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Mā te kitenga me te whakamahinga whai hua o ngā hua taiao ka āwhina i te whakapai ake i te oranga o te tangata. He whānuitia te whakamahinga o ngā matū aukati tipu tipu hei patu taru hei whakahaere i ngā taru. Nā te hiahia ki te whakamahi i ngā momo patu taru rerekē, me tautuhi ngā matū me ngā tikanga mahi hou. I roto i tēnei rangahau, i kitea e mātou he matū N-alkoxypyrrole hou, ko te coumamonamide, mai i Streptomyces werraensis MK493-CF1, ā, i whakatūria te tukanga hanga katoa. Mā roto i ngā whakamātautau mahi koiora, i kitea e mātou ko te waikawa urs-monoamic he takawaenga hanga o te urs-monoamide me te pūmanawa.aukati tipu tipu. Hei tāpiritanga, kua whakawhanakehia e mātou ngā momo matū waikawa urbenonic, tae atu ki te matū urbenyloxy (UDA), he nui te mahi patu otaota me te kore e pā kino ki te tipu o ngā pūtau HeLa. I kitea anō e mātou ka whakararuraru ngā matū waikawa urmotonic i ngā microtubules tipu; hei tāpiritanga, ka pā te KAND ki ngā miro actin, ā, ka puta te mate o ngā pūtau; he rerekē ēnei pānga maha-āhua i ērā o ngā aukati microtubule e mōhiotia ana, ā, e kī ana i tētahi tikanga mahi hou mō te waikawa ursonic, he painga nui tēnei i roto i te whanaketanga o ngā patu otaota hou.
Ko te kitenga me te whakamahinga whai hua o ngā hua taiao whai hua me ō rātou hua he huarahi hei whakapai ake i te kounga o te oranga tangata. Ko ngā matū matū tuarua i hangaia e ngā moroiti, ngā tipu me ngā pepeke kua arahi ki ngā whanaketanga nui i roto i te rongoā me te ahuwhenua. He maha ngā paturopi me ngā rongoā anti-leukemia kua whakawhanakehia mai i ngā hua taiao. Hei tāpiri, he maha ngā momongā patu pepeke, ka tangohia ngā patu harore me ngā patu taru mai i ēnei hua taiao hei whakamahi i roto i te ahuwhenua. Ina koa, he taputapu nui ngā patu taru whakahaere taru hei whakanui ake i ngā hua o te ahuwhenua hou, ā, kua whakamahia kētia ngā momo matū maha ki te hokohoko. He maha ngā tukanga pūtau i roto i ngā tipu, pērā i te photosynthesis, te metabolism waikawa amino, te hanganga pakitara pūtau, te whakahaere i te mitosis, te tohu phytohormone, te hanganga pūmua rānei, e kiia ana he whāinga noa o ngā patu taru. Ko ngā matū e aukati ana i te mahi microtubule he akomanga noa o ngā patu taru e pā ana ki te tipu o te tipu mā te pā ki te whakahaere mitotic2.
Ko ngā microtubules he wāhanga o te angaanga pūtau, ā, he whānuitia te tiaki i roto i ngā pūtau eukaryotic. Ko te heterodimer tubulin he mea hanga mai i te α-tubulin me te β-tubulin e hanga ana i ngā protofilaments microtubule rārangi, me te 13 protofilaments e hanga ana i te hanganga porotaka. He maha ngā mahi a ngā microtubules i roto i ngā pūtau tipu, tae atu ki te whakatau i te āhua o te pūtau, te wehenga pūtau, me te kawe ā-roto3,4. Kei roto i ngā pūtau tipu ngā microtubules i raro i te membrane plasma interphase, ā, e whakaarohia ana ko ēnei microtubules cortical te kaiwhakahaere i te whakaritenga o ngā microfibrils cellulose mā te whakahaere i ngā matatini cellulose synthase4,5. Ko ngā microtubules cortical o ngā pūtau epidermal pakiaka, kei roto i te rohe tere o te pito pakiaka, kei te taha, ā, ka whai ngā microfibers cellulose i ēnei microtubules ka whakawhāiti i te ahunga o te whānui o te pūtau, nā reira ka whakatairanga i te roa o te pūtau anisotropic. Nō reira, he hononga tata te mahi microtubule ki te āhua o te tipu. Ko ngā whakakapinga waikawa amino i roto i ngā ira e whakamārama ana i te tubulin ka puta he rerekētanga o ngā rarangi microtubule cortical me te tipu taha maui, taha matau rānei i roto i te Arabidopsis 6,7. Waihoki, ko ngā whakarerekētanga i roto i ngā pūmua e pā ana ki te microtubule e whakahaere ana i ngā mahi microtubule ka arahi hoki ki te tipu pakiaka he rerekē8,9,10,11,12,13. Hei tāpiri, ko te maimoatanga ki ngā patu taru e whakararuraru ana i te microtubule pērā i te disopyramide, e mōhiotia ana ko te pretilachlor, ka puta hoki te tipu pakiaka taha maui14. E whakaatu ana ēnei raraunga he mea nui te whakahaere tika o te mahi microtubule hei whakatau i te ahunga o te tipu o te tipu.
Kua kitea ngā momo aukati microtubule, ā, he nui ngā takoha a ēnei rongoā ki te rangahau pūtau pūtau, tae atu ki te ahuwhenua me te rongoā2. Ina koa, ko te oryzalin, ngā matū dinitroaniline, te disopyramide, ngā matū e pā ana ki te benzamide, me ō rātou momo rite ka taea te aukati i te mahi microtubule, ā, ka aukati i te tipu o te tipu. Nō reira, he whānuitia te whakamahinga hei patu taru. Heoi, i te mea he wāhanga nui ngā microtubule o ngā pūtau tipu me ngā pūtau kararehe, he paitini te nuinga o ngā aukati microtubule ki ngā momo pūtau e rua. Nō reira, ahakoa te whai huatanga e mōhiotia ana hei patu taru, he iti noa ngā kaihoko antimicrotubule e whakamahia ana mō ngā kaupapa mahi.
