CN104017798B - A kind of mutant of Momordica grosvenori SgCS gene and application thereof - Google Patents

A kind of mutant of Momordica grosvenori SgCS gene and application thereof Download PDF

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CN104017798B
CN104017798B CN201410245775.5A CN201410245775A CN104017798B CN 104017798 B CN104017798 B CN 104017798B CN 201410245775 A CN201410245775 A CN 201410245775A CN 104017798 B CN104017798 B CN 104017798B
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sgcs
yeast
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momordica grosvenori
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马小军
赵欢
唐其
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Guilin Jifosi Luohanguo Biotechnology Co ltd
Guilin Jifosi Siraitia Grosvenorii Co ltd
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Institute of Medicinal Plant Development of CAMS and PUMC
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Abstract

The present invention relates to the mutant protein of a kind of SgCS gene deriving from Momordica grosvenori, the nucleotide sequence of its coding, and this gene produces the application of momordica glycoside V in yeast host or plant host.

Description

A kind of mutant of Momordica grosvenori SgCS gene and application thereof
Technical field
The present invention relates to the mutant of a kind of SgCS gene deriving from Momordica grosvenori and in producing momordica glycoside V Purposes.
Background technology
Containing multiple saponin constituent in Momordica grosvenori (Siraitia grosvenorii), the most report and be isolated to more than 20 Planting Momordica grosvenori cucurbitane type tetracyclic triterpene type saponin constituent, wherein Momordia grosvenori aglycone IV, glycosides V and Simon glycosides I are to report up to now Mogroside in the highest 3 kinds of compositions of sugariness, respectively 392,425 and 563 times of sweetness of cane sugar.In all sweet glycosides, The content of momordica grosvenori glycoside V is the highest, accounts for the 20% of total saponin content；Simultaneously as main active component, momordica grosvenori glycoside V has Antibechic, eliminate the phlegm, Antispasmodic activity, have anticancer, anti-inflammatory, hypoglycemic waits effect, makes few in number excavating from Chinese medicine The novel sweetener with treatment function out.It is high that mogroside has sugariness, and heat is low, nontoxic, in good taste, the sweetest The special bitter taste of chrysanthemum glycosides, with the addition of momordica grosvenori glycoside V in the beverage that Coca-Cola Co. in 2008 newly releases, China in 2011 The Fructus Monordicae extract of 2 companies has also passed through the GRAS certification by U.S. FDA, successfully squeezes into American market.But due to The holding at high price of Momordia grosvenori aglycone extract on market, therefore cannot be with sugarcane in terms of food, health products, daily healthy articles for use Sugar competition.
But, the distribution of momordica grosvenori glycoside V has tissue specificity, only exists in ripening fruits, and content is the lowest only accounts for About the 1% of fruit dry weight, accounts for the 3-4 ‰ of fresh fruit content.The content that the means of conventional cross-breeding improve momordica grosvenori glycoside V is difficult Degree is big, and the cycle is long.First, wild Momordica grosvenori resource is the most almost become extinct, and cultivar unification is serious, and genetic background is narrow Narrow (genetic similarity is more than 0.9)；Secondly, the Momordica grosvenori of cross-pollination is height heterozygosis from the point of view of heredity angle, closely Hand over characters of progenies decline substantially, cultivate homozygous strain difficulty very big；3rd, the quality of Momordica grosvenori belongs to quantitative character, is subject to The controlled by multiple genes of minor effect, if the cultivation period that the merit on multiple parents concentrates on some kind is long；Again Person, as dioecious Momordica grosvenori, offspring's female-male proportion of its seedling is about 3:7, utilizes current means opening Judging its Sex Differentiation before spending, every strain plant floor space when plantation is about 4.5cm2, a large amount of combination high-quality shapes are sieved Menu strain seedling is by significant wastage soil and fund；Finally, the method for polyploid breeding is used to occur what fruit substantially diminished Problem.In recent years, improve Momordia grosvenori aglycone and become the research direction of hot topic containing quantifier elimination.According to statistics, sieve in 1 ‰ fruits is often improved Chinese fruit glycosides V content can reduce the extraction cost of 10%.It is therefore believed that start with from the biosynthetic relevant enzyme of momordica grosvenori glycoside V, By studying the gene function of relevant enzyme, regulate and control to solve the low difficult problem of Momordia grosvenori aglycone content, will be one the most directly, more Effective new way.
