Mun gode da ziyartar Nature.com. Sigar burauzar da kuke amfani da ita tana da ƙarancin tallafin CSS. Don samun sakamako mafi kyau, muna ba da shawarar ku yi amfani da sabuwar sigar burauzar ku (ko ku kashe Yanayin Daidaitawa a cikin Internet Explorer). A halin yanzu, don tabbatar da ci gaba da tallafi, muna nuna shafin ba tare da salo ko JavaScript ba.
Ganowa da amfani da kayayyakin halitta mai amfani na iya taimakawa wajen inganta rayuwar ɗan adam. Ana amfani da sinadarai masu hana ci gaban tsirrai sosai a matsayin magungunan kashe kwari don magance ciyayi. Saboda buƙatar amfani da nau'ikan magungunan kashe kwari daban-daban, akwai buƙatar gano mahadi tare da sabbin hanyoyin aiki. A cikin wannan binciken, mun gano wani sabon sinadarin N-alkoxypyrrole, coumamonamide, daga Streptomyces werraensis MK493-CF1 kuma mun kafa cikakken tsarin hadawa. Ta hanyar gwaje-gwajen ayyukan halittu, mun gano cewa urs-monoamic acid wani abu ne na roba na urs-monoamide kuma yana da yuwuwarMai hana ci gaban shukaBugu da ƙari, mun ƙirƙiro nau'ikan abubuwan da suka samo asali daga acid na urbenonic, gami da abubuwan da suka samo asali daga urbenyloxy (UDA), wanda ke da yawan aikin kashe ƙwayoyin cuta ba tare da yin mummunan tasiri ga ci gaban ƙwayoyin HeLa ba. Mun kuma gano cewa abubuwan da suka samo asali daga acid na urmotonic suna lalata ƙwayoyin cuta na shuka; ban da haka, KAND yana shafar filaments na actin kuma yana haifar da mutuwar ƙwayoyin halitta; Waɗannan tasirin da ke da fuskoki da yawa sun bambanta da na waɗanda aka sani daga masu hana ƙwayoyin cuta kuma suna ba da shawarar sabuwar hanyar aiki ga ursonic acid, wanda ke wakiltar babban fa'ida a cikin haɓaka sabbin magungunan kashe ƙwari.
Ganowa da amfani da kayayyakin halitta masu amfani da abubuwan da suka samo asali a aikace hanya ce ta inganta rayuwar ɗan adam. Abubuwan da ke haifar da ƙwayoyin cuta, shuke-shuke da kwari ke samarwa sun haifar da manyan ci gaba a fannin magani da noma. An ƙirƙiro magungunan rigakafi da yawa da magungunan rage radadi daga samfuran halitta. Bugu da ƙari, nau'ikanmagungunan kashe kwariAna fitar da magungunan kashe kwari da magungunan kashe kwari daga waɗannan samfuran halitta don amfani a noma. Musamman ma, magungunan kashe kwari masu mahimmanci kayan aiki ne don ƙara yawan amfanin gona a noma na zamani, kuma an riga an yi amfani da nau'ikan mahadi daban-daban a kasuwanci. Ana ɗaukar matakai da yawa na ƙwayoyin halitta a cikin tsire-tsire, kamar photosynthesis, metabolism na amino acid, haɗa bangon tantanin halitta, daidaita mitosis, siginar phytohormone, ko haɗa furotin, a matsayin abubuwan da ake amfani da su wajen maganin kashe kwari. Abubuwan da ke hana aikin microtubule sune nau'in maganin kashe kwari da suka shafi ci gaban shuka ta hanyar shafar ƙa'idar mitotic2.
Microtubules sassan cytoskeleton ne kuma ana kiyaye su sosai a cikin ƙwayoyin eukaryotic. Tubulin heterodimer ya ƙunshi α-tubulin da β-tubulin waɗanda ke samar da layukan microtubule protofilaments, tare da protofilaments 13 waɗanda ke samar da tsari mai silinda. Microtubules suna taka rawa da yawa a cikin ƙwayoyin shuka, gami da tantance siffar tantanin halitta, rarrabuwar tantanin halitta, da jigilar ƙwayoyin halitta3,4. Kwayoyin shuka suna ɗauke da microtubules a ƙarƙashin membrane na plasma na interphase, kuma ana tsammanin waɗannan abubuwan da ake kira cortical microtubules suna sarrafa tsarin ƙwayoyin cellulose microfibrils ta hanyar daidaita hadaddun ƙwayoyin cellulose synthase4,5. Ƙananan ƙwayoyin cortical na ƙwayoyin epidermal na tushen, waɗanda ke cikin yankin tsawaitawa cikin sauri na ƙarshen tushen, suna nan a gefe, kuma ƙananan ƙwayoyin cellulose suna bin waɗannan ƙananan ƙwayoyin kuma suna iyakance alkiblar faɗaɗa tantanin halitta, ta haka suna haɓaka tsawaita tantanin halitta na anisotropic. Saboda haka, aikin microtubule yana da alaƙa da yanayin shuka. Sauya amino acid a cikin kwayoyin halitta da ke samar da tubulin yana haifar da karkacewar jerin ƙwayoyin microtubule na cortical da kuma girma na gefen hagu ko dama a cikin Arabidopsis 6,7. Hakazalika, maye gurbi a cikin sunadaran da ke da alaƙa da microtubule waɗanda ke daidaita yanayin microtubule na iya haifar da gurɓataccen ci gaban tushe8,9,10,11,12,13. Bugu da ƙari, magani da magungunan kashe kwari masu lalata microtubule kamar disopyramide, wanda aka fi sani da pretilachlor, shi ma yana haifar da ci gaban tushen da ke daurewa na gefen hagu14. Waɗannan bayanai sun nuna cewa daidaitaccen tsarin aikin microtubule yana da mahimmanci don tantance alkiblar ci gaban shuka.
An gano nau'ikan magungunan hana ƙwayoyin cuta iri-iri, kuma waɗannan magunguna sun ba da gudummawa mai mahimmanci ga binciken ƙwayoyin cuta, da kuma noma da magani2. Musamman ma, oryzalin, mahaɗan dinitroaniline, disopyramide, mahaɗan da suka shafi benzamide, da kuma analogues ɗinsu na iya hana aikin ƙwayoyin cuta ta hanyar hana ci gaban tsirrai. Saboda haka, ana amfani da su sosai a matsayin maganin kashe kwari. Duk da haka, tunda ƙwayoyin cuta masu mahimmanci ne a cikin ƙwayoyin tsirrai da dabbobi, yawancin masu hana ƙwayoyin cuta suna da guba ga nau'ikan ƙwayoyin cuta guda biyu. Saboda haka, duk da amfanin da aka san su da shi a matsayin maganin kashe kwari, ana amfani da adadi mai yawa na magungunan hana ƙwayoyin cuta don dalilai na aiki.
