CN103710369A - Bifunctional enzyme gene for kelp mannose 6-phosphate isomerization and GDP (Guanosine Diphosphatemannose)-mannose pyrophosphorylation - Google Patents
Bifunctional enzyme gene for kelp mannose 6-phosphate isomerization and GDP (Guanosine Diphosphatemannose)-mannose pyrophosphorylation Download PDFInfo
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Abstract
The invention relates to the technical field of genetic engineering, in particular to a bifunctional enzyme gene for kelp mannose 6-phosphate isomerization and GDP (Guanosine Diphosphatemannose)-mannose pyrophosphorylation. A gene nucleotide sequence and an amino acid sequence of encoded protein are SEQ ID NO:1 and SEQ ID NO:2 respectively; the gene cloning technology is adopted to clone a gene sequence which constructs an eukaryotic expression vector, and enzyme activity detection is carried out on recombinant protein, so that the enzyme gene is proved to have a weak function of catalyzing fructose-6-phosphoric acid and mannose-6-phosphoric acid to achieve an allosteric effect, as well as remarkable activity of GDP-mannose pyrophosphorylase, and therefore, the enzyme gene belongs to a key enzyme encoding gene in the sodium alginate biosynthetic pathway. The bifunctional enzyme gene has important application value on improving contents of sodium alginate and fucoidin of algae such as kelp.
Description
Technical field
The present invention relates to the codase gene of a main laminaria phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme.Be particularly related to the nucleotide sequence of a main laminaria (Saccharina japonica) phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene, and proteins encoded and in ability and the application in improvement Important Economic composition proterties of synthetic algin and fucoidin.
Background technology
Sea-tangle is marine plant (algae) kind of whole world Main Cultivation.As a kind of ocean vegetables, sea-tangle has very high nutritive value, and meanwhile, the Main Economic compositions such as algin, fucoidin (fucoidin), can be used as starting material and be widely used in all trades and professions.Algin of many uses, is mainly used in the aspects such as foodstuffs industry, medical and health, textile industry, scientific research.Fucoidin not only can be used as bonding agent and the resistance vapor of metal ion, also has as anti HIV-1 virus, anticoagulation, antithrombotic, physiological function and the effect such as antitumor simultaneously, and be a kind of important medicinal substance.
Clone products synthesis related gene is also verified its function, discloses the relation between gene and product, auxiliaryly carries out breed improvement and has become International Agriculture breeding field and improve one of effective way of economic sector content; Meanwhile, utilize genetic engineering technique production active substance and economic product to become the core content of modern biotechnology industry development.
Algin (Algin) is the alkaline metal salts such as water miscible sodium alginate, potassium and water-insoluble alginic acid (Alginic acid) and the alginic acid salt of being combined with the above metal ion of divalence thereof; Its basic structure is by the irregular polysaccharide being formed by connecting of β-1-4 glycosidic link by α-Isosorbide-5-Nitrae-L-guluronic acid block (α-L-guluronicacid, G) and β-Isosorbide-5-Nitrae-D-MANNOSE aldehydic acid block (β-D-mannuronic, M).
Fucoidin (fucoidin sulfuric ester, fucoidan or fucan sulfate, FS) be a kind of polysaccharide being mainly present in brown alga cell walls and some marine invertebrates, by L-fucose and sulfate group, formed, also have in addition D-wood sugar, D-semi-lactosi or uronic acid.
The biogenetic derivation of algin and fucoidin is only Phaeophyceae algae, marine invertebrate and microorganism, as true sea-tangle (Saccharina japonica), Sargassum (Sargassum), azotobacter vinelandii (Azotobacter vinelandii), marine Pseudomonas (Pseudomonas sp.), the false single-cell bacteria (Pseudomonas aeruginosa) of verdigris, Halomonas marina etc.
