pā ma luna o ka medium agar e loaʻa ana nā nui i hōʻike ʻia o KAND a i ʻole DMSO a hoʻomoʻa ʻia i loko o kahi keʻena ulu no 14 mau lā.
E hoʻohana i nā mea kanu i hoʻoluʻu ʻia me 5 mg/ml propidium iodide (PI) e ʻike i ka hoʻonohonoho ʻana o ka meristem aʻa. Ua nānā ʻia nā hōʻailona PI e ka microscopy fluorescence me ka hoʻohana ʻana i kahi microscope scan laser confocal TCS SPE (Leica Microsystems).
Ua hana ʻia ke kaha ʻana i nā aʻa me ka β-glucuronidase (GUS) e like me ke kaʻina hana i wehewehe ʻia e Malami lāua ʻo Benfey36. Ua hoʻopaʻa ʻia nā hua liʻiliʻi i loko o ka 90% acetone i ka pō, ua kaha ʻia me ka 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid i loko o ka GUS buffer no 1 hola a ua waiho ʻia i loko o kahi hopena chloraldehyde hydrated. (8 g chloral hydrate, 2 ml wai a me 1 ml glycerol) a ua nānā ʻia e ka microscopy hoʻohālikelike ʻokoʻa me ka hoʻohana ʻana i kahi microscope Axio Imager M1 (Carl Zeiss).
Ua ana ʻia nā kihi aʻa ma nā mea kanu 7 lā i ulu ʻia ma nā papa i kau ʻia ma ke ʻano kū pololei. E ana i ke kihi o ke aʻa mai ke kuhikuhi o ka vector umekaumaha e like me ka mea i wehewehe ʻia ma ka hana 6.
Ua nānā ʻia ka hoʻonohonoho ʻana o nā microtubules cortical e like me ka mea i wehewehe ʻia, me nā hoʻololi liʻiliʻi i ka protocol 37. Ua hoʻohana ʻia ka antibody Anti-β-tubulin (KMX-1, Merk Millipore: MAB3408) a me Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) ma ke ʻano he antibodies mua a me ka lua ma 1:1000 a me 1:100 dilutions, kēlā me kēia. Ua loaʻa nā kiʻi Fluorescence me ka hoʻohana ʻana i kahi microscope scanning laser confocal TCS SPE (Leica Microsystems). E kiʻi i nā kiʻi Z-stack a hana i nā projections intensity kiʻekiʻe e like me nā kuhikuhi a ka mea hana.
Ua hana ʻia ka hoʻāʻo hoʻonui cell HeLa me ka hoʻohana ʻana i ka Cell Counting Kit 8 (Dojindo) e like me nā kuhikuhi a ka mea hana.
Ua kālailai ʻia ka ulu ʻana o E. coli DH5α ma ke ana ʻana i ka nui o nā cell i loko o ka moʻomeheu me ka hoʻohana ʻana i kahi spectrophotometer ma 600 nm (OD600).
Ua nānā ʻia ka hoʻonohonoho ʻana o Cytoskeletal i loko o nā pūnaewele transgenic BY-2 me ka hoʻohana ʻana i kahi microscope fluorescence i lako me kahi mea scan confocal CSU-X1 (Yokogawa) a me kahi kāmela sCMOS (Zyla, Andor Technology). Ua loiloi ʻia ka nui o Cytoskeletal e ka nānā ʻana i ke kiʻi, kahi i helu ai i ka pakeneka o nā pikela cytoskeletal ma waena o nā pikela cytoplasmic i nā kiʻi confocal me ka hoʻohana ʻana i ka polokalamu ImageJ e like me ka mea i wehewehe ʻia38,39.
No ka ʻike ʻana i ka make ʻana o nā cell i loko o nā cell BY-2, ua hoʻoulu ʻia kahi aliquot o ka hoʻokuʻu ʻana o nā cell me 0.05% Evans blue no 10 mau minuke ma ka mahana o ka lumi. ʻO ke koho ʻana o ka waihoʻoluʻu polū Evans o nā cell make e pili ana i ka hoʻopuka ʻana o ka dye mai nā cell ola e ka membrane plasma i hoʻopaʻa ʻia40. Ua nānā ʻia nā cell i hoʻoluʻu ʻia me ka hoʻohana ʻana i kahi microscope kahua ʻālohilohi (BX53, Olympus).
Ua ulu ʻia nā pūnaewele HeLa ma DMEM i hoʻohui ʻia me 10% FBS i loko o kahi incubator humidified ma 37°C a me 5% CO2. Ua mālama ʻia nā pūnaewele me 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), a i ʻole 100 ng/ml Nocodmaze (Sigma) no 6 h ma 37°C. Ua hoʻopaʻa ʻia nā pūnaewele me MetOH no 10 min a laila me ka acetate no 5 min ma ka mahana o ka lumi. Ua hoʻoulu ʻia nā pūnaewele paʻa me ka antibody mua β-tubulin (1D4A4, Proteintech: 66240-1) i hoʻoheheʻe ʻia i loko o 0.5% BSA/PBS no 2 mau hola, holoi ʻia i 3 mau manawa me TBST, a laila hoʻoulu ʻia me ka antibody kao Alexa Fluor. 488 1 hola. – ʻIole IgG (Thermo Fisher Scientific: A11001) a me 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) i hoʻoheheʻe ʻia i loko o 0.5% BSA/PBS. Ma hope o ka holoi ʻana me TBST ʻekolu manawa, ua ʻike ʻia nā ʻāpana i hoʻoluʻu ʻia ma kahi microscope Nikon Eclipse Ti-E i hoʻohuli ʻia. Ua hopu ʻia nā kiʻi me kahi kāmela Hamamatsu ORCA-R2 CCD i hoʻomaʻalili ʻia me ka hoʻohana ʻana i ka polokalamu MetaMorph (Molecular Devices).
Ka manawa hoʻouna: Iune-17-2024