Cloning, Functional Identification and Expression Analysis Of Chalkone Synthase Los Ntawm Cistanche Tubulosa

Abstract: Lub Hom Phiaj

Kawm txog kev ua haujlwm ntawmchalcone synthase los ntawm Cistanche tubulosathiab cov qauv qhia ntawm CtCHS gene hauv paj dawb, liab thiab ntshav.

Cov txheej txheemCtCHS noob tau cloned los ntawm RT-PCR thiab bioinformatics tsom xam tau ua. Cov pET-28a-CtCHS plasmid tau raug xa mus rau hauv Escherichia coli los ua kom muaj protein ntau los ntawm IPTG. CtCHS recombinant protein yog incubated nrog cov substrates p-coumaroyl CoA thiab malonyl CoA, thiab cov khoom tau kuaj pom los ntawm HPLC thiab MS. Lub pCAMBIA1300-35S-GFP-CtCHS plasmid raug xa mus rau hauv lub protoplast ntawm Arabidopsis thaliana los soj ntsuam cov subcellular localization ntawm cov protein CtCHS. Cov qauv qhia ntawm CtCHS gene hauv paj ntawm C. tubulosa nrog peb xim tau kuaj pom los ntawm qRT-PCR.

Cov txiaj ntsigIb tug CtCHS noob tau cloned. Qhov ntev ntawm qhov qhib kev nyeem ntawv (ORF) yog 1173 bp, encoding 390 amino acids, thiab qhov hnyav molecular ntawm cov protein yog 42 8000.

Organic CISTANCHE EXTRACT nrog 30% ECHINACOSIDE thiab 12% ACTEOSIDE

CtCHS muaj cov catalytic residues Cys164, His303, Asn336, uas tau khaws cia hauv chalcone synthase. Cov protein CtCHS tau qhia nyob rau hauv E. coli thiab purified kom tau cov recombinant protein. CtCHS protein tuaj yeem ua rau p-coumaroyl CoA thiab malonyl CoA los tsim cov naringenin chalcone. CtCHS protein feem ntau yog nyob hauv cytoplasm. CtCHS gene tau qhia ntau heev hauv cov paj liab thiab liab, thiab cov txheeb ze qhia txog qib yog 8.44 thiab 3.21 npaug siab dua li ntawm w hite paj, feem.

Kev xaus A CtCHS noob tau cloned los ntawm lub paj ntawm C. tubulosa thiab cov protein tau qhia. CtCHS protein muaj chalcone synthase muaj nuj nqi, thiab cov kev qhia ntawm CtCHS noob yog siab dua nyob rau hauv cov paj maub, qhia tias CtCHS noob yuav tswj cov paj xim ntawm C. tubulosa, uas nteg ib lub hauv paus rau kev tshawb fawb ntxiv ntawm kev tswj mechanism ntawm paj xim ntawm C. tubulosa.

Cov lus tseem ceeb: Cistanche tubulosa (Schenk) Wight; chalcone synthase; tsom xam kev ua enzyme; subcellular localization; kev tsom xam

Cistanche Tubulosa(SCHENK) WIGHT yog genus Pyreal tsob nroj, uas yog ib txwm faib nyob rau yav qab teb Xinjiang [1] hauv Hetian cheeb tsam ntawm Xinjiang [2]. Cov paj yeeb yog siab nyob rau hauv tshuaj muaj nqis. Nws cov nqaij stems yog ib qhov chaw ntawm 2020 version ntawm "Suav Pharmacopoeia". [3] Kev tshawb fawb ntawm cov yeeb nkab paj cordonus nqaij stems tau nce, tab sis muaj qee qhov kev tshawb fawb lom ntawm nws cov paj. Lub sij hawm flowering ntawm cov yeeb nkab paj yog lub Plaub Hlis Ntuj mus rau Tsib Hlis [4], feem ntau muaj peb xim dawb. , liab, thiab ntshav liab Cov yeeb nkab paj muaj xim nplua nuj, tab sis lawv cov paj xim txheej txheem txheej txheem tsis tau tshaj tawm cov chav kawm muaj feem xyuam rau hom, cov ntsiab lus, cov ntsiab lus, thiab kev faib tawm ntawm cov ntaub so ntswg cov qauv, cov xim, thiab kev tswj hwm ntawm ib puag ncig. thiab genetic yam [5] Cov xim hauv cov paj paj feem ntau suav nrog peb pawg: flavonoids, carotene, thiab beetine xim, ntau yam xim zoo nkauj heev [7] Raws li peb ntau yam ntawm Paeonia Lactiflora Pall.

