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

Abstract:Sucrose synthase plays lub luag haujlwm tseem ceeb hauv cov nroj tsuag cov piam thaj metabolism los ntawm kev tsim cov tshuaj uridine diphosphate (UDP)-glucose, uas ua haujlwm ua tus neeg pub khoom noj khoom haus glycosyl rau ntau yam metabolicprocesses. Nyob rau hauv txoj kev tshawb no, ib tug sucrose synthase noob npe hu ua CtSus tau cloned los ntawmCistanche tubulosa, ib txwm suav tshuaj paub txog nws cov nplua nujCov ntsiab lus ntawm ntau hom glycosides, raws li kev txheeb xyuas cov txiaj ntsig. Cov kab lus qhib ntawm CtSus yog 2 418 bp ntev encoding 805 amino acids. Kev soj ntsuam ib ntus tau qhia txog kev tswj hwm kev sib txuas nrog cov nroj tsuag sucrose synthase tsev neeg ntawm ob lub N-terminal thiab C-terminal cheeb tsam ntawm CtSus protein.Phylogenetic tsom xam pom tau tias CtSus qhia kev sib raug zoo nrog kev sib raug zoo nrog.sucrose synthaseslos ntawm lwm cov nroj tsuag hauv tib qhov kev txiav txim. Kev txheeb xyuas kev ua haujlwm ntawm CtSus tau ua los ntawm tag nrho cov cell catalysiscoupling nrog UGT71BD1, tus yam ntxwv glycosyltransferase enzyme. Cov txiaj ntsig tau pom tias qhov kev taw qhia ntawm CtSus tau txhim kho qhov hloov pauv ntawm glycosylation catalyzed los ntawm UGT71BD1. Kev qhia soluble ntawm CtSus hauv Escherichia coli tau ua tiav ntxiv los ntawm kev siv pCold ™ TF qhia vector. Hauv vitro enzymatic assayindicated cov haujlwm ntawm CtSus txhawm rau tsim cov UDP-glucose nyob rau hauv muaj sucrose thiab UDP. Realtime quantitative-polymerase saw cov tshuaj tiv thaiv tau pom tias cov qauv qhia ntawm CtSus gene nyob rau hauv qhov sib txawv ntawm C. tubulosa thiab ncua kev kawm cell kab lis kev cai ntawm C. tubulosa nyob rau hauv drought kev nyuaj siab correlated nrog theacumulation qauv soj ntsuam rau phenylethanol glycosides, feem. Tsis tas li ntawd, cov amino acids tseem ceeb thiab lawv cov kev sib cuam tshuam ntawm enzyme thiab substrate tau tshawb fawb raws li kev kwv yees cov qauv protein thiab cov txiaj ntsig ntawm molecular dockingresults. Zuag qhia tag nrho, peb qhov kev tshawb pom tau txheeb xyuas cov sucrose synthase CtSus lub luag haujlwm rau kev muab cov khoom siv glycosyl donorUDP-glucose thaum lub sijhawm biosynthesis ntawm glycoside cov khoom hauv C. tubulosa thiab kuj tau muab cov noob qoob uas tuaj yeem siv rau hauv engineering strain tsim rau glycoside cov khoom tsim.

Cov lus tseem ceeb: Cistanche tubulosa; sucrose synthase; glycosides; UDP-qabzib; enzymatic catalysis

