Cloning, Functional Identification and Expression Analysis Of Chalkone Synthase Los Ntawm Cistanche Tubulosa Ⅱ
Mar 25, 2024
3 Cov txiaj ntsig thiab kev tshuaj xyuas
3.1 Cloning ntawm CtCHS gene
Plaub kab lus piav qhia raws li "chalcone synthase" tau tshuaj xyuas siv cov ntaub ntawv sau tseg ntawm Cistanches tubulosa paj. Los ntawm NCBI qhov kev sib piv Blast, nws tau pom tias tsuas yog ib ntu muaj qhov ntev ntawm CHS gene thiab muaj FPKM ntau dua 10. Cov primers tshwj xeeb tau tsim los ntawm cov kab ke no, thiab ib feem ntawm 1200 bp tau txais los ntawm PCR amplification siv. Cistanches tubulosa paj cDNA ua tus qauv (Daim duab 1). Cov khoom PCR tau gel rov qab thiab txuas nrog pCE2TA / Blunt-Zero vector. Cov txiaj ntsig tau ua tiav tau zoo ib yam nrog kev txheeb xyuas cov ntawv sau tseg. Cov noob muaj npe hu ua CtCHS. Cov kab ke ntev yog 1173 bp thiab encodes 390 amino acids.
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3.2 Bioinformatics tsom xam ntawm CtCHS
3.2.1 CtCHS lub cev thiab cov khoom siv tshuaj lom neeg thiab kev tsom xam ntawm transmembrane
Cov software online ProtParam tau siv los txheeb xyuas lub cev thiab tshuaj lom neeg ntawm cov protein uas tau muab zais los ntawm CtCHS gene. CtCHS encodes 390 amino acids, cov qauv molecular yog C1908H3041N511O562S22, qhov txheeb ze molecular loj yog 42 836.54, theoretical isoelectric point yog 5.85, thiab nws tsis ruaj khov. Cov coefficient II yog 43.24, uas yog cov protein tsis ruaj khov, thiab tag nrho qhov nruab nrab hydrophilicity (GRAVY) yog −0.052, uas yog hydrophilic protein. Cov cuab yeej Prot Scale online tau siv ntxiv los txheeb xyuas cov protein hydrophilicity / hydrophobicity. Cov txiaj ntsig kev soj ntsuam tau pom tias tus nqi siab tshaj plaws ntawm CtCHS protein hydrophilicity / hydrophobicity yog 2.422 (342) thiab tus nqi qis tshaj yog −2.489 (119). Cov amino acid peptide saw encoded los ntawm CtCHS noob muaj ntau hydrophilic amino acid residues dua hydrophobic amino acid residues, yog li nws yog ib tug hydrophilic protein. Cov software online TMHMM tau siv los kwv yees cov qauv transmembrane ntawm cov protein. Cov protein CtCHS tsis muaj transmembrane domain.
Daim duab 1 PCR amplification ntawm CtCHS noob
3.2.2 Kev twv ua ntej ntawm cov qauv theem nrab thiab cov qauv tertiary ntawm CtCHS protein
Cov cuab yeej online SOPMA tau siv los kwv yees cov qauv protein thib ob. Cov txiaj ntsig tau pom hauv daim duab 2-A. 44.87% -helices, 31.03% random coils, 15.64% txuas chains thiab 8.46% -cov ntawv ua cov txheej txheem thib ob ntawm CtCHS protein. theem qauv. Lub software tsom xam hauv online SWISS-MODEL tau siv los kwv yees cov qauv tertiary ntawm cov protein, thiab cov qauv siv lead ua ntawm mov CHS1 tau siv los ua tus qauv los kwv yees peb-dimensional qauv ntawm CtCHS protein. Cov txiaj ntsig tau pom hauv daim duab 2-B. Qhov sib xws ntawm Cistanche tubulosa CtCHS thiab mov CHS1 yog 83.55%.
