Txhim kho ntawm Phenylethanoid Glycosides Biosynthesis hauv Cell Cultures Ntawm Cistanche Deserticola Los ntawm Osmotic Stress

Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791

Chun-Zhao Liu. Xi-Yu Cheng

AbstractCov txiaj ntsig ntawm osmotic kev ntxhov siab ntawm kev loj hlob ntawm tes thiab phenylethanoid glycosides (PeGs) biosynthesis tau tshawb xyuas hauv cov kab lis kev cai ntawm tes.Cistanche deserticolaYC Ma, cov suab puam tshuaj ntsuab cog nyob rau sab hnub poob ntawm Tuam Tshoj. Ntau yam kev pib sucrose tau cuam tshuam rau kev loj hlob ntawm tes thiab PeGs biosynthesis hauv cov kab lis kev cai raug tshem tawm, thiab qhov hnyav tshaj plaws qhuav thiab PeGs tsub zuj zuj mus txog 15.9 gl–1-DW thiab 20.7 mg g–1-DW feem ntawm thawj osmotic Kev ntxhov siab ntawm 300 mOsm kg-1 qhov twg sucrose concentration yog 175.3 mM. Stoichiometric tsom xam nrog sib txawv ua ke ntawm sucrose thiab non-metabolic qab zib (mannitol) los yog non-qab zib osmotic agents (PEG thiab NaCl) qhia tias osmotic kev nyuaj siab nws tus kheej yog ib qho tseem ceeb rau kev txhim kho PeGs biosynthesis nyob rau hauv cell suspension kab lis kev cai.Cistanche deserticola. Qhov siab tshaj plaws PeGs cov ntsiab lus ntawm 26.9 thiab 23.8 mg g–1-DW tau tom qab 21 hnub ntawm kev sib txuas ntawm 87.6 mM sucrose nrog 164.7 mM mannitol (303 mOsm kg–1) lossis 20 mM PEG feem, uas siab dua uas ntawm C. deserticola cell kab lis kev cai loj hlob nyob rau hauv thawj zaug sucrose concentration ntawm 175.3 mM tom qab 30 hnub. Kev txhawb nqa PeGs nyob rau hauv cov kab lis kev cai ntawm tes raug cuam tshuam rau qhov nce ntawm phenylalanine ammonium lyase (PAL) kev ua haujlwm vim yog kev ntxhov siab osmotic.

Ntsiab lus Cistanche deserticola, Suab puam tshuaj nroj tsuag, Phenylethanoid glycosides, Osmotic stress, Phenylalanine ammonia-lyase

Cistanchetubulosamuaj ntau yam teebmeem, nyem qhov no kom paub ntau ntxiv

Taw qhia

Cistanche deserticolaYC Ma, tshuaj ntsuab muaj txiaj ntsig hauv Suav, ua rau cov hauv paus hniav ntawm Haloxylon ammodendrun thiab loj hlob hauv thaj chaw suab puam ntawm sab hnub poob Tuam Tshoj. Phenylethanoid glycosides (PeGs), lub ntsiab bioactive Cheebtsam cais los ntawm C. deserticola, tau cim kev ua ub no nyob rau hauv scavenging dawb radicals, pab txhawb kev tiv thaiv, txhim kho kev sib deev muaj nuj nqi, thiab lwm yam. (Zong et al. 1996; Lu 1998; Wang et al. 2001). Vim tsis muaj kev tswj xyuas exploitation thiab kev siv ntawmCistanche deserticola, natural resources ntawmCistanche deserticola nyob ntawm ntug kev qaug zog. Raws li cov teeb meem no, PeGs ntau lawm los ntawm cell ncua kev coj noj coj ua ntawm C. deserticola tau raug suav hais tias yog ib qho kev cog lus los daws qhov tsis txaus.Cistanche deserticolacog cov ntaub ntawv (Lu thiab Mei 2003; Ouyang li al. 2003; Cheng et al. 2005a).