Streptomyces nau'in halittar Streptomyces ne, wanda ya haɗa da ƙwayoyin cuta masu narkewa a jiki, masu kama da na aerobic, masu kama da na gram-positive, kuma an san shi sosai saboda ikonsa na samar da nau'ikan metabolites na biyu. Saboda haka, ana ɗaukarsa ɗaya daga cikin mahimman hanyoyin samar da sabbin samfuran halitta masu aiki a fannin halitta. A cikin binciken da ake yi a yanzu, mun gano wani sabon sinadari mai suna coumamonamide, wanda aka ware daga Streptomyces werraensis MK493-CF1 da S. werraensis ISP 5486. Ta amfani da nazarin spectral da cikakken nazarin spectral, an gano tsarin coumamonamide kuma an tantance kwarangwal ɗin N-alkoxypyrrole na musamman. An gano cewa Ursmonic acid, wani tsaka-tsaki na ursmonoamide da abubuwan da ya samo asali, yana hana girma da tsiron shahararren shukar Arabidopsis thaliana. A cikin wani bincike na dangantaka tsakanin tsari da aiki, mun gano cewa wani sinadari mai dauke da C9 wanda aka canza zuwa ursonic acid, wanda ake kira nonyloxy derivative of ursonic acid (KAND), yana ƙara yawan tasirin hana girma da tsiro sosai. Abin lura shi ne, sabon maganin hana ci gaban shuka da aka gano shi ma ya shafi ci gaban taba da liverwort kuma bai kasance mai guba ga ƙwayoyin cuta ko ƙwayoyin HeLa ba. Bugu da ƙari, wasu abubuwan da aka samo daga urmotonic acid suna haifar da wani nau'in tushen da ya karkace, yana nuna cewa waɗannan abubuwan da aka samo daga gare su kai tsaye ko a kaikaice suna shafar ƙananan ƙwayoyin cuta. Daidai da wannan ra'ayin, lura da muka yi da ƙananan ƙwayoyin cuta da aka yiwa lakabi da immunohistochemically ko tare da sunadaran fluorescent sun nuna cewa maganin KAND yana cire ƙwayoyin cuta. Bugu da ƙari, magani da kumamotonic acid abubuwan da aka samo daga gare su sun lalata ƙananan ƙwayoyin cuta na actin. Don haka, mun gano sabon maganin hana ci gaban shuka wanda tsarin aikinsa na musamman ya haɗa da lalata ƙwayoyin cuta.
An ware nau'in MK493-CF1 daga ƙasa a Shinagawa-ku, Tokyo. nau'in MK493-CF1 ya samar da mycelium mai rassa mai kyau. An tantance jerin sassan kwayar halittar RNA ta 16S (1422 bp). Wannan nau'in yayi kama da na S. werranensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: nau'in da aka saba, 99.93%). Dangane da wannan sakamakon, an gano cewa wannan nau'in yana da alaƙa da nau'in nau'in S. werranensis. Saboda haka, mun sanya wa wannan nau'in suna na ɗan lokaci S. werranensis MK493-CF1. S. werranensis ISP 5486T shi ma yana samar da irin wannan mahaɗan halittu masu aiki. Tunda ba a yi bincike sosai kan samon samfuran halitta daga wannan ƙananan halittu ba, an gudanar da ƙarin bincike kan sinadarai. Bayan an noma S. werraensis MK493-CF1 a kan sha'ir ta hanyar fermentation mai ƙarfi a zafin jiki na 30°C na tsawon kwanaki 14, an cire maganin da kashi 50% na EtOH. An busar da samfurin 60 ml don samun 59.5 mg na ɗanyen samfurin. An yi wa ɗanyen samfurin da aka cire a cikin HPLC na baya don ba da N-methoxy-1H-pyrrole-2-carboxamide (1, mai suna coumamonamide, 36.0 mg). Jimlar adadin 1 shine kusan kashi 60% na ɗanyen samfurin. Saboda haka, mun yanke shawarar yin nazari dalla-dalla game da kaddarorin kumamotoamide 1.
Coumamonamide 1 fari ne mai launin fari kuma babban ƙudurin taro na spectrometry (HRESIMS) yana tabbatar da C6H8N2O2 (Hoto na 1). An siffanta ɓangaren pyrrole na wannan mahaɗin da δH 6.94 (1H, t, J = 2.8, 4.8 Hz, H-4), δH 6.78 (1H, d, J = 2.5, δH a cikin 1H NMR bakan: 4.5 Hz, H-5) da δH 6.78 (1H, d, J = 2.5 Hz, H-6), kuma 13C NMR bakan yana nuna kasancewar ƙwayoyin carbon sp2 guda huɗu. An tantance kasancewar ƙungiyar amide a matsayin C2 ta hanyar haɗin HMBC daga proton C-3 zuwa carbon carbon amide a δC 161.1. Bugu da ƙari, 1 H da 13 C NMR suna kololuwa a δH 4.10 (3H, S) da δC 68.3 suna nuna kasancewar ƙungiyoyin N-methoxy a cikin ƙwayar. Duk da cewa ba a tantance matsayin ƙungiyar methoxy daidai ba ta amfani da nazarin spectroscopic kamar enhanced difference spectroscopy da nuclear Overhauser abbreviation (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide ya zama mahaɗin farko da aka zaɓa.
Domin tantance tsarin 1 daidai, an yi cikakken haɗin kai (Hoto na 2a). Maganin 2-aminopyridine 2 da ake samu a kasuwa tare da m-CPBA ya haifar da N-oxide 3 daidai a cikin yawan amfanin ƙasa. Bayan 2-aminoazidation na 2, an gudanar da aikin cyclocondensation da Abramovich ya bayyana a cikin benzene a 90°C don samun 1-hydroxy-1H-pyrrole-2-carbonitrile 5 a cikin gram. Gudun 60% (matakai biyu). 15,16. Methylation da hydrolysis na 4 sannan suka ba 1-methoxy-1H-pyrrole-2-carboxylic acid (wanda ake kira "cumotonic acid", 6) a cikin yawan amfanin ƙasa mai kyau (70%, matakai biyu). A ƙarshe, amidation via acid chloride intermediate 6 ta amfani da ammonia mai ruwa-ruwa ya ba Kumamoto amide 1 a cikin yawan amfanin ƙasa 98%. Duk bayanan spectral na 1 da aka haɗa sun yi kama da na ware 1, don haka an tantance tsarin 1;
Haɗawa gabaɗaya da kuma nazarin ayyukan halittu na urbenamide da urbenic acid. (a) Haɗawa gaba ɗaya na Kumamoto amide. (b) An shuka tsire-tsire na Arabidopsis Columbia (Col) na daji na kwana bakwai a kan faranti na Murashige da Skoog (MS) waɗanda ke ɗauke da coumamonamide 6 ko coumamonamide 1 a yawan da aka nuna. Ma'aunin sikelin = 1 cm.
