CN109337884A - A kind of gene encoding for pyruvate kinase and its application - Google Patents
A kind of gene encoding for pyruvate kinase and its application Download PDFInfo
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- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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- C12Y207/0104—Pyruvate kinase (2.7.1.40)
Abstract
The invention discloses a kind of gene encoding for pyruvate kinase, and the gene order of the pyruvate kinase is as shown in SEQ ID NO.1.The encoding amino acid sequence of the gene encoding for pyruvate kinase is as shown in SEQ ID NO.2.The gene order of the pyruvate kinase derives from M. truncatula.The present invention clones gene encoding for pyruvate kinase from M. truncatula, and has carried out system identification to its function, it was found that the gene not only can form pyruvic acid by catalytic phosphatase enol pyruvic acid in vitro, may also participate in the biosynthesis of flavonoids.
Description
Technical field
The invention belongs to the technical fields of genetic engineering, and in particular to a kind of gene encoding for pyruvate kinase and application thereof.
Background technique
Pyruvate kinase (PK) is the key that one of enzyme is adjusted in glycolytic cycle.PK is by turning phosphorylation for phosphorus
The phosphate group of sour enol pyruvic acid (PEP) is transferred to ADP, forms the ATP of a molecule and the pyruvic acid of a molecule.Pyruvic acid
Kinases by glycolytic pathway it is most important to the control of metabolic fluxes, it is often more important that its substrate phosphoenolpyruvate and
Product acetone acid has been involved in a variety of metabolic processes.Such as: pyruvic acid enters the tricarboxylic acid cycle of mitochondria, and phosphoenolpyruvate third
For ketone acid as glycolysis product to the intermediate material of protein and fatty acid synthesis conversion, it is intracorporal multiple that content influences plant
Metabolic process.Phosphoenolpyruvate participates in generating twice the process of shikimic acid, is that phosphoenolpyruvate is made for the first time
DAHP is synthesized in the case where DAHP (3-Deoxy-D-arabinoheptulosonate7-phosphate) synthesizes enzyme effect for substrate,
The other is phosphoenolpyruvate and shikimic acid -3- phosphoric acid (Erythrose-4-phosphate) are in EPSP (5-
Enolpyruvylshikimate3-phosphate the process that EPSP is generated under enzyme effect) is synthesized.To sum up, phosphoenolpyruvate third
Ketone acid is the important bridge formula intermediate product for connecting two approach, and pyruvate kinase exactly influences phosphoenolpyruvate and contains
One key enzyme of amount.
Pyruvate kinase is present in all biologies, the pyruvate kinase of non-plant origin by numerous studies, but
Be pyruvate kinase in plant research it is also very poor, be to exist mostly pyruvate kinase in the form of multicopy in plant,
Expansion in this evolution materially increases the diversity of gene encoding for pyruvate kinase expression and protein active, to make
It obtains plant and adapts to various environmental conditions complicated and changeable for greater flexibility.Pyruvate kinase plays in development of plants and metabolic process
Multi-functional all has important meaning to complicated metabolism and growth course in plant is understood to the research of pyruvate kinase function
Justice.But the gene encoding for pyruvate kinase function in plant, especially model legume M. truncatula or unknown at present,
Multi-functional and its effect and potential application value in plant metabolism in view of pyruvate kinase, clone and identification
Plant gene encoding for pyruvate kinase with function, specificity and bioactivity has important theory and application value.
Summary of the invention
In order to solve the problems in the prior art, the present invention provides a kind of gene encoding for pyruvate kinase and its applications, realize
Purpose be that the present invention clones the gene encoding for pyruvate kinase with catalytic activity from leguminous plant M. truncatula, and to it
Function is identified, produces active pyruvic acid, while gene encoding for pyruvate kinase has also assisted in the metabolism of flavonoids,
So also for it is other biology in using biotechnology adjust flavonoids biosynthesis provide important genetic resources and skill
Art method.
To achieve the goals above, technical solution provided by the invention is that the present invention provides a kind of pyruvate kinase bases
Cause, which is characterized in that the gene order of the pyruvate kinase is as shown in SEQ ID NO.1.
Further, the encoding amino acid sequence of the gene encoding for pyruvate kinase is as shown in SEQ ID NO.2.
Further, the gene order of the pyruvate kinase derives from M. truncatula.
The present invention also provides a kind of plasmids comprising the gene encoding for pyruvate kinase.
The present invention also provides the gene encoding for pyruvate kinase to improve the application in Flavonoid Content.