Existing report to a certain degree predicts mogroside biosynthesis pathway, first through mevalonic acid (MVA) and Two approach of methylerythritol phosphorylation (MEP) form isopentenyl diphosphate (IPP) and 3,3-dimethylallylpyrophosphate (DMAPP), can mutually convert between IPP and DAMPP, MVA approach occurs in kytoplasm, and MEP approach occurs in plastid.This Being the total approach of triterpenoid saponin biosynthesis, wherein the 3-hydroxy-3-methylglutaric acid list acyl coenzyme A in MVA approach is also Protoenzyme (HMG-CoA reductase, HMGR) catalysis 3-hydroxy-3-methylglutaric acid list acyl coenzyme A (HMG-CoA) generates first hydroxyl Valeric acid (mevalonate, MVA), this single step reaction is the irreversible process that need to rely on NADP, and HMGR is this approach In crucial rate-limiting enzyme, can be suppressed by the selectivity of Lovastatin.IPP and DMAPP can be by ox base pyrophosphate synthase (GPS) catalysis forms ox base pyrophosphoric acid (GPP), IPP Yu GPP is under the catalytic action of farnesyl pyrophosphate synthase (FPS) And then form farnesyl pyrophosphate (FPP), the most again through the catalysis shape of squalene synthase (squalene synthase, SQS) Squalene (Squalene), SQS is the rate-limiting enzyme in isoprene approach, is the albumen that is combined with film of a class, is to have two Planting the enzyme of function, farnesyl pyrophosphate (FPP) condensation that first can be catalyzed 2 molecules generates presqualene diphosphonic acid (presqualene diphosphate, PSPP), then changes into spiny dogfish by PSPP in the presence of NADPH and Mg2+ Alkene, as sterol in plant and first precursor of triterpenoid.SQS is the key in triterpene substance biosynthesis pathway Enzyme, it expresses height and the strong and weak content height directly determining downstream product of activity, the most bacterium, yeast, glossy ganoderma, Animal, the mankind and plant obtain clone and separate.The continuation effect formation 2 of squalene epoxidase, 3-oxidosqualene, this thing Matter is phytosterol and the common precursor material of triterpenoid saponin synthesis, and cyclisation one step of oxidosqualene determines walking of downstream To, it is phytosterol or triterpenoid saponin.In Momordica grosvenori, cucurbit dienol synthase (cucurbitadienol synthase, CS) and cycloartenol synthase (cycloartenol synthase, CAS) gene belongs to squalene cyclase gene family, its Middle CS can be cyclized into cucurbit dienol, through a series of add hydroxyl, add glycosyl reaction generate Momordia grosvenori aglycone, be sweet glycosides synthesis In key one step；And CAS can be cyclized into cycloartenol, modifying through base portion on this basis and obtain phytosterol, CAS is Phytosterol and first key enzyme of steroid compound synthesis.
Producing by active strictly the regulating and controlling of rate-limiting enzyme of isoprenoid material, cucurbit dienol synthase gene initially exists Report in cucurbita pepo (Cucurbita pepo), it is possible to 2,3-oxidosqualene generates cucurbit dienol as substrate, catalysis, Prove that cucurbit dienol closes enzymatic reaction and is independently of another approach of cycloartenol synthase cyclisation formation sterol simultaneously, because of It is the biggest that this improves its catalysis potentiality.At present it has been reported that CS gene only in cucurbita pepo, cucumber and Momordica grosvenori, cucumber In also have turned out CS there is the function of squalene cyclase.
At least from the point of view of above-mentioned known path, the substrate of SgCAS competition SgCS, fight for its carbon skeleton, towards plant steroid The direction of alcohol is developed, the remote-effects synthesis of Momordia grosvenori aglycone, it therefore may be anticipated that, CAS gene and CS gene will be sieve The Chinese fruit biosynthetic key node of glycosides V.
Momordica grosvenori research in terms of molecular biology be more common in the method by molecular labeling study genetic diversity, Affiliations etc., slightly aobvious relative to the research of other medicinal plants slow, the research for Momordica grosvenori functional gene at present rarely has Report, although can retrieve Momordica grosvenori cucurbit dienol synthase (SgCS) in GENEBANK, Momordica grosvenori cycloartenol closes The sequential structure of enzyme (SgCAS), but, inventor passes through the means of molecular biology with this existing gene pairs protokaryon and eucaryon When host transforms, after finding that this gene proceeds to host, it is impossible to form activated albumen, accordingly, it would be desirable to by certain Molecular biological function improves or molecular evolution methods, improves this gene order and optimizes, to overcome momordica grosvenori glycoside V raw Key difficulties in thing route of synthesis.
Summary of the invention
Present invention firstly relates to a kind of mutant protein deriving from Momordica grosvenori SgCS gene, its function fragment or function Territory, described mutant protein amino acid sequence is as shown in SEQ ID NO.2.
The invention still further relates to the encoding gene of the mutant protein of described SgCS gene, its function fragment, described coding The nucleotide sequence of gene is preferably as shown in SEQ ID NO.1.