Da farko, mun tantance ayyukan halittu na urbenamide da tsaka-tsakinsa don iyawarsu ta daidaita girman shuka. Mun ƙara yawan ursmonamide 1 ko ursmonic acid 6 ga ƙwayoyin halittar MS agar matsakaici da kuma na Arabidopsis thaliana a wannan matsakaici. Waɗannan gwaje-gwajen sun nuna cewa yawan (500 μM) na 6 ya hana girman tushen (Hoto na 2b). Na gaba, mun samar da nau'ikan abubuwan da suka samo asali daban-daban ta hanyar maye gurbin matsayin N1 na 6 kuma mun yi nazarin alaƙar tsari da aiki a kansu (an bayyana tsarin haɗakar analog a cikin Bayanin Tallafi (SI)). An shuka ƙwayoyin Arabidopsis a kan matsakaici wanda ke ɗauke da nau'ikan ursonic acid 50 μM, kuma an auna tsawon tushen. kamar yadda aka nuna a hoton. Kamar yadda aka nuna a Hotuna 3a, b, da S1, ƙwayoyin coumamo suna da tsayi daban-daban na sarƙoƙin alkoxy masu layi (9, 10, 11, 12, da 13) ko manyan sarƙoƙin alkoxy (15, 16, da 17) a matsayin N1. Abubuwan da aka samo sun nuna babban hana ci gaban tushen. Bugu da ƙari, mun gano cewa amfani da 200 μM 10, 11, ko 17 ya hana germination (Hoto na 3c da S2).
Nazarin dangantakar tsari da ayyukan Kumamoto amide da mahaɗan da ke da alaƙa. (a) Tsarin tsari da haɗakar analogues. (b) Ƙididdige tsawon tushen tsirrai masu kwana 7 da aka girma a kan matsakaicin MS tare da ko ba tare da abubuwan da aka samo daga coumamonamide 50 μM ba. Taurari suna nuna manyan bambance-bambance tare da maganin sham (gwajin t, p)<0.05). n>18. An nuna bayanai a matsayin matsakaicin ± SD. nt yana nufin "ba a gwada ba" saboda fiye da kashi 50% na tsaban ba su tsiro ba. (c) Adadin yawan tsiron iri da aka yi wa magani da aka dasa na tsawon kwanaki 7 a cikin matsakaicin MS tare da ko ba tare da coumamonamide 200 μM da mahaɗan da suka shafi hakan ba. Alamun asterisks suna nuna manyan bambance-bambance tare da maganin sham (gwajin chi-square). n=96.
Abin sha'awa, ƙara sarƙoƙin gefen alkyl fiye da C9 ya rage aikin hana aiki, yana nuna cewa mahaɗan da ke da alaƙa da kumamotoic acid suna buƙatar sarƙoƙin gefe na wani girman don nuna ayyukansu na halitta.
Domin nazarin dangantakar tsari da ayyukan da aka yi ya nuna cewa an gyara C9 zuwa ursonic acid kuma wanda ba shi da sinadarin ursonic acid (wanda daga baya ake kira KAND 11) shine mafi ingancin maganin hana ci gaban shuka, mun gudanar da cikakken bayanin KAND 11. Maganin Arabidopsis tare da 50 μM KAND 11 kusan ya hana ci gaban shuka gaba ɗaya, yayin da ƙarancin yawan (40, 30, 20, ko 10 μM) na KAND 11 ya hana ci gaban tushen ta hanyar da ta dogara da kashi (Hoto na 4a, b). Don gwada ko KAND 11 yana shafar ci gaban tushen meristem, mun bincika tushen meristems da aka shafa da propidium iodide (PI) kuma mun auna girman yankin meristem. Girman meristem na tsirrai da aka girma a kan matsakaiciyar da ke ɗauke da 25 μM KAND-11 shine 151.1 ± 32.5 μm, yayin da girman meristem na tsirrai da aka girma a kan matsakaiciyar sarrafawa da ke ɗauke da DMSO shine 264.7 ± 30.8 μm (Hoto na 4c, d), wanda ke nuna cewa KAND-11 yana dawo da ayyukan ƙwayoyin halitta. yana yaɗuwa. Meristem na tushen. Daidai da wannan, maganin KAND 11 ya rage adadin alamar rarraba ƙwayoyin halitta CDKB2;1p::CDKB2;1-GUS a cikin tushen meristem (Hoto na 4e) 17. Waɗannan sakamakon sun nuna cewa KAND 11 yana hana ci gaban tushen ta hanyar rage ayyukan yaɗuwar ƙwayoyin halitta.
Binciken tasirin hana ƙwayoyin urbenonic acid (urbenyloxy derivatives) akan girma. (a) Tsirrai masu nau'in Col na daji na kwanaki 7 da aka girma a kan faranti na MS tare da yawan ƙwayoyin KAND 11 da aka nuna. Ma'aunin ma'auni = 1 cm. (b) Adadin tsawon tushen. Haruffa suna nuna bambance-bambance masu mahimmanci (gwajin Tukey HSD, p<0.05). n>16. An nuna bayanai a matsayin matsakaicin ± SD. (c) Na'urar hangen nesa ta Confocal na tushen propidium iodide mai launin daji wanda aka shuka a kan faranti na MS tare da ko ba tare da 25 μM KAND 11 ba. Fararen maƙallan suna nuna tushen meristem. Ma'aunin sikelin = 100 µm. (d) Adadin girman tushen meristem (n = 10 zuwa 11). An tantance bambance-bambancen ƙididdiga ta amfani da gwajin t (p)<0.05). Sandunan suna wakiltar matsakaicin girman meristem. (e) Na'urar hangen nesa ta bambancin tsangwama (DIC) ta wani tushen meristem wanda ke ɗauke da ginin CDKB2; 1pro: CDKB2; An yi wa 1-GUS fenti da fenti a kan tsire-tsire masu kwanaki 5 da aka shuka a kan faranti na MS tare da ko ba tare da gwajin KAND na 25 µM ba.
An ƙara gwada gubar da ke tattare da KAND 11 ta amfani da wani shukar dicotyledonous, taba (Nicotiana tabacum), da kuma wani babban ƙwayar shukar ƙasa, liverwort (Marchantia polymorpha). Kamar yadda yake a yanayin Arabidopsis, ƙwayoyin taba SR-1 da aka shuka a kan matsakaici mai ɗauke da 25 μM KAND 11 sun samar da gajerun saiwoyi (Hoto na 5a). Bugu da ƙari, iri 40 daga cikin 48 sun tsiro a kan faranti waɗanda ke ɗauke da 200 μM KAND 11, yayin da dukkan iri 48 suka tsiro a kan hanyar da aka yi wa mocked, wanda ke nuna cewa yawan KAND yana da mahimmanci (p<0.05; gwajin chi -square) ya hana tsiron taba. (Hoto na 5b). Bugu da ƙari, yawan KAND 11 wanda ya hana ci gaban ƙwayoyin cuta a cikin liverwort yayi kama da ingantaccen yawan da ke cikin Arabidopsis (Hoto na 5c). Waɗannan sakamakon sun nuna cewa KAND 11 na iya hana ci gaban nau'ikan tsire-tsire iri-iri. Sannan muka binciki yiwuwar gubar da ke tattare da mahaɗan beyar monoamide a cikin wasu halittu, wato ƙwayoyin HeLa na ɗan adam da nau'in Escherichia coli DH5α, a matsayin wakilan ƙwayoyin dabbobi mafi girma da na ƙwayoyin cuta, bi da bi. A cikin jerin gwaje-gwajen yaduwar ƙwayoyin halitta, mun lura cewa coumamonamide 1, coumamonamidic acid 6, da KAND 11 ba su shafi ci gaban ƙwayoyin HeLa ko E. coli a yawan da ke cikin 100 μM ba (Hoto na 5d,e).
