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Ganowa da amfani mai amfani na samfuran halitta na iya taimakawa inganta rayuwar ɗan adam.Ana amfani da sinadarai masu hana ci gaban shuka a ko'ina a matsayin maganin ciyawa don sarrafa ciyawa.Saboda buƙatar yin amfani da nau'o'in maganin herbicides daban-daban, akwai buƙatar gano mahadi tare da sababbin hanyoyin aiki.A cikin wannan binciken, mun gano wani labari N-alkoxypyrrole fili, coumamonamide, daga Streptomyces werraensis MK493-CF1 kuma ya kafa cikakken tsarin haɗin gwiwa.Ta hanyar tantance ayyukan nazarin halittu, mun gano cewa urs-monoamic acid shine tsaka-tsakin roba na urs-monoamide kuma mai yuwuwa.mai hana shuka girma.Bugu da ƙari, mun haɓaka nau'o'in urbenonic acid daban-daban, ciki har da urbenyloxy derivative (UDA), wanda ke da babban aikin herbicidal ba tare da mummunan tasiri ga ci gaban kwayoyin HeLa ba.Mun kuma gano cewa abubuwan da suka samo asali na urmotonic acid suna rushe microtubules na shuka;Bugu da ƙari, KAND yana rinjayar actin filaments kuma yana haifar da mutuwar kwayar halitta;Wadannan sakamako masu yawa sun bambanta da waɗanda aka sani da masu hanawa na microtubule kuma suna ba da shawarar sabon tsarin aiki don ursonic acid, wanda ke wakiltar muhimmiyar fa'ida a cikin ci gaban sabbin magungunan herbicides.
Ganowa da aikace-aikace a aikace na samfuran halitta masu fa'ida da abubuwan da suka samo asali, hanya ce ta inganta rayuwar ɗan adam.Na biyu metabolites samar da microorganisms, shuke-shuke da kwari sun haifar da babban ci gaba a magani da noma.Yawancin maganin rigakafi da magungunan cutar sankarar bargo an samo su daga samfuran halitta.Bugu da kari, daban-daban irimagungunan kashe qwari, magungunan kashe qwari da namun daji ana fitar da su daga waxannan kayayyakin da ake amfani da su wajen noma.Musamman magungunan magance ciyawa sune muhimman kayan aiki don haɓaka amfanin gona a noma na zamani, kuma an riga an yi amfani da nau'ikan mahadi daban-daban a kasuwa.Hanyoyi da yawa na salon salula a cikin tsire-tsire, kamar photosynthesis, amino acid metabolism, haɗin bangon tantanin halitta, ƙa'idodin mitosis, siginar phytohormone, ko haɗin furotin, ana ɗaukar maƙasudin maƙasudi na herbicides.Haɗaɗɗen da ke hana aikin microtubule rukuni ne na gama gari na herbicides waɗanda ke shafar ci gaban shuka ta hanyar tasirin mitotic regulation2.
Microtubules sune sassan cytoskeleton kuma ana kiyaye su sosai a cikin ƙwayoyin eukaryotic.Tubulin heterodimer ya ƙunshi α-tubulin da β-tubulin suna samar da ka'idodin microtubule na layi, tare da 13 protofilaments samar da tsarin silinda.Microtubules suna taka rawa da yawa a cikin ƙwayoyin tsire-tsire, ciki har da ƙayyade siffar tantanin halitta, rabon tantanin halitta, da jigilar intracellular3,4.Kwayoyin tsire-tsire sun ƙunshi microtubules a ƙarƙashin membrane na plasma na interphase, kuma waɗannan abubuwan da ake kira cortical microtubules ana tunanin su sarrafa tsarin microfibrils cellulose ta hanyar tsarin tsarin cellulose synthase complexes4,5.Cortical microtubules na tushen epidermal Kwayoyin, yanzu a cikin yankin na m elongation na tushen tip, suna located a kai tsaye, kuma cellulose microfibers bi wadannan microtubules da iyakance shugabanci na cell fadada, game da shi inganta anisotropic cell elongation.Sabili da haka, aikin microtubule yana da alaƙa da alaƙa da ilimin halittar shuka.Sauye-sauyen amino acid a cikin kwayoyin halittar tubulin suna haifar da skew na cortical microtubule arrays da haɓakar hagu- ko dama a cikin Arabidopsis 6,7.Hakazalika, maye gurbi a cikin sunadaran da ke da alaƙa da microtubule waɗanda ke daidaita ƙarfin microtubule kuma na iya haifar da gurɓataccen ci gaban tushen8,9,10,11,12,13.Bugu da ƙari, jiyya tare da microtubule masu lalata herbicides kamar disopyramide, wanda kuma aka sani da pretilachlor, kuma yana haifar da ci gaban tushen tushen gefen hagu na hagu14.Wadannan bayanan sun nuna cewa daidaitaccen tsari na aikin microtubule yana da mahimmanci don ƙayyade alkiblar ci gaban shuka.
An gano nau'ikan masu hana microtubule iri-iri, kuma waɗannan magungunan sun ba da gudummawa mai mahimmanci ga binciken cytoskeletal, da kuma aikin gona da magani2.Musamman ma, oryzalin, dinitroaniline mahadi, disopyramide, abubuwan da ke da alaƙa da benzamide, da analogs na su na iya hana aikin microtubule kuma ta haka ne hana ci gaban shuka.Saboda haka, ana amfani da su sosai azaman maganin ciyawa.Duk da haka, tun da microtubules wani muhimmin abu ne na kwayoyin shuka da dabba, yawancin masu hana microtubule sune cytotoxic ga nau'in tantanin halitta guda biyu.Sabili da haka, duk da ƙwarewar da aka sani da su azaman herbicides, ana amfani da ƙayyadaddun adadin magungunan antimicrotubule don dalilai masu amfani.