Nws zoo nkaus li dawb, thiab cov xim tseem ceeb ntawm cov xim muaj cov xim liab thiab zoo nkauj muaj peev xwm ua rau cov paj tam sim no li ntshav -liab [8]. Floral xim yog ib qho tseem ceeb ntawm ntau cov nroj tsuag ornamental, thiab nws kuj yog qhov tseem ceeb tshaj plaws uas nyiam cov kab paj yeeb [9]. Cov kev tshawb fawb tau pom tias nws tau dhau los Qhov siab tshaj plaws ntawm kev mus ntsib kab yog qhov tob ntshav cistanche cog [10]. Rau kev tshawb fawb txog kev tshawb fawb txog kev tshawb fawb ntawm cov noob caj noob ces ntawm cistanoids hauv lub raj paj cistanosaurus paj, nws yog qhov tsim nyog los qhia meej txog lub raj paj cordon xim tswj lub cev, muab lub hauv paus theoretical rau kev txhim kho cov xim ntawm genetic engineering technology, txhawb nws cov xim nplua nuj, nce kev pom, expanding daim ntawv thov Scope.

Txoj kev biosynthetic ntawmflavonoidspib nrog covphenylpropanoid txoj kev. Phenylalanine yog thawj zaug hloov mus rau hauv cinnamic acid nyob rau hauv qhov kev txiav txim ntawm phenylalanine ammonialyase (PAL), thiab cinnamic acid yog 4-hydroxylated los ntawm cinnamic acid. Cov enzyme (cinnamic acid-4-hydroxylase, C4H) thiab 4-coumarate coenzyme A ligase (4-coumarate coenzyme A ligase, 4CL) catalyze cov tshuaj tiv thaiv los tsim 4-coumaroyl-CoA [11]. Ib qho molecule ntawm 4-coumaroyl-CoA thiab peb lub molecules ntawm malonyl-CoA yog catalyzed los ntawm chalcone synthase (CHS) los tsim naringenin chalcone, uas muajflavonoid cov ntsiab lus.Lub cev pob txha ntawm chalcone isomerase, flavanone-3-hydroxylase, dihydroflavonol 4-reductase, thiab lwm yam. tsim muaj ntau yam flavonoids xws li isoflavones, flavonols, anthocyanins, thiab lwm yam. [12]. Raws li ib qho tseem ceeb enzyme nyob rau hauv txoj kev flavonoid biosynthetic, CHS tswj cov ntsiab lus ntawm flavonoids hauv cov nroj tsuag thiab tseem ua lub luag haujlwm tseem ceeb hauv kev tswj cov paj xim [13]. Piv txwv li, post-transcriptional gene silencing ntawm Gentiana scabra Bunge CHS tuaj yeem cuam tshuam cov biosynthesis ntawm anthocyanins thiab ua rau cov xim dawb ntawm corolla lobes [14]. CHS gene silencing ntawm Actinidia eriantha Benth. CHS txo cov ntsiab lus anthocyanin hauv cov paj thiab ua rau cov xim ntawm cov nplaim paj liab ua kom sib zog [15]. Yog li ntawd, hauv txoj kev tshawb no, CtCHS noob tau cloned los ntawm Cistanche deserticola paj, thiab bioinformatics tsom xam, prokaryotic qhia thiab purification, enzyme kev tsom xam, subcellular localization tsom xam thiab qhia kev tsom xam nyob rau hauv peb xim ntawm paj tau ua rau nws los tshawb txog lub functionality ntawm CtCHS. cov protein thiab cov qauv qhia ntawm CtCHS noob tau siv los tshawb nrhiav qhov kev sib raug zoo ntawm CtCHS thiab Cistanche deserticola paj xim, uas tau tsim lub hauv paus rau kev kawm txog kev tswj hwm ntawm Cistanche deserticola paj xim.