Hana girman KAND 11 a cikin halittun da ba na Arabidopsis ba. (a) An shuka shuke-shuken taba SR-1 na daji masu makonni biyu a kan faranti na MS da aka sanya a tsaye waɗanda ke ɗauke da 25 μM KAND 11. (b) An shuka shuke-shuken taba SR-1 na daji masu makonni biyu a kan faranti na MS da aka sanya a kwance waɗanda ke ɗauke da 200 μM KAND 11. (c) Ƙwayoyin liverwort na Tak-1 na daji masu makonni biyu waɗanda aka shuka a kan faranti na Gamborg B5 tare da yawan KAND 11 da aka nuna. Kibiyoyi ja suna nuna spores waɗanda suka daina girma a cikin lokacin shiryawa na makonni biyu. (d) Gwajin yaduwar ƙwayoyin halitta na ƙwayoyin HeLa. An auna adadin ƙwayoyin da ke rayuwa a tazara ta lokaci mai tsawo ta amfani da kayan ƙidayar ƙwayoyin halitta 8 (Dojindo). A matsayin sarrafawa, an yi wa ƙwayoyin HeLa magani da 5 μg/ml actinomycin D (Act D), wanda ke hana kwafi na RNA polymerase kuma yana haifar da mutuwar ƙwayoyin halitta. An yi nazarin a cikin sau uku. (e) Gwajin yaduwar ƙwayoyin E. coli. An yi nazarin girman E. coli ta hanyar auna OD600. A matsayin magani, an yi wa ƙwayoyin cuta magani da 50 μg/ml ampicillin (Amp), wanda ke hana haɗakar bangon ƙwayoyin cuta. An yi nazarin sau uku.
Domin fahimtar yadda tasirin gubar da ke tattare da sinadarai masu alaƙa da uramide ke haifarwa, mun sake yin nazarin abubuwan da suka samo asali daga acid urbenic tare da tasirin hana matsakaita. kamar yadda aka nuna a hoton. Kamar yadda aka nuna a cikin Hotuna na 2b, 6a, tsire-tsire da aka shuka a kan faranti na agar waɗanda ke ɗauke da babban taro (200 μM) na urmotonic acid 6 sun samar da gajarta da tushen hagu masu lanƙwasa (θ = - 23.7 ± 6.1), yayin da na tsire-tsire da aka shuka a kan hanyar sarrafawa, tsire-tsire sun samar da kusan tushen madaidaiciya (θ = - 3.8 ± 7.1). An san cewa wannan haɓakar da ke tattare da rashin aiki na ƙwayoyin cuta na cortical14,18. Daidai da wannan binciken, magungunan da ke lalata microtubule disopyramide da oryzalin sun haifar da karkatar da tushen iri ɗaya a ƙarƙashin yanayin girmarmu (Hotuna na 2b, 6a). A lokaci guda, mun gwada abubuwan da suka samo asali daga urmotonic acid kuma muka zaɓi da yawa daga cikinsu waɗanda, a wasu tarin, suka haifar da haɓakar tushen da ke tattare da rashin daidaituwa. Haɗaɗɗun 8, 9, da 15 sun canza alkiblar girman tushen a 75 μM, 50 μM, da 40 μM, bi da bi, suna nuna cewa waɗannan mahaɗan suna iya lalata microtubules yadda ya kamata (Hoto na 2b, 6a). Mun kuma gwada mafi ƙarfin ursolic acid derivative, KAND 11, a ƙaramin taro (15 µM) kuma mun gano cewa amfani da KAND 11 ya hana girman tushen kuma alkiblar girman tushen bai daidaita ba, kodayake suna da saurin gangara zuwa hagu (Hoto na C3). . Saboda yawan magungunan microtubule-destabilizing wani lokacin yana hana girman shuka maimakon haifar da karkata tushen, daga baya mun tantance yuwuwar cewa KAND 11 yana shafar microtubules ta hanyar lura da ƙananan ƙwayoyin cortical a cikin ƙwayoyin epidermal na tushen. Immunohistochemistry ta amfani da anti-β-tubulin antibodies a cikin ƙwayoyin epidermal na tushen shuka da aka yi wa magani da 25 μM KAND 11 ya nuna ɓacewar kusan dukkanin ƙwayoyin cortical a cikin ƙwayoyin epidermal a cikin yankin tsawaitawa (Hoto na 6b). Waɗannan sakamakon sun nuna cewa kumamotonic acid da abubuwan da ya samo asali suna aiki kai tsaye ko a kaikaice akan microtubules don wargaza su kuma waɗannan mahadi sabbin masu hana ƙwayoyin cuta ne.
Ursonic acid da abubuwan da suka samo asali suna canza ƙwayoyin cortical microtubules a cikin Arabidopsis thaliana. (a) An auna kusurwar karkata tushen a gaban nau'ikan abubuwan da suka samo asali na urmotonic acid a yawan da aka nuna. An kuma yi nazarin tasirin abubuwa guda biyu da aka sani suna hana microtubules: disopyramide da oryzalin. A cikin akwatin yana nuna ma'aunin da ake amfani da shi don auna kusurwar girma na tushen. Alamar tauraro tana nuna manyan bambance-bambance tare da maganin sham (gwajin t, p)<0.05). n>19. Madaurin sikelin = 1 cm. (b) Ƙananan ƙwayoyin cortical a cikin ƙwayoyin epidermal a yankin tsawaitawa. An nuna ƙananan ƙwayoyin a cikin nau'in Arabidopsis na daji ko tushen Col da aka girma a kan faranti na MS tare da ko ba tare da 25 μM KAND 11 ba ta hanyar yin amfani da ƙwayoyin rigakafi na farko na β-tubulin da ƙwayoyin rigakafi na biyu na Alexa Fluor. Madaurin sikelin = 10 µm. (c) Tsarin mitotic na ƙananan ƙwayoyin cuta a cikin tushen meristem. An nuna ƙananan ƙwayoyin cuta ta amfani da tabo na immunohistochemical. An ƙidaya tsarin mitotic, gami da yankunan prophase, spindles, da phragmoplasts, daga hotunan confocal. Kibiyoyi suna nuna tsarin ƙananan ƙwayoyin cuta na mitotic. Taurari suna nuna bambance-bambance masu mahimmanci tare da maganin sham (gwajin t, p<0.05). n>9. Madaurin sikelin = 50 µm.