Streptomyces shine jinsin dangin Streptomyces, wanda ya haɗa da aerobic, gram-positive, kwayoyin filamentous kuma an san shi sosai don ikonsa na samar da nau'ikan metabolites masu yawa.Saboda haka, ana la'akari da ɗayan mahimman tushen sabbin samfuran halitta masu aiki da ilimin halitta.A cikin binciken na yanzu, mun gano wani sabon fili da ake kira coumamonamide, wanda aka keɓe daga Streptomyces werraensis MK493-CF1 da S. werraensis ISP 5486. Yin amfani da bincike mai zurfi da cikakken bincike mai zurfi, an kwatanta tsarin coumamonamide da kuma musamman N-alkoxypyrrole skeleton. aka ƙaddara.kira.Ursmonic acid, tsaka-tsakin roba na ursmonoamide da abubuwan da suka samo asali, an samo shi don hana haɓakawa da haɓakar sanannen tsirin Arabidopsis thaliana.A cikin nazarin dangantakar aiki-tsari, mun gano cewa wani fili tare da C9 wanda aka canza zuwa ursonic acid, wanda ake kira nonyloxy derivative na ursonic acid (KAND), yana haɓaka tasirin hanawa akan girma da haɓaka.Musamman ma, sabon da aka gano mai hana ci gaban shuka shima ya shafi ci gaban taba da liverwort kuma ba cytotoxic ga kwayoyin cuta ko kwayoyin HeLa ba.Bugu da ƙari, wasu abubuwan da aka samo na urmotonic acid suna haifar da gurɓataccen tushen phenotype, yana nuna cewa waɗannan abubuwan da aka samo asali sun shafi microtubules kai tsaye ko a kaikaice.Daidai da wannan ra'ayin, abubuwan da muka lura na microtubules masu lakabi ko dai immunohistochemically ko tare da sunadaran sunadaran suna nuna cewa maganin KAND yana lalata microtubules.Bugu da ƙari, jiyya tare da abubuwan da aka samo kumamotonic acid ya rushe actin microfilaments.Don haka, mun gano sabon mai hana ci gaban shuka wanda keɓantaccen tsarin aiki ya haɗa da lalata cytoskeleton.
MK493-CF1 an keɓe shi daga ƙasa a Shinagawa-ku, Tokyo.Iri MK493-CF1 ya samar da reshe mai kyau na stromal mycelium.An ƙaddara jerin ɓangaren 16S ribosomal RNA gene (1422 bp).Wannan nau'in yana da kama da S. werraensis (NBRC 13404T = ISP 5486, 1421/1422 bp, T: nau'i na al'ada, 99.93%).Dangane da wannan sakamakon, an ƙaddara cewa wannan nau'in yana da alaƙa da nau'in nau'in S. werraensis.Saboda haka, mun ba da sunan wannan nau'in na ɗan lokaci S. werraensis MK493-CF1.S. werraensis ISP 5486T kuma yana samar da mahaɗan bioactive iri ɗaya.Tun da an sami ɗan ƙaramin bincike na farko don samun samfuran halitta daga wannan ƙananan ƙwayoyin cuta, an ƙara yin binciken sinadarai.Bayan noman S. werraensis MK493-CF1 akan matsakaicin sha'ir ta hanyar fermentation mai ƙarfi a 30 ° C na kwanaki 14, an fitar da matsakaici tare da 50% EtOH.An bushe 60 ml na samfurin don samun 59.5 MG na danyen mai.An ƙaddamar da cirewar ɗanyen don juyawa lokaci na HPLC don ba N-methoxy-1H-pyrrole-2-carboxamide (1, mai suna coumamonamide, 36.0 mg).Jimlar adadin 1 shine kusan 60% na tsantsar danyen mai.Saboda haka, mun yanke shawarar yin nazari daki-daki game da kaddarorin kumamotoamide 1.
Coumamonamide 1 shine farin amorphous foda da babban ƙuduri mai girma (HRESIMS) ya tabbatar da C6H8N2O2 (Fig. 1).Rukunin pyrrole da aka maye gurbin C2 na wannan fili yana 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), da kuma 13C NMR bakan yana nuna kasancewar sp2 carbon atoms guda hudu.Kasancewar ƙungiyar amide a matsayin C2 an kimanta ta hanyar haɗin gwiwar HMBC daga proton C-3 zuwa carbonyl amide carbonyl a δC 161.1.Bugu da ƙari, 1 H da 13 C NMR kololuwa a δH 4.10 (3H, S) da δC 68.3 suna nuna kasancewar ƙungiyoyin N-methoxy a cikin kwayoyin halitta.Ko da yake har yanzu ba a ƙayyade ainihin matsayi na ƙungiyar methoxy ba ta amfani da bincike na spectroscopic kamar ingantaccen spectroscopy spectroscopy da makaman nukiliya overhauser abbreviation (NOEDF), N-methoxy-1H-pyrrole-2-carboxamide ya zama fili na farko na dan takara.
Don ƙayyade daidaitaccen tsari na 1, an yi jimlar ƙira (Fig. 2a).Jiyya na 2-aminopyridine 2 na kasuwanci tare da m-CPBA ya haifar da daidaitaccen N-oxide 3 a cikin yawan amfanin ƙasa.Bayan 2-aminoazidation na 2, cyclocondensation dauki da Abramovich ya bayyana an gudanar da shi a cikin benzene a 90 ° C don samun 1-hydroxy-1H-pyrrole-2-carbonitrile 5 da ake so a cikin grams.Gudun 60% (matakai biyu).15,16.Methylation da hydrolysis na 4 sa'an nan kuma ya ba 1-methoxy-1H-pyrrole-2-carboxylic acid (wanda ake kira "cumotonic acid", 6) a cikin yawan amfanin ƙasa (70%, matakai biyu).A ƙarshe, amidation ta hanyar matsakaicin acid chloride 6 ta amfani da ammonia mai ruwa ya ba Kumamoto amide 1 a cikin 98% yawan amfanin ƙasa.Duk bayanan da aka haɗa na 1 sun kasance daidai da 1 keɓe, don haka an ƙayyade tsarin 1;
Gabaɗaya kira da bincike na ayyukan nazarin halittu na urbenamide da urbenic acid.(a) Jimlar hadawar Kumamoto amide.(b) An shuka tsiron daji na kwana bakwai na Arabidopsis Columbia (Col) akan faranti na Murashige da Skoog (MS) waɗanda ke ɗauke da coumamonamide 6 ko coumamonamide 1 a adadin da aka nuna.Ma'aunin Sikeli = 1 cm.
Da farko, mun tantance ayyukan nazarin halittu na urbenamide da tsaka-tsakinsa don iyawarsu don daidaita ci gaban shuka.Mun ƙara nau'o'i daban-daban na ursmonamide 1 ko ursmonic acid 6 zuwa MS agar matsakaici da kuma al'ada Arabidopsis thaliana seedlings akan wannan matsakaici.Wadannan ƙididdigar sun nuna cewa babban taro (500 μM) na 6 ya hana ci gaban tushen (Fig. 2b).Bayan haka, mun ƙirƙiri abubuwan haɓaka daban-daban ta hanyar maye gurbin N1 matsayi na 6 kuma mun yi nazarin dangantakar aiki-tsari akan su (an kwatanta tsarin haɗin analog a cikin Bayanin Tallafi (SI)).An shuka tsiron Arabidopsis akan matsakaici mai ɗauke da 50 μM ursonic acid, kuma an auna tsayin tushen.kamar yadda aka nuna a hoton.Kamar yadda aka nuna a cikin Figures 3a, b, da S1, coumamo acid suna da tsayi daban-daban na sarƙoƙi na alkoxy na layi (9, 10, 11, 12, da 13) ko manyan sarƙoƙi na alkoxy (15, 16, da 17) a matsayi na N1.Abubuwan da aka samo asali sun nuna gagarumin hana ci gaban tushen.Bugu da ƙari, mun gano cewa aikace-aikacen 200 μM 10, 11, ko 17 ya hana germination (Figs. 3c da S2).