DICHEN CISTANCHE CULTIVATES BASES hauv YUTIAN, HOTAN, XINJIANG

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Cistanche tubulosa (Schenk) R. WightNws yog perennial herbaceous parasitic nroj tsuag ntawm genus Cistanche nyob rau hauv tsev neeg Orobanchaceae. Nws yog ib txwm tsis tshua muaj thiab muaj txiaj ntsig cov khoom siv tshuaj hauv Suav teb, ib txwm nyob hauv Xinjiang thiab Inner Mongolia. Nws yog qab zib, qab ntsev thiab sov hauv qhov, thiab muaj cov haujlwm ntawmtonifying raum yang, pab essence thiab ntshav, thiab moistening cov hnyuv thiabtshem tawm cem quav. Nws yog lub npe hu ua "suab puam ginseng"[1]. Kev tshawb fawb niaj hnub tau pom tiasCistanche tubulosamuajntau yam tshuaj lom neegxws liphenylethanoid glycosides, iridoid ether terpenesthiab lawv glycosides, lignans thiab lawv glycosides, oligosaccharide esters, polysaccharides, polyols, oligosaccharides, sterols thiab lawv glycosides, monoterpenoid glycosides, qab zib, amino acids thiab volatile roj [2,3], tshwj xeeb tshaj yog glycoside tebchaw yog cov khoom xyaw tseem ceeb tshaj plaws. Ntawm lawv, phenylethanoid glycoside tebchaw muaj cov ntsiab lus siab tshaj plaws hauv cov khoom siv tshuaj thawj zaug thiab muaj ntau yam kev ua haujlwm tshuaj xws li neuroprotection thiab anti-aging [4]. Lub biosynthesis ntawm cov lus hais saum toj no glycoside tebchaw koom nrog ntau cov kauj ruam ntawm glycosylation cov tshuaj tiv thaiv, uas yog catalyzed los ntawmglycosyltransferases hauv cov nroj tsuag, thiab covmuab cov active glycosyl pubyog qhov tseem ceeb hauv txoj kev no. Cov neeg mob ntshav qab zib feem ntau muaj xws li uridine diphosphate (UDP), thymidine diphosphate (TDP), thiab guanosine diphosphate (GDP). Ntawm lawv, UDP-glucose yog cov nquag nquag glycosyl pub dawb hauv cov nroj tsuag. Nws tuaj yeem tsis tsuas yog lees paub ncaj qha los ntawm glucosyltransferase txhawm rau txhawm rau txhawm rau hloov cov tshuaj tiv thaiv glucosyl, tab sis kuj tuaj yeem siv los ua tus thawj coj los koom rau hauv kev sib txuas ntawm lwm hom glycosyl pub dawb. Piv txwv li, UDP-glucose tuaj yeem siv los ua lub substrate los tsim UDP-rhamnose nyob rau hauv lub catalysis ntawm 4,6-dehydratase, 3,5-isomerase thiab 4-reductase[5]; UDP-glucose tuaj yeem siv los ua cov substrate los ua kom cov synthesis ntawm UDP-xylose los ntawm UDP-glucose dehydrogenase thiab UDP-xylose synthase; UDP-xylose tuaj yeem ua ntxiv los ntawm UDP-xylose epimerase los tsim UDP-arabinose[6]; UDP-glucose thiab UDP-galactose yog diastereomers thiab tuaj yeem hloov mus rau ib leeg los ntawm epimerase [7,8]. Yog li, kev sib txuas ntawm UDP-qabzib muaj qhov cuam tshuam tseem ceeb ntawm kev muab cov glycosyl nquag pub rau hauv cov nroj tsuag thiab kev hloov pauv glycosylation ntawm cov metabolites theem nrab.