Ko Streptomyces tētahi momo o te whānau Streptomyces, kei roto ko ngā huakita aerobic, gram-positive, filamentous, ā, e mōhiotia whānuitia ana mō tōna kaha ki te whakaputa i te whānuitanga o ngā metabolites tuarua. Nō reira, e kiia ana ko tētahi o ngā puna nui rawa atu o ngā hua taiao hou e kaha ana ki te koiora. I roto i te rangahau o nāianei, i kitea e mātou tētahi matū hou e kiia nei ko te coumamonamide, i wehea mai i Streptomyces werraensis MK493-CF1 me S. werraensis ISP 5486. Mā te whakamahi i te tātaritanga spectral me te tātaritanga spectral katoa, i tautuhia te hanganga o te coumamonamide, ā, i whakatauhia tōna angaanga N-alkoxypyrrole ahurei. te hanganga. Ko te waikawa Ursmonic, he takawaenga hanga o te ursmonoamide me ōna hua, i kitea he aukati i te tipu me te whakatipu o te tipu tauira rongonui Arabidopsis thaliana. I roto i tētahi rangahau whanaungatanga hanganga-ngohe, i kitea e mātou he matū me te C9 i whakarerekētia ki te waikawa ursonic, e kiia nei ko te nonyloxy derivative of ursonic acid (KAND), ka whakanui ake i te pānga aukati ki te tipu me te whakatipu. He mea nui, ko te aukati tipu tipu hou i kitea i pā ki te tipu o te tupeka me te atewort, ā, kāore i paitini ki ngā huakita, ki ngā pūtau HeLa rānei. Heoi anō, ko ētahi o ngā matū waikawa urmotonic ka puta he momo pakiaka kua whakapēhia, e tohu ana ka pā tika, ka pā ā-kore rānei ēnei matū ki ngā microtubules. E rite ana ki tēnei whakaaro, ko ā mātou kitenga i ngā microtubules kua tohua mā te immunohistochemical, mā te fluorescent rānei, e tohu ana ko te maimoatanga KAND ka tango i te polymerization o ngā microtubules. Hei tāpiri, ko te maimoatanga me ngā matū waikawa kumamotonic i whakararu i ngā microfilaments actin. Nō reira, kua kitea e mātou he matū aukati tipu tipu hou ko tōna tikanga mahi ahurei ko te whakangaromanga o te angaanga pūtau.
I wehea te momo MK493-CF1 i te oneone i Shinagawa-ku, Tokyo. I hangaia e te momo MK493-CF1 he mycelium stromal manga pai. I whakatauhia te raupapatanga wāhanga o te ira RNA ribosomal 16S (1422 bp). He tino rite tēnei momo ki te S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: momo noa, 99.93%). I runga i tēnei hua, i whakatauhia he whanaunga tata tēnei momo ki te momo momo o S. werraensis. Nō reira, i tapaina e mātou tēnei momo ko S. werraensis MK493-CF1. Ka whakaputahia hoki e te S. werraensis ISP 5486T ngā matū koiora ōrite. Nā te mea he iti noa ngā rangahau tōmua mō te whiwhi hua taiao mai i tēnei moroiti, i mahia ētahi atu rangahau matū. I muri i te whakatipu i te S. werraensis MK493-CF1 ki runga i te papa parei mā te whakapūkara totoka i te 30°C mō ngā rā 14, ka tangohia te papa ki te 50% EtOH. 60 ml o te tauira i whakamaroketia kia whiwhi ai i te 59.5 mg o te tango mata. I tukuna te tango mata ki te HPLC wāhanga whakamuri kia puta ai te N-methoxy-1H-pyrrole-2-carboxamide (1, ko te coumamonamide te ingoa, 36.0 mg). Ko te tapeke o te 1 he tata ki te 60% o te tango mata. Nō reira, i whakatau mātou ki te ako taipitopito i ngā āhuatanga o te kumamotoamide 1.
He paura mā kore-āhua te Coumamonamide 1, ā, e whakaūtia ana te C6H8N2O2 e te ine taumaha papatipu taumira teitei (HRESIMS) (Pikitia 1). Ko te wāhanga pyrrole kua whakakapia ki te C2 o tēnei pūhui e tohuhia ana e δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH i roto i te ine taumaha 1H NMR: 4.5 Hz, H-5) me δH 6.78 (1H, d, J = 2.5 Hz, H-6), ā, e whakaatu ana te ine taumaha 13C NMR i te aroaro o ngā ngota waro sp2 e whā. I aromatawaitia te aroaro o tētahi rōpū amide i te tūranga C2 mā te hononga HMBC mai i te pūmua C-3 ki te waro amide carbonyl i δC 161.1. Hei tāpiritanga, ko ngā tihi 1 H me te 13 C NMR i te δH 4.10 (3H, S) me te δC 68.3 e tohu ana i te aroaro o ngā rōpū N-methoxy i roto i te ngota. Ahakoa kāore anō kia whakatauhia te tūranga tika o te rōpū methoxy mā te whakamahi i te tātaritanga spectroscopy pērā i te enhanced difference spectroscopy me te nuclear Overhauser abbreviation (NOEDF), ko te N-methoxy-1H-pyrrole-2-carboxamide te matū tuatahi.
Hei whakatau i te hanganga tika o te 1, i whakahaerehia he hanganga katoa (Pikitia 2a). Ko te maimoatanga o te 2-aminopyridine 2 e wātea ana i te hokohoko me te m-CPBA i hua ake ai te N-oxide 3 e rite ana i roto i te hua tau. I muri i te 2-aminoazidation o te 2, ko te tauhohenga cyclocondensation i whakaahuahia e Abramovich i whakahaerehia i roto i te benzene i te 90°C hei whiwhi i te 1-hydroxy-1H-pyrrole-2-carbonitrile 5 e hiahiatia ana i roto i te karamu. Tere 60% (e rua ngā wāhanga). 15,16. Ko te methylation me te hydrolysis o te 4 i puta ake ai te 1-methoxy-1H-pyrrole-2-carboxylic acid (e kiia nei ko te "cumotonic acid", 6) i roto i te hua pai (70%, e rua ngā taahiraa). Hei whakamutunga, ko te amidation mā te waikawa chloride intermediate 6 mā te whakamahi i te ammonia wai i puta ai te Kumamoto amide 1 i roto i te hua 98%. Ko ngā raraunga spectral katoa o te 1 i hangaia he rite ki te 1 motuhake, nō reira i whakatauhia ai te hanganga o te 1;
Te whakahiato whānui me te tātari i te mahi koiora o te urbenamide me te waikawa urbenic. (a) Te whakahiatotanga katoa o te amide Kumamoto. (b) I whakatipuria ngā tipu Arabidopsis Columbia (Col) momo mohoao e whitu-rā te pakeke ki runga i ngā pereti Murashige me Skoog (MS) kei roto ko te coumamonamide 6, ko te coumamonamide 1 rānei i ngā kukū kua tohua. Pae tauine = 1 cm.
Tuatahi, i aromatawaihia e mātou ngā mahi koiora o te urbenamide me ōna takawaenga mō tō rātou kaha ki te whakarerekē i te tipu o te tipu. I tāpirihia e mātou ngā kukū rerekē o te ursmonamide 1, te waikawa ursmonic 6 rānei ki te reo MS agar, ā, i whakatipuria ngā tipu Arabidopsis thaliana ki runga i tēnei reo. I whakaatuhia e ēnei whakamātautau he nui ngā kukū (500 μM) o te 6 i aukati i te tipu o te pakiaka (Pikitia 2b). Muri iho, i hangaia e mātou ngā momo pānga mā te whakakapi i te tūranga N1 o te 6, ā, i whakahaerehia he rangahau whanaungatanga hanganga-mahi ki runga i a rātou (kei roto i te Mōhiohio Tautoko (SI) te whakaahuatanga o te tukanga hangahanga anaro). I whakatipuria ngā tipu Arabidopsis ki runga i tētahi reo kei roto ko te 50 μM pānga waikawa ursonic, ā, i inehia te roa o te pakiaka. e ai ki te pikitia. E ai ki ngā Pikitia 3a, b, me te S1, he rerekē te roa o ngā mekameka alkoxy raina (9, 10, 11, 12, me te 13) ngā mekameka alkoxy nui rānei (15, 16, me te 17) o ngā waikawa coumamo i te tūranga N1. I whakaatuhia e ngā hua i ahu mai he aukati nui ki te tipu o te pakiaka. I tua atu, i kitea e mātou ko te tono o te 200 μM 10, 11, 17 rānei i aukati i te whakatipu (Pikitia 3c me te S2).