Different in the algin of different biogenetic derivations and fucoidin structure, but its building-up process is consistent substantially.It is comparatively detailed that the biosynthetic pathway of microorganism algin and fucoidin is studied, as pseudomonas (Pseudomonas aeruginosa), vinelandii (Azotobacter vinelandii) and γ-mycetozoan (Gammaproteo bacteria).In pseudomonas, synthetic path consists of polystep reaction, phosphomannose isomerase (AlgA), mannose-phosphate mutase (AlgC) and GDP-mannitol dehydrogenase (AlgD) are responsible for the precursor substance GDP-mannuronic acid (Guanosine diphosphate mannuronic acid, GDP-ManA) of synthetic algin and fucoidin.And this synthetic path remains indefinite in brown alga.The scholars such as Michel carry out bioinformatic analysis to long capsule water cloud (Ectocarpus siliculosus) genomic data, and the algin of the long capsule water cloud of having recombinated and fucoidin synthesize path.In its genome database, searched the gene PMI(phosphomannose isomerase with AlgA, AlgC, AlgD and AlgG homology, Mannose-6-phosphate isomerase), PMM(Phosphomannomutase), GMD(GDP-D-mannose dehydratase) and MC5E(Mannuronan C-5-epimerase).In the microorganisms such as bacterium, AlgA is a kind of bifunctional enzyme gene, it is the Type2 type of PMI, the first step and the three-step reaction of energy catalytic synthesis, but scholar has only searched single functional enzyme gene PMI in long capsule water cloud, do not find the enzyme gene GMP(GDP-D-mannose pyrophosphorylase of catalysis three-step reaction, GDP-D-mannose pyrophosphorylase, MPG), see Fig. 2.
The brown algas such as sea-tangle are algin and fucoidin natural origin, and it is exactly the main raw material that algin and fucoidin extract that the feature of its mcroorganism amount and high-content makes it all the time.But the gene pathway research for its synthetic algin and fucoidin is very weak, the codase gene that is converted into the reaction of GDP-seminose for its crucial catalysis Man-6-P still belongs to unknown, thereby the breed improvement of the brown alga plants such as sea-tangle based on gene clone cannot be realized.
Summary of the invention
For the technical problem existing in currently available technology, the invention provides a kind of phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene, a kind of phosphomannose isomery of its codified and GDP-seminose pyrophosphorylation bifunctional enzyme albumen, described albumen only has faint catalysis fructose-6-phosphate and Man-6-P allosteric enzyme (PMI) activity, but have enzyme (MPG) activity that significant catalysis Man-6-P and GDP-seminose transform, described gene and proteins encoded thereof can be used for improving algin and the synthetic content of fucoidin.
One of object of the present invention is to provide a kind of phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene, described gene is phosphomannose isomery and the GDP-seminose pyrophosphorylation bifunctional enzyme gene of separating from sea-tangle (Saccharina japonica), called after SjPMI4; The nucleotide sequence of described gene is as shown in SEQ ID NO:1.
Two of object of the present invention is to provide a kind of albumen of described genes encoding, nucleotide sequence coded by shown in SEQ ID NO:1 of described albumen, and its aminoacid sequence is as shown in SEQ ID NO:2; It only has faint catalysis fructose-6-phosphate and Man-6-P allosteric is active, but has the activity of significant catalysis Man-6-P and the conversion of GDP-seminose.
Three of the object of the invention is described gene and the application of proteins encoded in synthetic algin and fucoidin thereof.
Four of the object of the invention is described gene and the application of proteins encoded in improvement economic sector proterties thereof.
The present invention is by the method for gene clone, a phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene from sea-tangle, have been cloned into, and the albumen that has proved by experiment its coding only has faint catalysis fructose-6-phosphate and Man-6-P allosteric is active, but having the activity that significant catalysis Man-6-P and GDP-seminose transform, is the key gene of synthetic algin and fucoidin.Clone and analyze sea-tangle phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene contributes to deep understand the brown alga algins such as sea-tangle and fucoidin synthesis mechanism also can provide genetic resources for algin and fucoidin genetically engineered and molecular breeding simultaneously.The present invention is separated to phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme gene first from sea-tangle (Saccharina japonica), and by carrier for expression of eukaryon, recombinant protein is carried out to enzymic activity detection, confirmed that it has faint catalysis fructose-6-phosphate and Man-6-P allosteric active function, but there is the active function that significant catalysis Man-6-P and GDP-seminose transform simultaneously, there is the using value of algin and fucoidin genetically engineered and molecular breeding.
Accompanying drawing explanation
Fig. 1 is usingd GDP-seminose as the algin of precursor and the synthetic path schematic diagram of fucoidin.
Fig. 2 is the biological function of phosphomannose isomerase (PMI).
Fig. 3 is the pcr amplification figure of SjPMI4 gene cDNA total length of the present invention.
Fig. 4 is SjPMI4 gene of the present invention and encoding amino acid sequence (being respectively initiator codon and terminator codon in square frame) thereof.