Osmotic kev ntxhov siab yog ib qho tseem ceeb cuam tshuam rau cov nroj tsuag kev loj hlob, kev loj hlob, morphogenesis, thiab tsim cov metabolites theem nrab. Txhim kho kev tsim cov metabolites theem nrab los ntawm cov kab lis kev cai hauv vitro nyob rau hauv kev pom zoo osmotic kev nyuaj siab tau raug tshaj tawm nyob rau hauv ob peb hom tshuaj ntsuab (Kim li al. 2001; Wang et al. 1999; Wu et al. 2005). Sucrose yog tus neeg sawv cev kev ntxhov siab ib txwm siv rau hauv cov xwm txheej no, thiab tseem ua haujlwm tseem ceeb ntawm cov pa roj carbon thiab lub zog. Yog li ntawd, nws yog ib qho nyuaj heev los cais cov nyhuv ntawm osmotic kev nyuaj siab thiab cov khoom noj khoom haus lub luag hauj lwm ntawm cov suab thaj ua carbon. Kev nce ntawm qhov pib osmotic kev ntxhov siab nrog nonmetabolic mannitol, sorbitol, thiab PEG kuj txhim kho paclitaxel tsub zuj zuj hauv Taxus Chinensis cell kab lis kev cai, saponin thiab polysaccharide tsub zuj zuj hauv Panax notoginseng cell kab lis kev cai, thiab eleutherosides tsub zuj zuj hauv Eleutherococcus sessiliflorus 9. et al. 2001; Shohael et al. 2006). Lawv cov txiaj ntsig tau qhia tias qhov cuam tshuam ntawm carbohydrate concentration thiab osmotic stress yog txawv raws li cov nroj tsuag hom thiab cov metabolites theem nrab.

Lub hom phiaj ntawm txoj kev tshawb fawb tam sim no yog tsom rau cov teebmeem osmotic ntawm sucrose, mannitol, PEG, thiab NaCl ntawm kev loj hlob ntawm tes thiab PeGs biosynthesis hauvCistanche deserticolacell suspension cultures. PAL kev ua haujlwm hauv cov kab lis kev cai raug ncua raug tshawb xyuas nyob rau hauv kev sib txawv ntawm cov neeg ua haujlwm osmotic thawj zaug, thiab cov txheej txheem ua tau ntawm kev txhim kho PeGs tsub zuj zuj los ntawm kev nce osmotic kev ntxhov siab yog tham txog.

Cov ntaub ntawv thiab cov txheej txheem

Cog cov ntaub ntawv thiab callus induction

Noob ntawmCistanche deserticolaYC Ma tau sau los ntawm thaj chaw suab puam hauv Xinjiang xeev ntawm Tuam Tshoj thiab khaws cia hauv 4C ua ntej siv. Botanical tus kheej tau lees paub los ntawm kev sib piv rau cov qauv siv ntawm lub koom haum ntawm Botany, Suav Academy ntawm Sciences, PR Tuam Tshoj. Cov noob tau deg sterilized los ntawm dipping hauv 70 feem pua ​​​​ethanol rau 30 s, ces muab tso rau hauv 20 feem pua ​​aqueous tov ntawm 5.4 feem pua ​​​​sodium hypochlorite hauv dej rau 20 feeb, tom qab ntawd peb yaug nrog dej tsis muaj tshuaj. Calli raug induced los ntawm nto-sterilized noob ntawmCistanche deserticolantawm MS (Murashige thiab Skoog 1962) nruab nrab ntxiv nrog 1.0 mg l–1 naphthaleneacetic acid (NAA), 2.0 mg l–1 6-benzyl adenine ({{7} }BA), 0.25 mg l–{10}}, 4-dichlorophenoxyacetic acid (2, 4-D), 6 gl–1 agar (PhytoTechnology LaboratoriesTM, Product ID: A181) thiab 30 gl-1 sucrose rau 60 hnub hauv lub txee loj hlob hauv qhov tsaus ntuj ntawm 25C. Cov kab lis kev cai callus raug ntxias los ntawm cov noob cog tau tom qab subcultured rau cov khoom nruab nrab saum toj no ntawm ib ntus ntawm 30 hnub (Cheng li al. 2005a).