The present invention also provides the plasmids to improve the application in Flavonoid Content.
In above-mentioned technical proposal, the present invention clones gene encoding for pyruvate kinase from M. truncatula, and carries out to its function
System identification, it was found that the gene can generate active recombinant protein in bacterium, participate in the biosynthesis of flavonoids.
Detailed description of the invention
Fig. 1 is MtPK1 and the multiple alignment of other plant PK amino acid sequences is analyzed；
Fig. 2 is the phylogenetic analysis of plant pyruvate kinase；
Fig. 3 is the subcellular localization of MtPK1；
Fig. 4 is the SDS-PAGE glue figure of recombinant protein MtPK1；
Fig. 5 is influence of the pH to recombination MtPK1 albumen reaction rate；
Fig. 6 is influence of the PEP concentration to recombination MtPK1 albumen reaction rate；
Fig. 7 is influence of the ADP concentration to recombination MtPK1 albumen reaction rate；
Fig. 8 is the distribution expression pattern of MtPK1；
Fig. 9 is the transgenic line that PCR identification of M tPK1 is overexpressed in M. truncatula；
Figure 10 is R108, NF0791 and the relative amount for being overexpressed anthocyanidin in strain.
Specific embodiment
The present invention is made further instructions below in conjunction with attached drawing.
Embodiment 1
Present invention finds a kind of gene encoding for pyruvate kinase MtPK1, and gene order is as shown in SEQ ID NO.1, SEQ
ATG is initiation codon in sequence shown in ID NO.1, and TGA is terminator codon.The protein of gene coding, amino acid
Sequence is as shown in SEQ ID NO.2.Its purposes is the heterogenous expression pyruvic acid in other biologies, crosses scale in M. truncatula
Up to can produce flavonoids.
The identification method of gene encoding for pyruvate kinase of the present invention the following steps are included:
(1) clone of MtPK1 full length gene segment
PCR reaction is carried out according to the Phusion high-fidelity DNA polymerase specification that Thermo Fischer Scient Inc. provides
(table 1, table 2).
Table 1Phusion enzyme reaction system
The sequence of Forward primer therein are as follows: ATGATGGCAGAGAAGAAACC；The sequence of Reverse primer
It is classified as: TCATTTCACAGTCAAGATTTTG
Table 2.PCR program
(2) the glue recycling of purpose band
The specification provided according to Takara biomedical technology (Beijing) company carries out target fragment and A is added to react.According to
Following table configures reaction system (table 3), 72 DEG C of reaction 15min.Add A after the reaction was completed, recycles purpose band.
The Ago-Gel DNA QIAquick Gel Extraction Kit specification pair provided according to Beijing CoWin Bioscience Co., Ltd.
Purpose band carries out glue recycling.The weight for weighing centrifuge tube cuts the DNA of single goal band from Ago-Gel, is put into
In centrifuge tube, simultaneously calculated for gel net weight is weighed.
1) 100 μ L BufferPG/100mg gels are added.
2) 50 DEG C of water-baths incubate, and centrifuge tube to the blob of viscose that leniently turns upside down therebetween is completely dissolved.
3) 200 μ l BufferPS are added, into adsorption column to activate pillar.Room temperature 13000g is centrifuged 1min, outwells collection
Waste liquid in pipe.
4) it is down to room temperature to sol solution, is added into adsorption column, stand 2min.Room temperature 13000g is centrifuged 1min, outwells receipts
Waste liquid in collector.
5) 450 μ l BufferPW are added into adsorption column, stand 5min.Room temperature 13000g is centrifuged 1min.Outwell collecting pipe
In waste liquid.
6) room temperature 13000g is centrifuged 1min.Adsorption column is put into new centrifuge tube, is vacantly dripped to adsorbed film middle position
Add 50 μ l Buffer EB, is placed at room temperature for 2min.Room temperature 13000g is centrifuged 1min.Collected by agarose gel electrophoresis determines
The stripe size of DNA, and concentration, -20 DEG C of preservations are measured with NanoDrop 2000.
Table 3 recycles segment and adds A system
(3) connection of pCXSN and target DNA segment
Purpose recycling segment and Xcm I enzyme are carried out according to the specification that Takara biomedical technology (Beijing) company provides
The pCXSN carrier connection recycled after cutting.Reaction system (table 4) is configured according to following table, flicks mixing, 16 DEG C of metal baths are stayed overnight.
