Gene Cloning, Functional Identification, Structural And Expression Analysis Of Sucrose Synthase Los Ntawm Cistanche Tubulosa Ⅲ

4 Kev tsom xam ntawm CtSus nyob rau hauv ntau qhov chaw ntawm Cistanche tubulosa thiab cell kab lis kev cai system nyob rau hauv drought kev nyuaj siab

4.1 Kev tsom xam ntawm CtSus hauv ntau qhov chaw ntawm Cistanche tubulosa

Hauv vitro, tag nrho-cell hloov pauv kev sim thiab kev sim tshuaj enzymatic catalytic cov tshuaj tiv thaiv tau lees paub tias cov protein encoded los ntawm CtSus gene tuaj yeem ua rau cov synthesis ntawm UDP-glucose. Txhawm rau tshawb nrhiav ntxiv txog kev sib raug zoo ntawm cov noob no thiab biosynthesis ntawm glycoside tebchaw hauv Cistanche tubulosa, qhov qhia theem ntawm cov noob no nyob rau ntau qhov chaw ntawm Cistanche tubulosa tau txheeb xyuas.

HIGH QUALITY CISTANCHE HERBA nrog 10-98% ECHINACOSIDE

Echinacoside is the most representative glycoside compound in Cistanche tubulosa, and its content can reach more than 30% of the dry weight of Cistanche tubulosa plants [23]. The research group previously measured the content of echinacoside in different parts of Cistanche tubulosa plants. Specifically, the content of echinacoside in different tissues is as follows: haustoria>underground part>>aerial ib feem; ntawm lawv, cov ntsiab lus ntawm echinacoside hauv haustoria yog qhov siab tshaj plaws.

Lub sijhawm tiag tiag fluorescence quantitative PCR tau ua tiav siv cDNA los ntawm ntau qhov chaw ntawm Cistanche tubulosa ua cov qauv, thiab cov txiaj ntsig tau txheeb xyuas los ntawm 2-ΔΔCT txoj kev thiab kev tsom xam qhov sib txawv. Cov txiaj ntsig tau pom hauv daim duab 4A. Kev qhia theem ntawm CtSus noob nyob rau hauv lub haustoria yog qhov siab tshaj plaws, 1.5 npaug ntawm qhov saum toj kawg nkaus ntawm lub dav hlau, thiab cov theem qhia nyob rau hauv cov av hauv av yog qhov siab tshaj qhov nyob rau hauv cov huab cua, uas yog raws li nyob rau hauv cov qauv ntawm phenylethanoid glycosides. sawv cev los ntawm echinacoside nyob rau hauv ntau qhov chaw ntawm Cistanche tubulosa.

Daim duab 4 Tus txheeb ze qhia theem ntawm CtSus nyob rau ntau qhov chaw ntawm C. tubulosa thiab ncua kev tshem tawm cell kho los ntawm PEG6000. A: Tus txheeb ze qhia theem ntawm CtSus nyob rau ntau qhov chaw ntawm C. tubulosa; B: Relativeexpression theem ntawm CtSus nyob rau hauv C. tubulosa suspension hlwb kho los ntawm PEG6000 ntawm lub sijhawm sib txawv. n=3, 𝑥̅± s.*P ＜ 0.05,***P ＜ 0.001