Tsim thiab tu cov kab lis kev cai ntawm tes

Lub ncua kev kawm callus kab lis kev cai (3. Lub raj mis uas muaj 100 ml ntawm cov kua nruab nrab saum toj no ntawm lub tshuab nqus dej ntawm 110 rpm ntawm 25C hauv qhov tsaus ntuj ntawm lub sijhawm subculture ntawm 18 hnub. Qhov nruab nrab pH raug kho rau 5.8 nrog 1 M NaOH lossis 1 M HCl ua ntej autoclaving. Kev ncua kev hu xov tooj kab lis kev cai subcultured tom qab kaum zaug tau siv los ua inoculum rau cov kab lis kev cai hauv qab no. Txhua qhov kev sim lub raj mis tau ua tiav hauv 250 ml Erlenmeyer flasks uas muaj 50 ml kua nruab nrab nrog 30 gl–1 sucrose thiab inoculated nrog 1.5 g tshiab qhov hnyav ntawm 18-hnub qub cell sucrose kab lis kev cai. Cov Erlenmeyer flasks tau incubated ntawm ib tug rotary shaker (110 rpm) ntawm 25C nyob rau hauv qhov tsaus ntuj.

Kev sim osmotic stress

Txhawm rau tshawb xyuas cov txiaj ntsig ntawm thawj zaug sucrose concentration ntawmKev loj hlob ntawm tes thiab PeGs txuam nrog,Cistanche deserticolaKev ncua kev coj noj coj ua tau cog rau hauv MS nruab nrab nrog 87.6,175.3, thiab 262.9 mM sucrose. Rau osmotic kev nyuaj siab,Cistanche deserticolaCov kab lis kev cai raug tshem tawm tau tsim nyob rau hauv MSnruab nrab muaj 87.6 mM sucrose ua ke nrog164,7 mM mannitol Lwm cov tshuaj osmotic (PEG 4, 000 thiabNaCl) tau ntxiv rau qhov sib npaug ntawm osmolarity ntawm87.6 mM 2000 0. Qhov sib npaug osmotic concentrationsCov neeg sawv cev osmotic tau sim txiav txim siab ua PEG4, 000 ntawm 20 mM thiab NaCl ntawm 50 mM, feem. Triplicate flasks tau siv rau hauv txhua qhov kev sim thiab txhua qhov txiaj ntsigyog cov txhais tau tias ntawm triplicate flasks ± SD.

Kev tsom xam

Cistanche deserticolakab lis kev cai nyob rau hauv ntau yam kev ntxhov siab osmotic tau pom los ntawm Nikon AFX-DX lub teeb tsom iav (Nikon, Nyiv) hauv qab 400· magnifications. Lub osmolarity ntawm kab lis kev cai nruab nrab yog ntsuas nrog lub vapor siab osmometer (Fiske One-Ten Osmometer, MA, USA). Rau kev txiav txim siab tshiab qhov hnyav (FW), lubCistanche deserticolacov hlwb tau maj mam nias rau ntawm daim ntawv lim kom tshem tawm cov dej ntau dhau thiab hnyav. Tom qab ntawd, cov hlwb tau qhuav hauv qhov cub ntawm 60C rau 24 teev thiab qhov hnyav qhuav (DW) tau sau tseg. Qhov seem qab zib concentration tau txiav txim siab los ntawm kev siv phenol thiab concentrated sulfuric acid, siv cov piam thaj raws li tus qauv (Dubois li al. 1956).

PeGs tau muab rho tawm los ntawm cov hlwb qhuav nrog methanol rau 15 min ntawm 60C hauv lub da dej ultrasonic (KQ2200, Shanghai Cany Precision Instrument Co., Ltd, Tuam Tshoj) nrog zaus ntawm 40 kHz thiab lub zog ntawm 100 W. Cov lim tau concentrated thiab cov residue yaj nyob rau hauv dej distilled thiab ces dhau los ntawm ib tug macro reticular resin kem (AB-8, tshuaj cov khoom ntawm Nankai University, Tianjin, Suav teb). Cov methanol eluate tau sau thiab tag nrho cov ntsiab lus PeGs tau soj ntsuam ntawm 333 nm los ntawm kev siv echinacoside raws li tus qauv (Du thiab Liu 1993a; Du et al. 1993b).

Cell viability tau kwv yees los ntawm kev txo qis ntawm 2, 3, 5-triphenyl tetrazolium chloride (TTC) (Steponkus thiab Lanphear 1967). Lub viability Performance index yog txhais raws li qhov absorbance ntsuas ib gram ntawm cov ntaub so ntswg tshiab. Kev txiav txim siab ntawm PAL kev ua haujlwm yog ua raws li txoj hauv kev ntawm Koukol thiab Conn (1961). Ib chav tsev ntawm PAL kev ua (U) txhais tau tias yog tus nqi ntawm absorbance variation ntawm 0.01.