Nazarin tsarin-aiki dangantaka na Kumamoto amide da mahadi masu alaƙa.(a) Tsari da tsarin kira na analogues.(b) Ƙididdigar tushen tsayin tsire-tsire na kwanaki 7 da aka girma akan matsakaicin MS tare da ko ba tare da 50 μM coumamonamide abubuwan da suka samo asali ba.Asterisks suna nuna bambance-bambance masu mahimmanci tare da maganin sham (t gwaji, p<0.05).n>18. Ana nuna bayanai azaman ma'anar ± SD.nt yana nufin "ba a gwada ba" saboda fiye da kashi 50% na tsaba ba su tsiro ba.(c) Ƙididdigar yawan germination na tsaba da aka kula da su na tsawon kwanaki 7 a cikin MS matsakaici tare da ko ba tare da 200 μM coumamonamide da mahadi masu dangantaka ba.Asterisks suna nuna bambance-bambance masu mahimmanci tare da maganin sham (gwajin chi-square).n=96.
Abin sha'awa shine, ƙarar sarƙoƙi na gefen alkyl fiye da C9 ya rage ayyukan hanawa, yana nuna cewa kumamotoic acid mahadi suna buƙatar sassan gefe na wani girman girman don nuna ayyukan ilimin halitta.
Saboda nazarin dangantakar aiki-tsari ya nuna cewa an canza C9 zuwa ursonic acid da kuma abin da aka samo asali na ursonic acid (wanda ake kira KAND 11) shine mafi tasiri mai hana ci gaban shuka, mun gudanar da cikakken bayanin KAND 11. Maganin Arabidopsis. tare da 50 μM KAND 11 kusan gaba ɗaya ya hana germination, yayin da ƙananan ƙididdiga (40, 30, 20, ko 10 μM) na KAND 11 sun hana ci gaban tushen girma a cikin hanyar dogara da kashi (Fig. 4a, b).Don gwada ko KAND 11 yana shafar tushen tushen meristem, mun bincika tushen meristems da aka lalata tare da propidium iodide (PI) kuma auna girman yanki na meristem.Girman meristem na tsire-tsire da aka girma a kan matsakaici mai dauke da 25 μM KAND-11 shine 151.1 ± 32.5 μm, yayin da girman girman ƙwayar tsire-tsire da aka girma akan matsakaicin kulawa wanda ke dauke da DMSO shine 264.7 ± 30.8 μm (Fig. 4c, d) , wanda ke nuna cewa KAND-11 yana mayar da aikin salula.yadawa.Tushen Meristem.Daidai da wannan, maganin KAND 11 ya rage yawan adadin CDKB2; 1p :: CDKB2; 1-GUS siginar a cikin tushen meristem (Fig. 4e) 17.Wadannan sakamakon sun nuna cewa KAND 11 yana hana ci gaban tushen ta hanyar rage ayyukan yaduwar kwayar halitta.
Binciken tasirin hanawa na abubuwan da suka samo asali na urbenonic acid (wanda aka samo asali na urbenyloxy) akan girma.(a) 7-day-day-nau'in daji Col seedlings girma a kan faranti MS tare da nunin taro na KAND 11. Scale bar = 1 cm.(b) Ƙididdigar tsayin tushen.Haruffa suna nuna bambance-bambance masu mahimmanci (Gwajin Tukey HSD, p<0.05).n>16. Ana nuna bayanai azaman ma'anar ± SD.(c) Confocal microscopy na propidium iodide-stained daji-nau'in Tushen Col girma a kan faranti MS tare da ko ba tare da 25 μM KAND 11. Farin bango yana nuna tushen meristem.Ma'aunin Sikeli = 100 µm.(d) Ƙididdigar girman tushen meristem (n = 10 zuwa 11).An ƙaddara bambance-bambancen ƙididdiga ta amfani da t-test (p<0.05).Sanduna suna wakiltar matsakaicin girman meristem.(e) Bambance-bambancen tsangwama (DIC) microscopy na tushen meristem mai dauke da ginin CDKB2;1 pro: CDKB2;1-GUS tabo da tabo akan tsire-tsire masu kwanaki 5 da aka girma akan faranti na MS tare da ko ba tare da 25 µM KAND assay ba.
An ƙara gwada phytotoxicity na KAND 11 ta amfani da wani shuka dicotyledonous, taba (Nicotiana tabacum), da kuma babban tsarin shukar ƙasa, liverwort (Marchantia polymorpha).Kamar yadda yake a cikin Arabidopsis, taba SR-1 seedlings da aka girma akan matsakaici mai ɗauke da 25 μM KAND 11 ya samar da guntu tushen (Fig. 5a).Bugu da ƙari, 40 na tsaba 48 da aka bazu a kan faranti mai ɗauke da 200 μM KAND 11, yayin da duk nau'ikan 48 sun bazu akan kafofin watsa labarai da aka yi wa izgili, wanda ke nuna cewa yawan adadin KAND yana da mahimmanci (p).<0.05;chi test -square) ya hana germination na taba.(Hoto na 5b).Bugu da ƙari, ƙaddamar da KAND 11 wanda ya hana ƙwayar ƙwayar cuta a cikin hanta ya kasance daidai da tasiri mai tasiri a cikin Arabidopsis (Fig. 5c).Wadannan sakamakon sun nuna cewa KAND 11 na iya hana ci gaban tsirrai iri-iri.Sa'an nan kuma muka bincika yiwuwar cytotoxicity na bear monoamide mahadi a cikin wasu kwayoyin halitta, wato Human HeLa Kwayoyin da Escherichia coli iri DH5a, a matsayin wakilan mafi girma dabba da kwayoyin Kwayoyin, bi da bi.A cikin jerin gwaje-gwaje na ƙwayoyin cuta, mun lura cewa coumamonamide 1, coumamonamidic acid 6, da KAND 11 ba su shafi ci gaban HeLa ko E. coli Kwayoyin a ƙananan 100 μM (Fig. 5d, e).