3.2.3 Sib piv sib piv thiab phylogenetic ntoo tsom xam thiab kev siv
DNAMAN software ua homology sib piv ntawm CtCHS thiab CHS amino acid sequences ntawm lwm hom. Cov txiaj ntsig tau pom hauv daim duab 3-C. CtCHS amino acid sequence yog raws li Sesamum indicum L. (XP_011091402.1) thiab Antirrhinum majus L. (P06515. 1), Mazus pumilus (NL Burman)Steenis (AAN05791.1), Erythranthe lewisiiG. L. Nesom & NS Fraga (AHJ80976.1), Perilla frutescens (L.) Britton (O04111.1) and Arabidopsis thaliana (L.) Heynh. (NP_196897.1). Cov amino acid sequences ntawm CHS yog qhov zoo sib xws, nrog rau qhov zoo sib xws yog 90.93%, 90.68%, 90.18%, 89.42%, 89.42%, thiab 81.61%.
NATURAL ORGANIC CISTANCHE EXTRACT nrog 30% ECHINACOSIDE thiab 12% ACTEOSIDE
Tsis tas li ntawd, ntau qhov kev sib txuas ua ke kuj tau pom tias CtCHS tau khaws cov catalytic residues Cys164, His303, thiab Asn336. Txhawm rau kom nkag siab ntxiv txog txoj haujlwm hloov pauv ntawm Cistanche tubulosa CtCHS hauv cov nroj tsuag CHS tsev neeg, MEGA-X software tau siv los tsim ib tsob ntoo phylogenetic ntawm CtCHS thiab CHS cov proteins ntawm lwm cov nroj tsuag. Cov txiaj ntsig tau pom hauv daim duab 3-DE lewisii AHJ80976.1) ElCHS muaj feem cuam tshuam nrog.
3.3 CtCHS prokaryotic qhia thiab purification
Cov pET-28a-CtCHS plasmid tau hloov mus rau hauv E. coli qhia hom BL21 (DE3), thiab cov protein qhia tau tshwm sim los ntawm IPTG ntawm qhov kub thiab txias. Cov protein tau huv si siv cov nickel ion affinity chromatography kem, thiab cov protein purified tau kuaj pom los ntawm 10% SDSPAGE. Cov txiaj ntsig tau pom nyob rau hauv daim duab 3. CtCHS protein band tshwm nyob ze 40, 000, uas zoo ib yam nrog cov txheeb ze molecular loj ntawm CtCHS protein ntawm 42,800.
3.4 CtCHS enzyme kev txheeb xyuas
Txhawm rau txheeb xyuas qhov ua haujlwm ntawm CtCHS, cov protein CtCHS purified tau tsim nrog malonyl-CoA, 4-coumaroyl-CoA thiab cov khoom raug kuaj pom siv HPLC thiab MS. Nyob rau hauv tej yam ntuj tso, naringenin-chalkone tsis ruaj tsis khov nyob rau hauv kev daws thiab yooj yim cyclized rau hauv naringenin. Yog li ntawd, naringenin-chalcone thiab naringenin raug xaiv raws li tus qauv tswj.
Cov txiaj ntsig ntawm HPLC tau pom hauv daim duab 4-A thiab B. Lub sijhawm khaws cia ntawm cov tshuaj tiv thaiv enzyme zoo ib yam li tus qauv naringenin. Ob leeg muaj qhov nqus siab tshaj plaws ntawm 289 nm hauv UV nqus spectrum, uas txawv ntawm lub sijhawm khaws cia ntawm naringenin-chalcone. Lub sij hawm thiab UV absorption spectra txawv, tab sis raws li kev tswj tsis zoo, cov khoom tom qab incubation ntawm high-temperature inactivated CtCHS protein nrog lub substrate tsis muaj naringenin, chalcone thiab naringenin. Kev tshawb nrhiav ntxiv los ntawm huab hwm coj spectrometry, thawj qib huab hwm coj spectrum (Daim duab 4-}C, D) pom tias cov khoom siv tshuaj tiv thaiv enzyme muaj [MH]-peak m/z271.060 97, thaum naringenin muaj [MH ]-peak m/z 271 060 91.
Muab piv rau MS2 spectra (Daim duab 5-E, F), CtCHS enzyme cov tshuaj tiv thaiv cov khoom thiab naringenin ob leeg muaj cov yam ntxwv fragment peaks ntawm m/z 107, 151, thiab 177, uas zoo ib yam nrog cov ntaub ntawv qhia [19], qhia tias CtCHS muaj chalcone complex. Enzymatic kev ua si, nws muaj peev xwm catalyze cov tshuaj tiv thaiv ntawm malonyl-CoA thiab 4-coumaroyl-CoA rau hauv naringenin-chalcone.