Duk da cewa Ursa tana da ikon kawo cikas ga aikin microtubule, ana sa ran tsarin aikinta zai bambanta da na microtubule depolymerizing agents na yau da kullun. Misali, yawan magungunan microtubule depolymerizing kamar disopyramide da oryzalin yana haifar da faɗaɗa anisotropic na ƙwayoyin epidermal, yayin da KAND 11 ba ya haifar da hakan. Bugu da ƙari, amfani da KAND 11 da disopyramide tare ya haifar da haɗin gwiwar amsawar ci gaban tushen da disopyramide ya haifar kuma an lura da hana ci gaban da KAND 11 ya haifar (Hoto na S4). Mun kuma yi nazarin martanin disopyramide 1-1 (phs1-1) mai saurin amsawa ga KAND 11. phs1-1 yana da maye gurbin maki tubulin kinase mara canonical kuma yana samar da gajerun tushe lokacin da aka yi masa magani da disopyramide9,20. Shuke-shuken phs1-1 da aka girma akan matsakaiciyar agar wanda ke ɗauke da KAND 11 suna da gajerun tushe kamar waɗanda aka girma akan disopyramid (hoto na S5).
Bugu da ƙari, mun lura da tsarin ƙwayoyin mitotic microtubule, kamar su yankunan prophase, spindles, da phragmoplasts, a cikin tushen tushen tsirrai da aka yi wa magani da KAND 11. Daidai da abin da aka lura da CDKB2;1p::CDKB2;1-GUS, an lura da raguwa mai yawa a cikin adadin ƙwayoyin mitotic microtubules (Hoto na .6c).
Domin gane gubar da ke tattare da sinadarin KAND 11 a lokacin da aka gano ƙarancin ƙwayoyin halitta, mun yi maganin ƙwayoyin dakatarwar taba BY-2 tare da KAND 11 kuma muka lura da martaninsu. Da farko mun ƙara KAND 11 zuwa ƙwayoyin BY-2 da ke bayyana TagRFP-TUA6, wanda ke sanya wa ƙananan ƙwayoyin cuta alama, don tantance tasirin KAND 11 akan ƙananan ƙwayoyin cuta. An tantance yawan ƙwayoyin cuta na cortical ta amfani da nazarin hoto, wanda ya ƙididdige kashi na ƙwayoyin cuta na cytoskeletal tsakanin ƙwayoyin cytoplasmic. Sakamakon gwajin ya nuna cewa bayan an yi amfani da 50 μM ko 100 μM KAND 11 na tsawon awa 1, yawan ya ragu sosai zuwa 0.94 ± 0.74% ko 0.23 ± 0.28%, bi da bi, yayin da yawan ƙwayoyin da aka yi wa magani da DMSO, ya kai 1.61 ± 0.34% (Hoto na 7a). Waɗannan sakamakon sun yi daidai da abin da aka lura a cikin Arabidopsis cewa maganin KAND 11 yana haifar da depolymerization na ƙananan ƙwayoyin cuta na cortical (Hoto na 6b). Mun kuma bincika layin BY-2 tare da filaments na actin masu lakabin GFP-ABD bayan magani tare da yawan KAND 11 iri ɗaya kuma mun lura cewa maganin KAND 11 ya katse filaments na actin. Jiyya tare da 50 μM ko 100 μM KAND 11 na tsawon awa 1 ya rage yawan filament na actin zuwa 1.20 ± 0.62% ko 0.61 ± 0.26%, bi da bi, yayin da yawan ƙwayoyin da aka yi wa magani da DMSO ya kasance 1.69 ± 0.51% (Hoto na 2). 7b). Waɗannan sakamakon sun bambanta da tasirin propyzamide, wanda ba ya shafar filaments na actin, da latrunculin B, wani actin depolymerizer wanda ba ya shafar microtubules (SI Hoto na S6). Bugu da ƙari, jiyya da coumamonamide 1, coumamonamide acid 6, ko KAND 11 bai shafi ƙananan ƙwayoyin cuta a cikin ƙwayoyin HeLa ba (SI Figure S7). Don haka, ana kyautata zaton tsarin aikin KAND 11 ya bambanta da na waɗanda aka sani suna kawo cikas ga cytoskeleton. Bugu da ƙari, bincikenmu na ƙananan ƙwayoyin BY-2 da aka yi wa magani da KAND 11 ya nuna farkon mutuwar ƙwayoyin halitta yayin maganin KAND 11 kuma ya nuna cewa yawan ƙwayoyin matattu masu launin shuɗi na Evans bai ƙaru sosai ba bayan minti 30 na maganin KAND 11, yayin da bayan mintuna 90 na magani da 50 μM ko 100 μM KAND, adadin ƙwayoyin matattu ya ƙaru zuwa 43.7% ko 80.1%, bi da bi (Hoto na 7c). Idan aka haɗa waɗannan bayanai, waɗannan bayanai sun nuna cewa sabon sinadarin ursolic acid KAND 11 wani abu ne mai hana ƙwayoyin cuta na musamman a cikin tsire-tsire tare da tsarin aiki da ba a san shi ba a da.
KAND yana shafar ƙwayoyin cortical microtubules, actin filaments, da kuma wanzuwar ƙwayoyin BY-2 na taba. (a) Ganin ƙananan ƙwayoyin cortical a cikin ƙwayoyin BY-2 a gaban TagRFP-TUA6. An duba ƙwayoyin BY-2 da aka yi wa magani da KAND 11 (50 μM ko 100 μM) ko DMSO ta hanyar amfani da na'urar duba ƙwayoyin halitta. An ƙididdige yawan ƙwayoyin cortical microtubule daga ƙananan ƙwayoyin halitta 25 masu zaman kansu. Haruffa suna nuna bambance-bambance masu mahimmanci (gwajin Tukey HSD, p.<0.05). Ma'aunin sikelin = 10 µm. (b) Filayen actin na cortical a cikin ƙwayoyin BY-2 da aka gani a gaban GFP-ABD2. An duba ƙwayoyin BY-2 da aka yi wa magani da KAND 11 (50 μM ko 100 μM) ko DMSO ta hanyar na'urar hangen nesa ta confocal. An ƙididdige yawan filaye na actin na cortical daga micrographs na ƙwayoyin halitta 25 masu zaman kansu. Haruffa suna nuna bambance-bambance masu mahimmanci (gwajin Tukey HSD, p<0.05). Madaurin sikelin = 10 µm. (c) An lura da matattun ƙwayoyin BY-2 ta hanyar fenti mai launin shuɗi na Evans. An duba ƙwayoyin BY-2 da aka yi wa magani da KAND 11 (50 μM ko 100 μM) ko DMSO ta hanyar amfani da na'urar hangen nesa mai haske. n=3. Madaurin sikelin = 100 µm.