Hana haɓakar KAND 11 a cikin ƙwayoyin da ba Arabidopsis ba.(a) An shuka tsire-tsire masu nau'in daji na SR-1 mai tsawon mako biyu akan faranti MS a tsaye mai ɗauke da 25 μM KAND 11. (b) An shuka tsire-tsire masu nau'in daji na SR-1 na sati biyu akan a kwance. MS faranti dauke da 200 μM KAND 11. (c) Makonni biyu na daji irin Tak-1 liverwort buds girma a kan Gamborg B5 faranti tare da nuna taro na KAND 11. Ja kiban nuna spores cewa daina girma a cikin makonni biyu shiryawa. lokaci.(d) Gwajin yaɗuwar sel na ƙwayoyin HeLa.An auna adadin ƙwayoyin da za a iya amfani da su a ƙayyadaddun lokaci ta amfani da kit ɗin ƙidayar tantanin halitta 8 (Dojindo).A matsayin sarrafawa, an bi da ƙwayoyin HeLa tare da 5 μg/ml actinomycin D (Dokar D), wanda ke hana rubutun RNA polymerase kuma yana haifar da mutuwar tantanin halitta.An yi nazari ne sau uku.(e) E. coli cell proliferation assay.An yi nazarin girma E. coli ta hanyar auna OD600.A matsayin sarrafawa, an bi da ƙwayoyin sel tare da 50 μg / ml ampicillin (Amp), wanda ke hana haɗin bangon ƙwayoyin ƙwayoyin cuta.An yi nazari ne sau uku.
Don ƙaddamar da tsarin aikin cytotoxicity wanda ke haifar da mahadi masu alaƙa da uramide, mun sake nazarin abubuwan urbenic acid tare da matsakaicin tasirin hanawa.kamar yadda aka nuna a hoton.Kamar yadda aka nuna a cikin Figures 2b, 6a, tsire-tsire da aka girma a kan faranti na agar da ke dauke da adadi mai yawa (200 μM) na urmotonic acid 6 sun samar da guntu da tushen hagu (θ = - 23.7 ± 6.1), yayin da tsire-tsire masu girma a kan matsakaicin kulawa, da seedlings samar da kusan madaidaiciya tushen (θ = - 3.8 ± 7.1).An san wannan haɓakar haɓakar ƙaƙƙarfan ƙaƙƙarfan haɓaka ta haifar da rashin aiki na cortical microtubules14,18.Daidai da wannan binciken, magungunan microtubule-destabilizing disopyramide da oryzalin sun haifar da irin wannan tushen karkatar da su a ƙarƙashin yanayin girma (Figs. 2b, 6a).A lokaci guda, mun gwada abubuwan da suka samo asali na urmotonic acid kuma mun zaɓi da yawa daga cikinsu waɗanda, a wasu ƙididdiga, haifar da ci gaban tushen tushen.Matsakaicin 8, 9, da 15 sun canza jagorancin tushen girma a 75 μM, 50 μM, da 40 μM, bi da bi, yana nuna cewa waɗannan mahadi na iya lalata microtubules yadda ya kamata (Fig. 2b, 6a).Mun kuma gwada mafi ƙarancin ursolic acid, KAND 11, a ƙaramin taro (15 µM) kuma mun gano cewa aikace-aikacen KAND 11 ya hana ci gaban tushen da kuma cewa jagorar ci gaban tushen bai yi daidai ba, kodayake suna son gangara zuwa hagu. Hoto C3)..Saboda mafi yawan ƙwayoyin microtubule-destabilizing kwayoyi wani lokaci suna hana ci gaban shuka maimakon haifar da karkatar da tushe, daga baya mun tantance yiwuwar KAND 11 yana rinjayar microtubules ta hanyar lura da microtubules na cortical a cikin tushen epidermal sel.Immunohistochemistry ta amfani da magungunan anti-β-tubulin a cikin kwayoyin epidermal na tushen seedling da aka bi da su tare da 25 μM KAND 11 ya nuna bacewar kusan dukkanin microtubules na cortical a cikin kwayoyin epidermal a cikin yankin elongation (Fig. 6b).Wadannan sakamakon sun nuna cewa kumamotonic acid da abubuwan da suka samo asali suna aiki kai tsaye ko a kaikaice akan microtubules don rushe su kuma waɗannan mahadi sune masu hanawa na microtubule.
Ursonic acid da abubuwan da suka samo asalinsa suna canza cortical microtubules a cikin Arabidopsis thaliana.(a) Tushen karkata an auna shi a gaban nau'ikan abubuwan urmotonic acid daban-daban a adadin da aka nuna.Sakamakon mahadi guda biyu da aka sani don hana microtubules: disopyramide da oryzalin an kuma bincikar su.Sakin yana nuna ma'aunin da ake amfani da shi don auna kusurwar girma tushen.Asterisks suna nuna bambance-bambance masu mahimmanci tare da maganin sham (t gwaji, p<0.05).n>19. Ma'auni na ma'auni = 1 cm.(b) Microtubules na cortical a cikin kwayoyin epidermal a cikin yankin elongation.Microtubules a cikin nau'in daji na Arabidopsis Col Tushen da aka girma akan faranti na MS tare da ko ba tare da 25 μM KAND 11 an hango su ta hanyar lalatawar rigakafi ta hanyar amfani da β-tubulin na farko na rigakafi da Alexa Fluor-conjugated secondary antibodies.Ma'aunin Sikeli = 10 µm.(c) Tsarin mitotic na microtubules a cikin tushen meristem.An hango microtubules ta amfani da tabo immunohistochemical.Siffar mitotic, gami da guraben da ya dace, sanduna, da phragmoplasts, an ƙidaya su daga hotuna masu kama da juna.Kibau suna nuna sifofin microtubule mitotic.Asterisks suna nuna bambance-bambance masu mahimmanci tare da maganin sham (t gwaji, p<0.05).n>9. Ma'auni ma'auni = 50 µm.
Ko da yake Ursa yana da ikon rushe aikin microtubule, ana sa ran tsarin aikin sa zai bambanta da na al'ada microtubule depolymerizing agents.Misali, mafi girman adadin microtubule depolymerizing jamiái irin su disopyramide da oryzalin suna haifar da haɓakar anisotropic na ƙwayoyin epidermal, yayin da KAND 11 baya.Bugu da ƙari, haɗin gwiwar KAND 11 da disopyramide sun haifar da haɓakar haɓakar haɓakar haɓakar haɓakar disopyramide da KAND 11-induced inhibition girma (Fig. S4).Mun kuma bincikar amsawar hypersensitive disopyramide 1-1 (phs1-1) mutant zuwa KAND 11. phs1-1 yana da maye gurbi na tubulin kinase wanda ba na canonical ba kuma yana haifar da guntu tushen lokacin da aka bi da shi tare da disopyramide9,20.phs1-1 tsire-tsire masu tsire-tsire waɗanda aka girma akan matsakaiciyar agar mai ɗauke da KAND 11 suna da guntu tushen kama da waɗanda aka girma akan disopyramid (fig. S5).