3.5 Subcellular localization tsom xam
Txhawm rau kawm txog lub luag haujlwm ntawm CtCHS cov protein hauv cov hlwb, pCAMBIA1300-35S-GFP-CtCHS plasmid tau tsim thiab hloov mus rau Arabidopsis thaliana protoplasts los soj ntsuam nws cov subcellular localization. Cov txiaj ntsig tau pom hauv daim duab 5. Raws li kev tswj hwm, pCAMBIA1300-35S-GFP protein tau muab faib rau hauv cov nucleus thiab cytoplasm, thaum pCAMBIA1300-35S-GFP-CtCHS fusion protein feem ntau yog muab faib rau hauv cytoplasm, qhia tau hais tias cov protein CtCHS tuaj yeem ua haujlwm hauv cytoplasm ntawm cov nroj tsuag. Muaj nuj nqi.
3.6 CtCHS nthuav qhia qauv tsom xam
qRT-PCR tau siv los txheeb xyuas qhov txheeb ze ntawm CtCHS hauv Cistanche deserticola paj nrog peb xim: dawb, ntshav thiab liab. Cov txiaj ntsig tau pom nyob rau hauv daim duab 6. Qhov kev qhia ntawm CtCHS hauv paj liab thiab paj liab tau nce siab dua li cov paj dawb. kom muaj nuj nqis. Piv nrog rau cov paj dawb, cov lus qhia theem ntawm CtCHS hauv paj liab yog 8.44 npaug siab dua, thiab qib qhia ntawm CtCHS hauv paj liab yog 3.21 npaug siab dua. Cov txiaj ntsig no qhia tau hais tias kev hloov pauv hauv CtCHS qhia qib yuav cuam tshuam rau qhov sib txawv ntawm paj xim ntawm Cistanche deserticola. Qhov siab dua qhov kev qhia theem, qhov tsaus xim paj. , qhov qis ntawm qhov qhia tawm, qhov sib zog ntawm lub paj xim.
****P＜0 000 1Fig. 6 Tus txheeb ze qhia theem ntawm CtCHS hauv paj nrog cov xim sib txawv
4 Kev sib tham
CHS yog aQhov tseem ceeb enzyme nyob rau hauv txoj kev flavonoid biosyntheticthiab muaj feem rau the kev cai ntawm paj ximnyob rau hauv ntau yam nroj tsuag. Piv txwv li, paj xim ntawmDianthus chinensisL[20] thiab Petunia × hybrida Hort. exVim[22] txhua tus raug cuam tshuam los ntawm CHS. tswj.Cistanches tubulosaFeem ntau muaj peb lub paj xim: dawb, liab, thiab ntshav, tab sis nws cov paj xim txheej txheem tseem tsis tau tshaj tawm. Hauv txoj kev tshawb no, CtCHS noob tau cloned siv RT-PCR los ntawmTranscriptome cov ntaub ntawv tsom xam ntawm Cistanches tubulosa paj, thiab nws
Bioinformatics tsom xamtau ua, thiab cov txiaj ntsig tau pom tias CtCHS muaj qhov zoo sib xws thiab kev sib raug zoo ntawm caj ces nrog cov amino acid ib ntus ntawm ElCHS ntawm Physalis nigra (AHJ80976.1). CHS yog hom III polyketide synthase (PKSIII), thiab cov kev tshawb fawb tau pom tias CHS muaj Cys-His-Asn catalytic triad [23]. Nyob rau hauv txoj kev tshawb no, amino acid ib theem zuj zus tsom xam pom tias CtCHS tau khaws cia catalytic residues Cys164, His303, thiab Asn336, qhia tias CtCHS tej zaum yuav muaj ib tug raug chalcone synthase muaj nuj nqi.
muaj peev xwm. Kev tshuaj xyuas enzyme ua haujlwm tau ua los ntawm kev siv prokaryotic qhia ntawm CtCHS protein hauv Escherichia coli thiab kuaj pom los ntawm HPLC thiab MS. Nws tau pom tias cov protein CtCHS tuaj yeem ua rau 4-coumaroyl-CoA thiab malonyl-CoA los tsim cov naringenin-chalcone.