Kev sib xyaw ntawm UDP-glucose hauv cov nroj tsuag tuaj yeem ua tiav los ntawm txoj kev synthase, phosphorylase txoj kev thiab kinase txoj kev. Ntawm lawv, sucrose synthase tuaj yeem ncaj qha catalyze cov tshuaj tiv thaiv ntawm UDP thiab sucrose los tsim UDP-glucose thiab fructose, nrog rau cov tshuaj tiv thaiv rov qab [9]. Hauv kev nkag siab dav, sucrose synthase belongs rau glycosyltransferase -4 subfamily, uas suav nrog ntau yam glycosyltransferases, xws li sucrose synthesis phosphatase, trehalose synthase thiab trehalose phosphorylase [10]. Sucrose synthase tau pom thawj zaug hauv cov kab mob nplej [11]. Txij thaum ntawd los, cov kws tshawb fawb tau txheeb xyuas ntau pua cov noob sucrose synthase los ntawm ntau hom nroj tsuag xws li mung taum (Vigna radiata), mov (Oryza sativa), pob kws (Zea mays), taum mog (Glycine max) thiab txiv duaj (Amygdalus persica). Lawv koom nrog ntau yam txheej txheem metabolic hauv cov nroj tsuag, suav nrog sucrose thauj thiab faib, cov hmoov txhuv nplej siab, cellulose synthesis thiab cell phab ntsa muaj pes tsawg leeg, biotic thiab abiotic stress, thiab lwm yam, thiab ua lub luag haujlwm tseem ceeb hauv kev faib cov pa roj carbon monoxide [12]. Tsis tas li ntawd, raws li kev tshawb fawb ntawm kev ua haujlwm ntawm lub cev, cov kws tshawb fawb kuj tau siv cov sucrose synthase los ua cov noob qoob rau cov enzymatic synthesis ntawm glycoside compounds, thiab ua tiav cov txiaj ntsig zoo. Piv txwv li, Masada et al. [13] co-expressed sucrose synthase gene AtSus-1 los ntawm Arabidopsis thaliana thiab glycosyltransferase gene CaUGT2 los ntawm Catharanthus roseus hauv vitro, thiab siv ib lub lauj kaub dual enzyme system los catalyze glycosylation cov tshuaj tiv thaiv ntawm curcumin, uas ua rau kom muaj zog. tawm los ntawm curcumin glycoside los ntawm 7 zaug. Tib lub sijhawm, tus nqi ntawm UDP-glucose pub siv tsuas yog ib feem kaum ntawm thawj tus nqi.

Bungaruang et al. [14] tsim ib qho kev sib txuas catalytic uas siv sucrose synthase GmSuSy los ntawm taum pauv thiab glycosyltransferase OsUGT los ntawm mov. Lawv ua tiav glycosylation hloov kho ntawm nothofagin los ntawm kev ntxiv me me ntawm UDP (1.0 mmol·L-1) thiab sucrose. Piv nrog rau ib qho enzyme catalysis ntawm OsUGT, kev tsim cov khoom lag luam glycosylation tau nce los ntawm peb qhov kev txiav txim loj, los ntawm μmol·L-1 mus rau mol·L-1.

Qhov kev nplua nuj thiab ntau haiv neeg ntawm glycoside tebchaw hauv Cistanche tubulosa qhia tias nws yuav tsum muaj lub zog sucrose synthase cuam tshuam nrog kev hloov pauv glycosylation ntawm cov metabolites theem nrab, tab sis cov enzyme no tsis tau tshaj tawm hauv cov nroj tsuag ntawm genus Cistanche. Hauv daim ntawv no, Cistanche tubulosa, cov khoom siv tshuaj muaj nyob hauv cov tshuaj pharmacopoeia, tau siv los ua cov khoom siv. Los ntawm kev txheeb xyuas cov ntaub ntawv pov thawj thiab kev sib piv transcriptome tsom xam ntawm qhov sib txawv, cov txheej txheem molecular biology tau siv rau kuv thiab txheeb xyuas cov noob sucrose synthase CtSus. Bioinformatics tsom xam, tag nrho-cell biotransformation, prokaryotic qhia thiab purification, nyob rau hauv vitro enzyme catalytic kev ua si tsom, thiab gene qhia tsom xam nyob rau hauv ntau qhov chaw ntawm Cistanche tubulosa thiab cog cov ntaub so ntswg kab lis kev cai kab lis kev cai nyob rau hauv drought kev nyuaj siab tau ua los tshawb txog lub luag hauj lwm ntawm CtSus thiab nws correlation nrog. Lub biosynthesis ntawm glycoside tebchaw nyob rau hauv Cistanche tubulosa, nteg lub hauv paus rau txoj kev tshawb no ntawm biosynthetic txoj kev ntawm glycoside tebchaw nyob rau hauv Cistanche tubulosa.