Te ako i te whanaungatanga hanganga-mahi o te amide Kumamoto me ngā matū e pā ana. (a) Kaupapa hanganga me te hanga o ngā mea rite. (b) Te ine i te roa o te pakiaka o ngā tipu 7-rā te pakeke i whakatipuria ki runga i te reo MS me te kore rānei he 50 μM coumamonamide derivatives. Ko ngā whetū e tohu ana i ngā rerekētanga nui me te maimoatanga rūpahu (whakamātautau t, p< 0.05). n>18. E whakaaturia ana ngā raraunga hei toharite ± SD. Ko te tikanga o te nt he "kāore i whakamatautauria" nā te mea neke atu i te 50% o ngā purapura kāore i pihi. (c) Te ine i te tere pihi o ngā purapura i rongoatia i whakatipuria mō ngā rā e 7 i roto i te reo MS me te 200 μM coumamonamide me ngā matū e pā ana, kāore rānei. Ko ngā whetū e tohu ana i ngā rerekētanga nui me te maimoatanga rūpahu (whakamātautau chi-square). n=96.
He mea whakamere, ko te tāpiri o ngā mekameka taha alkyl he roa ake i te C9 i whakaiti i te mahi aukati, e kī ana me whai mekameka taha o tētahi rahi ngā matū e pā ana ki te waikawa kumamotoic kia whakaatu i tā rātou mahi koiora.
Nā te mea i whakaatuhia e te tātaritanga whanaungatanga hanganga-ngohe kua whakarerekētia a C9 ki te waikawa ursonic, ā, ko te nonyloxy derivative o te waikawa ursonic (e kiia nei ko KAND 11) te aukati tipu tipu tino whai hua, i whakahaerehia e mātou he whakaahuatanga taipitopito ake o KAND 11. Ko te maimoatanga o Arabidopsis ki te 50 μM KAND 11 i tata kore e aukati i te whakatipu, engari ko ngā kukū iti (40, 30, 20, 10 μM rānei) o KAND 11 i aukati i te tipu pakiaka i runga i te tikanga e whakawhirinaki ana ki te horopeta (Pikitia 4a, b). Hei whakamātautau mēnā ka pā te KAND 11 ki te oranga o te meristem pakiaka, i tirotirohia e mātou ngā meristem pakiaka i pania ki te propidium iodide (PI) ā, i ine i te rahi o te rohe meristem. Ko te rahi o te meristem o ngā tipu i whakatipuria ki runga i tētahi reo kei roto ko te 25 μM KAND-11 he 151.1 ± 32.5 μm, ko te rahi ia o te meristem o ngā tipu i whakatipuria ki runga i tētahi reo whakahaere kei roto ko te DMSO he 264.7 ± 30.8 μm (Pikitia 4c, d), e tohu ana ka whakahokia mai e KAND-11 te mahi pūtau. horapa. Meristem pakiaka. E rite ana ki tēnei, i whakaitihia e te maimoatanga KAND 11 te nui o te tohu wehenga pūtau CDKB2;1p::CDKB2;1-GUS i roto i te meristem pakiaka (Pikitia 4e) 17. E tohu ana ēnei hua ka aukati a KAND 11 i te tipu pakiaka mā te whakaiti i te mahi tipu pūtau.
Te tātari i te pānga aukati o ngā matū waikawa urbenonic (ngā matū urbenyloxy) ki te tipu. (a) Ngā tipu Col momo-mohoao 7-rā te pakeke i whakatipuria ki runga i ngā pereti MS me ngā kukū kua tohua o KAND 11. Pae tauine = 1 cm. (b) Te ine i te roa o te pakiaka. E tohu ana ngā reta i ngā rerekētanga nui (whakamātautau Tukey HSD, p< 0.05). n>16. Ka whakaaturia ngā raraunga hei toharite ± SD. (c) Te karuārai kanohi o ngā pakiaka Col momo-mohoao kua pania ki te propidium iodide i whakatipuria ki runga i ngā pereti MS me te 25 μM KAND 11, kāore rānei. Ko ngā pūrua mā e tohu ana i te meristem pakiaka. Pae tauine = 100 µm. (d) Te ine i te rahi o te meristem pakiaka (n = 10 ki te 11). I whakatauhia ngā rerekētanga tatauranga mā te whakamahi i te whakamātautau-t (p< 0.05). Ko ngā pae e tohu ana i te rahi toharite o te meristem. (e) Te karuārai rerekētanga rerekētanga (DIC) o te meristem pakiaka kei roto te hanganga CDKB2; 1pro: CDKB2; 1-GUS i pania, i pania hoki ki ngā tipu 5-rā te pakeke i whakatipuria ki runga i ngā pereti MS me te whakamātautau 25 µM KAND, kāore rānei.
I whakamātauria anō te paitini o te KAND 11 mā te whakamahi i tētahi atu tipu dicotyledonous, te tupeka (Nicotiana tabacum), me tētahi tauira tipu whenua nui, te atewort (Marchantia polymorpha). Pērā i te Arabidopsis, ko ngā tipu tupeka SR-1 i whakatipuria ki runga i te reo kei roto ko te 25 μM KAND 11 i whakaputa i ngā pakiaka poto ake (Pikitia 5a). Hei tāpiri, 40 o ngā purapura 48 i pihi ake ki runga i ngā pereti kei roto ko te 200 μM KAND 11, engari ko ngā purapura 48 katoa i pihi ake ki runga i te reo kua tukatukahia, e tohu ana he nui ake ngā kukū o te KAND (p< 0.05; whakamātautau chi -tapawhā) i aukati i te whakatipu tupeka. (Pikitia 5b). Hei tāpiri, ko te kukū o KAND 11 i aukati i te tipu huakita i roto i te atewort he rite ki te kukū whai hua i roto i te Arabidopsis (Pikitia 5c). E whakaatu ana ēnei hua ka taea e KAND 11 te aukati i te tipu o ngā momo tipu. Kātahi ka tirotirohia e mātou te kaha pea o ngā matū e pā ana ki te pea monoamide i roto i ētahi atu rauropi, arā, ko ngā pūtau HeLa tangata me te momo Escherichia coli DH5α, hei māngai mō ngā pūtau kararehe me ngā pūtau huakita teitei ake. I roto i tētahi raupapa whakamātautau tipu pūtau, i kitea e mātou kāore te coumamonamide 1, te waikawa coumamonamidic 6, me te KAND 11 i pā ki te tipu o ngā pūtau HeLa, E. coli rānei i ngā kukū o te 100 μM (Pikitia 5d,e).