4.2 Kev tsom xam ntawm CtSus hauv Cistanche deserticola ncua kev kawm hlwb nyob rau hauv drought kev nyuaj siab

Kev tshawb fawb ua ntej ntawm qhov project tau pom tias kev nyuaj siab ntxhov siab vim los ntawm PEG6000 tuaj yeem ua rau muaj kev cuam tshuam ntawm phenylethanol glycosides hauv Cistanche deserticola ncua kev kawm hlwb. Los ntawm 3 mus rau 9 hnub tom qab induction, cov ntsiab lus echinaceaside tau nce ntau. Txij hnub tim 12 txog rau hnub tim 15, qhov kev loj hlob ntawm echinacoside cov ntsiab lus tau qeeb thiab mus txog qhov muaj nqis tshaj plaws nyob rau hnub 15th. Tom qab ntawd, raws li lub sijhawm kab lis kev cai nce, cov ntsiab lus echinacoside tau nce ntau. Cov ntsiab lus ntawm fructoside maj mam txo qis [24]. Raws li qhov kev tshawb fawb no, daim ntawv no tau siv cDNA ntawm cov tsis kho Cistanche deserticola ncua kev kawm hlwb thiab PEG6000-induced Cistanche deserticola suspension hlwb raws li cov qauv los ua lub sij hawm real-time fluorescence quantitative PCR nrhiav kom tau kuaj CtSus noob nyob rau hauv Cistanche deserticola suspension hlwb. drought kev nyuaj siab. Kev hloov hauv cov theem qhia. Cov txiaj ntsig tau pom hauv daim duab 4B. Nyob rau hauv Cistanche deserticola ncua kev kawm hlwb induced los ntawm PEG6000, qhov kev qhia ntawm CtSus tau nce ntau heev nyob rau hnub 6th tom qab induction, mus txog tus nqi siab tshaj plaws nyob rau hnub 9, thiab tom qab ntawd poob rov qab mus rau tib theem raws li kev tswj hwm. Pawg tib theem. Cov txiaj ntsig saum toj no qhia tau hais tias kev nyuaj siab ntxhov siab tuaj yeem ua rau muaj kev nthuav qhia ntawm CtSus gene hauv Cistanche deserticola ncua kev kawm xovtooj ntawm tes, uas zoo ib yam nrog cov qauv sib xyaw ntawm echinaceaside nyob rau hauv kev ntxhov siab. Txawm li cas los xij, lub ncov qhia ntawm CtSus noob tshwm sim ua ntej lub ncov ntawm echinaceaside cov ntsiab lus, vim hais tias cov nquag glycosyl pub synthesized los ntawm CtSus catalysis yog ib qho tseem ceeb precursor yuav tsum tau rau ntau-kauj ruam glycosylation cov tshuaj tiv thaiv nyob rau hauv lub tom ntej biosynthetic pathway ntawm echinaceaside. , nws yog speculated tias tom qab raug drought kev nyuaj siab, cov kab mob yuav nyiam mus nqa cov noob muaj feem xyuam rau cov thawj metabolism kom ua tiav cov tsub zuj zuj ntawm nquag pub, thiab tom qab ntawd ua tiav qhov tseem ceeb theem nrab metabolism hauv cov khoom metabolic.

5 Kawm txog cov qauv peb-dimensional ntawm CtSus protein thiab kev tshuaj xyuas ntawm cov chaw tseem ceeb

Raws li kev ua haujlwm ntawm CtSus hauv catalyzing zus tau tej cov glycosyl pub UDP-qabzib, cov qauv hauv paus ntawm CtSus catalytic kev ua haujlwm tau kawm ntxiv. Cov cuab yeej online SOPMA tau siv los kwv yees tus qauv thib ob ntawm cov protein. Cov txiaj ntsig tau pom tias cov qauv thib ob ntawm CtSus muaj 55.28% -helices, 25.47% random coils, 12.80% txuas ntxiv strands, thiab 6.46% -tig (Daim duab 5A), qhia tias -helices yog qhov tseem ceeb tshaj plaws theem nrab cov txheej txheem hauv CtSus protein, ua raws los ntawm random coils, uas kuj suav nrog ntau feem ntawm cov protein. Txuas ntxiv strands thiab -tig yog faib thoob plaws hauv cov protein. Raws li cov kev tshawb fawb uas twb muaj lawm, sucrose synthase feem ntau muaj nyob rau hauv daim ntawv ntawm ib tug tetramer, uas yog xam tau tias yog nws active daim ntawv. Yog li, daim ntawv no ntxiv siv AlphaFold2 los kwv yees tus qauv ntawm CtSus protein thiab tau txais nws cov qauv peb sab ntawm cov protein tetramers. PDB (Protein Data Bank) kev sib piv cov ntaub ntawv pom tau tias qhov sib txawv ntawm Arabidopsis thaliana sucrose synthase AtSus1 (PDBID 3S28) thiab CtSus tuaj yeem ncav cuag 77.93%. Cov qauv CtSus tau kwv yees tau muab piv nrog AtSus1 cov qauv peb sab, thiab lub hauv paus txhais tau tias square deviation (RMSD) tus nqi tom qab protein superposition yog 1.11 Å, qhia tias cov qauv spatial ntawm ob qhov zoo sib xws (Daim duab 5B).