Cov ntaub ntawv thiab cov txheej txheem

Cov ntaub ntawv tshiab Cistanche tubulosa cov nroj tsuag tau sau los ntawm Hotan City, Xinjiang, Tuam Tshoj, thiab kev txheeb xyuas kab mob lom tau ua los ntawm xibfwb Tu Pengfei ntawm Peking University; lub Cistanche tubulosa ncua kev kawm cell kab lis kev cai yog tsim los ntawm peb pawg neeg tshawb fawb nyob rau hauv thaum ntxov theem [15]; E. coliFast-T1 tau yuav los ntawm Nanjing Novozymes Biotech Co., Ltd.; E. coli BL21 (DE3) tau yuav los ntawm Beijing Quanshijin Biotechnology Co., Ltd.; pCold™ I thiab pCold™ TF vectors tau yuav los ntawm Bio-Tech (Beijing) Co., Ltd.; UDP-glucose tswj tau yuav los ntawm Xi'an Qiyue Biotechnology Co., Ltd., thiab aesculin thiab resveratrol substrates tau yuav los ntawm Shanghai Yuanye Biotechnology Co., Ltd.

DICHEN CISTANCHE CULTIVATES BASES hauv HOTAN, XINJIANG

RNA extraction thiab cDNA synthesis RNA tau muab rho tawm raws li cov lus qhia ntawm OMEGA RNA Plant Kit (Cat R6827-01, Omega Bio-Tek). Agarose gel electrophoresis thiab Nanodrop 2000 spectrophotometer tau siv los xyuas qhov purity, concentration thiab kev ncaj ncees ntawm RNA. Cov qauv RNA tsim nyog tau thim rov qab siv cov khoom siv thim rov qab TransScript® OneStep gDNA Tshem tawm thiab cDNA Synthesis SuperMix (Cat AT311-02, Beijing Quanshijin Biotechnology Co., Ltd.) los ua ke thawj cov saw ntawm cDNA.

Mining ntawm sucrose synthase noob thiab cloning ntawm CtSus genes Cov amino acid ib ntus ntawm qhov ua haujlwm tau txheeb xyuas Arabidopsis thaliana sucrose synthase AtSus1 (GenBank accession naj npawb: AED92894.1) [16] tau siv los ua tus qauv. Sucrose synthase noob tau soj ntsuam los ntawm cov ntaub ntawv transcriptome ntawm C. tubulosa los ntawm lub zos Blastp sib lawv liag kev sib raug zoo, ua ke nrog transcriptome noob annotation, FPKM (gene expression abundance) tsom xam thiab sib piv cov ntaub ntawv transcriptome tsom xam. Cov primers tshwj xeeb tau tsim los raws li cov ntaub ntawv sib txuas ntawm cov noob caj noob ces muab los ntawm cov ntaub ntawv thib ob ntawm C. tubulosa (Table 1). C. tubulosa cDNA tau siv los ua tus qauv thiab PCR amplification ntawm lub hom phiaj noob tau ua los ntawm kev siv lub siab ncaj ncees PCR enzyme (Cat P505-d1, Nanjing Novozymes Biotech Co., Ltd.). PCR amplification khoom tau huv si siv FastPure Gel DNA Extraction Mini Kit (Cat DC201-01, Nanjing Novozymes Biotech Co., Ltd.) thiab ces cloned raws li cov khoom siv cloning ceev (Cat C112-01, Nanjing Novogene Biotech Co., Ltd.) tau txuas nrog pCE2 TA / Blunt-Zero cloning vector raws li kev khiav hauj lwm cov lus qhia, pauv mus rau Fast-T1 muaj peev xwm hlwb, thiab kab lis kev cai thaum hmo ntuj ntawm 37 degree. Cov plasmid tau muab rho tawm thiab xa mus rau Beijing Liuhe BGI Genomics Co., Ltd.