1 Cov khoom siv thiab cov cuab yeej siv

1.1 Cov khoom siv

Cistanche tuberosa paj tau sau los ntawm Yutian County, Hotan Prefecture, Xinjiang thaum lub Tsib Hlis 2018. Lub hauv paus ntsiab lus random tau ua raws li lub sijhawm ua piv txwv, thiab Cistanche tuberosa paj nrog lub cev zoo thiab cov nroj tsuag qhov siab tau raug xaiv. Peb hom Cistanche deserticola nrog peb lub paj xim dawb, liab, thiab ntshav tau coj thiab txheeb xyuas tias Cistanche tubulosa (Schenk) Wight los ntawm xibfwb Tu Pengfei ntawm Peking University Tsev Kawm Ntawv ntawm Pharmacy. Lawv tau khov sai sai hauv cov kua nitrogen, khaws cia rau ntawm cov dej khov qhuav, thiab thauj mus rau Beijing. E. coli DH5 chemically competent hlwb tau yuav los ntawm Nanjing Novezan Biotechnology Co., Ltd., thiab E. coli BL21 (DE3) chemically competent hlwb tau muas los ntawm Beijing Quanshijin Biotechnology Co., Ltd.; pET-28a, pCAMBIA1300-35S-GFP Lub vector tau yuav los ntawm Wuhan Transduction Biology Laboratory Co., Ltd. Malonyl-CoA tau yuav los ntawm Sigma Company, 4-coumaroyl-CoA, naringenin -chalcone tau yuav los ntawm Shanghai Yuanye Biotechnology Co., Ltd., thiab naringenin tau yuav los ntawm Shanghai McLean Biochemical Technology Co., Ltd.

1.2 Cov cuab yeej

Nanodrop 2000C spectrophotometer (Thermo Fisher Company, USA); gradient PCR ntsuas (Eppendorf Company, Lub teb chaws Yelemees); CFX 96 fluorescence quantitative PCR instrument, UVP gel imager, gel electrophoresis instrument, protein electrophoresis instrument (BioRad Company, USA); EnSpire

Microplate nyeem ntawv (PerkinElmer Company, USA); qhov kub thiab txias incubator (Shanghai Jinghong Experimental Equipment Co., Ltd.); DHZ-D loj-muaj peev xwm puv-kub oscillator (Taicang Experimental Equipment Factory); THZ-82A dej da dej tas li oscillator (Jiangsu Kexi Instrument Co., Ltd.); AIRTECH ultra-huv workbench (Suzhou Antai Cua Technology Co., Ltd.); high-pressure steam sterilizer (TOMY Company, Nyiv); Milli Q dej ntshiab (Millipore, United States); LC-20A high-efficiency kua Phase chromatograph (Shimadzu Company, Nyiv); UPLC-Q-Exactive-Orbitrap high-resolution mass spectrometry (Thermo Fisher Company, USA); FV1200 laser confocal microscope (Olympus Company, Nyiv).

2 Kev sim cov txheej txheem

2.1 RNA extraction thiab cDNA synthesis. Cistanche deserticola paj tau hauv av hauv cov kua nitrogen thiab tom qab ntawd raug RNA rho tawm kev soj ntsuam.