Te aukati i te tipu o te KAND 11 i roto i ngā rauropi ehara i te Arabidopsis. (a) I whakatipuria ngā tipu tupeka SR-1 mohoao e rua wiki te pakeke ki runga i ngā pereti MS poutū kei roto ko te 25 μM KAND 11. (b) I whakatipuria ngā tipu tupeka SR-1 mohoao e rua wiki te pakeke ki runga i ngā pereti MS poutū kei roto ko te 200 μM KAND 11. (c) Ngā puku atewort Tak-1 mohoao e rua wiki te pakeke i whakatipuria ki runga i ngā pereti Gamborg B5 me ngā kukū kua tohua o te KAND 11. Ko ngā pere whero e tohu ana i ngā pua i mutu te tipu i roto i te wā whakatipuranga e rua wiki. (d) Te whakamātautau tipu pūtau o ngā pūtau HeLa. I inehia te maha o ngā pūtau ora i ngā wā wā kua whakaritea mā te whakamahi i te kete tatau pūtau 8 (Dojindo). Hei mana whakahaere, i rongoatia ngā pūtau HeLa ki te 5 μg/ml actinomycin D (Act D), e aukati ana i te tuhi RNA polymerase ka mate te pūtau. I mahia ngā tātaritanga i roto i ngā tāruarua e toru. (e) Te whakamātautau tipu pūtau E. coli. I tātarihia te tipu E. coli mā te ine i te OD600. Hei mana whakahaere, i rongoatia ngā pūtau ki te ampicillin (Amp) 50 μg/ml, e aukati ana i te hanganga o te pakitara pūtau huakita. I whakahaerehia ngā tātaritanga i roto i ngā tāruatanga e toru.
Hei whakamārama i te tikanga mahi o te paitini pūtau i puta mai i ngā matū e pā ana ki te uramide, i tātarihia anō e mātou ngā matū waikawa urbenic me ngā pānga aukati ngawari, e whakaaturia ana i te pikitia. E whakaaturia ana i roto i ngā Pikitia 2b, 6a, ko ngā tipu i whakatipuria ki runga i ngā pereti agar kei roto ngā kukū teitei (200 μM) o te waikawa urmotonic 6 i whakaputa i ngā pakiaka poto ake me te piko maui (θ = – 23.7 ± 6.1), engari ko ngā tipu i whakatipuria ki runga i te papa whakahaere, he tata tika ngā pakiaka i puta i ngā tipu (θ = – 3.8 ± 7.1). E mōhiotia ana ko tēnei tipu whakarara āhuatanga ka puta mai i te kore mahi a ngā microtubules cortical14,18. E rite ana ki tēnei kitenga, ko ngā rongoā whakakore-microtubule disopyramide me te oryzalin i whakaoho i te whakarara pakiaka rite tonu i raro i ā mātou tikanga tipu (Pikitia 2b, 6a). I taua wā anō, i whakamatauria e mātou ngā matū waikawa urmotonic, ā, i tīpakohia e mātou ētahi o aua mea, i ētahi kukū, i whakaoho i te tipu pakiaka whakarara. I whakarerekētia e ngā matū 8, 9, me te 15 te ahunga o te tipu o te pakiaka i te 75 μM, 50 μM, me te 40 μM, ia, e tohu ana ka taea e ēnei matū te whakakore i ngā microtubules (Pikitia 2b, 6a). I whakamātauria hoki e mātou te matū tino kaha o te waikawa ursolic, te KAND 11, i te kukū iti iho (15 µM) ā, i kitea ko te tono o te KAND 11 i aukati i te tipu o te pakiaka, ā, he koretake te ahunga o te tipu o te pakiaka, ahakoa i te nuinga o te wā ka piki ki te taha maui (Pikitia C3). . Nā te mea ko te kukū teitei ake o ngā rongoā whakakore microtubule i ētahi wā ka aukati i te tipu o te tipu kaua ki te whakapiko i te pakiaka, i muri mai ka aromatawaihia e mātou te tūponotanga ka pā te KAND 11 ki ngā microtubules mā te tirotiro i ngā microtubules cortical i roto i ngā pūtau epidermal pakiaka. I whakaatuhia e te immunohistochemistry mā te whakamahi i ngā paturopi anti-β-tubulin i roto i ngā pūtau epidermal o ngā pakiaka tipu i rongoatia ki te 25 μM KAND 11 te ngaronga o te nuinga o ngā microtubules cortical i roto i ngā pūtau epidermal i te rohe roa (Pikitia 6b). E whakaatu ana ēnei hua he mahi tika, he mahi ā-kore rānei te waikawa kumamotonic me ōna hua ki ngā microtubule hei whakararuraru i a rātou, ā, he aukati microtubule hou ēnei pūhui.
Ka whakarerekē te waikawa Ursonic me ōna huanga i ngā microtubules cortical i roto i te Arabidopsis thaliana. (a) Ko te koki piko pakiaka i inehia i te aroaro o ngā momo huanga waikawa urmotonic i ngā kukū kua tohua. I tātarihia hoki ngā pānga o ngā matū e rua e mōhiotia ana hei aukati i ngā microtubules: te disopyramide me te oryzalin. E whakaatu ana te whakaurunga i te paerewa i whakamahia hei ine i te koki tipu pakiaka. E tohu ana ngā whetū i ngā rerekētanga nui me te maimoatanga rūpahu (whakamātautau t, p< 0.05). n>19. Pae tauine = 1 cm. (b) Ngā microtubules cortical i roto i ngā pūtau epidermal i te rohe roa. Ko ngā microtubules i roto i ngā pakiaka Arabidopsis Col momo-mohoao i tipuhia i runga i ngā pereti MS me te 25 μM KAND 11, kāore rānei, i tirohia mā te tae immunohistochemical mā te whakamahi i ngā paturopi tuatahi β-tubulin me ngā paturopi tuarua Alexa Fluor-conjugated. Pae tauine = 10 µm. (c) Hanganga mitotic o ngā microtubules i roto i te meristem pakiaka. I tirohia ngā microtubules mā te whakamahi i te tae immunohistochemical. Ko ngā hanganga mitotic, tae atu ki ngā rohe prophase, ngā spindles, me ngā phragmoplasts, i tatauhia mai i ngā whakaahua confocal. Ko ngā pere e tohu ana i ngā hanganga microtubule mitotic. Ko ngā whetū e tohu ana i ngā rerekētanga nui me te maimoatanga rūpahu (whakamātautau t, p< 0.05). n>9. Pae tauine = 50 µm.