Table 1 Nucleotide sib lawv liag ntawm primers siv nyob rau hauv txoj kev tshawb no. qPCR: Real-time quantitative-polymerasechain cov tshuaj tiv thaiv

Bioinformatics tsom xam ntawm sucrose synthase CtSus los ntawm Cistanche tubulosa

Cov software online ProtParam (https://web.expasy.org/protparam/) tau siv los tshuaj xyuas cov khoom ntawm lub cev ntawm cov protein; online software TMHMM (https://services.healthtech.dtu.dk/services/TMHMM{0}}.0/) yog siv los kwv yees cov qauv transmembrane ntawm cov protein; online software SOPMA (https://npsa-rabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html)) tau siv los ua kwv yees tus qauv thib ob ntawm cov protein; online software SMART (http://smart.embl.de/) tau siv los kwv yees qhov chaw khaws cia ntawm cov protein amino acid ib ntus; lub software DANMAN tau siv los sib piv cov amino acid sequences ntawm CtSus nrog cov sucrose synthases los ntawm lwm hom; Cov ntoo phylogenetic tau tsim los ntawm kev tshuaj xyuas pawg siv cov neeg nyob sib ze koom nrog txoj kev siv software MEGA11, nrog bootstrap rov ua dua ntawm 1,000 zaug.

Kev tsom xam ntawm CtSus nyob rau hauv ntau qhov chaw ntawm Cistanche tubulosa thiab nyob rau hauv drought kev nyuaj siab

Real-time fluorescence quantitative PCR primers rau CtSus thiab cov khoom siv hauv cov noob DNAj tau tsim los siv DNAMAN software [17] (Table 1). Tus txheeb ze qhia theem ntawm CtSus noob nyob rau hauv ncua kev kawm ntawv hlwb ntawm Cistanche tubulosa nyob rau hauv ntau qhov chaw ntawm Cistanche tubulosa thiab nyob rau hauv drought kev nyuaj siab tau soj ntsuam los ntawm real-time fluorescence quantitative PCR. Real-time fluorescence quantitative PCR tsom xam tau ua tiav siv TransStart® Top Green qPCR SuperMix Kit (Cat AQ131-01, Beijing Quanshijin Biotechnology Co., Ltd.). Qhov kev pab cuam amplification tau txais txoj hauv kev ob kauj ruam: 94 degree rau 30 s; 94 degree rau 5 s, 60 degree rau 30 s, rov ua dua rau 40 cycles; Nws tau khiav ntawm Bio-Rad CFX Connect™ Real-Time PCR Detection System (Bio-Rad Laboratory, Hercules, CA, USA). Txoj kev 2–ΔΔ CT tau siv los txheeb xyuas qhov txheeb ze ntawm CtSus gene hauv ntau pawg sim. GraphPadPrism 9 tau siv los ntsuas qhov tseem ceeb thiab kos duab kos duab.

Kev tsim kho ntawm prokaryotic qhia vector thiab heterologous qhia ntawm CtSus gene Raws li cov txiaj ntsig ntawm noob caj noob ces, cov primers tshwj xeeb uas muaj vector homology caj npab thiab qhov chaw txwv (Table 1) tau tsim los rau PCR amplification. Lub hom phiaj gene CtSus tau tsim los rau hauv pET-28a, pET-24b, pCold™ I thiab pCold™ TF qhia vectors siv seamless cloning thev naus laus zis siv ClonExpress Ultra One Step Cloning Kit (CatC115-01 , Nanjing Novogene Biotech Co., Ltd.). Cov plasmids ntawm kev ua tiav kev ua tiav recombinant qhia vectors tau muab rho tawm thiab hloov mus rau hauv Escherichia coli qhia muaj peev xwm ntawm tes Transetta (DE3). Cov hlwb raug coj mus rau hauv LB kua nruab nrab uas muaj 50 µg·mL-1 kanamycin sulfate (nrog pET-28a thiab pET-24b raws li kev qhia vectors) lossis 1{{ 22}}0 µg·mL-1 ampicillin (nrog pCold™ I thiab pCold™ TF raws li kev qhia vectors) thiab 50 ug·mL-1 chloramphenicol, raws li, ntawm 37 degree thiab 200 r·min{{ 15}} nrog co kom txog thaum A600 tus nqi ntawm cov tshuaj tua kab mob mus txog 0.4-0.6. Ntxiv isopropyl-beta-Dthiogalactopyranoside (IPTG) ntawm qhov kawg siab ntawm 0.5 mmol·L-1 los txhawb kev qhia cov protein. Incubate nyob rau hauv ib tug tas li kub shaker ntawm 23 degree (nrog pET-28a thiab pET-24b raws li qhia vectors) los yog 15 degree (nrog pCold™ I thiab pCold™ TF raws li qhia vectors), 180 r· min-1 rau 16 h (nrog pET28a thiab pET-24b raws li qhia vectors) los yog 24 h (nrog pCold™ I thiab pCold™ TF raws li qhia vectors), ces sau cov kab mob los ntawm centrifugation.