Bugu da ƙari, mun lura da tsarin microtubule mitotic, irin su prophase zones, spindles, da phragmoplasts, a cikin tushen meristem na tsire-tsire da aka bi da shi tare da KAND 11. Daidai da abubuwan lura don CDKB2; 1p :: CDKB2; 1-GUS, raguwa mai yawa a cikin an lura da adadin microtubules mitotic (Fig. 6c).
Don siffanta cytotoxicity na KAND 11 a ƙudurin subcellular, mun bi da ƙwayoyin dakatarwa ta taba BY-2 tare da KAND 11 kuma mun lura da martanin su.Mun fara ƙara KAND 11 zuwa ƙwayoyin BY-2 masu bayyana TagRFP-TUA6, waɗanda ke ba da alamar microtubules, don tantance tasirin KAND 11 akan microtubules na cortical.An tantance yawan ƙananan ƙwayoyin cortical ta amfani da nazarin hoto, wanda ya ƙididdige yawan adadin pixels cytoskeletal tsakanin pixels cytoplasmic.Sakamakon binciken ya nuna cewa bayan jiyya tare da 50 μM ko 100 μM KAND 11 don 1 hour, yawancin ya ragu sosai zuwa 0.94 ± 0.74% ko 0.23 ± 0.28%, yayin da yawancin kwayoyin da aka bi da su tare da DMSO, sun kai 1.634 ± % (Hoto 7a).Wadannan sakamakon sun yi daidai da lura a cikin Arabidopsis cewa KAND 11 magani yana haifar da depolymerization na cortical microtubules (Fig. 6b).Mun kuma bincika layin BY-2 tare da GFP-ABD-labeled actin filaments bayan jiyya tare da irin wannan maida hankali na KAND 11 kuma mun lura cewa KAND 11 magani ya rushe actin filaments.Jiyya tare da 50 μM ko 100 μM KAND 11 don 1 h ya rage yawan adadin actin filament zuwa 1.20 ± 0.62% ko 0.61 ± 0.26%, bi da bi, yayin da yawa a cikin kwayoyin DMSO da aka kula da su shine 1.69 ± 0.51% (Figure 0.51%).7b).Wadannan sakamakon sun bambanta da tasirin propyzamide, wanda ba ya shafar actin filaments, da kuma latrunculin B, wani actin depolymerizer wanda baya rinjayar microtubules (SI Figure S6).Bugu da ƙari, jiyya tare da coumamonamide 1, coumamonamide acid 6, ko KAND 11 bai shafi microtubules a cikin kwayoyin HeLa ba (SI Figure S7).Don haka, tsarin aikin KAND 11 an yi imanin ya bambanta da na sanannun masu rushewar cytoskeleton.Bugu da kari, binciken mu na sel BY-2 da aka bi da su tare da KAND 11 ya bayyana farkon mutuwar tantanin halitta a lokacin jiyya na KAND 11 kuma ya nuna cewa adadin matattun kwayoyin halitta na Evans blue-ba su karu sosai bayan 30 min na maganin KAND 11, yayin da bayan minti 90 na jiyya tare da 50 μM ko 100 μM KAND, adadin matattu ya karu zuwa 43.7% ko 80.1%, bi da bi (Fig. 7c).A hade, waɗannan bayanan suna nuna cewa sabon ursolic acid wanda aka samu KAND 11 shine ƙayyadaddun ƙwayar cytoskeletal mai hana shuka tare da tsarin aikin da ba a san shi ba a baya.
KAND yana shafar microtubules na cortical, filament actin, da yuwuwar taba BY-2 Kwayoyin.(a) Kallon microtubules na cortical a cikin kwayoyin BY-2 a gaban TagRFP-TUA6.Kwayoyin BY-2 da aka bi da su tare da KAND 11 (50 μM ko 100 μM) ko DMSO an bincika su ta hanyar microscopy.An ƙididdige yawan ƙananan ƙananan ƙwanƙwasa cortical daga micrographs na sel masu zaman kansu 25.Haruffa suna nuna bambance-bambance masu mahimmanci (Gwajin Tukey HSD, p<0.05).Ma'aunin Sikeli = 10 µm.(b) Filament actin na cortical a cikin sel BY-2 da aka gani a gaban GFP-ABD2.Kwayoyin BY-2 da aka bi da su tare da KAND 11 (50 μM ko 100 μM) ko DMSO an bincika su ta hanyar microscopy.An ƙididdige yawan adadin filament na cortical actin daga micrographs na sel masu zaman kansu 25.Haruffa suna nuna bambance-bambance masu mahimmanci (Gwajin Tukey HSD, p<0.05).Ma'aunin Sikeli = 10 µm.(c) Duban matattun ƙwayoyin BY-2 ta Evans blue tabo.Kwayoyin BY-2 da aka bi da su tare da KAND 11 (50 μM ko 100 μM) ko DMSO an bincika su ta hanyar microscopy mai haske.n=3.Ma'aunin Sikeli = 100 µm.
Ganowa da amfani da sabbin kayayyaki na halitta ya haifar da gagarumin ci gaba a fannoni daban-daban na rayuwar dan Adam, ciki har da magunguna da noma.An gudanar da bincike na tarihi don samun mahadi masu amfani daga albarkatun kasa.Musamman ma, an san actinomycetes da amfani a matsayin maganin rigakafi na antiparasitic don nematodes saboda ikon su na samar da nau'o'in metabolites daban-daban irin su avermectin, sinadarin gubar na ivermectin da bleomycin da abubuwan da suka samo asali, ana amfani da su don magani a matsayin wakili na anticancer21,22.Hakazalika, an gano nau'ikan mahadi na herbicidal daga actinomycetes, wasu daga cikinsu an riga an yi amfani da su a kasuwa1,23.Sabili da haka, nazarin metabolites na actinomycete don ware samfuran halitta tare da ayyukan ilimin halitta da ake so ana ɗaukar dabarun tasiri.A cikin wannan binciken, mun gano wani sabon fili, coumamonamide, daga S. werraensis kuma mun samu nasarar haɗa shi.Ursonic acid shine tsaka-tsakin roba na urbenamide da abubuwan da suka samo asali.Yana iya haifar da siffa na curling tushen, nuna matsakaici zuwa karfi herbicidal ayyuka, da kuma kai tsaye ko a kaikaice lalata shuka microtubules.Duk da haka, tsarin aikin urmotonic acid zai iya bambanta da na masu hanawa na microtubule na yanzu, tun da KAND 11 kuma ya rushe actin filaments kuma yana haifar da mutuwar tantanin halitta, yana ba da shawarar tsarin tsari wanda urmotonic acid da abubuwan da suka samo asali suna tasiri da yawa na tsarin cytoskeletal..