Ganowa da amfani da sabbin kayayyakin halitta ya haifar da ci gaba mai yawa a fannoni daban-daban na rayuwar ɗan adam, ciki har da magani da noma. An gudanar da bincike na tarihi don samun mahadi masu amfani daga albarkatun ƙasa. Musamman ma, actinomycetes an san su da amfani a matsayin maganin kashe ƙwayoyin cuta ga ƙwayoyin cuta saboda ikonsu na samar da metabolites daban-daban na biyu kamar avermectin, babban mahaɗin ivermectin da bleomycin da abubuwan da suka samo asali, waɗanda ake amfani da su a magani azaman maganin hana ciwon daji21,22. Haka kuma, an gano nau'ikan mahaɗan kashe ƙwayoyin cuta daga actinomycetes, waɗanda wasu daga cikinsu an riga an yi amfani da su a kasuwanci1,23. Saboda haka, ana ɗaukar nazarin metabolites na actinomycete don ware samfuran halitta tare da ayyukan halittu da ake so a matsayin dabara mai tasiri. A cikin wannan binciken, mun gano wani sabon mahaɗi, coumamonamide, daga S. werraensis kuma mun yi nasarar haɗa shi. Ursonic acid matsakaici ne na urbenamide da abubuwan da suka samo asali. Yana iya haifar da curling na tushen halitta, yana nuna matsakaicin aiki zuwa ƙarfi na kashe ƙwayoyin cuta, da kuma lalata ƙwayoyin cuta na tsire-tsire kai tsaye ko a kaikaice. Duk da haka, tsarin aikin urmotonic acid na iya bambanta da na masu hana microtubule da ke akwai, tunda KAND 11 kuma yana wargaza filaments na actin kuma yana haifar da mutuwar ƙwayoyin halitta, yana nuna wata hanya ta tsari wacce urmotonic acid da abubuwan da ya samo asali ke tasiri ga tsarin cytoskeletal iri-iri.
Ƙarin bayani game da urbenonic acid zai taimaka wajen fahimtar tsarin aikin urbenonic acid. Musamman ma, burin na gaba shine a tantance ikon ursonic acid na ɗaurewa da ƙananan microtubules don tantance ko ursonic acid da abubuwan da ya samo asali suna aiki kai tsaye akan microtubules da kuma rage su, ko kuma ko aikinsu yana haifar da rashin daidaituwar microtubule. Bugu da ƙari, a yanayin da microtubules ba kai tsaye bane, gano wurin aiki da abubuwan da kwayoyin ursonic acid ke hari akan ƙwayoyin shuka zai taimaka wajen fahimtar halayen mahadi masu alaƙa da hanyoyin da za a iya inganta ayyukan kashe ƙwayoyin cuta. Gwajin aikinmu na bioactivity ya bayyana ikon cytotoxic na musamman na ursonic acid akan ci gaban tsire-tsire kamar Arabidopsis thaliana, taba da liverwort, yayin da ƙwayoyin E. coli ko HeLa ba su shafi ba. Ƙarancin guba ga ƙwayoyin dabbobi fa'idar abubuwan da ke haifar da ursonic acid ne idan an haɓaka su azaman maganin kashe ƙwayoyin cuta don amfani a filayen noma. Hakika, tunda microtubules tsari ne na gama gari a cikin eukaryotes, hana su zaɓi a cikin tsire-tsire babban buƙata ne ga magungunan kashe ƙwayoyin cuta. Misali, propyzamide, wani abu mai hana ƙwayoyin cuta na microtubule depolymerizing wanda ke ɗaure kai tsaye zuwa tubulin kuma yana hana polymerization, ana amfani da shi azaman maganin kashe ƙwayoyin cuta saboda ƙarancin gubarsa ga ƙwayoyin dabbobi24. Sabanin disopyramide, benzamides masu alaƙa suna da takamaiman abubuwan da aka nufa. Baya ga ƙwayoyin cuta na tsire-tsire, RH-4032 ko benzoxamide suma suna hana ƙwayoyin cuta na dabbobi ko oomycetes, bi da bi, kuma ana amfani da zalilamide azaman maganin kashe ƙwayoyin cuta saboda ƙarancin gubarsa25,26,27. Beyar da aka gano da abubuwan da aka nufa suna nuna gubar ƙwayoyin cuta masu zaɓi akan tsire-tsire, amma ya kamata a lura cewa ƙarin gyare-gyare na iya canza takamaiman abubuwan da aka nufa, wanda zai iya samar da ƙarin abubuwan da aka nufa don sarrafa fungi ko oomycetes masu cutarwa.
Abubuwan da ke tattare da urbenonic acid da abubuwan da suka samo asali suna da amfani ga ci gaban su a matsayin maganin kashe kwari da kuma amfani da su a matsayin kayan aikin bincike. Muhimmancin cytoskeleton wajen sarrafa siffar ƙwayoyin shuka an san shi sosai. Nazarin da aka yi a baya ya nuna cewa tsire-tsire sun haɓaka hanyoyin haɗaka na tsarin ƙwayoyin cuta ta hanyar sarrafa yanayin ƙwayoyin cuta don sarrafa yanayin ƙwayoyin cuta yadda ya kamata. An gano adadi mai yawa na ƙwayoyin cuta waɗanda ke da alhakin daidaita ayyukan ƙwayoyin cuta, kuma binciken da ya shafi hakan har yanzu yana ci gaba da gudana3,4,28. Fahimtarmu ta yanzu game da yanayin ƙwayoyin cuta a cikin ƙwayoyin shuka ba ta bayyana cikakken hanyoyin tsarin ƙwayoyin cuta ta cortical ba. Misali, kodayake disopyramide da oryzalin na iya cire ƙwayoyin cuta ta hanyar ƙwayoyin cuta, disopyramide yana haifar da mummunan karkacewar tushe yayin da oryzalin yana da ɗan tasiri kaɗan. Bugu da ƙari, maye gurbi a cikin tubulin, wanda ke daidaita ƙwayoyin cuta, yana haifar da dextrorotation a cikin tushe, yayin da paclitaxel, wanda kuma ke daidaita yanayin ƙwayoyin cuta ta microtubule, ba ya yi. Saboda haka, nazarin da gano manufofin ƙwayoyin cuta na ursolic acid ya kamata ya samar da sabbin fahimta game da daidaita yanayin ƙwayoyin cuta ta cortical na tsire-tsire. Haka nan, kwatancen sinadarai masu tasiri a nan gaba wajen haɓaka gurɓataccen ci gaba, kamar disopyramide, da kuma sinadarai marasa inganci, kamar oryzalin ko kumamotoric acid, za su ba da alamu kan yadda gurɓataccen ci gaba ke faruwa.
A gefe guda kuma, sake fasalin ƙwayoyin halittar da suka shafi tsaro wata hanya ce ta bayyana gubar ursonic acid. Kamuwa da cuta ko shigar da wani abu mai haifar da cutar a cikin ƙwayoyin halittar shuka wani lokaci yana haifar da lalata ƙwayoyin halittar da mutuwar ƙwayoyin halittar da ke biyo baya29. Misali, an ruwaito cewa cryptoxanthin da aka samo daga oomycete yana wargaza ƙwayoyin halittar microtubules da actin filaments kafin mutuwar ƙwayoyin halittar taba, kamar abin da ke faruwa da maganin KAND30,31. Kamanceceniya tsakanin martanin kariya da martanin ƙwayoyin halittar da ursonic acid ke haifarwa ya sa muka yi hasashen cewa suna haifar da tsarin ƙwayoyin halittar gama gari, kodayake tasirin ursonic acid mafi sauri da ƙarfi fiye da cryptoxanthin ya bayyana. Duk da haka, bincike ya nuna cewa wargajewar ƙwayoyin halittar actin yana haifar da mutuwar ƙwayoyin halittar kwatsam, wanda ba koyaushe yake tare da rushewar ƙwayoyin halittar microtubule29 ba. Bugu da ƙari, har yanzu za a gani ko ko dai mai cutar ko mai haifar da cutar yana haifar da gurɓataccen tushen, kamar yadda abubuwan da ke haifar da ursonic acid ke yi. Don haka, ilimin kwayoyin halitta da ke haɗa martanin kariya da cytoskeleton matsala ce mai kyau da za a magance. Ta hanyar amfani da ƙananan ƙwayoyin halitta masu nauyin da ke da alaƙa da ursonic acid, da kuma nau'ikan abubuwan da ke da ƙarfi daban-daban, suna iya samar da damammaki don kai hari ga hanyoyin ƙwayoyin halitta da ba a sani ba.