Cov txiaj ntsig thiab kev sib tham

Cov txiaj ntsig ntawm thawj zaug sucrose concentration ntawm kev loj hlob ntawm tes thiab PeGs biosynthesis hauv cell suspension kab lis kev cai ntawmCistanche deserticola.Cov lus teb ntawmCistanche deserticolacell suspension kab lis kev cai rau thawj zaug sucrose concentration ntawm 87.6 thiab 262.9 mM raug kuaj. Raws li pom nyob rau hauv daim duab 1, cov hlwb loj hlob thiab sau PeGs sai ntawm qhov qis pib sucrose concentration ntawm 87.6 hli, thiab cov cell loj hlob thiab PeGs tsub zuj zuj tau suppressed pom tseeb ntawm qhov siab pib sucrose concentration ntawm 262.9 mM. Qhov piv ntawm qhov hnyav tshiab rau qhov hnyav qhuav poob qis thaum pib sucrose concentration nce ntawm 87.6 los ntawm 262.9 mM (cov ntaub ntawv tsis qhia). Qhov siab tshaj plaws qhuav qhov hnyav ntawm 15.9 gl–1 thiab PeGs cov ntsiab lus ntawm 20.7 mg g–1-DW tau nyob rau hnub 30 ntawm qhov pib sucrose concentration ntawm 175.3 mM uas osmolarity yog li ntawm 300 mOsm kg–1. Raws li kev soj ntsuam microscopic, cov phab ntsa ntawm tes tau raug cais tawm ntawm lawv cov cytoplasm ntawm qhov siab tshaj plaws osmotic kev nyuaj siab ntawm 388 mOsm kg-1 qhov twg thawj zaug sucrose concentration yog 262.9 mM (Fig. 2), thiab qhov siab tshaj osmotic kev nyuaj siab yuav cuam tshuam cov metabolism ntawm cov hlwb thiab ua rau. Kev loj hlob hnyav thiab kev poob qis ntawm PeGs biosynthesis. Ib qho tshwm sim zoo sib xws kuj tau pom nyob rau hauv kev ncua kev coj noj coj ua ntawm T. Chinensis ncua kev kawm cell kab lis kev cai, thiab qhov zoo tshaj plaws sucrose concentration rau paclitaxel ntau lawm yog 175.3 mM (Kim li al. 2001). Txawm li cas los xij, thawj zaug sucrose concentration ntawm 204.5 mM tau txais txiaj ntsig zoo rau kev loj hlob ntawm E. sessiliflorus cell kab lis kev cai. Qhov siab dua sucrose concentration ntawm 262.9 mM txhim kho biosynthesis ntawm eleutherosides, phenol, thiab flavonoids hauv Eleutherococcus kab lis kev cai (Shohael li al. 2006). Nws yog tseeb hais tias thawj zaug sucrose concentration yog ib qho tseem ceeb rau kev loj hlob thiab theem nrab metabolites tsub zuj zuj ntawm cov nroj tsuag cell kab lis kev cai thiab nws cov nyhuv yog txuam nrog ib tug tshwj xeeb cell kab.

Cov nyhuv ntawm cov tshuaj osmotic sib txawv ntawm kev loj hlob ntawm tes thiab PeGs biosynthesis hauv cell suspension kab lis kev cai ntawm C. deserticola

Txhawm rau nkag siab txog lub luag haujlwm osmotic ntawm metabolic sucrose thiab lwm yam tsis-metabolic chemical substrates ntawm PeGs biosynthesis hauv cell ncua kev coj noj coj ua ntawmCistanche deserticolaIb qho kev sim tau ua tiav siv sucrose ua ke nrog cov tshuaj uas tsis yog metabolic osmotic (mannitol, PEG, thiab NaCl). Hauv cov kev sim no, 164.7 mM mannitol, 20 mM PEG, thiab 50 mM NaCl tau ntxiv rau qhov nruab nrab uas muaj 87.6 mM sucrose thiab ua rau thawj osmolarity nyob ib ncig ntawm 300 mOsm kg-1, uas yog qhov sib npaug osmotic mob piv rau nruab nrab nrog 175.3 mM. sucrose.