Tag Nrho RNA tau muab rho tawm los ntawm cov khoom siv (Norgen, Cat 17200), RNA concentration thiab zoo tau kuaj pom los ntawm NanoDrop 2000C, thiab RNA cov qauv nrog A260 / A280 piv ntawm 1.8 thiab 2.1 tau xaiv rau kev rov qab hloov pauv ntawm cDNA. M-MLV thim rov qab transcriptase (Promega, M170B) tau siv los thim rov qab-transcript tag nrho RNA tsim nyog rau hauv cDNA. Cov kev sim ua haujlwm tau ua raws li cov lus qhia.

2.2 Cloning ntawm CtCHS gene

Raws li cov ntaub ntawv Cistanche deserticola paj transcriptome ntawm peb cov kev tshawb fawb pawg [16], CtCHS nrog ib tug ua tiav qhib nyeem ntawv raug tshuaj xyuas, thiab SnapGene software tau siv los tsim cov primers tshwj xeeb raws li cov noob kab ke (Table 1). PCR amplification tau ua tiav siv Cistanche deserticola paj cDNA ua tus qauv. Lub tshuab ua kom nrov nrov yog 2 × Phanta Max Buffer 25 μL, dNTP Mix (10 mmol / L) 1 μL, sab sauv thiab sab hauv qab primers (10 μmol / L) 2 μL txhua, cDNA 2 μL, Phanta Max Super- Fidelity

DNA Polymerase 1 μL, ddH2O 17 μL. Cov kev mob tshwm sim yog: pre-denaturation ntawm 95 degree rau 3 min; 25 cycles ntawm denaturation ntawm 95 degree rau 15 s, annealing ntawm 55 degree rau 15 s, thiab extension ntawm 72 degree rau 1 min; thiab cov tshuaj tiv thaiv txuas ntxiv ntawm 72 degree rau 5 min. Cov khoom PCR tau kuaj pom los ntawm 1% agarose gel electrophoresis, thiab cov khoom siv DNA purification (Novizan, Cat DC301-01) tau siv rau gel rov qab, thiab tom qab ntawd cov DNA rov qab tau huv los ntawm cov khoom siv cloning ceev (Novizan, Cat. C601-01). Cov DNA tawg tau ligated nrog pCE2 TA / Blunt-Zero vector, hloov mus rau Escherichia coli DH5 cov hlwb muaj peev xwm, thiab tom qab culturing ib hmos ntawm 37 degree, ib leeg clone strains raug xaiv thiab xa mus rau lub tuam txhab rau sequencing.

Table 1 Primer sequences

2.3 Bioinformatics tsom xam

Siv ProtParam online software los txheeb xyuas cov khoom muaj protein ntau lub cev thiab tshuaj lom neeg; siv Prot Scale online cuab tam los txheeb xyuas cov protein hydrophilicity/hydrophobicity; siv cov cuab yeej online SOPMA los kwv yees cov qauv protein thib ob; siv online tsom xam software SWISS-MODEL los kwv yees protein tertiary qauv; siv online software TMHMM twv seb cov qauv transmembrane ntawm cov protein; siv DNAMAN software los sib piv homology ntawm CtCHS nrog rau CHS amino acid sequences ntawm lwm hom; siv MEGA-X software los tsim ib tsob ntoo phylogenetic, siv cov neeg zej zog koom nrog (NJ) thiab kev kho Poisson Qhov kev ncua deb yog xam los ntawm txoj kev Bootstrap, thiab tus naj npawb ntawm Bootstrap repetitions yog 1000 zaug.