Ƙarin cikakkun bayanai na urbenonic acid zai taimaka wajen fahimtar tsarin aikin urbenonic acid.Musamman ma, manufa ta gaba ita ce kimanta ikon ursonic acid don ɗaure zuwa rage ƙananan microtubules don sanin ko ursonic acid da abubuwan da suka samo asali suna aiki kai tsaye a kan microtubules da depolymerize su, ko kuma aikin su ya haifar da lalata microtubule.Bugu da ƙari, a cikin yanayin da microtubules ba manufa ta kai tsaye ba, gano wurin da ake aiki da kwayoyin halitta na ursonic acid a kan kwayoyin shuka zai taimaka kara fahimtar kaddarorin mahadi masu dangantaka da hanyoyin da za a iya inganta aikin herbicidal.Binciken mu na bioactivity ya bayyana iyawar cytotoxic na musamman na ursonic acid akan ci gaban tsire-tsire irin su Arabidopsis thaliana, taba da liverwort, yayin da babu E. coli ko ƙwayoyin HeLa.Kadan ko babu guba ga ƙwayoyin dabba shine fa'idar ursonic acid idan an haɓaka su azaman maganin ciyawa don amfani a buɗe wuraren noma.Lalle ne, tun da microtubules sune tsarin gama gari a cikin eukaryotes, zaɓin zaɓin su a cikin tsire-tsire shine mabuɗin buƙatu don maganin herbicides.Misali, propyzamide, wani microtubule depolymerizing wakili wanda ke daure kai tsaye zuwa tubulin kuma yana hana polymerization, ana amfani dashi azaman maganin ciyawa saboda ƙarancin guba ga ƙwayoyin dabba24.Ya bambanta da disopyramide, benzamides masu alaƙa suna da takamaiman takamaiman manufa.Baya ga microtubules shuka, RH-4032 ko benzoxamide kuma yana hana microtubules na sel dabbobi ko oomycetes, bi da bi, kuma ana amfani da zalilamide azaman fungicides saboda ƙarancin phytotoxicity25,26,27.Sabuwar beyar da aka gano da abubuwan da aka samo ta suna nuna cytotoxicity na zaɓi akan tsire-tsire, amma yana da kyau a lura cewa ƙarin gyare-gyare na iya canza ƙayyadaddun manufar su, mai yuwuwar samar da ƙarin abubuwan haɓaka don sarrafa fungi ko oomycetes.
Abubuwan musamman na urbenonic acid da abubuwan da suka samo asali suna da amfani don haɓaka su azaman maganin ciyawa da amfani azaman kayan aikin bincike.Muhimmancin cytoskeleton wajen sarrafa siffar tantanin halitta an san shi sosai.Binciken da aka yi a baya ya nuna cewa tsire-tsire sun samo asali ne na hadaddun hanyoyin haɗin gwiwar cortical microtubule ta hanyar sarrafa motsin microtubule don sarrafa ƙwayoyin cuta da kyau.An gano adadi mai yawa na kwayoyin da ke da alhakin daidaita ayyukan microtubule, kuma bincike mai alaka yana ci gaba da gudana3,4,28.Fahimtarmu na yanzu game da haɓakar ƙwayoyin microtubule a cikin ƙwayoyin shuka ba ta cika cikakken bayanin hanyoyin ƙungiyar microtubule na cortical ba.Misali, ko da yake duka disopyramide da oryzalin na iya lalata microtubules, disopyramide yana haifar da murdiya mai tsanani yayin da oryzalin yana da ɗan ƙaramin tasiri.Bugu da ƙari, maye gurbi a cikin tubulin, wanda ke daidaita microtubules, kuma yana haifar da dextrorotation a cikin tushen, yayin da paclitaxel, wanda kuma yana ƙarfafa ƙarfin microtubule, baya.Sabili da haka, yin nazari da gano maƙasudin kwayoyin halitta na ursolic acid ya kamata ya ba da sababbin fahimta game da ka'idojin microtubules na tsire-tsire.Haka nan, kwatancen sinadarai a nan gaba waɗanda ke da tasiri wajen haɓaka gurɓataccen ci gaba, irin su disopyramide, da sinadarai marasa inganci, irin su oryzalin ko kuma kumamotoric acid, za su ba da haske kan yadda gurɓatacciyar girma ke faruwa.
A gefe guda, sake fasalin cytoskeletal da ke da alaƙa da tsaro shine wata yuwuwar bayyana cytotoxicity na ursonic acid.Kamuwa da cuta na pathogen ko shigar da elicitor a cikin kwayoyin halitta wani lokaci yana haifar da lalata cytoskeleton da mutuwar kwayar halitta na gaba29.Misali, an ba da rahoton cryptoxanthin da aka samu oomycete ya rushe microtubules da actin filaments kafin mutuwar tantanin taba, kama da abin da ke faruwa tare da maganin KAND30,31.Kamanceceniya tsakanin martanin tsaro da martanin salon salula wanda ursonic acid ya jawo ya sa mu yi tunanin cewa suna haifar da tsarin salula na yau da kullun, kodayake tasirin ursonic acid mai sauri da ƙarfi fiye da cryptoxanthin ya bayyana.Duk da haka, bincike ya nuna cewa rushewar actin filaments yana inganta mutuwar kwayar halitta ba tare da bata lokaci ba, wanda ba koyaushe yana tare da rushewar microtubule29 ba.Bugu da kari, ya rage a gani ko dai kwayar cutar ko kuma mai fitar da kwayar cutar tana haifar da gurbataccen ci gaban tushen, kamar yadda abubuwan da ake samu na ursonic acid ke yi.Don haka, ilimin kwayoyin da ke haɗa martanin tsaro da cytoskeleton matsala ce mai ban sha'awa da za a magance.Ta hanyar yin amfani da kasancewar ƙananan mahadi masu nauyin kwayoyin da ke da alaƙa da ursonic acid, da kuma nau'ikan abubuwan da suka samo asali masu ƙarfi daban-daban, za su iya ba da dama ga hanyoyin da ba a san su ba.