Fig. 5 is that after SjPMI4 gene transformation yeast of the present invention, PCR detects positive colony pcr amplification figure.
Fig. 6 is SDS-PAGE detection figure after SjPMI4 gene transformation yeast expression product purification of the present invention.
Fig. 7 is the Western-Blot detection figure of SjPMI4 gene transformation yeast expression product of the present invention.
Fig. 8 is the differing temps of the SjPMI4 gene transformation yeast expression product of the present invention detection figure to enzymic activity.
Fig. 9 is the pH of the SjPMI4 gene transformation yeast expression product of the present invention detection figure to enzymic activity.
Figure 10 is the detection figure of the different metal ions enzyme activity of SjPMI4 gene transformation yeast expression product of the present invention.
Figure 11 is the different concentration of substrate enzymic activity variation diagrams of SjPMI4 gene transformation yeast expression product of the present invention
Figure 12 is the different concentration of substrate double reciprocal curve figure of SjPMI4 gene transformation yeast expression product of the present invention.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these examples are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted specific experiment condition in the following example, conventionally according to normal condition, molecular cloning experiment guide (Sambrook J, et al.2008.Molecular Cloning:A Laboratory Manual, 3rd Ed.) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: the cloning and analysis of full length gene coding region
Sea-tangle gathers from Rongcheng City, Shandong Province, and acquisition time is in July, 2011.Adopt Trizol method to extract the total RNA of Female Gametophytes of Laminaria Japonica, use the PrimeScript II1 of TAKARA company
stit is template that Strand cDNA Synthesis test kit be take the first chain cDNA of the total RNA reverse transcription of kelp gametophyte, utilizes cDNA end rapid amplifying technology, uses BDSMART
tMrACE cDNA Ampl ification Kit carries out RACE-PCR amplification, primer is respectively 5 '-AGGGCTACGAGCACAAGGAAAAGGGGG-3 ' and 5 '-GAC CTCTACAAGGATGACAACCACAAGC-3 ', RACE-PCR amplification program is: 94 ℃ of 30sec, 72 ℃ of 3min, 5 circulations; 94 ℃ of 30s, 70 ℃ of 30s, 72 ℃ of 3min, 5 circulations; 94 ℃ of 30s, 68 ℃ of 30s, 72 ℃ of 3min, 20 circulations; 72 ℃ of 10min.According to RACE sequencing result, adopt TouchdownPCR technology and regular-PCR technology to carry out the amplification of the CDS full length sequence of sea-tangle PMI gene, amplimer comprises 3 groups of (5 '-CGACCCGACCTCTACAAGGATGACAACC-3 ' and 5 '-TCATTGGCAGCCATGAAGAACGACTCTC-3 '; 5 '-CGAGACCGAGGAGGGCAAGATGTAC-3 ' and 5 '-CTCATTGGCAGC CATGAAGAACGAC-3 '; 5 '-GCCCTTCGTCACACACACGAC-3 ' and 5 '-CGGTGCAGGTTGAACACAATCT-3 ').PCR product is after 1% agarose gel electrophoresis detects, and the blob of viscose that cutting contains object band under ultraviolet lamp, is used sepharose to reclaim test kit and reclaim object fragment, in-20 ℃ of preservations.The object fragment reclaiming, in 16 ℃ of metal baths, spend the night and be connected to cloning vector pMD19-T, and be transformed in competent escherichia coli cell E.coli Top10, coat on the LB solid medium that contains 100mg/mL Amp, 37 ℃ of incubated overnight, after the blue hickie screening of IPTG/X-gal, a picking 4-10 positive colony checks order.Sequencing result, by sequence alignment, has been separated to a sea-tangle PMI gene, called after SjPMI4.SjPMI4CDS sequence total length is 1020bp, and its nucleotide sequence is as shown in SEQ ID NO:1, and 339 amino acid of encoding, take ATG as initiator codon, and T AA is terminator codon, is the 3'UTR sequence of 588bp afterwards.
Preparation and the analysis of embodiment 2:SjPMI4 proteins encoded
Sea-tangle SjPMI4PCR product is after 1% agarose gel electrophoresis detection, under ultraviolet lamp, cut object band, sepharose reclaims, reclaim product S jPMI4 and pPICZ α A plasmid and carry out EcoRI and NotI double digestion, after 37 ℃ of metal bath 3-4h, with 1% agarose gel electrophoresis, detect, use sepharose to reclaim test kit and reclaim.Object fragment SjPMI4 is connected with plasmid pPICZ α A, and 16 ℃ are spent the night, the recombinant plasmid called after pPICZ α A-PMI4 building.