CHS koom nrog hauv kev sib txuas ntawm flavonoids hauv ntau cov nroj tsuag, tab sis nws qhov chaw subcellular txawv ntawm ntau hom. Piv txwv li, Carthamus tinctorius L. CtCHS1[24], Morus atropurpurea Roxb. MaCHS[25], Eupatorium adenophorum Spreng. EaCHS[26] txhua tus nyob hauv cytoplasm, thaum Paeonia delavayi var. lutea (Delavay ex Franch.) Finet thiab Gagnep. PdCHS
[27], eggplant Solanum melongena L. SmCHS [28] yog nyob rau hauv lub cytoplasm thiab nucleus, thiab nyob rau hauv grape Vitis vinifera L. VvCHS nyob rau hauv lub cytoplasm, nucleus thiab cell phab ntsa [29]. Hauv txoj kev tshawb no, pCAMBIA1300-35S-GFP-CtCHS plasmid tau siv los hloov Arabidopsis thaliana protoplasts thiab nws tau pom tias CtCHS protein feem ntau nyob hauv cytoplasm, thiab tuaj yeem ua rau cov khoom tsim ntawm naringenin-chalcone hauv cytoplasm rau txhawb kev sib sau ntawm flavonoids.
Txoj kev phenylpropanoid ceg ntawm 4-coumaroyl-CoA mus rau hauv txoj kev flavonoid synthesis thiab lignan biosynthesis txoj kev. CHS catalyzes 4-coumaroyl-CoA thiab malonyl-CoA los tsim cov naringenin thiab chalcone los pib flavonoids. Txoj hauv kev biosynthetic ntawm cov khoom sib txuas zoo li yog thawj qhov tseem ceeb enzyme hauv txoj kev no [30]. Yog li ntawd, CHS qhia tau zoo heev rau cov nroj tsuag kom khaws cov flavonoids ntau dua thiab tsim cov paj tsaus. Piv txwv li, qhia theem ntawm CHS nyob rau hauv ntshav-paj ntau yam ntawm Hosta plantaginea (Lam.) Aschers yog siab dua li ntawm cov paj dawb. Hosta HpCHS tau hloov mus rau hauv cov luam yeeb kom tau Piv nrog rau cov yeeb yam qus, cov nroj tsuag overexpression muaj cov nplooj tsaus nti thiab siab dua tag nrho cov ntsiab lus anthocyanin [31].
Hauv txoj kev tshawb no, theem qhia ntawm CtCHS noob muaj feem xyuam nrog cov xim ntawm Cistanches tubulosa paj. Nws yog heev nthuav nyob rau hauv maub liab paj paj thiab liab paj, thiab yog tsawg nyob rau hauv lub teeb xim dawb paj. Qhov kev qhia txawv ntawm CtCHS yuav yog vim qhov txawv ntawm Cistanches tubulosa paj. Ib qho ntawm cov laj thawj uas muaj qhov sib txawv suits thiab xim. Hauv cov ntsiab lus, txoj kev tshawb no cloned CtCHS gene los ntawm Cistanche deserticola paj thiab ua bioinformatics tsom rau nws. CtCHS recombinant protein tau txais los ntawm exogenous qhia hauv Escherichia coli. Kev tshuaj ntsuam xyuas enzyme ua pov thawj tias cov protein CtCHS tuaj yeem ua rau 4-coumaroyl coenzyme. A thiab malonyl-coenzyme A tsim cov naringenin-chalkone. Nws tau pom tias CtCHS cov noob tau nthuav tawm hauv cov paj liab thiab paj liab thiab qis hauv paj dawb. Cov txiaj ntsig no qhia tau hais tias CtCHS noob tuaj yeem tswj hwm kev tsim cov paj sib txawv hauv Cistanche deserticola, uas muaj txiaj ntsig zoo rau kev tshawb fawb ntxiv ntawm cov txheej txheem tswj ntawm Cistanche tubulosa paj xim thiab muab cov ntaub ntawv siv rau kev tshawb fawb tom ntej ntawm lub luag haujlwm ntawm CtCHS hauv kev tswj hwm ntawm paj xim hauv Cistanche deserticola.