A hade tare, ganowa da aikace-aikacen sabbin mahadi waɗanda ke daidaita haɓakar haɓakar microtubule za su samar da hanyoyi masu ƙarfi don magance hadaddun hanyoyin ƙwayoyin ƙwayoyin cuta waɗanda ke ƙulla ƙayyadaddun sifar tantanin halitta.A cikin wannan mahallin, urmotonic acid da aka haɓaka kwanan nan, wanda ke rinjayar microtubules da actin filaments kuma ya haifar da mutuwar kwayar halitta, na iya ba da damar da za a iya ƙaddamar da haɗin kai tsakanin kulawar microtubule da sauran hanyoyin.Don haka, nazarin sinadarai da nazarin halittu ta amfani da urbenonic acid zai taimaka mana mu fahimci hanyoyin sarrafa kwayoyin halitta waɗanda ke sarrafa cytoskeleton shuka.
Inoculate S. werraensis MK493-CF1 a cikin 500 ml baffled Erlenmeyer flask dauke da 110 ml na iri matsakaicin iri wanda ya kunshi 2% (w/v) galactose, 2% (w/v) Essence manna, 1% (w/v) Bacto abun da ke ciki .-soyton (Thermo Fisher Scientific, Inc.), 0.5% (w / v) tsantsa masara (KOGOSTCH Co., Ltd., Japan), 0.2% (w / v) (NH4) 2SO4 da 0.2% CaCO3 a cikin ruwa mai tsabta.(pH 7.4 kafin haifuwa).An sanya al'adun iri akan jujjuyawar juyi (180 rpm) a 27 ° C na kwanaki 2.Production namo via m jihar fermentation.An canza al'adun iri (7 ml) a cikin 500 ml K-1 flask wanda ya ƙunshi 40 g na matsakaicin samarwa wanda ya ƙunshi 15 g na sha'ir da aka matse (MUSO Co., Ltd., Japan) da 25 g na ruwa mai narkewa (pH ba a daidaita shi ba). kafin haifuwa).).An gudanar da fermentation a 30 ° C a cikin duhu don kwanaki 14.An fitar da kayan fermentation tare da 40 ml / kwalban EtOH da centrifuged (1500 g, 4 ° C, 10 min).An fitar da ma'aunin al'ada (60 ml) tare da cakuda 10% MeOH/EtOAc.An ƙafe Layer Layer a ƙarƙashin matsa lamba don samun ragowar (59.5 MG), wanda aka yiwa HPLC tare da gradient elution (0-10 minutes: 90%) akan ginshiƙi na baya (SHISEIDO CAPCELL PAK C18 UG120, 5 μm, ID). 10 mm × Tsawon 250 mm) H2O/CH3CN, Minti 10-35: 90% H2O/CH3CN zuwa 70% H2O/CH3CN (gradient), 35-45 mintuna: 90% H2O/EtOH, 45-155 mintuna: 90% H2O / EtOH zuwa 100% EtOH (gradient (gradient), 155-200 min: 100% EtOH) a cikin nauyin 1.5 ml / min, coumamonamide (1, 36.0 mg) an ware shi azaman farin foda amorphous.
Kumamotoamide (1);1H-NMR (500 MHz, CDCl3) δ 6.93 (t, J = 2.5 Hz, 1H), 6.76 (dd, J = 4.3, 1.8 Hz 1H), 6.05 (t , J = 3.8 Hz, 1H).), 4.08 (s, 3H);13C-NMR (125 MHz, CDCl3) δ 161.1, 121.0, 119.9, 112.2, 105.0, 68.3;ESI-HRMS [M+H]+: [C6H9N2O2]+ ƙididdige ƙima: 141.0659, ƙimar ƙima: 141.0663, IR νmax 3451, 3414, 3173, 2938, 1603, 15973-153 cm
An samo tsaba na Columbia (Col-0) daga Cibiyar Albarkatun Halitta ta Arabidopsis (ABRC) tare da izini don amfani da bincike.Col-0 tsaba an yada su kuma ana kiyaye su a ƙarƙashin yanayin dakin gwaje-gwajenmu kuma an yi amfani da su azaman tsire-tsire na Larabawa na daji.Arabidopsis tsaba an haifuwa saman kuma an haɓaka su a cikin rabin ƙarfi Murashige da matsakaicin Skoog mai ɗauke da 2% 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 23 ° C da haske mai tsayi.T. Hashimoto (Cibiyar Kimiyya da Fasaha ta Nara) ta samar da iri na phs1-1 mutant.
T. Hashimoto (Cibiyar Kimiyya da Fasaha ta Nara) ta samar da iri iri SR-1 kuma ana amfani da su azaman tsire-tsire na taba.An shafe tsaba na taba a saman kuma an jika shi cikin ruwa maras kyau na tsawon dare uku don inganta haɓaka, sannan a sanya shi a cikin maganin rabin ƙarfi wanda ya ƙunshi 2% sucrose, 0.05% (w/v) MES, da 0.8% gellan danko (Fujifilm Wako Pure Chemical) Murshige.da Skoog matsakaici) tare da pH 5.7 kuma an sanya shi a 23 ° C ƙarƙashin haske akai-akai.
T. Kohchi (Jami'ar Kyoto) ne ya ba da Strain Tak-1 kuma an yi amfani da shi azaman ma'auni na gwaji don nazarin hanta.An samo Gemma daga tsire-tsire masu tsire-tsire da aka haifuwa sannan kuma a sanya shi akan Gamborg B5 matsakaici (Fujifilm Wako Pure Chemical) mai dauke da 1% sucrose da 0.3% gellan danko kuma a sanya shi a 23 ° C a ƙarƙashin haske mai ci gaba.
Taba BY-2 Kwayoyin (Nicotiana tabacum L. cv. Bright Yellow 2) S. Hasezawa (Jami'ar Tokyo) ne ya samar da ita.Kwayoyin BY-2 sun diluted 95-ninka a cikin gyare-gyaren Linsmeier da Skoog matsakaici kuma an kara su mako-mako tare da 2,4-dichlorophenoxyacetic acid 32.An gauraya dakatarwar tantanin halitta akan jujjuyawar girgiza a 130 rpm a 27°C a cikin duhu.Wanke sel tare da ƙarar matsakaicin matsakaici sau 10 kuma a sake dawowa cikin matsakaici iri ɗaya.BY-2 transgenic cell Lines a tsaye tsaye suna bayyana alamar microtubule TagRFP-TUA6 ko alamar filament alama GFP-ABD2 a ƙarƙashin ƙwayar ƙwayar ƙwayar ƙwayar cuta ta farin kabeji 35S an samar da su kamar yadda aka bayyana33,34,35.Ana iya kiyaye waɗannan layukan tantanin halitta da aiki tare ta amfani da hanyoyin kama da waɗanda aka yi amfani da su don ainihin layin tantanin halitta BY-2.