UPLC-Q-Exactive-Orbitrap MS tau siv los tshawb nrhiav ntxiv. Cov txheej txheem ua kua yog Waters Acquity UPLC BEH Sheild RP18 kem (100 mm × 3.0 mm, 1.7 μm), kem kub 40 degree, naringenin , chalcone thiab naringenin. Lub ntim ntim ntawm cov tshuaj tswj yog 4 μL, thiab lub ntim ntim ntawm cov tshuaj tiv thaiv enzyme yog 7 μL. Dej (A)-acetonitrile (B) yog siv raws li theem mobile. elution gradient yog: 0 txog 5 min, 30% B; 5 rau 15 min. , 30% ~ 60% B; 15 ~ 20 min, 60% ~ 100% B; 20 ~ 25 feeb, 100% B; 25 ~ 31 min, 100% ~ 30% B. Tus nqi ntws tawm yog 0.3 mL / min. Qhov loj spectrometry tej yam kev mob yog electrospray ion qhov chaw, nitrogen li carrier gas, sheath gas siab 3.5 MPa, auxiliary gas siab 1.0 MPa, tshuaj tsuag siab 3500 V, capillary kub 350 degree, auxiliary gas cua sov kub 200 degree, zoo thiab tsis zoo ion hom, scanning ion range m/z yog 100-1500, Full MS daws teeb meem yog 35 000, dd-MS2 daws teeb meem yog 17 500, (N)

CE / steppednce teem rau 35, 60.

2.6 Subcellular localization ntawm CtCHS protein

Raws li CtCHS ib ntus thiab lub hauv paus ntsiab lus ntawm seamless cloning, primers nrog enzyme txiav qhov chaw Kpn Ⅰ thiab BamH Ⅰ tau tsim, thiab cov ntsiab lus sib thooj tau nthuav dav los ntawm PCR. Lub pCAMBIA1300-35S-GFP plasmid tau zom nrog kev txwv endonucleases Kpn Ⅰ thiab BamH Ⅰ, thiab lub hom phiaj fragment thiab vector tau txuas los ntawm cov khoom siv cloning seamless (Novizan, Cat C115-01), thiab tom qab ntawd xa mus. rau hauv E. coli DH5 cov hlwb muaj peev xwm. , xaiv ib hom clone rau sequencing, thiab rho tawm plasmids los ntawm cov hom raug. Cov pCAMBIA1300-35S-GFP-CtCHS thiab pCAMBIA 1300-35S-GFP plasmids tau hloov mus ua Arabidopsis thaliana protoplasts siv PEG4000, thiab kab lis kev cai nyob rau hauv qis qis rau 8 mus rau 10 teev. Lub subcellular localization ntawm CtCHS protein tau pom nyob rau hauv lub laser confocal microscope.

2.7 CtCHS nthuav qhia qauv tsom xam

Raws li CtCHS ib ntus, DNAMAN tau siv los tsim lub sijhawm tiag tiag fluorescence quantitative primers, nrog F-box ua cov khoom siv sab hauv. Cov primer sequences muaj nyob rau hauv Table 1. Cov txheeb ze qhia ntawm CtCHS nyob rau hauv Cistanche deserticola paj ntawm cov xim sib txawv tau kuaj pom los ntawm real-time fluorescence quantitative PCR. Siv TransStart Green qPCR SuperMix (tag nrho kub, AQ101-02) los ntsuas nrog SYBR Green fluorescent dye method, cov tshuaj tiv thaiv kab mob: 2 × TransStart Green qPCR SuperMix 5 μL, sab sauv thiab nqes dej primers (10

μmol/L) {{0}}.2 μL txhua, cDNA template 0.5 μL, nucleotidase-dawb dej 4.1 μL, ob-kauj ruam txoj kev siv rau cov tshuaj tiv thaiv, thiab cov txheej txheem tshuaj tiv thaiv yog raws li txoj kev ntawm Dong Xianjuan et al. [18]. Txhua tus qauv muaj 3 cov khoom siv roj ntsha, thiab qhov kev sim tau rov ua dua peb zaug. Cov ntaub ntawv sim tau txheeb xyuas siv Excel rau 2

Tus txheeb ze qhia ntawm CtCHS tau suav nrog siv −ΔΔCt txoj kev, thiab GraphPad Prism 8 tau siv los ua qhov tseem ceeb tsom xam thiab kos duab histograms.