The invention still further relates to the mutant of described Momordica grosvenori SgCS gene at catalysis squalene substrate synthesis cucurbit diene Application in alcohol, described application includes, by described mutant gene by genetic engineering means proceed to eucaryon yeast host, Plant host, makes described mutant high expressed, to be catalyzed MF59 substrate synthesis cucurbit dienol.Described host is preferably ferment Female IVF, arabidopsis, tobacco, Momordica grosvenori.
The present invention is further directed to the application in synthesis momordica glycoside V of the described mutant gene, and described should With including, by raising the expression of this gene in host, increasing the biosynthesis of momordica glycoside V, described host is energy Enough yeast by specific biosynthesis pathway synthesis momordica glycoside V or plant host.Described host is preferably yeast IVF, arabidopsis, tobacco, Momordica grosvenori.
Accompanying drawing explanation
Fig. 1, the structure of recombinant yeast expression vector: bacterium colony PCR detection after 1A. yeast recombinant plasmid transformed Escherichia coli (M:DL2000DNA molecular weight marker；4,13,17,20,21,22 and 23: positive colony)；1B. recombinant plasmid pYES2-SgCS's Double digestion electrophoresis detection (M:DL2000DNA molecular weight marker；1,2: the endonuclease bamhi of recombinant plasmid).
The secondary metabolite of Fig. 2, the GC-MS method detection yeast containing pYES2-SgCS recombinant plasmid: 2A: negative right According to；2B: cucurbit dienol standard items；2C: the yeast secondary metabolite containing recombinant plasmid.
The secondary metabolite of Fig. 3, the GC-MS method detection yeast containing unmutated SgCS: 3A: comparison；3B: do not dash forward GC-MS detection after the white Coding Sequence Transformed yeast of a kink of preserved egg
Fig. 4, bacterium colony PCR verify recombinant plasmid SgCS-ORF-pBI121.
Fig. 5, transformed plant screening on resistance culture base, 5A: transgenic Arabidopsis plants；5B: the plan of non-transgenosis South mustard adjoining tree.
Fig. 6, the arabidopsis of process LAN SgCS gene compares with the phenotype of wildtype Arabidopsis thaliana, 6A: process LAN SgCS gene Arabidopsis；6B: wildtype Arabidopsis thaliana.
Fig. 7, SgCS gene is quantitative fluorescence analysis result in wild type and transgenic arabidopsis.
Detailed description of the invention
Embodiment 1, the bioinformatic analysis of SgCS gene and the acquisition of mutant sequence
Using SgCS-ORF sequence as analysis of material, utilize the online software of ExPAEy Proteomics Server Protparam (http://web.expasy.org/protparam/) carries out physicochemical property to the albumen of SgCS gene code Prediction, the online software of Interproscan (http://www.ebi.ac.uk/Tools/pfa/iprscan/) predicted protein Conserved domain, utilizes the albumen that SgCS is encoded by the online software of SOPMA (http://www.ibcp.fr/predict.html) Carry out the prediction of secondary structure, carry out three through online software SWISS MODEL (http://swissmodel.expasy.org/) The prediction of level structure, by SignalP 4.1Server (http://www.cbs.dtu.dk/services/SignalP/), PSORT (http://wolfpsort.org/) and TMHMM Server v.2.0 software (http://www.cbs.dtu.dk/ Services/TMHMM/) signal peptide of this albumen, Subcellular Localization and the position in cross-film district are predicted.Utilize the BLAST of NCBI Analyze albumen and the homology of this albumen in other species of this gene code, and by MEGA6 software building Neighbor- Joining systematic evolution tree.(such as can be made by the method that protein structure is carried out mutation forecasting being combined with chemical modification method Evaluate and future position mutations on protein function by SIFT (Sorting Intolerant From Tolerant) program Impact etc.), it is thus achieved that through the mutein sequence of the function optimization of program prediction,