Ahakoa kei a Ursa te kaha ki te whakararuraru i te mahi a te microtubule, e tika ana kia rerekē tana tikanga mahi i ngā kaihoko depolymerizing microtubule noa. Hei tauira, ko te nui ake o ngā kukū o ngā kaihoko depolymerizing microtubule pēnei i te disopyramide me te oryzalin ka puta te whānui anisotropic o ngā pūtau epidermal, engari kāore a KAND 11. Hei tāpiri, ko te whakamahinga ngātahi o te KAND 11 me te disopyramide i hua ake ai he urupare tipu pakiaka i puta mai i te disopyramide, ā, i kitea te aukati tipu i puta mai i te KAND 11 (Pikitia S4). I tātarihia hoki e mātou te urupare o te mutant disopyramide 1-1 (phs1-1) hypersensitive ki te KAND 11. He mutant tubulin kinase point non-canonical tō te phs1-1, ā, he poto ake ngā pakiaka i te wā e rongoatia ana ki te disopyramide9,20. Ko ngā tipu mutant phs1-1 i tipuhia ki runga i te reo agar kei roto ko te KAND 11 he poto ake ngā pakiaka rite ki ērā i tipuhia ki runga i te disopyramid (pikitia S5).
Hei tāpiritanga, i kitea e mātou ngā hanganga microtubule mitotic, pērā i ngā rohe prophase, ngā spindle, me ngā phragmoplast, i roto i te meristem pakiaka o ngā tipu i rongoatia ki te KAND 11. E ai ki ngā kitenga mō CDKB2;1p::CDKB2;1-GUS, i kitea he hekenga nui o te maha o ngā microtubule mitotic (Pikitia .6c).
Hei whakaahua i te paitini pūtau o KAND 11 i te taumira pūtau-iti, i tukinotia e mātou ngā pūtau whakatārewatanga BY-2 tupeka ki te KAND 11, ā, i tirohia tā rātou urupare. I tāpirihia tuatahitia e mātou te KAND 11 ki ngā pūtau BY-2 e whakaatu ana i te TagRFP-TUA6, e tohu ana i ngā microtubules mā te fluorescence, hei aromatawai i te pānga o KAND 11 ki ngā microtubules cortical. I aromatawaihia te mātotoru o ngā microtubules cortical mā te whakamahi i te tātari whakaahua, i ine i te ōrau o ngā pika cytoskeletal i waenga i ngā pika cytoplasmic. I whakaatuhia e ngā hua whakamātautau i muri i te maimoatanga ki te 50 μM, 100 μM rānei KAND 11 mō te 1 hāora, i heke nui te mātotoru ki te 0.94 ± 0.74%, 0.23 ± 0.28% rānei, ia, ko te mātotoru o ngā pūtau i tukinotia ki te DMSO, i eke ki te 1.61 ± 0.34% (Pikitia 7a). He rite ēnei hua ki te kitenga i roto i te Arabidopsis e whakaoho ana te maimoatanga KAND 11 i te depolymerization o ngā microtubules cortical (Pikitia 6b). I tirotirohia hoki e mātou te raina BY-2 me ngā miro actin kua tohua ki te GFP-ABD i muri i te maimoatanga me te kukū ōrite o KAND 11, ā, i kitea ko te maimoatanga KAND 11 i whakararu i ngā miro actin. Ko te maimoatanga me te 50 μM, 100 μM rānei KAND 11 mō te 1 h i tino whakaiti i te kiato miro actin ki te 1.20 ± 0.62%, 0.61 ± 0.26% rānei, ia, engari ko te kiato i roto i ngā pūtau i rongoatia ki te DMSO he 1.69 ± 0.51% (Pikitia 2). 7b). He rerekē ēnei hua ki ngā pānga o te propyzamide, kāore e pā ki ngā miro actin, me te latrunculin B, he depolymerizer actin kāore e pā ki ngā microtubules (Pikitia SI S6). Hei tāpiri, ko te maimoatanga me te coumamonamide 1, te waikawa coumamonamide 6, te KAND 11 rānei kāore i pā ki ngā microtubules i roto i ngā pūtau HeLa (Pikitia SI S7). Nō reira, e whakaponohia ana he rerekē te tikanga mahi a te KAND 11 i tērā o ngā kaiwhakararu anga pūtau e mōhiotia ana. Hei tāpiri, i whakaatuhia e tā mātou tirotiro mōkitokito o ngā pūtau BY-2 i rongoatia ki te KAND 11 te tīmatanga o te mate pūtau i te wā o te maimoatanga KAND 11, ā, i whakaatuhia kāore i tino piki ake te ōwehenga o ngā pūtau mate kua pania ki te kikorangi a Evans i muri i te 30 meneti o te maimoatanga KAND 11, engari i muri i te 90 meneti o te maimoatanga me te 50 μM, 100 μM rānei o te KAND, kua piki ake te maha o ngā pūtau mate ki te 43.7%, ki te 80.1% rānei, ia (Pikitia 7c). Ina huihuia, e tohu ana ēnei raraunga ko te KAND 11, he matū hou i ahu mai i te waikawa ursolic, he aukati anga pūtau motuhake ki te tipu me tētahi tikanga mahi kāore i te mōhiotia i mua.
Ka pā te KAND ki ngā microtubules cortical, ngā miro actin, me te oranga o ngā pūtau BY-2 tupeka. (a) Te tirohanga o ngā microtubules cortical i roto i ngā pūtau BY-2 i te aroaro o te TagRFP-TUA6. I tirotirohia ngā pūtau BY-2 i rongoatia ki te KAND 11 (50 μM, 100 μM rānei) DMSO rānei mā te karuārai confocal. I tatauhia te mātotoru o ngā microtubules cortical mai i ngā micrograph o ngā pūtau motuhake e 25. E tohu ana ngā reta i ngā rerekētanga nui (whakamātautau Tukey HSD, p< 0.05). Pae tauine = 10 µm. (b) Ngā miro actin cortical i roto i ngā pūtau BY-2 i tirohia i te aroaro o GFP-ABD2. Ko ngā pūtau BY-2 i rongoatia ki te KAND 11 (50 μM, 100 μM rānei) DMSO rānei i tirohia mā te karuārai confocal. I tatauhia te kiato o ngā miro actin cortical mai i ngā whakaahua iti o ngā pūtau motuhake e 25. E tohu ana ngā reta i ngā rerekētanga nui (whakamātautau Tukey HSD, p< 0.05). Pae tauine = 10 µm. (c) Te mātakitaki i ngā pūtau BY-2 mate mā te tae kikorangi a Evans. Ko ngā pūtau BY-2 i rongoatia ki te KAND 11 (50 μM, 100 μM rānei) DMSO rānei i tirotirohia mā te karuārai kanapa. n=3. Pae tauine = 100 µm.