Idan aka haɗa su wuri ɗaya, ganowa da amfani da sabbin mahaɗan da ke daidaita yanayin ƙwayoyin cuta zai samar da hanyoyi masu ƙarfi don magance hadaddun hanyoyin ƙwayoyin cuta da ke ƙarƙashin tantance siffar ƙwayoyin cuta. A cikin wannan mahallin, sinadarin urmotonic da aka haɓaka kwanan nan, wanda ke shafar ƙwayoyin cuta da filaments na actin kuma yana haifar da mutuwar ƙwayoyin cuta, na iya samar da dama don fahimtar alaƙar da ke tsakanin sarrafa ƙwayoyin cuta da waɗannan sauran hanyoyin. Don haka, nazarin sinadarai da halittu ta amfani da acid na urbenonic zai taimaka mana mu fahimci hanyoyin sarrafa ƙwayoyin cuta da ke sarrafa ƙwayoyin cuta na tsirrai.
A yi wa S. werraensis MK493-CF1 allura a cikin kwalbar Erlenmeyer mai girman milimita 500 da aka cika da ruwa mai nauyin milimita 110 na iri wanda ya ƙunshi galactose 2% (w/v), man Essence 2% (w/v), 1% (w/v) abun da ke cikin Bacto. -soyton (Thermo Fisher Scientific, Inc.), 0.5% (w/v) cirewar masara (KOGOSTCH Co., Ltd., Japan), 0.2% (w/v) (NH4)2SO4 da 0.2% CaCO3 a cikin ruwan da aka cire ion. (pH 7.4 kafin a tace). An yi amfani da iri a kan injin girgiza mai juyawa (180 rpm) a zafin jiki na 27°C na tsawon kwana 2. Ana noma shi ta hanyar yin fermentation mai ƙarfi. An mayar da shukar iri (7 ml) zuwa cikin kwalbar K-1 mai nauyin 500 ml wanda ke ɗauke da gram 40 na kayan samarwa wanda ya ƙunshi gram 15 na sha'ir da aka matse (MUSO Co., Ltd., Japan) da gram 25 na ruwan da aka cire daga ion (pH ba a daidaita shi ba kafin a tace shi). An yi ta jiƙawa a zafin jiki na 30°C a cikin duhu na tsawon kwanaki 14. An cire kayan jiƙawar da 40 ml/kwalba EtOH kuma an sanya shi a cikin centrifuge (1500 g, 4°C, minti 10). An cire ruwan da ke sama (60 ml) da cakuda 10% MeOH/EtOAc. An cire Layer ɗin halitta a ƙarƙashin matsin lamba mai raguwa don samun ragowar (59.5 mg), wanda aka yi wa HPLC tare da cirewar gradient (0-10 minti: 90%) a kan ginshiƙin juyi na baya (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID 10 mm × tsawon 250 mm) H2O/CH3CN, mintuna 10–35: 90% H2O/CH3CN zuwa 70% H2O/CH3CN (gradient), mintuna 35–45: mintuna 90% H2O/EtOH, mintuna 45–155: 90% H2O/EtOH zuwa 100% EtOH (gradient (gradient), mintuna 155–200: 100% EtOH) a cikin ƙimar kwarara na 1.5 ml/min, an ware coumamonamide (1, 36.0 mg) a matsayin farin foda mara tsari.
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]+ ƙimar da aka ƙididdige: 141.0659, ƙimar da aka auna: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 1593, 1537 cm–1.
An samo tsaban Columbia (Col-0) daga Cibiyar Albarkatun Halittu ta Arabidopsis (ABRC) tare da izinin yin bincike. An yaɗa iri na Col-0 kuma an kula da su a ƙarƙashin yanayin dakin gwaje-gwajenmu kuma an yi amfani da su azaman tsire-tsire na Arabidopsis na daji. An tsaftace iri na Arabidopsis a saman kuma an noma su a cikin matsakaicin ƙarfi na Murashige da Skoog wanda ke ɗauke da kashi 2% na sucrose (Fujifilm Wako Pure Chemical), 0.05% (w/v) 2-(4-morpholino)ethanesulfonic acid (MES) (Fujifilm Wako Pure Chemical). ) da 1.5% agar (Fujifilm Wako Pure Chemical), pH 5.7, a zafin jiki na 23 °C da haske mai ɗorewa. T. Hashimoto (Cibiyar Kimiyya da Fasaha ta Nara) ne ya samar da tsaba na mutant na phs1-1.
T. Hashimoto (Cibiyar Kimiyya da Fasaha ta Nara) ne ya samar da irin SR-1 kuma aka yi amfani da shi azaman tsire-tsire na taba na daji. An tsaftace tsaban taba a saman kuma an jiƙa su a cikin ruwan da ba shi da tsafta na tsawon dare uku don haɓaka tsiro, sannan aka sanya su a cikin ruwan da ke ɗauke da kashi 2% na sucrose, 0.05% (w/v) MES, da 0.8% gellan gum (Fujifilm Wako Pure Chemical) Murashige. da Skoog medium) tare da pH 5.7 kuma aka sanya su a zafin jiki na 23°C a ƙarƙashin haske mai ɗorewa.
T. Kohchi (Jami'ar Kyoto) ne ya samar da Tak-1 na Strain kuma an yi amfani da shi a matsayin sashin gwaji na yau da kullun don nazarin liverwort. An samo Gemma daga tsire-tsire masu noma da aka yi musu magani sannan aka shafa shi a kan matsakaiciyar Gamborg B5 (Fujifilm Wako Pure Chemical) wanda ke ɗauke da 1% sucrose da 0.3% gellan gum sannan aka saka shi a zafin jiki na 23°C a ƙarƙashin haske mai ci gaba.
S. Hasezawa (Jami'ar Tokyo) ne ya samar da ƙwayoyin taba BY-2 (Nicotiana tabacum L. cv. Bright Yellow 2). An narkar da ƙwayoyin BY-2 sau 95 a cikin Linsmeier da Skoog da aka gyara kuma ana ƙara su a kowane mako da 2,4-dichlorophenoxyacetic acid 32. An haɗa dakatarwar ƙwayoyin halitta a kan abin girgiza mai juyawa a 130 rpm a 27°C a cikin duhu. A wanke ƙwayoyin halitta da sau 10 na ƙarar sabo kuma a sake dasa su a cikin matsakaici ɗaya. An samar da layukan ƙwayoyin halitta na BY-2 waɗanda ke bayyana alamar microtubule TagRFP-TUA6 ko alamar actin filament GFP-ABD2 a ƙarƙashin ƙwayar cuta ta cauliflower mosaic 35S promoter kamar yadda aka bayyana33,34,35. Ana iya kiyaye waɗannan layukan ƙwayoyin halitta kuma a daidaita su ta amfani da hanyoyin kama da waɗanda aka yi amfani da su don layin ƙwayar BY-2 na asali.