According in GengBank log in SgCS gene cDNA sequence design rite-directed mutagenesis primer (as shown in table 1 below, small letter Letter representation carries out the base of point mutation)
The design of table 1. mutant primer
First round PCR reacts: using cDNA as template, with SgCS-F1, SgCS-R1 and SgCS-F2, SgCS-R2 enters respectively Performing PCR expands, and obtains SgCS-ORF1 and SgCS-ORF2 fragment, separates detection through agarose gel electrophoresis, cuts glue respectively and reclaims. 2 kinds of glue reclaim product mix as the second template taking turns PCR, obtain mutator with primer SgCS-F1 and SgCS-R2 amplification Fragment, connects the named pMD19T-SgCS of carrier T, converts competent escherichia coli cell, selects positive colony and tests through order-checking Card, the gene order of the sudden change obtained is as follows:
This sequential structure is as shown in SEQ ID NO.1:
ATGTGGAGGTTAAAGGTCGGAGCAGAAAGCGTTGGGGAGAATGATGAGAAATGGTTGAAGAGCATAAGCAATCACTT GGGACGCCAAGTGTGGGAGTTCTGTCCGGATGCCGGCACCCAACAACAGCTCTTGCAAGTCCACAAAGCTTGTAAAG CTTTCCACGATGACCGTTTCCACCGAAAGCAATCTTCCGATCTCTTTATCACTATTCAGTATGGAAAGGAAGTAGAA AATGGTGGAAAGACAGCGGGAGTGAAATTGAAAGAAGGGGAAGAGGTGAGGAAAGAGGCAGTAGAGAGTAGCTTAGA GAGGGCATTAAGTTTCTACTCAAGCATCCAGACAAGCGATGGGAACTGGGCTTCGGATCTTGGGGGGCCCATGTTTT TACTTCCGGGTCTGGTGATTGCCCTCTACGTTACAGGCGTCTTGAATTCTGTTTTATCCAAGCACCACCGGCAAGAG ATGTGCAGATATGTTTACAATCACCAGAATGAAGATGGGGGGTGGGGTCTCCACATCGAGGGCCCAAGCACCATGTT TGGTTCC GCACTGAATTATGTTGCACTCAGGCTGCTTGGAGAAGACGCCAACGCCGGGGCAATGCCAAAAGCACGT GCTTGGATCTTGGACCACGGTGGCGCCACCGGAATCACTTCCTGGGGCAAATTGTGGCTTTCTGTACTTGGAGTCTA CGAATGGAGTGGCAATAATCCTCTTCCACCCGAATTTTGGTTATTTCCTTACTTCCTACCATTTCATCCAGGAAGAA TGTGGTGCCATTGTCGAATGGTTTATCTACCAATGTCATACTTATATGGAAAGAGATTTGTTGGGCCAATCACACCC ATAGTTCTGTCTCTCAGAAAAGAACTCTACGCAGTTCCATATCATGAAATAGACTGGAATAAATCTCGCAATACATG TGCAAAGGAGGATCTGTACTATCCACATCCCAAGATGCAAGATATTCTGTGGGGATCTCTCCACCACGTGTATGAAC CCTTGTTTACTCGTTGGCCTGCCAAACGCCTGAGAGAAAAGGCTTTGCAGACTGCAATGCAACATATTCACTATGAA GATGAGAATACCCGATATATATGCCTTGGCCCTGTCAACAAGGTACTCAATCTGCTTTGTTGTTGGGTTGAAGATCC CTACTCCGACGCCTTCAAACTTCATCTTCAACGAGTCCATGACTATCTCTGGGTTGCTGAAGATGGCATGAAAATGC AGGGTTATAATGGGAGCCAGTTGTGGGACACTGCTTTCTCCATCCAAGCAATCGTATCCACCAAACTTGTAGACAAC TATGGCCCAACCTTAAGAAAGGCACACGACTTCGTTAAAAGTTCTCAGATTCAGCAGGACTGTCCTGGGGATCCTAA TGTTTGGTACCGTCACATTCATAAAGGTGCATGGCCATTTTCAACTCGAGATCATGGATGGCTCATCTCTGACTGTA CAGCAGAGGGATTAAAGGCTGCTTTGATGTTATCCAAACTTCCATCCGAAACAGTTGGGGAATCATTAGAACGGAAT CGCCTTTGCGATGCTGTAAACGTTCTCCTTTCTTTGCAAAACGATAATGGTGGCTTTGCATCATATGAGTTGACAAG ATCATACCCTTGGTTGGAGTTGATCAACCCCGCAGAAACGTTTGGAGATATTGTCATTGATTATCCGTATGTGGAGT GCACCTCAGCCACAATGGAAGCACTGACGTTGTTTAAGAAATTACATCCCGGCCATAGGACCAAAGAAATTGATACT GCTATTGTCAGGGCGGCCAACTTCCTTGAAAATATGCAAAGGACGGATGGCTCTTGGTATGGATGTTGGGGGGTTTG CTTCACGTATGCGGGGTGGTTTGGCATAAAGGGATTGGTGGCTGCAGGAAGGACATATAATAATTGCCTTGCCATTC GCAAGGCTTGCGATTTTTTACTATCTAAAGAGCTGCCCGGCGGTGGATGGGGAGAGAGTTACCTTTCATGTCAGAAT AAGGTATACACAAATCTTGAAGGAAACAGACCGCACCTGGTTAACACGGCCTGGGTTTTAATGGCCCTCATAGAAGC TGGCCAGGCTGAGAGAGACCCAACACCATTGCATCGTGCAGCAAGGTTGTTAATCAATTCCCAGTTGGAGAATGGTG ATTTCCCCCAACAGGAGATCATGGGAGTCTTTAATAAAAATTGCATGATCACATATGCTGCATACCGAAACATTTTT CCCATTTGGGCTCTTGGAGAGTATTGCCATCGGGTTTTGACTGAATAA