Daim duab 5 Kev tshawb nrhiav tus qauv ntawm CtSus. A: Predicted theem nrab qauv ntawm CtSus siv SOPMA.Blue: helix; Ntshav: Random coil; Liab: Extended strand; Ntsuab: ntawv. B: Peb-dimensionalstructure alignment ntawm AtSus1 (hauv xim xiav) thiab CtSus (hauv xim ntsuab). Ob leeg tau pom tias yog tetramer.C: Cov seem tseem ceeb nyob rau hauv lub substrate binding hnab tshos ntawm AtSus1 (nyob rau hauv xiav xim) thiab CtSus (nyob rau hauv ntsuab colourwith labeled residues); D: Binding conformations alignment ntawm UDP thiab fructose hauv AtSus1 (hauv bluecolor) thiab CtSus (hauv xim ntsuab); E: Kev sib cuam tshuam ntawm UDP thiab CtSus qhia hauv 2D daim duab ntsuas los ntawm Discovery Studio Client

Cov ntaub ntawv tshaj tawm cov protein-ligand crystal complex qauv ntawm Arabidopsis AtSus1 nrog UDP thiab fructose (PDBID3S29) tau siv los ua tus qauv [16] los tshuaj xyuas hom kev khi ntawm CtSus nrog UDP thiab fructose. Cov txiaj ntsig molecular docking tau qhia hauv daim duab 5C. Nws tuaj yeem pom tau tias cov hnab ris hauv substrate khi ntawm AtSus1 thiab CtSus zoo sib xws hauv cov ntsiab lus ntawm cov amino acid hom, spatial faib, thiab teeb tsa, thiab qhov sib tshooj yog siab, ua pov thawj tias qhov sib lawv liag ntawm sucrose synthase yog kev txuag hauv cov nroj tsuag. Kev sib haum xeeb ntawm ob lub ligands, UDP thiab fructose, nyob rau hauv cov protein substrate binding hnab tshos, yog qhia nyob rau hauv daim duab 5D. Lub molecular docking qhov zoo tshaj plaws conformation ntawm UDP thiab CtSus overlaps zoo nrog cov conformation ntawm UDP nyob rau hauv lub AtSus1-UDP siv lead ua complex, ua pov thawj qhov tseeb ntawm cov molecular docking tau. Kev sib cuam tshuam ntawm UDP thiab cov tseem ceeb amino acid residues nyob rau hauv cov protein substrate binding hnab yog qhia nyob rau hauv daim duab 5E. UDP thiab CtSus feem ntau yog ua ke los ntawm hydrogen bonds thiab hydrophobic kev sib tshuam. Cov tseem ceeb amino acid residues nyob rau hauv lub substrate binding hnab ris muaj xws li Leu294, Gly301, Met576, Arg578, Lys583, Gln646, Asn652, Leu677, Thr678, thiab Glu681.