Raws li pom nyob rau hauv daim duab 3a, b, qhov sib ntxiv ntawm mannitol (tsis-metabolic qab zib osmotic tus neeg saib xyuas) thiab PEG (non-permeable osmotic tus neeg saib xyuas) me ntsis inhibited cell loj hlob tus nqi tab sis ho txhim kho PeGs biosynthesis nyob rau hauvCistanche deserticolacell suspension cultures. Qhov siab tshaj plaws PeGs cov ntsiab lus ntawm 26.9 thiab 23.8 mg g–1-DW tau txais, feem, nyob rau hnub 21 thaum cov hlwb tau loj hlob hauv kev sib txuas ntawm 87.6 mM sucrose nrog 164.7 mM mannitol (303 mOsm kg–1) lossis 20 mM PEG (299 mOsm kg-1). Cov nyiaj PeGs tau siab dua li ntawmCistanche deserticolacell kab lis kev cai loj hlob nyob rau hauv MS kua nruab nrab nrog ib tug thawj sucrose concentration ntawm 175.3 mM nyob rau hnub 30. Qab zib tau noj maj mam nyob rau hauv lub sij hawm ntawm kev kawm.Cistanche deserticolakab lis kev cai ntawm tes, thiab tom qab ntawd cov piam thaj uas ua rau osmotic kev ntxhov siab poob qis raws li (Fig. 3c, d). Osmotic kev ntxhov siab ntawm cov kua nruab nrab nrog qhov pib sucrose concentration ntawm 175.3 mM poob los ntawm 280 mus rau 110 mOsm kg-1 thaum PeGs hauv cov kab lis kev cai ntawm tes pib sau rau hnub 9 thiab mus txog lawv qhov siab tshaj plaws nyob rau hnub 30 (cov ntaub ntawv tsis qhia). Cov tshuaj uas tsis yog-metabolic osmotic (mannitol thiab PEG) tsis tau noj nyob rau hauv chav kawm ntawm cell kab lis kev cai, tso cai osmotic kev nyuaj siab ntawm cov kua nruab nrab yuav tsum tau khaws cia ntawm 200 thiab 280 mOsm kg-1, ib tug osmotic siab haum rau PeGs biosynthesis. Cov txiaj ntsig no tau pom tias kev ntxhov siab osmotic nws tus kheej ua lub luag haujlwm tseem ceeb hauv kev txhim kho PeGs biosynthesis hauvCistanche deserticolacell kab lis kev cai.

Qhov sib ntxiv ntawm NaCl tau tshaj tawm los txhim kho indole alkaloids thiab saponin biosynthesis raws li hauv cell kab lis kev cai ntawm Catharanthus roseus thiab Panax ginseng (Smith li al. 1987; Wu et al. 2005), tab sis kev kho mob ntawm NaCl suppressed lub cell loj hlob ntawm T. Chinensis. thaum uas qhia tsis txawv hauv palitaxel biosynthesis (Kim li al. 2001). Yog li ntawd, yuav tsum muaj kev tshawb xyuas ntxaws rau txhua kis. Qhov sib ntxiv ntawm osmotic sib npaug ntawm 50 mM NaCl suppressed cell loj hlob thiab PeGs biosynthesis. Qhov tshwm sim no yuav yog vim qhov permeable ntawm NaCl uas tsis muaj txiaj ntsig rau cov kab lis kev cai ntawm tes ntawm cov nroj tsuag suab puam no uas tuaj yeem tiv taus kev ntxhov siab heev.

Fig. 3 Cov nyhuv ntawm kev sib txuas ntawm sucrose thiab lwm yam tsis-metabolic osmotic agents ntawm cell loj hlob (a), PeGs tsub zuj zuj (b), qab zib noj (c), thiab osmotic kev nyuaj siab hloov (d) nyob rau hauv lub chav kawm ntawm C. deserticola cell kab lis kev cai. Tus nqi yog txhais tau tias ntawm triplicate flasks ± SD

Cov nyhuv ntawm cov tshuaj osmotic sib txawv ntawm kev muaj peev xwm ntawm tes thiab PAL kev ua haujlwm hauv cell ncua kev coj noj coj ua ntawm Cistanche deserticola