Use restriction endonuclease DraI to carry out linearization process recombinant plasmid pPICZ alpha A-PMI4, the distilled water with sterilizing after purifying dissolves, and by electric shock, transforms in pichia pastoris phaff X-33 competent cell.The cell culture of having hatched is coated and contained different concns Zeocin
tMyPD flat board on, every 50-200 μ L coating one flat plate; Flat board is placed in to the incubator dark culturing 2-4 days of 28 ℃, until single bacterium colony occurs.
With boiling, freeze the method for boiling and prepare pichia pastoris phaff pcr template, (according to the sequencing result having obtained, design is with forward and reverse primer of restriction enzyme site, containing the forward primer 5 '-CAGC of EcoRI restriction enzyme site to utilize primer
gAATTCaTGAGACGCCTGAATTGC-3 ' and containing the reverse primer 5 '-TAT of NotI restriction enzyme site
gCGGCCGCtATCAAACTCCAAATCC-3 ') carry out the screening of positive colony, PCR detects recon.PCR product detects in 1% agarose gel electrophoresis, and gel image analysis instrument imaging automatically, is used above-mentioned primer amplification PMI4CDS full length sequence, obtains the PCR product that length is about 1000bp, in the same size with expection.Picking electrophoresis detection is inserted the correct cloning and sequencing of band, detects and has or not sudden change, and whether frameshit frame changes.Get the bacterium liquid that the correct positive colony of order-checking preserves and be inoculated in 50mL BMGY substratum, 28 ℃, 250rpm/min cultivates, and 24h is 0.5% to adding methyl alcohol to final concentration in fermented liquid.The broken bacterium liquid of ultrasonic method, collect the supernatant liquor after fragmentation, membrane filtration with 0.45 μ m, after Ni column purification, the protein sample of collecting is put into dialysis tubing and is dialysed, and to remove unwanted ion, obtains restructuring PMI4 albumen, its aminoacid sequence, as shown in SEQ ID NO:2, adopts SDS-PAGE, Western-Blot to detect the expression of recombinant protein.
The functional verification of embodiment 3:SjPMI4 proteins encoded
PMI enzyme activity determination: with reference to the two step coupling methods of (2008) such as MarutaT, and slightly transform.Reaction system is as follows: the NADP+ that contains 0.5mM in the damping fluid of the Tris-HCl of 50mM pH7.5,5mM MgCl2,1U/mL PGI(Glucose-6-phosphate isomerase), 1U/mL G6PDH(Glucose-6-phosphate dehydrogenase), the M-6-P(Mannose-6-phosphate of 1mM) and the restructuring PMI4 albumen of appropriate embodiment 3 preparations, total reaction system is 300 μ L, adds substrate M-6-P initial action.Initial action after hatching 2min after above-mentioned system of removing substrate is mixed under relevant temperature condition, take corresponding damping fluid as blank, the variation of assaying reaction 0min, 6min and 12min light absorption value respectively at 340nm place, each reaction arranges 4 Duplicate Samples.After testing, PMI4 albumen PMI enzymic activity is lower, and enzyme is lived as 0.81U/mg, and optimal reactive temperature is 15 ℃, and optimal pH is 8.5, and this enzyme is cold-adapted enzyme, basic protein.Zn
2+, Cu
2+and Mn
2+enzymic activity is had to restraining effect; And Ca
2+and Mg
2+enzyme is lived substantially without impact; Co
2+there is the effect that promotes that enzyme is lived.
MPG enzyme assay: the damping fluid of 1ml50mM pH7.6Tris-HCl, 4mM glucose, 1mM ADP, 1mMNADP, 10mM MnCl
2, 1U hexokinase, 1U nucleoside-50-diphospho-kinase, 1U glucose-6-phosphate dehydrogenase (G6PD) and concentration range are 0.05 to 5mM GDP-seminose.The trisodium phosphate that reaction is 2mM by the restructuring PMI4 albumen that adds embodiment 3 to prepare and final concentration is initial.After above-mentioned system is mixed, under relevant temperature condition, hatch initial action, take corresponding damping fluid as blank, the variation of assaying reaction 0min, 3min and 6min light absorption value respectively at 340nm place, each reaction arranges 4 Duplicate Samples.Enzyme is lived as 3.28U/mg, and Km value is 32.44mM, and optimal reactive temperature is 40 ℃, and optimal pH is 7.0.This enzyme is high temperature enzyme, neutral protein; Mn
2+, Ca
2+, Cu
2+and Mg
2+can promote enzyme to live, Zn
2+suppress its activity.