Table 4T4DNA connection enzyme system
(4) a small amount of extractions of plasmid
1) 50 μ L bacterium solutions are taken to be inoculated in LB liquid medium of the 5mL containing Carb, 37 DEG C of overnight shaking cultures.
2) bacterium solution being incubated overnight is added in 2mL sterile centrifugation tube, 12000g is centrifuged 1min, outwells supernatant.
3) 250 μ L P1 (confirmation addition RNAse Ι) are drawn to be added in above-mentioned bacterial sediment, keep thallus abundant with pipette tips piping and druming
It mixes.
4) 250 μ L P2 are drawn to be added in centrifuge tube, leniently turns upside down 7 times, cracks bacterium sufficiently.
5) 350 μ L N3 are drawn to be added in centrifuge tube, immediately gently overturns 10 times, makes to be sufficiently mixed.
6) room temperature 12000g is centrifuged 5min.
7) supernatant is drawn into adsorption column, and room temperature 12000g is centrifuged 1min.
8) waste liquid in collecting pipe is outwelled, 150 μ L PB are added into adsorption column.
9) room temperature 12000g is centrifuged 1min.
10) 400 μ L PW, room temperature 12000g are added into adsorption column and are centrifuged 1min.
11) adsorption column is put into a new 1.5mL centrifuge tube, draws the centre that adsorbed film is vacantly added in 50 μ L EB
Position is placed at room temperature for 5min.Room temperature 12000g is centrifuged 1min, -20 DEG C of preservation plasmids.
Gained transformant extracts plasmid and carries out double digestion identification.Positive transformant is accredited as after clone spreads cultivation, sample presentation
Sequencing.
(5) conversion of Escherichia coli (DH5 α and M15) competent cell
1) competent escherichia coli cell is taken to be placed on ice to melt.
2) plus 5 μ L plasmids, mixing are placed on 30min on ice.
3) 42 DEG C of thermal shock 1min take out rapidly, and are gently placed in 2min on ice immediately.
4) 500 μ L LB liquid mediums are added, mix.37 DEG C, shaken cultivation 1h.
5) bacterium solution is coated on the LB solid medium containing Carb with the spreading rod of sterilizing, super-clean bench seals after drying.
6) 37 DEG C, overnight stand culture.
7) bacterium solution PCR identification is carried out with sterile pipette tips picking monoclonal.
By carrying out clone and sequencing analysis discovery to MtPK1 gene, MtPK1 overall length is 2629bp, contains 3 exons
With 2 intrones, a protein sequence containing 497 amino acid is encoded, the isoelectric point (pI) of the protein prediction is 8.14,
Relative molecular mass is about 55.68kD.
Embodiment 2
According to the annotation in NCBIgenbank and KEGG SSDB database, respectively from arabidopsis, rice and M. truncatula
The full length sequence of 13,10 and 12 pyruvate kinases is obtained, sequence alignment and evolutionary analysis are used for.
Using DNAMAN carry out amino acid sequence homologous compare analysis, and according to 31.0 database of Pfam to it contained by
Conserved domain is analyzed；Systematic evolution tree is constructed according to Neighbor-joining method using Mega7 software.
The present invention has carried out bioinformatic analysis to the amino acid sequence that MtPK1 is encoded.By the amino acid sequence of MtPK1
Multiple sequence alignments analysis is carried out with the amino acid sequence of its homologous protein, compares analysis the result is shown in Figure 1, pyruvic acid swashs in Fig. 1
The β barrel-like structure domain of enzyme and α/β structural domain are respectively at 8-350 and 367-493 amino acid position；Solid line represents pyruvate kinase
Conservative domain activity site.Consensus indicates the completely the same amino acid of sequence, and conservative amino is emphasized with black.
By the result of Fig. 1 it is found that all pyruvate kinases are all containing a conservative Pyruvate kinase activity site: [LIVAC]-x-
[LIVM]-[LIVM]-[SAPCV]-K-[LIV]-E-[NKRST]-x-[DEQHS]-[GSTA]-[LIVM].The site is rich in bad
Propylhomoserin may play main catalytic action.Importantly, in 31.0 database of Pfam to structural domain contained by these sequences
It carries out analysis to find, other than the Pyruvate kinase activity site conservative containing one, have in the N-terminal of these pyruvate kinases
One β barrel-like structure domain (PF00224.14), the C-terminal at them have a α/β structural domain (PF02887.9), further illustrate
They belong to pyruvate kinase family.