Its encoding amino acid sequence structure is as shown in SEQ ID NO.2:
MWRLKVGAESVGENDEKWLKSISNHLGRQVWEFCPDAGTQQQLLQVHKACKAFHDDRFHRKQSSDLFITIQYGKEVE NGGKTAGVKLKEGEEVRKEAVESSLERALSFYSSIQTSDGNWASDLGGPMFLLPGLVIALYVTGVLNSVLSKHHRQE MCRYVYNHQNEDGGWGLHIEGPSTMFGSALNYVALRLLGEDANAGAMPKARAWILDHGGATGITSWGKLWLSVLGVY EWSGNNPLPPEFWLFPYFLPFHPGRMWCHCRMVYLPMSYLYGKRFVGPITPIVLSLRKELYAVPYHEIDWNKSRNTC AKEDLYYPHPKMQDILWGSLHHVYEPLFTRWPAKRLREKALQTAMQHIHYEDENTRYICLGPVNKVLNLLCCWVEDP YSDAFKLHLQRVHDYLWVAEDGMKMQGYNGSQLWDTAFSIQAIVSTKLVDNYGPTLRKAHDFVKSSQIQQDCPGDPN VWYRHIHKGAWPFSTRDHGWLISDCTAEGLKAALMLSKLPSETVGESLERNRLCDAVNVLLSLQNDNGGFASYELTR SYPWLELINPAETFGDIVIDYPYVECTSATMEALTLFKKLHPGHRTKEIDTAIVRAANFLENMQRTDGSWYGCWGVC FTYAGWFGIKGLVAAGRTYNNCLAIRKACDFLLSKELPGGGWGESYLSCQNKVYTNLEGNRPHLVNTAWVLMALIEA GQAERDPTPLHRAARLLINSQLENGDFPQQEIMGVFNKNCMITYAAYRNIFPIWALGEYCHRVLTE
Embodiment 2, utilizes Yeast expression SgCS gene to produce cucurbit dienol
Yeast expression carrier pYES2, yeast strain IVF are purchased from Invitrogen company；Yeast extract, peptone, fine jade Cosmetics is purchased from Sigma company；Other conventional medication are import or domestic AR.
YPD solid culture based formulas: 1% yeast extract, 1% peptone, 2% glucose, 2% agar powder
Yeast Extraction buffer: weigh HEPES 1.192g, 75mM EDTA (ethylenediamine tetra-acetic acid), 0.1% (v/v) Triton X-100 (Triton X-100), regulates pH to 7.4, is surely dissolved in 100mL.
SD solid medium: 0.67% yeast lacks ispol without amino acid media, 0.077%Ura (Clotech), 2% galactolipin
SDG fluid nutrient medium: 0.67% yeast is without amino acid media, and 0.077%Ura lacks ispol, 2% Galactolipin
1. the structure of Yeast expression carrier pYES2-SgCS
With the positive colony containing the SgCS gene suddenlyd change as template, design is containing restriction enzyme site (HindIII and BamHI) Primer, primer sequence is as follows:
Upstream primer: 5 '-AAGCTTATGTGGAGGTTAAAGGTC-3 '
Downstream primer: 5 '-GGATCCTTATTCAGTCAAAACCCG-3 '
The PCR clone SgCS full-length gene fragment containing HindIII and BamHI.Agarose gel electrophoresis separates detection, and glue returns Receive purpose band, connect pMD19-T vector Escherichia coli, select positive colony and pass through sequence verification.
Containing GAL1 promoter on Yeast expression carrier pYES2, extract the correct positive colony plasmid of order-checking and yeast table Reaching vector plasmid, with HindIII and BamHI, 2 kinds of plasmids are carried out double digestion reaction at 37 DEG C, system is as follows:
After endonuclease reaction 3h, digestion products carrying out gel electrophoresis separation, gel reclaims the purpose band in 2 kinds of products, Being attached reaction by proper proportion under the effect of T4DNA ligase, reaction system is as follows:
16 DEG C of reactions are overnight.Product full dose after connection converts bacillus coli DH 5 alpha competent cell, containing Amp (50mg/ L) screening on LB solid medium, picking list bacterium colony carries out bacterium colony PCR detection, empirical tests, obtains 7 sun in 23 single bacterium colonies Sex clone (see Fig. 1 a), obtains following result (see Fig. 1 b) through being digested qualification.Finally that positive colony is correct through sequence verification, To the monoclonal that order-checking is correct, named SgCS-pYES2, bacterium solution adds isopyknic 30% glycerine (final concentration 15%) Freezen protective is in-80 DEG C.