Kev sib tham

Kev hloov kho glycosylation yog ib qho tseem ceeb ntawm kev txhim kho lub cev thiab kev ua haujlwm lom neeg ntawm cov khoom ntuj lossis tshuaj ua ntej. Piv nrog rau cov txheej txheem tshuaj ib txwm muaj, kev hloov kho enzymatic glycosylation muaj qhov zoo ntawm cov tshuaj tiv thaiv me me, muaj zog xaiv, thiab ib puag ncig tus phooj ywg. Txawm li cas los xij, glycosylation cov tshuaj tiv thaiv ntawm glycosyltransferase yuav tsum muaj ntau ntawm UDP-qab zib pub dawb, uas yog kim thiab nyuaj kom tau, ua rau qhov tseeb tias glycosyltransferases tsis tuaj yeem siv dav hauv kev tsim khoom. Sucrose synthase tuaj yeem ua rau cov tshuaj tiv thaiv rov qab: sucrose + UDP ⇌ UDP-glucose + fructose thiab tuaj yeem tsim lub voj voog UDP-glucose txuas ntxiv los ntawm kev sib txuas cov tshuaj tiv thaiv nrog glycosyltransferase. Cistanche tubulosa yog nplua nuj nyob rau hauv ntau yam glycoside tebchaw ntawm ntau hom qauv, sawv cev los ntawm phenylethanol glycoside tebchaw, tawm tswv yim hais tias cov nquag glycosyl pub synthesis txoj hauv nws lub cev nrog sucrose synthase muaj ib tug muaj zog metabolism, tab sis qhov cuam tshuam sucrose synthase los ntawm Cistanche nroj tsuag tsis tau. tau qhia. Nyob rau hauv txoj kev tshawb no, ib tug sucrose synthase noob CtSus tau cloned los ntawm Cistanche tubulosa thawj zaug. Cov protein encoded los ntawm cov noob no muaj cov chaw khaws cia ntawm cov nroj tsuag sucrose synthase. Los ntawm kev sib piv cov sequence ntawm sucrose synthases los ntawm lwm cov nroj tsuag, nws tau pom tias cov amino acid sequence zoo sib xws ntawm nws thiab sucrose synthases los ntawm cov nroj tsuag ntawm tib qhov kev txiav txim yog ntau tshaj 90%, qhia ib tug high degree ntawm sequence kev txuag ntawm sucrose synthases los ntawm cov nroj tsuag. Molecular evolution tsom xam pom tias CtSus belongs rau dicotyledonous cog sucrose synthase ceg thiab muaj feem xyuam nrog cov sucrose synthase PrSus los ntawm P. ramosa ntawm tsev neeg Orobanchaceae.

Txhawm rau tshawb nrhiav cov haujlwm catalytic ntawm CtSus, qhov kev tshawb fawb no tau ua ke nrog glycosyltransferase UGT71BD1, uas nws cov haujlwm tau raug txheeb xyuas yav dhau los los ntawm pab pawg tshawb fawb, los tsim kom muaj dual-plasmid co-expression system. Los ntawm tag nrho-cell catalytic thwmsim, cov xwm txheej tau ua tiav yam tsis tau ntxiv UDP-qab zib pub ntxiv. Glycosylation cov tshuaj tiv thaiv ntawm coumarin compound cinnamon thiab stilbene compound resveratrol. Piv nrog rau pawg tswj hwm, qhov sib ntxiv ntawm CtSus tau nce qhov kev hloov pauv ntawm UGT71BD1- catalyzed glycosylation cov tshuaj tiv thaiv. Nyob rau lub hauv paus no, qhov kev tshawb fawb no tau tsim ib qho CtSus recombinant qhia plasmid thiab ua tiav cov lus qhia ntawm cov protein recombinant hauv E. coli. Hauv vitro, enzymatic catalytic cov tshuaj tiv thaiv qhia tau hais tias nyob rau hauv lub xub ntiag ntawm sucrose thiab UDP, CtSus tuaj yeem ua rau lub cim ntawm UDP-glucose, thiab tom qab qhov ua rau muaj kev cuam tshuam cov cim npe uas muaj nyob rau hauv recombinant protein raug tshem tawm, cov khoom ntawm CtSus catalyzing tiam UDP. -glucose tau txais. Tus nqi tau raug kho kom zoo dua. Kev hloov pauv tag nrho ntawm tes thiab hauv vitro enzymatic catalytic cov tshuaj tiv thaiv tau lees paub qhov kev ua ntawm CtSus raws li sucrose synthase catalytic active qab zib pub UDP-glucose. Txhawm rau tshawb nrhiav ntxiv txog kev sib raug zoo ntawm CtSus gene thiab biosynthesis ntawm glycosides hauv Cistanche tuberosum, qhov kev qhia ntawm CtSus nyob rau hauv ntau qhov chaw ntawm Cistanche tuberosum tau soj ntsuam los ntawm lub sijhawm tiag tiag fluorescence quantitative PCR thwmsim. Cov txiaj ntsig tau pom tias cov noob tau nthuav tawm hauv haustoria ntawm Cistanche tuberosum. Qhov kev qhia siab tshaj plaws. Cistanche deserticola yog cov kab mob cab thiab tsis tuaj yeem tau txais cov as-ham uas xav tau rau kev loj hlob thiab kev loj hlob los ntawm photosynthesis. Yog li ntawd, nws yuav tsum tau muaj kab mob parasitic ntawm cov hauv paus hniav ntawm tus tswv tsev thiab cia siab rau tus tswv tsev kom tau txais cov as-ham los tswj kev loj hlob. Hauv cov nroj tsuag, sucrose feem ntau muab lub zog thiab cov pa roj carbon qhov chaw pub [12]. Txawm li cas los xij, sucrose tsis tuaj yeem siv ncaj qha los ntawm cov hlwb thiab xav tau ntxiv decomposed. Haustoria yog tus choj txuas rau Cistanche deserticola thiab tus tswv tsev cog, thiab ua lub luag haujlwm tseem ceeb hauv nws txoj kev loj hlob. tseem ceeb heev