An haɓaka ƙwayoyin HeLa a cikin Dulbecco's modified Eagle's medium (DMEM) (Life Technologies) wanda aka haɓaka tare da 10% ƙwayar ƙwayar ƙwayar tayi, 1.2 U/ml penicillin, da 1.2 μg/ml streptomycin a cikin incubator 37°C tare da 5% CO2.
Dukkanin gwaje-gwajen da aka siffanta a cikin wannan rubutun an yi su bisa ga ka'idojin kiyaye lafiyar halittu na Jafananci.
An narkar da mahadi a cikin dimethyl sulfoxide (DMSO; Fujifilm Wako Pure Chemical) azaman mafita na jari kuma an diluted a cikin MS matsakaici don Arabidopsis da taba ko Gamborg B5 matsakaici don hanta.Don ƙididdigar hana haɓakar tushen ci gaban, fiye da tsaba 10 a kowace faranti an shuka su akan matsakaicin agar mai ɗauke da mahadi da aka nuna ko DMSO.An shuka iri a cikin ɗakin girma na kwanaki 7.An dauki hoton shukar kuma an auna tsawon tushen.Don gwajin germination na Arabidopsis, ana shuka tsaba 48 a kowace faranti akan matsakaicin agar mai ɗauke da 200 μM fili ko DMSO.An shuka tsaba na Arabidopsis a cikin ɗakin girma kuma an ƙidaya adadin da aka shuka kwanaki 7 bayan germination (dag).Don tantance ƙwayar taba, ana shuka tsaba 24 a kowace faranti akan matsakaicin agar mai ɗauke da 200 μM KAND ko DMSO.An shuka tsaba na taba a cikin ɗakin girma kuma an ƙidaya adadin da aka shuka bayan kwanaki 14.Don gwajin hana haɓakar haɓakar hanta, embryos 9 daga kowane farantin an sanya su a kan matsakaicin agar wanda ke ɗauke da ƙima na KAND ko DMSO kuma an sanya su cikin ɗakin girma na kwanaki 14.
Yi amfani da tsire-tsire masu launin 5 mg/ml propidium iodide (PI) don ganin ƙungiyar tushen meristem.An lura da siginar PI ta microscopy mai kyalli ta amfani da microscope na TCS SPE confocal Laser (Leica Microsystems).
An yi tabon tushen tarihi tare da β-glucuronidase (GUS) bisa ga ka'idar da Malami da Benfey36 suka bayyana.An saita seedlings a cikin 90% acetone na dare, wanda aka lalata tare da 0.5 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-glucuronic acid a cikin GUS buffer na 1 hour kuma an sanya shi a cikin maganin chloraldehyde mai ruwa.(8 g chloral hydrate, 2 ml ruwa da 1 ml glycerol) kuma ana lura da shi ta hanyar bambance-bambancen tsangwama ta hanyar microscopy ta amfani da microscope Axio Imager M1 (Carl Zeiss).
An auna kusurwoyin tushen akan tsire-tsire masu kwanaki 7 da aka girma akan faranti a tsaye.Auna kusurwar tushen daga alkiblar vector kamar yadda aka bayyana a mataki na 6.
An lura da tsari na microtubules na cortical kamar yadda aka bayyana, tare da ƙananan gyare-gyare ga yarjejeniya 37.Anti-β-tubulin antibody (KMX-1, Merk Millipore: MAB3408) da Alexa Fluor 488-conjugated anti-mouse IgG (Thermo Fisher Scientific: A32723) an yi amfani dashi azaman rigakafi na farko da sakandare a 1: 1000 da 1: 100 dilutions, bi da bi.An samo hotuna masu walƙiya ta amfani da TCS SPE confocal laser scanning microscope (Leica Microsystems).Nemi hotuna-tari na Z kuma ƙirƙirar matsakaicin tsinkaye bisa ga umarnin masana'anta.
An yi gwajin yaɗuwar cell HeLa ta amfani da Kit ɗin Ƙididdigar Cell 8 (Dojindo) bisa ga umarnin masana'anta.
An yi nazarin haɓakar E. coli DH5α ta hanyar auna yawan ƙwayar salula a cikin al'ada ta amfani da spectrophotometer a 600 nm (OD600).
An lura da ƙungiyar Cytoskeletal a cikin sel BY-2 transgenic ta amfani da microscope mai kyalli wanda aka sanye da na'urar tantancewa ta CSU-X1 (Yokogawa) da kyamarar sCMOS (Zyla, Fasahar Andor).An tantance yawan ƙwayar huhu ta hanyar nazarin hoto, wanda ya ƙididdige yawan adadin pixels cytoskeletal tsakanin pixels cytoplasmic a cikin hotuna masu ɓoye ta amfani da software na ImageJ kamar yadda aka bayyana38,39.
Don gano mutuwar tantanin halitta a cikin sel BY-2, an samar da aliquot na dakatarwar tantanin halitta tare da 0.05% Evans blue na mintuna 10 a zafin jiki.Zaɓaɓɓen Evans blue tabo na matattun sel ya dogara ne akan fitar da rini daga sel masu dacewa ta hanyar ƙwayar plasma mara kyau40.An lura da tabo ta hanyar amfani da microscope mai haske (BX53, Olympus).
Kwayoyin HeLa sun girma a cikin DMEM an ƙara su tare da 10% FBS a cikin incubator mai humidified a 37 ° C da 5% CO2.An bi da kwayoyin halitta tare da 100 μM KAND 11, kumamonamic acid 6, kumamonamide 1, 100 ng/ml colcemid (Gibco), ko 100 ng/ml Nocodmaze (Sigma) don 6 h a 37 ° C.An gyara sel tare da MetOH na minti 10 sannan tare da acetate don 5 min a zazzabi na ɗaki.Kafaffen sel an haɗa su tare da β-tubulin primary antibody (1D4A4, Proteintech: 66240-1) diluted a cikin 0.5% BSA/PBS na tsawon sa'o'i 2, an wanke sau 3 tare da TBST, sannan an haɗa shi da Alexa Fluor goat antibody.488 1 h.- Mouse IgG (Thermo Fisher Scientific: A11001) da 15 ng/ml 4′,6-diamidino-2-phenylindole (DAPI) diluted a cikin 0.5% BSA/PBS.Bayan wankewa da TBST sau uku, an lura da tabo akan na'urar hangen nesa ta Nikon Eclipse Ti-E.An ɗauki hotuna tare da sanyaya Hamamatsu ORCA-R2 CCD kamara ta amfani da software na MetaMorph (Na'urorin Molecular).
Lokacin aikawa: Juni-17-2024