Lysis buffer (20 mmol·L-1 NaH2PO4-Na2HPO4, 0.5 mol·L-1 NaCl, 20 mmol·L{10} } imidazole, 3% glycerol, 100 mmol·L-1 phenylmethylsulfonyl fluoride, 1 mg·mL-1 lysozyme, pH 7.4) ntxiv, thiab cov hlwb tau tawg rau 8 min thiab centrifuged. Lub supernatant thiab precipitate tau coj raws li. Lub supernatant tau adsorbed los ntawm HisTrap FF affinity kem (Cat 17531901, Cytiva) uas muaj Ni Sepharose 6 Fast Flow muab tub lim kom adsorb lub hom phiaj protein uas muaj Nws tag. Tom qab elution nrog ntau qhov sib txawv ntawm imidazole, txhua feem raug coj mus rau SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) nrhiav pom. Cov feem uas muaj cov hom phiaj protein tau muab sib xyaw ua ke, concentrated los ntawm centrifugation thiab desalted los ntawm ultrafiltration tube (Millipore), thiab khaws cia rau hauv tsis muaj [muaj 50 mmol·L-1 Tris-HCl (pH 7.5), 0.2 mol·L{{ 30}NaCl, 1 mmol·L-1 dithiothreitol, 3% glycerol] rau cov tshuaj tiv thaiv hauv vitro enzymatic catalysis tom qab.

CtSus tag nrho-cell catalysis Lub pCold™ I-CtSus plasmid thiab cov lus qhia plasmid pET-28a-UGT71BD1[18] ntawm glycosyltransferase UGT71BD1, uas tau txheeb xyuas los ntawm pab pawg tshawb fawb ua ntej, tau ib txhij hloov mus rau hauv kev qhia muaj peev xwm. E. coli Transetta (DE3). Cov kab mob zoo recombinant uas muaj CtSus thiab UGT71BD1 cov noob tau raug xaiv thiab muab tso rau hauv LB nruab nrab (qhov nruab nrab loj hlob) uas muaj 50 ug·mL-1 kanamycin sulfate, 100 ug ·mL-1 ampicillin thiab 50 ug·mL-1 chloramphenicol ntawm qhov ratio ntawm 1:100 (V/V). Cov kab lis kev cai tau coj los ntawm 37 degree kom txog thaum A600 tus nqi ntawm cov tshuaj tua kab mob yog 0. Cov kab lis kev cai tau txuas ntxiv ntawm 15 degree thiab 180 r·min-1 rau 24 teev. Cov kab lis kev cai tau centrifuged ntawm 4 degree thiab 4 000 r·min-1. Sau cov hlwb. Inoculate cov hlwb sau rau hauv M9 nruab nrab (fermentation nruab nrab) ntawm qhov sib piv ntawm 40 mL kab lis kev cai nruab nrab ib gram ntawm cov kab mob, thiab ntxiv 75 μmol·L-1 sucrose thiab 25 μmol·L-1 aesculetin (1) los yog resveratrol (2) substrates rau pawg tshuaj tiv thaiv. Txuas ntxiv co kab lis kev cai rau 24 h, centrifuge ntawm 4000 × g, 4 degree rau 30 min, ntxiv methanol rau ultrasonically tawg lub precipitate, ntxiv 2 zaug lub ntim ntawm methanol rau supernatant, centrifuge ntawm 4000 × g, 4 degree rau 30 min, coj cov supernatant thiab ua ke, evaporate cov kuab tshuaj nyob rau hauv lub siab txo, re-solve nyob rau hauv methanol, lim los ntawm 0.22 μm lim membrane, thiab ntes cov tshuaj tiv thaiv cov khoom siv Agilent 1260 siab ua kua chromatograph. CAPCELL PAK C18 COLUMN (250 mm × 4.6 mm, 5 µm) tau siv los ua kem analytical kem, 0.1% formic acid dej (A) thiab methanol (B) tau siv los ua theem mobile, thiab elution gradient yog: {{61 }} min, 30%-90% B; 25-27 min, 90%-100% B; 27-33 min, 100% B. Qhov loj spectrometry tsis yog hom zoo thiab tsis zoo ion, thiab tsis siv neeg ntau theem MS1, MS2, thiab MS3 tag nrho scans.