Although the invention describes concrete example, having is a bit significantly to those skilled in the art, can the present invention be made various changes and be changed under the premise without departing from the spirit and scope of the present invention.Therefore, claims have covered all these changes within the scope of the present invention.
Claims (4)
1. the encode gene of phosphomannose isomery and GDP-seminose pyrophosphorylation bifunctional enzyme in sea-tangle, is characterized in that, the nucleotide sequence of described gene is as shown in SEQ ID NO:1.
2. the albumen by genes encoding described in claim 1, it is characterized in that, the aminoacid sequence of described albumen is as shown in SEQ ID NO:2, it only has faint catalysis fructose-6-phosphate and Man-6-P allosteric is active, but has the activity of significant catalysis Man-6-P and the conversion of GDP-seminose.
3. the application of bifunctional enzyme gene claimed in claim 1 in synthetic algin and fucoidin.
4. the application of albumen in synthetic algin and fucoidin described in claim 2.
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Cited By (6)
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| CN107267533A (en) * | 2017-07-26 | 2017-10-20 | 青岛海大蓝科生物科技有限公司 | The GMD genes and its protein and purposes of the dehydrogenase of GDP mannoses 6 are encoded in long capsule water cloud |
| CN107287170A (en) * | 2017-08-16 | 2017-10-24 | 青岛海大蓝科生物科技有限公司 | UGD genes, its protein and purposes in one main laminaria |
| CN107354164A (en) * | 2017-07-26 | 2017-11-17 | 青岛海大蓝科生物科技有限公司 | UGD genes, its protein and the purposes of the dehydrogenase of GDP glucose 6 are encoded in long capsule water cloud |
| CN107502615A (en) * | 2017-10-16 | 2017-12-22 | 中国海洋大学 | The gene and its protein and purposes of the dehydratase of GDP mannoses 4,6 are encoded in sea-tangle |
| CN107828750A (en) * | 2017-11-22 | 2018-03-23 | 青岛海大蓝科生物科技有限公司 | One main laminaria GDP fucoses synzyme and its encoding gene and purposes |
| CN108034000A (en) * | 2017-11-17 | 2018-05-15 | 四川大学 | Chinese cymbidium mannose-binding protein |
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| CN107267533A (en) * | 2017-07-26 | 2017-10-20 | 青岛海大蓝科生物科技有限公司 | The GMD genes and its protein and purposes of the dehydrogenase of GDP mannoses 6 are encoded in long capsule water cloud |
| CN107354164A (en) * | 2017-07-26 | 2017-11-17 | 青岛海大蓝科生物科技有限公司 | UGD genes, its protein and the purposes of the dehydrogenase of GDP glucose 6 are encoded in long capsule water cloud |
| CN107287170A (en) * | 2017-08-16 | 2017-10-24 | 青岛海大蓝科生物科技有限公司 | UGD genes, its protein and purposes in one main laminaria |
| CN107287170B (en) * | 2017-08-16 | 2020-02-21 | 青岛海大蓝科生物科技有限公司 | UGD gene in kelp, protein and application thereof |
| CN107502615A (en) * | 2017-10-16 | 2017-12-22 | 中国海洋大学 | The gene and its protein and purposes of the dehydratase of GDP mannoses 4,6 are encoded in sea-tangle |
| CN107502615B (en) * | 2017-10-16 | 2020-05-05 | 中国海洋大学 | Gene encoding GDP-mannose-4,6-dehydratase in kelp, and protein and use thereof |
| CN108034000A (en) * | 2017-11-17 | 2018-05-15 | 四川大学 | Chinese cymbidium mannose-binding protein |
| CN107828750A (en) * | 2017-11-22 | 2018-03-23 | 青岛海大蓝科生物科技有限公司 | One main laminaria GDP fucoses synzyme and its encoding gene and purposes |
| CN107828750B (en) * | 2017-11-22 | 2020-11-10 | 青岛海大蓝科生物科技有限公司 | Kelp GDP fucose synthetase, and coding gene and application thereof |
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