Nā te kitenga me te whakamahinga o ngā hua taiao hou kua arahi ki ngā whanaketanga nui i roto i ngā āhuatanga maha o te oranga tangata, tae atu ki te rongoā me te ahuwhenua. Kua mahia ngā rangahau o mua hei tiki i ngā matū whai hua mai i ngā rauemi taiao. Ina koa, e mōhiotia ana he whai hua ngā actinomycetes hei paturopi ārai-parasitic mō ngā nematodes nā tō rātou kaha ki te whakaputa i ngā momo matū tuarua pērā i te avermectin, te matū matua o te ivermectin me te bleomycin me ōna hua, e whakamahia ana hei rongoā hei kaihoko ārai-matepukupuku21,22. Waihoki, kua kitea he maha ngā momo matū patu otaota mai i ngā actinomycetes, ko ētahi o ēnei kua whakamahia kētia hei arumoni1,23. Nō reira, ko te tātari i ngā matū patu actinomycete hei wehe i ngā hua taiao me ngā mahi koiora e hiahiatia ana e kiia ana he rautaki whai hua. I roto i tēnei rangahau, i kitea e mātou he matū hou, ko te coumamonamide, mai i S. werraensis, ā, i angitu te hanga. Ko te waikawa Ursonic he takawaenga hanga o te urbenamide me ōna hua. Ka taea e ia te whakapiko i te pakiaka, te whakaatu i te mahi patu otaota ngawari ki te kaha, me te kino tika, kore tika rānei i ngā microtubules tipu. Heoi anō, tera pea he rerekē te tikanga mahi a te waikawa urmotonic i tērā o ngā aukati microtubule o nāianei, nā te mea ka whakararuraru hoki te KAND 11 i ngā miro actin ka mate ai ngā pūtau, e tohu ana i tētahi tikanga whakahaere e pā ai te waikawa urmotonic me ōna hua ki te whānuitanga o ngā hanganga cytoskeletal.
Mā te whakaahua taipitopito atu i te waikawa urbenonic ka āwhina i te mārama ake ki te tikanga mahi a te waikawa urbenonic. Ko te whāinga e whai ake nei ko te aromatawai i te kaha o te waikawa ursonic ki te here ki ngā microtubules kua whakaitihia hei whakatau mena ka mahi tika te waikawa ursonic me ōna hua ki ngā microtubules me te tango i te polymerization, mena ka hua ake rānei tā rātou mahi i te whakararuraru o te microtubule. Hei tāpiri, i te wā kāore ngā microtubules i te ūnga tika, mā te tautuhi i te wāhi mahi me ngā whāinga ngota o te waikawa ursonic i runga i ngā pūtau tipu ka āwhina i te mārama ake ki ngā āhuatanga o ngā pūhui e pā ana me ngā huarahi pea hei whakapai ake i te mahi patu otaota. I whakaatuhia e tā mātou whakamātautau bioactivity te kaha cytotoxic ahurei o te waikawa ursonic ki te tipu o ngā tipu pēnei i te Arabidopsis thaliana, te tupeka me te atewort, i te mea kāore i pāngia ngā pūtau E. coli me ngā pūtau HeLa. He iti, he kore rānei te paitini ki ngā pūtau kararehe he painga o ngā hua waikawa ursonic mena ka whakawhanakehia hei patu taru hei whakamahi i roto i ngā mara ahuwhenua tuwhera. Ina hoki, i te mea he hanganga noa ngā microtubules i roto i ngā eukaryotes, ko tā rātou aukati whiriwhiri i roto i ngā tipu he whakaritenga matua mō ngā patu taru. Hei tauira, ko te propyzamide, he kaihoko tango-pūmua microtubule e herea tika ana ki te tubulin me te aukati i te whakapūmua, e whakamahia ana hei patu taru nā te mea he iti te paitini ki ngā pūtau kararehe24. He rerekē ki te disopyramide, he rerekē ngā āhuatanga motuhake o ngā benzamide e pā ana. Haunga ngā microtubule tipu, ka aukatihia hoki e te RH-4032, te benzoxamide rānei ngā microtubule o ngā pūtau kararehe, oomycetes rānei, ā, ka whakamahia te zalilamide hei patu harore nā te mea he iti te paitini o ngā pūtau kararehe25,26,27. Ko te pea hou nei me ōna hua e whakaatu ana i te paitini o ngā tipu, engari he mea tika kia mōhio ko ngā whakarerekētanga atu ka whakarerekē pea i te motuhake o ō rātou hua, ka whakarato pea i ētahi atu hua hei whakahaere i ngā harore pathogenic, oomycetes rānei.
He mea whai hua ngā āhuatanga ahurei o te waikawa urbenonic me ōna hua hei whanaketanga hei patu taru, hei whakamahinga hoki hei taputapu rangahau. E mōhiotia whānuitia ana te hiranga o te angaanga pūtau ki te whakahaere i te āhua o ngā pūtau tipu. Kua whakaatuhia e ngā rangahau o mua kua whanakehia e ngā tipu ngā tikanga uaua o te whakaritenga microtubule cortical mā te whakahaere i ngā hihiri microtubule kia tika ai te whakahaere i te morphogenesis. He maha ngā ngota e kawenga ana mō te whakahaere i te mahi microtubule kua tautuhia, ā, kei te haere tonu ngā rangahau e pā ana3,4,28. Kāore tō tātou māramatanga o nāianei mō ngā hihiri microtubule i roto i ngā pūtau tipu e tino whakamārama ana i ngā tikanga o te whakaritenga microtubule cortical. Hei tauira, ahakoa ka taea e te disopyramide me te oryzalin te tango i te polymerization o ngā microtubule, ka puta te he o te pakiaka i te disopyramide, engari he iti noa te pānga o te oryzalin. I tua atu, ko ngā whakarerekētanga i roto i te tubulin, e whakau ana i ngā microtubule, ka puta hoki te dextrorotation i roto i ngā pakiaka, engari ko te paclitaxel, e whakau ana hoki i ngā hihiri microtubule, kāore. Nō reira, ko te ako me te tautuhi i ngā whāinga ngota o te waikawa ursolic me whakarato i ngā māramatanga hou ki te whakahaere i ngā microtubule cortical tipu. Waihoki, mā ngā whakatauritenga ā muri ake nei o ngā matū e whai hua ana ki te whakatairanga i te tipu hē, pērā i te disopyramide, me ngā matū kāore i te tino whai hua, pērā i te oryzalin, te waikawa kumamotoric rānei, ka homai he tohu ki te āhua o te tipu hē.
I tētahi atu taha, ko ngā whakarerekētanga o ngā angaanga pūtau e pā ana ki te tiaki he mea anō hei whakamārama i te paitini o te waikawa ursonic. Ko te pangia e te mate urutā, te whakaurunga rānei o te kaiwhakaoho ki roto i ngā pūtau tipu ka pakaru te angaanga pūtau, ā, ka mate te pūtau i muri mai29. Hei tauira, kua pūrongohia te cryptoxanthin i ahu mai i te oomycete ka whakararu i ngā microtubules me ngā miro actin i mua i te matenga o ngā pūtau tupeka, he rite ki te mea e tupu ana me te maimoatanga KAND30,31. Ko ngā āhuatanga ōrite i waenga i ngā urupare tiaki me ngā urupare pūtau i puta mai i te waikawa ursonic i ārahi i a mātou ki te whakapae ka whakaohohia e rātou ngā tukanga pūtau noa, ahakoa he tere ake, he kaha ake hoki te pānga o te waikawa ursonic i te cryptoxanthin. Heoi, kua whakaatuhia e ngā rangahau ko te whakararu i ngā miro actin ka whakatairanga i te mate pūtau ohorere, kāore i te haere tahi me te whakararu i te microtubule29. Hei tāpiri, me kite tonu mēnā ko te mate urutā, ko te kaiwhakaoho rānei te take o te tipu pakiaka kua hē, pērā i ngā hua o te waikawa ursonic. Nō reira, ko te mōhiotanga ngota e hono ana i ngā urupare tiaki me te angaanga pūtau he raruraru whakamere hei aro atu. Mā te whakamahi i te aroaro o ngā pūhui taumaha-iti e pā ana ki te waikawa ursonic, me te whānuitanga o ngā momo matū e pā ana ki ngā kaha rerekē, ka whakaratohia pea he whai wāhitanga ki te arotahi ki ngā tikanga pūtau kāore i te mōhiotia.