The chadogram shown according to fig. 2, these pyruvate kinases are obviously polymerized to two main clusters: cytoplasm pyruvate kinase
(Cytosolic PK) and plastid pyruvate kinase (Plastidial PK) encodes the Gene A t3g49160 of class pyruvate kinase
Alone become cluster.PKc isodynamic enzyme is consistently polymerized to two tuftlets (Cytosolic-1 and Cytosolic-2), and PKp isodynamic enzyme also gathers
At two tuftlets (Plastidial- α and Plastidial- β).Cytoplasm pyruvate kinase MtPK1 and MtPK2, MtPK4 gather one
Cluster, their affiliation is most close, and amino acid sequence homology is up to 99%.It is distributed in 4 cytoplasms third of Cytosolic-2
Pyruvate kinase (MtPK5, MtPK6, MtPK9 and MtPK10) and its sequence homology are up to 68%.Cytoplasm pyruvic acid is belonged to swash
The homology of the OsPK1 (Os11g0148500) and MtPK1 of enzyme are also 68%.
Embodiment 3
The coded sequence of MtPK1, PCR are cloned with primer MtPK1SalF and MtPK1BamR with restriction enzyme site
Product is connected into the carrier pJIT163-hGFP after same digestion with restriction enzyme after SalI and BamHI digestion and is melted with constructing
Close gene M tPK1-hGFP.Correct structure will be sequenced and import arabidopsis leaf protoplast.25 DEG C of incubation 16h, Lycra laser are swept
Laser Scanning Confocal Microscope detection fluorescence is retouched using pJIT163-hGFP as positive control.
Belong to cytoplasm pyruvate kinase in order to further determine MtPK1, the present invention constructs the driving of CAMV 35S promoter
MtPK1-hGFP amalgamation and expression transient expression vector, and be transformed into arabidopsis mesophyll protoplast, utilize Leica
The positioning scenarios of confocal laser scanning microscope fluorescin.The green florescent signal of GFP is high-visible in cytosolic domain,
And the autofluorescence region of chloroplaset is almost without green fluorescence.Top layer is instantaneous in protoplasts of Arabidopsis thaliana broken by ultrasonic cell in Fig. 3
GFP is expressed, bottom is transient expression MtPK1-GFP fusion protein, passes through Leica confocal laser scanning microscope green fluorescence
The positioning scenarios of albumen.GFP and Bright field respectively indicates green fluorescence and bright field.Fig. 3's the result shows that: MtPK1
It is the pyruvate kinase of cytoplasm positioning, this utilizes arabidopsis, rice and the building of M. truncatula pyruvate kinase with front
Phylogenetic analysis and the cytoplasm positioning result that speculates is consistent.
Embodiment 4
The Function Identification method of gene encoding for pyruvate kinase of the present invention coding albumen the following steps are included:
Step (1), (2) are the same as (1), (2) the step of embodiment 1
(1) connection of pQE30 carrier and target DNA segment.
Digestion purpose band after the recovery and carrier are attached (table 5) according to following table reaction system.
Table 5T4DNA connection enzyme system
(2) induction, purifying of recombinant protein and the measurement of enzyme activity
1) take 20 μ L bacterium solutions in the LB liquid medium that 2mL is added to Carb, 37 DEG C of overnight shaking cultures.
2) bacterium solution being incubated overnight is added to 50% glucose of 300mL Carb containing 100mg/L and autoclave sterilization
In LB liquid medium, 37 DEG C of shaking table cultures to OD600Value is 0.5-0.6, takes 1mL bacterium solution, collects thallus as control.
3) 1M IPTG, the final concentration of 0.3mM of 300 μ L are added in 300mL bacterium solution, for 24 hours in 16 DEG C of cultures.
4) 4 DEG C, 9000g is centrifuged 5min, collects thallus.
5) lysozyme of the lysis buffer and 1mg/mL of 10 times of volumes is added, stands 30min on ice.
6) albumen is discharged to clasmatosis 10min with Ultrasonic Cell Disruptor.
7) 4 DEG C, 9000g is centrifuged 30min, collects supernatant.
8) nickel column for activating 8 times of column volume washbuffer of sample addition, after sample all flows through affine column packing,
Foreign protein is rinsed with the washbuffer of 20 times of column volumes, finally elutes target protein using 5 times of column volume elutebuffer.
Collecting protein is in the pipe of Millpore (30kDa), and 4 DEG C, 4000g is centrifuged 15min protein concentrate.