[25], yog li qhov kev qhia siab ntawm sucrose synthase hauv haustoria ntawm Cistanche deserticola yog qhov tsim nyog. Kev qhia siab ntawm CtSus nyob rau hauv haustoria kuj zoo ib yam nrog cov qauv loj ntawm phenylethanoid glycosides hauv haustoria. Tsis tas li ntawd, los ntawm fluorescence quantitative PCR tsom xam ntawm kev hloov pauv hauv CtSus gene qhia theem hauv Cistanche deserticola ncua kev kawm hlwb ntawm lub sijhawm sib txawv nyob rau hauv kev ntxhov siab, nws tau pom tias kev nyuaj siab ntxhov siab tuaj yeem ua rau muaj kev nthuav qhia CtSus noob nyob rau hauv ncua kev kawm cell kab, uas yog. raws li echinaceaside. Cov qauv sib xyaw ua ke hauv cov kab hluav taws xob raug tshem tawm nyob rau hauv kev ntxhov siab drought zoo ib yam. Cov txiaj ntsig saum toj no qhia tias CtSus koom nrog txoj hauv kev biosynthetic ntawm phenylethanoid glycosides sawv cev los ntawm echinacea hauv Cistanche tulipis hauv vivo. Nws yog ib qho ntawm ntau txoj hauv kev biosynthetic. Thawj kauj ruam glycosylation cov tshuaj tiv thaiv muab cov nquag glycosyl pub UDP-glucose. Hauv luv, qhov kev tshawb fawb no

Txoj kev tshawb no tau txheeb xyuas cov noob sucrose synthase tshiab hauv Cistanche deserticola, uas tau ua rau hauv vitro enzymatic synthesis ntawm active glycosyl pub thiab muab cov noob caj noob ces tshiab rau kev tsim cov kab mob engineering rau biosynthesis ntawm Cistanche deserticola glycosides.

Cov neeg sau kev koom tes: Tian Weisheng yog lub luag haujlwm rau kev tshuaj xyuas bioinformatics, kev tshuaj xyuas kev qhia, kev tshuaj xyuas enzyme thiab kev sau ntawv ntawm thawj cov qauv ntawm CtSus gene; Yan Yaru yog lub luag haujlwm rau kev tshuaj ntsuam gene thiab cloning; Cui Xiaoxue thiab Huang Wenqian tau koom nrog hauv kev tshuaj xyuas bioinformatics thiab qhia kev tsom xam; Wang Yingxia thiab Zhao Saijing koom nrog vector tsim thiab enzyme Kev tsom xam; Li Jun thiab Shi Shepo feem ntau yog coj cov enzyme kev tsom xam thiab kev qhia qhia; Tu Pengfei thiab Liu Xiao yog lub luag haujlwm rau kev tsim daim ntawv tswv yim, coj kev sim, thiab sau ntawv thiab kho cov ntawv. Txhua tus kws sau ntawv tau koom nrog kho cov ntawv.

Cov ntaub ntawv

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