Kev kuaj pom ntawm CtSus hauv vitro enzyme catalytic kev ua ub no Lub recombinant protein tau los ntawm cov saum toj no-hais HisTrap FF kem affinity chromatography tau raug tshem tawm tag siv Factor Xa. Lub enzyme digestion system yog raws li nram no: cov recombinant protein tau hloov mus rau ib tug concentration ntawm 1 mg·mL-1, thiab 1 μL Factor Xa tau ntxiv rau 50 μL ntawm cov recombinant protein nyob rau hauv ib tug buffer ntawm 20 mmol·L-1 Tris-HCl (pH 8.0), 100 mmol·L-1 NaCl, thiab 2 mmol·L-1

CaCl2 ntawm 23 degree rau 6 h. Cov tshuaj tiv thaiv kab mob yog 100 μL. Cov khoom lag luam ntawm cov enzyme digestion tau raug rau cov protein sib cais los ntawm HisTrap FF affinity chromatography kom tau lub hom phiaj protein nrog tag tshem tawm rau tom ntej hauv vitro enzyme catalysis tshua. Cov tshuaj tiv thaiv enzyme yog raws li nram no: 10mmol·L-1 UDP, 100 mmol·L-1 sucrose, 50 mmol·L-1 MgCl2, 60 ug CtSus protein, Tris-HCl (pH) 7.0) ntxiv rau 100 μL, reacted hauv da dej ntawm 30 degree rau 6 h, thiab tom qab kev kho mob thiab HPLC txoj kev tsom xam tau ua raws li tau piav qhia hauv kev siv [19].

Kev kwv yees peb-dimensional qauv thiab kev soj ntsuam molecular docking ntawm CtSus protein AlphaFold2 tau siv los kwv yees peb-dimensional qauv ntawm CtSus protein [20]. Lub ligand molecule tau hydrogenated thiab them siv AutoDock cuab yeej 1.5.6 software. Lub substrate binding hnab tshos tau txais los ntawm kev sib tw nrog cov qauv protein AtSus1 (PDBID 3S28). Lub sab chains ntawm cov amino acid residues nyob ib ncig ntawm lub kab noj hniav tau teem kom hloov tau.

Lub thawv kab sib chaws tau teeb tsa rau 40 × 40 × 40 Å3, thiab daim phiaj sib nrug yog 0.375 Å. Molecular docking tau ua hauv AutoDock Vina [21]. PyMOL 2.0 software tau siv los pom cov qauv nrog qhov zoo tshaj plaws affinity (kcat·mol-1

).

Kev txheeb xyuas txheeb cais GraphPad Prism 9 tau siv rau kev txheeb xyuas kev txheeb xyuas. Cov ntaub ntawv sim tau qhia tias txhais tau tias ± tus qauv sib txawv. Qhov kev xeem t tau siv rau kev sib piv ntawm ob pawg, thiab ib txoj kev tsom xam ntawm qhov sib txawv yog siv rau kev sib piv ntawm ntau pawg. Qhov sib txawv tau suav tias yog qhov tseem ceeb thaum P < 0.05.