Hauv peb cov kev tshawb fawb yav dhau los (Cheng li al. 2005b, 2006), elicitor ntxiv tau txhawb nqa PeGs biosynthesis hauvCistanche deserticolakab lis kev cai ntawm tes, thiab kev txhawb nqa tau cuam tshuam nrog kev ua haujlwm ntawm PAL, thawj qhov tseem ceeb enzyme hauv PeGs biosynthesis (Hahlbrock thiab Grisebach 1979). Raws li pom nyob rau hauv daim duab 4, qhov sib ntxiv ntawm non-metabolic 164.7 mM mannitol los yog 20 mM PEG rau hauv MS kua nruab nrab uas muaj 87.6 mM sucrose me ntsis txo cell viability, tab sis ho txhim kho PAL kev ua si uas siab tshaj qhov induced los ntawm thawj zaug sucrose concentration ntawm 175,3m wb. Txhim kho PeGs tsub zuj zuj hauvCistanche deserticolaKev ncua ntawm tes kab lis kev cai tau hais txog qhov nce ntawm PAL kev ua haujlwm txhawb nqa los ntawm kev ntxhov siab osmotic nws tus kheej. Thawj qhov sucrose concentration ntawm 175.3 mM hauv MS kua nruab nrab ua rau txo qis hauv cell viability hauv 15 hnub thiab inhibited PAL kev ua haujlwm hauv 21 hnub. Tom qab ib lub sij hawm ntawm kev noj qab haus huv los ntawm kev loj hlobCistanche deserticolakab lis kev cai ntawm tes, ob qho tib si cell viability thiab PAL kev ua ntawm cov kab lis kev cai tau nce thiab ncav cuag lawv qhov siab tshaj plaws nyob rau hnub 24 thiab hnub 30, feem. Txhim kho PAL kev ua haujlwm los ntawm kev ntxhov siab osmotic kuj tau pom los txhim kho saponin biosynthesis hauv P. ginseng cell kab lis kev cai thiab alkaloid tsub zuj zuj hauv C. roseus cultured cells (Godoy-Hernandez li al. 2000; Wu et al. 2005). Txawm li cas los xij, qhov sib ntxiv ntawm NaCl permeable rau hauv MS kua nruab nrab uas muaj 87.6 mM sucrose txo qis cell viability thiab inhibited PAL kev ua si thaum lub sij hawm tag nrho cov kab lis kev cai. Yog li ntawd, ob qho tib si cell loj hlob thiab PeGs biosynthesis tau pom tseeb suppressed.

Daim duab 4 Variation ntawm cell viability (a) thiab PAL kev ua (b) nyob rau hauv C. deserticola cell kab lis kev cai nyob rau hauv txawv osmotic kev nyuaj siab nyob rau hauv lub chav kawm ntawmCistanche deserticolacell kab lis kev cai. Tus nqi yog txhais tau tias ntawm triplicate flasks ± SD

Cistanche deserticolayog hom suab puam thiab kev tshawb nrhiav ntawm cov suab puam hom kab lis kev cai hauv vitro cell kab lis kev cai tau nthuav tawm cov kev cov nyom tshiab thiab cov cib fim rau kev nkag siab txog cov nroj tsuag physiology thiab ecological adaptations. Nyob rau hauv txoj kev tshawb no, peb tau tsim ib qho system nrog lub peev xwm los kawm txog osmotic stress-mediated signal transduction biosynthesis pathway, thiab lub system muab lub hauv paus rau loj-scale zus tau tej cov PeGs siv lubCistanche deserticolacell kab lis kev cai los ntawm ib tug tshiab osmotic kev nyuaj siab-tswj lub tswv yim.

Kev lees paub

Cov kws sau ntawv lees paub txog kev txhawb nqa nyiaj txiag los ntawm Innovation Research Program ntawm Suav Academy ntawm Sciences.

Los ntawm: 'Txhim kho ntawm phenylethanoid glycosides biosynthesis nyob rau hauv cell kab lis kev cai ntawm Cistanche deserticola los ntawm osmotic kev nyuaj siab' los ntawm Chun-Zhao Liu. Xi-Yu Cheng

---Plant Cell Rep (2008) 27:357–362 DOI 10.1007/s00299-007-0443-3

Cov ntaub ntawv

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