An noma ƙwayoyin HeLa a cikin maganin Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) wanda aka ƙara masa 10% na sinadarin shanu na tayi, penicillin 1.2 U/ml, da 1.2 μg/ml streptomycin a cikin injin incubator mai zafin 37°C tare da 5% CO2.
An yi duk gwaje-gwajen da aka bayyana a cikin wannan rubutun bisa ga ƙa'idodi da jagororin kare lafiyar halittu na Japan.
An narkar da mahadi a cikin dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) a matsayin maganin ajiya sannan a narkar da su a cikin maganin MS don Arabidopsis da taba ko kuma maganin Gamborg B5 don liverwort. Don gwajin hana ci gaban tushen, an shuka iri sama da 10 a kowace faranti a kan maganin agar wanda ke ɗauke da mahadi da aka nuna ko DMSO. An dasa iri a cikin ɗakin girma na tsawon kwanaki 7. An ɗauki hotunan tsirrai kuma an auna tsawon tushen. Don gwajin germination na Arabidopsis, an shuka iri 48 a kowace faranti a kan maganin agar wanda ke ɗauke da mahadi 200 μM ko DMSO. An shuka tsaban Arabidopsis a cikin ɗakin girma kuma an ƙidaya adadin tsirrai da suka tsiro kwanaki 7 bayan tsiro (dag). Don gwajin germination na taba, an shuka iri 24 a kowace faranti a kan maganin agar wanda ke ɗauke da 200 μM KAND ko DMSO. An shuka tsaban taba a cikin ɗakin girma kuma an ƙidaya adadin tsirrai da suka tsiro bayan kwana 14. Don gwajin hana haɓakar hanta, an yi wa amfrayo guda 9 daga kowanne faranti fenti a kan matsakaicin agar wanda ke ɗauke da yawan KAND ko DMSO da aka nuna kuma aka saka su a cikin ɗakin girma na tsawon kwanaki 14.
Yi amfani da tsire-tsire masu launin propidium iodide (PI) 5 mg/ml don ganin tsarin tushen meristem. An lura da siginar PI ta hanyar amfani da na'urar hangen nesa ta amfani da na'urar hangen nesa ta TCS SPE confocal laser scanning microscope (Leica Microsystems).
An yi amfani da β-glucuronidase (GUS) wajen yin tabon asalin halitta bisa ga ka'idar da Malami da Benfey36 suka bayyana. An daure 'ya'yan itacen a cikin kashi 90% na acetone cikin dare ɗaya, an yi musu fenti da 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid a cikin GUS buffer na tsawon awa 1 sannan aka sanya su a cikin ruwan chloraldehyde mai tsafta. (8 g na chloral hydrate, 2 ml na ruwa da glycerol 1 ml) kuma an lura da su ta hanyar amfani da na'urar microscope ta Axio Imager M1 (Carl Zeiss).
An auna kusurwoyin tushe a kan tsirrai masu kwanaki 7 da aka shuka a kan faranti da aka sanya a tsaye. Auna kusurwoyin tushen daga alkiblar vector na nauyi kamar yadda aka bayyana a mataki na 6.
An lura da tsarin ƙwayoyin cuta na cortical kamar yadda aka bayyana, tare da ƙananan gyare-gyare ga ka'idar 37. An yi amfani da antibody na anti-β-tubulin (KMX-1, Merk Millipore: MAB3408) da Alexa Fluor 488-conjugated anti-linux IgG (Thermo Fisher Scientific: A32723) a matsayin antibodies na farko da na sakandare a 1:1000 da 1:100 dilutions, bi da bi. An samo hotunan haske ta amfani da na'urar daukar hoton laser confocal TCS SPE (Leica Microsystems). Sami hotunan Z-stack kuma ƙirƙirar hasashen ƙarfi mafi girma bisa ga umarnin masana'anta.
An yi gwajin yaduwar ƙwayoyin HeLa ta amfani da Kayan Ƙidayar Kwayoyin Halitta 8 (Dojindo) bisa ga umarnin masana'anta.
An yi nazarin girman E. coli DH5α ta hanyar auna yawan ƙwayoyin halitta a cikin al'ada ta amfani da na'urar auna hoto a 600 nm (OD600).
An lura da tsarin cytoskeletal a cikin ƙwayoyin BY-2 masu canzawa ta amfani da na'urar hangen nesa mai haske wacce aka sanye da na'urar daukar hoton confocal ta CSU-X1 (Yokogawa) da kyamarar sCMOS (Zyla, Andor Technology). An tantance yawan cytoskeletal ta hanyar nazarin hoto, wanda ya ƙididdige kashi na cytoskeletal pixels tsakanin cytoplasmic pixels a cikin hotunan confocal ta amfani da software na ImageJ kamar yadda aka bayyana38,39.
Domin gano mutuwar ƙwayoyin halitta a cikin ƙwayoyin BY-2, an saka wani ɓangare na dakatarwar ƙwayoyin halitta da kashi 0.05% na Evans blue na tsawon mintuna 10 a zafin ɗaki. Tabon ƙwayoyin halitta masu launin shuɗi na Evans blue ya dogara ne akan fitar da rini daga ƙwayoyin halitta masu rai ta hanyar membrane na plasma mai cike da sinadarai40. An lura da ƙwayoyin halitta masu launi ta amfani da na'urar hangen nesa mai haske (BX53, Olympus).
An girma ƙwayoyin HeLa a cikin DMEM tare da ƙarin 10% FBS a cikin incubator mai laushi a 37°C da 5% CO2. An yi wa ƙwayoyin magani da 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), ko 100 ng/ml Nocodmaze (Sigma) na tsawon awanni 6 a 37°C. An gyara ƙwayoyin halitta da MetOH na tsawon mintuna 10 sannan aka yi da acetate na tsawon mintuna 5 a zafin ɗaki. An haɗa ƙwayoyin halitta da β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) a cikin 0.5% BSA/PBS na tsawon awanni 2, an wanke sau 3 da TBST, sannan aka haɗa da Alexa Fluor akuya antibody. 488 awa 1. – IgG na linzamin kwamfuta (Thermo Fisher Scientific: A11001) da 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) an narkar da su a cikin 0.5% BSA/PBS. Bayan an wanke su da TBST sau uku, an ga ƙwayoyin halitta masu tabo a kan na'urar hangen nesa ta Nikon Eclipse Ti-E. An ɗauki hotuna da kyamarar Hamamatsu ORCA-R2 CCD mai sanyaya ta amfani da manhajar MetaMorph (Na'urorin Molecular).
Lokacin Saƙo: Yuni-17-2024