2. Momordica grosvenori pYES2-SgCS genetic transformation yeast IVF
Converting in recombinant plasmid SgCS-pYES2 to yeast strain IVF, concrete operation step is as follows:
1) frozen yeast strain IVF in-80 DEG C of refrigerators is taken out, on YPD solid medium, draws plate activated strains, 30 DEG C of incubators are cultivated.Picking list colony inoculation is in the YPD fluid nutrient medium of 2mL, and under the conditions of 30 DEG C, 250rpm shakes Overnight incubation, reaches more than 1 to OD600.Draw bacterium solution to be forwarded in the fresh YPD fluid nutrient medium of 20mL, control OD600 low In 0.3, then continue to cultivate 3h in 30 DEG C of incubators, can be used to convert as OD600=0.4～0.6.
2) bacterium solution being transferred in the centrifuge tube of sterilizing, at 4 DEG C 2,500rpm is centrifuged 5min and collects thalline, abandons supernatant.Add Entering the resuspended thalline of appropriate aqua sterilisa, at 4 DEG C 2,500rpm is centrifuged 5min, collects thalline, abandons supernatant.
3) in thalline, addition 1X LiAc-1XTE, with resuspended bacterium solution, makes the final concentration of 4% (v/ of 1X LiAc-1XTE V), it is dispensed in centrifuge tube, often pipe 100 μ L.
4) conversion fluid system is as shown in the table, fully mixes.
5) conversion fluid 200rpm shaken cultivation 30min in 30 DEG C of incubators after mixing.
6) in conversion fluid, add the DMSO88 μ L of sterilizing, mix gently, then at 42 DEG C of water-bath thermal shock 7min.
7) abandoning supernatant after 12,000rpm high speed centrifugation 5s, add the resuspended thalline of 1XTE of 1mL, repeated centrifugation abandons supernatant.
8) add the aqua sterilisa suspension thalline of 50 μ L, coat on SD solid medium flat board, 30 DEG C of incubators stand Cultivate 36-48h.
9) picking list bacterium colony, is inoculated in the liquid yeast culture medium SDG that 5mL contains galactolipin, shakes at 200rpm 30 DEG C Swing cultivation 36-48h, fully induce the expression of SgCS albumen.
3. extract the secondary metabolite of yeast
When detecting the OD600=0.8-1.0 of culture medium, 4 DEG C 3,000rpm is centrifuged 5min and collects thalline and weigh.At yeast In thalline, add Yeast protein Extraction buffer according to the ratio of 2mL/g, be subsequently adding isopyknic bead, on earthquake device Being immediately placed on ice after concussion 1min, this step is repeated 5 times, with fully cracking yeast thalline.Under the conditions of 4 DEG C 12,000rpm from Heart 10min, in the absorption supernatant containing Yeast protein to the new centrifuge tube of precooling, this step is repeated once.
Yeast self contains 2,3-oxidosqualene, if SgCS gene can successfully be expressed in yeast pYES2, that The albumen that SgCS expresses can be with 2, and 3-oxidosqualene is substrate, and catalysis generates cucurbit dienol.Therefore we use GC- The method of MS detects whether the generation of cucurbit dienol.First extract the secondary metabolite in yeast, collect 50mL's Yeast culture, centrifugal collection thalline；With lysate (20%KOH-50% ethanol) the resuspended thalline of 2mL, by bacteria suspension in 95 DEG C water-bath 45min, about 10min concussion mixing is once.Solution is cooled down after terminating by water-bath, adds 2mL n-hexane, mixes, 4, 500rpm is centrifuged 30s, draws upper strata supernatant and is placed in new centrifuge tube, and this step is repeated once.Blow concentration by nitrogen to collect Upper liquid, the isopropanol adding 300 μ L dissolves chromatography thing, by membrane filtration, finally carries out GC-MS detection, and result shows, is containing Have in the yeast of pYES-SgCS recombinant plasmid, equally when about 19.80 minutes retention times, occur in that and cucurbit dienol mark The peak that quasi-product are consistent, and in the appearance compareing not this peak in yeast without recombinant plasmid.(see Fig. 2).
Compared to SaCS gene after the sudden change constructed by the present invention, use identical transgenic method and yeast host, turn Entering not carry out to suddenly change after SgCS gene, the testing result for yeast secondary metabolite shows, unmutated SgCS gene, Identical yeast host cannot synthesize cucurbit dienol (see Fig. 3).