Ina huihuia, ko te kitenga me te whakamahinga o ngā matū hou e whakarerekē ana i te hihiri o te microtubule ka whakarato i ngā tikanga kaha hei aro atu ki ngā tikanga ngota matatini e whai pānga ana ki te whakatau i te āhua o te pūtau tipu. I roto i tēnei horopaki, ko te matū hou kua whakawhanakehia ko te waikawa urmotonic, e pā ana ki ngā microtubule me ngā miro actin, ā, e whakaoho ana i te mate o te pūtau, ka whakarato pea i tētahi whai wāhitanga ki te wetewete i te hononga i waenga i te whakahaere microtubule me ēnei atu tikanga. Nō reira, ko te tātari matū me te koiora mā te whakamahi i te waikawa urbenonic ka āwhina i a tātou ki te mārama ki ngā tikanga whakahaere ngota e whakahaere ana i te anga pūtau tipu.
Whakatōkia te S. werraensis MK493-CF1 ki roto i tētahi ipu Erlenmeyer kua whakakōpirihia 500 mL kei roto ko te 110 mL o te ranunga purapura kei roto ko te 2% (w/v) galactose, 2% (w/v) te whakapiri Essence, 1% (w/v) te hanganga Bacto. -soyton (Thermo Fisher Scientific, Inc.), 0.5% (w/v) te tangohanga kānga (KOGOSTCH Co., Ltd., Hapani), 0.2% (w/v) (NH4)2SO4 me te 0.2% CaCO3 ki roto i te wai whakakore iona. (pH 7.4 i mua i te whakahoromata). I whakatōkia ngā ahurea purapura ki runga i tētahi rūrū hurihuri (180 rpm) i te 27°C mō ngā rā e 2. Te whakatipu whakaputa mā te whakatōnga āhua totoka. I whakawhitia te ahurea purapura (7 ml) ki roto i tētahi pounamu K-1 500 ml kei roto ko te 40 karamu o te rauemi whakaputa kei roto ko te 15 karamu o te parei pēhi (MUSO Co., Ltd., Hapani) me te 25 karamu o te wai tango-ion (kāore anō kia whakatikatikaina te pH i mua i te whakahoromata). I mahia te whakapūkara i te 30°C i roto i te pōuri mō ngā rā 14. I tangohia te rauemi whakapūkara ki te 40 ml/pounamu EtOH, ā, i whakapūkaratia (1500 karamu, 4°C, 10 meneti). I tangohia te wai whakapūkara (60 ml) ki te ranunga o te 10% MeOH/EtOAc. I mimitia te paparanga rauropi i raro i te pēhanga iti kia whiwhi ai i tētahi toenga (59.5 mg), i tukuna ki te HPLC me te whakapūtanga rōnaki (0–10 meneti: 90%) i runga i te pou āhua whakamuri (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × roa 250 mm) H2O/CH3CN, 10–35 meneti: 90% H2O/CH3CN ki te 70% H2O/CH3CN (rōnaki), 35–45 meneti: 90% H2O/EtOH, 45–155 meneti: 90% H2O/EtOH ki te 100% EtOH (rōnaki (rōnaki), 155–200 meneti: 100% EtOH) i te tere rere o te 1.5 ml/min, i wehea te coumamonamide (1, 36.0 mg) hei paura mā kore āhua.
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]+ uara i tatauhia: 141.0659, uara i inehia: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
I whiwhihia ngā purapura Columbia (Col-0) mai i te Arabidopsis Biological Resource Center (ABRC) me te whakaaetanga mō te whakamahinga rangahau. I whakatipuria, i tiakina hoki ngā purapura Col-0 i raro i ngā āhuatanga taiwhanga, ā, i whakamahia hei tipu Arabidopsis momo mohoao. I whakahoromatahia ngā purapura Arabidopsis ki te mata, ā, i whakatōkia ki roto i te reo Murashige me Skoog haurua-kaha kei roto ko te 2% sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino)ethanesulfonic acid (MES) (Fujifilm Wako Pure Chemical). ) me te 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, i te 23 °C me te mārama tonu. Nā T. Hashimoto (Nara Institute of Science and Technology) i whakarato ngā purapura o te phs1-1 mutant.
Nā T. Hashimoto (Nara Institute of Science and Technology) i whakarato ngā purapura o te momo SR-1, ā, i whakamahia hei tipu tupeka mohoao. I whakahoromatatia ngā purapura tupeka ki te mata, ā, i rumakina ki te wai whakahoromata mō ngā pō e toru hei whakatairanga i te whakatipu, kātahi ka whakanohoia ki roto i tētahi otinga haurua-kaha kei roto ko te 2% sucrose, 0.05% (w/v) MES, me te 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige. me te Skoog medium) me te pH 5.7, ā, i whakatōkia ki te 23°C i raro i te mārama tonu.
Nā T. Kohchi (Te Whare Wānanga o Kyoto) i whakarato te momo Tak-1, ā, i whakamahia hei waeine whakamātautau paerewa mō te rangahau atewort. I tangohia mai a Gemma i ngā tipu kua whakatōkia, kātahi ka pania ki runga i te reo Gamborg B5 (Fujifilm Wako Pure Chemical) kei roto ko te 1% sucrose me te 0.3% gellan gum, ā, i whakatōkia ki te 23°C i raro i te mārama tonu.
Nā S. Hasezawa (Te Whare Wānanga o Tōkyō) i whakarato ngā pūtau BY-2 tupeka (Nicotiana tabacum L. cv. Kōwhai Marama 2). I whakawaiwaitia ngā pūtau BY-2 kia 95 ngā wā ki roto i te reo Linsmeier me Skoog kua whakarerekētia, ā, i tāpirihia ia wiki ki te waikawa 2,4-dichlorophenoxyacetic 32 . I whakaranua te whakatārewatanga pūtau ki runga i te rūrū hurihuri i te 130 rpm i te 27°C i te pōuri. Horoia ngā pūtau ki te 10 ngā wā te nui o te reo hou, ā, i whakatārewatia anō ki roto i te reo kotahi. I hangaia ngā raina pūtau whakawhiti BY-2 e whakaatu pūmau ana i te tohu microtubule TagRFP-TUA6, te tohu actin filament GFP-ABD2 rānei i raro i te kaiwhakatairanga huaketo mosaic cauliflower 35S e ai ki te whakaahuatanga 33,34,35. Ka taea te pupuri me te tukutahi i ēnei raina pūtau mā te whakamahi i ngā tikanga e rite ana ki ērā i whakamahia mō te raina pūtau BY-2 taketake.