Embodiment 3, the structure of SgCS gene plant over-express vector and Agrobacterium-Mediated Transformation arabidopsis
Select wildtype Arabidopsis thaliana Arabidopsis thaliana (Columbia type) as the material of genetic transformation, intend South canola seed first soaking disinfection 15 minutes in the alcohol of 70-75%, then wash 2 times, then in sterilizing in absolute ethyl alcohol It is dried about 1 minute on filter paper.Vernalization treatment 4-7 days in 4 DEG C of refrigerators, dibbling is in vermiculite Nutrition Soil (ratio of 1:1) On cultivate in greenhouse to blooming.PBI121 plant expression vector (Clontech company).
The plant over-express vector of 1.SgCS gene builds；
1) XbaI and SalI is selected to build plant expression vector as the restriction enzyme site of upstream and downstream primer.Utilize Primer Premier 5.0 Software for Design contains the primer of restriction enzyme site, and upstream and downstream primer is as follows:
Contained the SgCS gene open reading frame of restriction enzyme site XbaI and SalI by PCR amplification with cDNA for template.
2) PCR primer being connected on pMD19-T carrier, through blue hickie screening, picking list bacterium colony does bacterium colony PCR checking, Select positive colony sequence verification.
3) order-checking is determined errorless single bacterium colony, expand to cultivate and extract plasmid.Utilize XbaI and SalI to even at 37 DEG C The carrier T connecing purpose fragment carries out double digestion reaction with pBI121 carrier simultaneously, and reaction system is as follows:
4), after endonuclease reaction terminates, first detection is separated through agarose gel electrophoresis, then by SgCS ORFs purpose bar Band and pBI121 carrier segments carry out cutting glue respectively and reclaim, and on Nanodrop, the concentration of product is reclaimed in detection.
5) by the connection of SgCS ORFs purpose fragment Yu pBI121 carrier segments, reaction system is as follows:
React at 16 DEG C overnight with reaction thoroughly.
6) take and all connect product, be transformed in the competent escherichia coli cell of DH5 α.37 DEG C of quiescent culture, picking list Bacterium colony, with the upstream and downstream primer SgCS process LAN 1-1 primer cloning ORFs and SgCS process LAN 1-2 as primer, is carried out Bacterium colony PCR verifies, chooses positive colony sequence verification.
7) check order the named SgCS-ORF-pBI121 of correct Positive recombinant clones.
2. the plant over-express vector SgCS-ORF-pBI121 of restructuring converts Agrobacterium GV3101；
1) take out frozen in the Agrobacterium GV3101 competent cell of-80 DEG C of refrigerators, thawed on ice.
2) the restructuring Subcellular Localization plasmid SgCS-pcYFP taking 10 μ L joins in 50 μ L competent cells, full dose sucking-off It is transferred in the electric revolving cup (BIO-RAD) of 0.2cm.
3) carry out on electroporation MicroPulser (BIO-RAD) electroporated, use Ec2 transform bacteria pattern, voltage For 2.5kV, the time is 4.5ms, treats that electricity revolving cup band metal both sides completely attach to the sheet metal of electroporation, and electric shock is until hearing " click " has saved electricity conversion.
4) in electricity revolving cup, add the LB fluid nutrient medium of 400 μ L, pressure-vaccum repeatedly.Repeating this step once, full dose proceeds to In EP pipe.
5) EP pipe is slightly centrifuged, abandons part supernatant, take appropriate bacterium solution and coat the LB solid culture containing Kan (50mg/L) On base, 28 DEG C of quiescent culture.Convert Agrobacterium GV3101 through bacterium colony PCR screening positive strain (Fig. 3), through sequence verification with The SgCS gene order known is consistent, it was demonstrated that the plant over-express vector of SgCS gene successfully constructs, named SgCS-ORF- pBI121。
The most agriculture bacillus mediated recombinant plasmid SgCS-ORF-pBI121 arabidopsis thaliana transformation；
Use classical Floral Dip method, arabidopsis thaliana transformation.
1) choosing the growth growing way of about 4 weeks healthy and strong, arabidopsis to be bloomed, with standby, removed top bud, is promoted it Lateral bud grows.
2) Agrobacterium that picking contains recombinant plasmid SgCS-ORF-pBI121 join 150mL containing rifampin (), to block that mould In the LB culture medium of element (), 230rpm shakes bacterium about 16-18 hour, to OD value between 1.2-1.6.Under greenhouse, 6,000rpm is centrifuged 5 minutes, abandon supernatant enrichment thalline.
3) by Agrobacterium thalline 300mL penetrating fluid (50g/L sucrose solution, the Silwet L-77 of 400-500 μ L) weight Outstanding, reach about 0.8 to OD value.
4) bacterium solution resuspended in previous step is poured in beaker, arabidopsis flowerpot is inverted, is buckled on beaker, it is ensured that be all Bud is immersed in bacterium solution, shakes Arabidopsis plant gently, soaks the about 30-60 second.
5) taking out plant, be placed in dark chest, keep high humility, lucifuge is cultivated 1 day.Then under normal lighting conditions Continue to cultivate.Converted once every 5-6 days, treat that full-bloom stage terminates.
6) after seed maturity, sowing is deposited in drier.
7) the arabidopsis seed gathered in the crops is after sterilization, and point is sowed at the MS after the sterilizing containing kanamycins (50 μ g/mL) On culture medium, it is placed in vernalization in the refrigerator of 4 DEG C and within 3-5 days, promotes to sprout, it is possible to that sprouts on resistance culture base is transgenosis Resistance seedling (see Fig. 5 A, B), then moves on to greenhouse and normally cultivates.
8), after green resistance seedling being grown on screening and culturing base, transplant and continue to cultivate to vermiculite Nutrition Soil, transgenosis The wildtype Arabidopsis thaliana of arabidopsis and comparison was sowed in the same time, and similarity condition is cultivated, and observes the phenotypic difference of the two.Wild The phenotype of type and transgenic arabidopsis has significant difference, wild type lotus throne base portion to have the multi-branched of similar tiller, and converts The lotus throne base portion of the arabidopsis of SgCS-ORF-pBI121 only has major branch, does not has branch (see Fig. 6 A, B), gathers in the crops transgenic arabidopsis Seed.
4. transgenic arabidopsis is real-time fluorescence quantitative PCR analysis
Utilize EASYspin Plus plant RNA rapid extraction kit, according to operating instruction, extract the wild type in 4-6 week With the total serum IgE of transgenic arabidopsis, after DNase I digested genomic dna, reverse transcription uses precious biological PrimeScriptTM RT Master Mix, 10 μ L reaction systems are as follows:
5X PrimeScript RT Master Mix 2μL
Total RNA ≤0.5μg
RNasw Free ddH2O polishing is to 10 μ L
Carrying out reverse transcription reaction after soft mixing, condition is as follows:
37 DEG C of 15min (reverse transcription reaction), 85 DEG C of 5s (inactivation reaction of reverse transcriptase).
With Actin as reference gene, internal reference upstream and downstream primer is respectively,
Upstream primer ' CAGATGCCCAGAAGTCTTGTTCC3 ' and downstream primer ' CGATTCCTGGACCTGCCTCATC '.
Utilizing Primer Premier 5 Software for Design SgCS gene for the primer of fluorescent quantitation, upstream and downstream primer is respectively For ' TGAGAAATGGTTGAAGAGC ' and ' CCATTTTCTACTTCCTTTCCAT '
Use the method detection SgCS gene expression in wild type and transgenic arabidopsis of real-time fluorescence quantitative PCR Amount, reaction system is 25 μ L.
Response procedures be 95 DEG C 30 seconds, 95 DEG C 5 seconds, 59 DEG C 10 seconds, 72 DEG C 10 seconds, 40 circulations, 65 DEG C-95 DEG C detections are molten Solution curve.Use 2-ΔΔctMethod carries out relative quantitative assay to the differential expression of genes of interest SgCS Yu reference gene Actin.
The fluorescent quantitation result of SgCS gene is shown in that Fig. 6, result show, in transgenic arabidopsis, SgCS gene is able to excess Express.
Finally, it should be noted that above example is only used for helping skilled in the art to understand the essence of the present invention, It is not construed as limiting the scope of the present invention.

Claims (4)

1. one kind derives from Momordica grosvenori SgCS albumen, it is characterised in that its amino acid sequence is as shown in SEQ ID NO.2.
2. the nucleotide sequence of coding albumen described in claim 1, it is characterised in that described sequence such as SEQ ID NO.1 Shown in.
3. the albumen described in claim 1, the nucleotides sequence described in claim 2 are listed in the application produced in cucurbit dienol, It is characterized in that, by engineered method, this albumen of high expressed in eucaryon host, catalysis squalene cyclisation, become calabash Reed dienol, described eucaryon host is yeast host or plant host, and described yeast is yeast strain IVF, and described plants Thing is arabidopsis or Momordica grosvenori.
4. the albumen described in claim 1, the nucleotides sequence described in claim 2 are listed in the application produced in momordica glycoside V, It is characterized in that, by engineered method, the expression of the described albumen in rise eucaryon host, thus improve Momordica grosvenori The yield of sweet glycosides V, described eucaryon host is yeast host or plant host, and described yeast is yeast strain IVF, described Plant is arabidopsis or Momordica grosvenori.
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