I whakatōngia ngā pūtau HeLa ki roto i te Dulbecco's Modified Eagle's medium (DMEM) (Life Technologies) i tāpirihia ki te 10% fetal bovine serum, 1.2 U/ml penicillin, me te 1.2 μg/ml streptomycin i roto i te incubator 37°C me te 5% CO2.
I mahia ngā whakamātautau katoa i whakaahuatia i roto i tēnei tuhinga i runga anō i ngā ture me ngā aratohu haumaru koiora a Hapanihi.
I whakarewaina ngā matū ki roto i te dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) hei otinga taketake, ā, i whakaranua ki roto i te reo MS mō te Arabidopsis me te reo tupeka, te reo Gamborg B5 rānei mō te atewort. Mō te whakamātautau aukati tipu pakiaka, neke atu i te 10 ngā kākano mō ia pereti i ruia ki runga i te reo agar kei roto ngā matū kua tohua, te DMSO rānei. I whakatōngia ngā kākano ki roto i te rūma tipu mō ngā rā e 7. I whakaahuahia ngā tipu, ā, i inehia te roa o ngā pakiaka. Mō te whakamātautau whakatipu Arabidopsis, 48 ngā kākano mō ia pereti i ruia ki runga i te reo agar kei roto te matū 200 μM, te DMSO rānei. I whakatipuria ngā kākano Arabidopsis ki roto i te rūma tipu, ā, i tatauhia te maha o ngā tipu kua pihi ake i te 7 rā i muri i te pihinga (dag). Mō te whakamātautau whakatipu tupeka, 24 ngā kākano mō ia pereti i ruia ki runga i te reo agar kei roto te 200 μM KAND, te DMSO rānei. I whakatipuria ngā kākano tupeka ki roto i te rūma tipu, ā, i tatauhia te maha o ngā tipu kua pihi ake i muri i ngā rā e 14. Mō te whakamātautau aukati tipu o te atewort, e 9 ngā kukune mai i ia pereti i whakatakotoria ki runga i te reo agar kei roto ngā kukū kua tohua o te KAND, o te DMSO rānei, ā, i whaowhina ki roto i te rūma tipu mō ngā rā 14.
Whakamahia ngā tipu kua pania ki te 5 mg/ml propidium iodide (PI) hei tiro i te whakaritenga o ngā pakiaka. I tirohia ngā tohu PI mā te karuārai fluorescence mā te whakamahi i te karuārai matawai taiaho TCS SPE confocal (Leica Microsystems).
I mahia te tae histochemical o ngā pakiaka ki te β-glucuronidase (GUS) i runga anō i te tikanga i whakaahuahia e Malami rāua ko Benfey36. I whakapūmauhia ngā tipu ki te 90% acetone i te pō, i tae ki te 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid i roto i te GUS buffer mō te 1 hāora, ā, i whakanohoia ki roto i te otinga chloraldehyde waikura (8 karamu chloral hydrate, 2 ml wai me te 1 ml glycerol) ā, i tirohia mā te karuārai rerekētanga rerekētanga mā te whakamahi i te karuārai Axio Imager M1 (Carl Zeiss).
I inehia ngā koki pakiaka i runga i ngā tipu e 7-rā te pakeke i whakatipuria ki runga i ngā pereti poutū. Inehia te koki o te pakiaka mai i te ahunga o te irahiko kaha e ai ki te whakaahuatanga i te taahiraa 6.
I kitea te whakaritenga o ngā microtubules cortical e ai ki te whakaahuatanga, me ngā whakarerekētanga iti ki te kawa 37. I whakamahia te paturopi anti-β-tubulin (KMX-1, Merk Millipore: MAB3408) me te Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) hei paturopi tuatahi me te tuarua i ngā waimehatanga 1:1000 me te 1:100. I tangohia ngā whakaahua fluorescence mā te whakamahi i te karuārai matawai laser confocal TCS SPE (Leica Microsystems). Tikina ngā whakaahua Z-stack ka waihanga i ngā whakaaturanga kaha mōrahi e ai ki ngā tohutohu a te kaihanga.
I whakahaerehia te whakamātautau tipu pūtau HeLa mā te whakamahi i te Cell Counting Kit 8 (Dojindo) e ai ki ngā tohutohu a te kaihanga.
I tātarihia te tipu o E. coli DH5α mā te ine i te kiato pūtau i roto i te ahurea mā te whakamahi i te ine ine i te 600 nm (OD600).
I tirohia te whakaritenga o te angaanga i roto i ngā pūtau BY-2 whakawhiti ira mā te whakamahi i tētahi karuārai fluorescence kua rite ki tētahi taputapu matawai confocal CSU-X1 (Yokogawa) me tētahi kāmera sCMOS (Zyla, Andor Technology). I aromatawaihia te kiato o te angaanga mā te tātari whakaahua, i ine i te ōrau o ngā pika angaanga i roto i ngā pika cytoplasmic i roto i ngā whakaahua confocal mā te whakamahi i te pūmanawa ImageJ e ai ki te whakaahuatanga38,39.
Hei kite i te matenga o ngā pūtau i roto i ngā pūtau BY-2, i whakatōkia tētahi wāhanga o te whakatārewatanga pūtau ki te kikorangi Evans 0.05% mō te 10 meneti i te pāmahana rūma. Ko te tae kikorangi Evans whiriwhiri o ngā pūtau mate e whakawhirinaki ana ki te putanga mai o te tae mai i ngā pūtau ora e te kiriuhi toto ora40. I tirohia ngā pūtau kua tae mā te whakamahi i tētahi karuārai kanapa (BX53, Olympus).
I whakatipuria ngā pūtau HeLa i roto i te DMEM me te tāpiritanga ki te 10% FBS i roto i te incubator makuku i te 37°C me te 5% CO2. I rongoatia ngā pūtau ki te 100 μM KAND 11, te waikawa kumamonamic 6, te kumamonamide 1, te 100 ng/ml colcemid (Gibco), te 100 ng/ml Nocodmaze (Sigma) rānei mō te 6 hāora i te 37°C. I whakapūmautia ngā pūtau ki te MetOH mō te 10 meneti, kātahi ki te acetate mō te 5 meneti i te pāmahana rūma. I whakatōngia ngā pūtau whakapūmau ki te paturopi tuatahi β-tubulin (1D4A4, Proteintech: 66240-1) i whakaranua ki te 0.5% BSA/PBS mō te 2 hāora, i horoia kia 3 ngā wā ki te TBST, kātahi ka whakatōngia ki te paturopi koati Alexa Fluor. 488 1 hāora. – IgG kiore (Thermo Fisher Scientific: A11001) me te 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) i whakaranua ki te 0.5% BSA/PBS. I muri i te horoinga ki te TBST e toru ngā wā, i tirohia ngā pūtau kua pania ki runga i te karuārai Nikon Eclipse Ti-E kua hurihia. I hopukina ngā whakaahua ki te kāmera Hamamatsu ORCA-R2 CCD kua whakamataohia mā te whakamahi i te pūmanawa MetaMorph (Molecular Devices).
Wā tuku: Pipiri-17-2024