9) 1mL 100mM Tris-HCl is added, 4 DEG C, 4000g is centrifuged 15min, to wash away salt and imidazoles.
10) Coomassie brilliant blue is added with the ratio of 500:20 to be dyed, λ595nmMeasure the concentration of recombinant protein；Meanwhile
SDS-PAGE electrophoresis confirms the size and purity of recombinant protein.
11) measurement of Pyruvate kinase activity: 500 μ L enzyme activity reaction systems: 50mM HEPES-KOH (pH 6.4), 25mM
KCl, 12mM MgCl2, 2mM PEP, 1mMADP, 1mM DTT, 5% (w/v) PEG8000,0.15mM NADH and 2units/ml
Lactic dehydrogenase.Under the conditions of 28 DEG C of room temperature, 5min is reacted, in λ340nmMeasure light absorption value.
12) production of NADH standard curve: the NADH of 0,12.5,25,50,75,100 and 150nmol of various concentration is prepared
Solution, in λ340nmMeasure the variation of light absorption value.Standard curve is made according to the concentration of light absorption value and NADH.According to the standard of NADH
The activity of curve calculating pyruvate kinase.
It is in order to which the Pyruvate kinase activity to MtPK1 albumen detects, MtPK1 is gene constructed on pQE30 carrier,
The expression of fusion protein is carried out using prokaryotic expression system.Fusion protein is isolated and purified using nickel ion affine resin,
Purity (Fig. 4) through SDS-PAGE electrophoresis confirmation destination protein.Under the conditions of existing for the ADP, with K+And Mg2+As co-factor,
Pyruvate kinase can be catalyzed PEP and be converted to pyruvic acid, in the case that reduced coenzyme Ⅰ (NADH) there are, pyruvic acid is taken off by lactic acid
Hydrogen enzyme (LDH) is converted into lactic acid.NADH has strong light absorption at 340nm, calculates pyruvic acid according to the change of light absorption value
The activity of kinases.The result shows that when substrate and co-factor are all in saturation state, the recombination egg with His label that is purified
It is white to show very extensive pH spectrum, and reach optimal pH (Fig. 5) in pH 6.5 or so.In addition, in optimal pH, MtPK1 pairs
The kinetic constant Km value of PEP (Fig. 6) and ADP (Fig. 7) is 149 μM and 106 μM respectively.To sum up show that MtPK1 is one conservative
And the cytoplasm pyruvate kinase that has active catalytic activity.
Embodiment 5
(1) conversion of Agrobacterium competent cell
1) the Agrobacterium competent cell for taking -80 DEG C of refrigerators to save is in freeze thawing on ice.
2) 2 μ L plasmids are added into competence, flicks mixing, is placed in 15min on ice.
3) it is put in liquid nitrogen, quick-frozen 1min；37 DEG C, heat shock 5min.
4) LB liquid medium of 500 μ L not added with antibiotic, 28 DEG C of shaken cultivation 3h are added into aforesaid liquid.
5) with sterilizing spreading rod by bacterium solution be coated on plus corresponding antibiotic LB solid medium on, after sealing 28 DEG C it is quiet
Set culture.
6) picking monoclonal carries out bacterium colony PCR, after confirmation culture bacterium colony is positive, -80 DEG C of preservation strains.
(2) conversion of transgenic plant
1) activation of bacterial strain: 20 μ L are drawn after the pCXSN-MtPK1/EHA105 bacterial strain freeze thawing of -80 DEG C of preservations and are inoculated in 2mL
In LB culture medium containing antibiotic, 28 DEG C of overnight shaking cultures.
1) expand culture: the bacterium for taking 1mL to activate is inoculated in the antibiotic LB culture medium of 100mL, and 28 DEG C of shaken cultivations are extremely
OD600=0.6.
2) take 5-8 weeks M. truncatula tender leaf add 1 drop Tween-20 (surfactant) put tap water undershoot be washed till it is still
Foam.
3) 10% hypochlorite disinfectant 15min of 30mL is added；Meanwhile 6000g centrifugation 5min collects the bacterial strain shaken, uses
Sterile SH3a liquid is resuspended, spare.
4) blade 3-4 times sterilized with aseptic water washing, until being remained without sodium hypochlorite.
5) blade is cut out into wound with scalpel, the blade cut and bacterium solution is mixed, 1h is infected.
6) it is laid in SH3a solid medium, dark place co-cultures 2d.
7) it moves in the SH3a culture medium of added with antibiotic, callus induction.
8) callus is moved on SH9 solid medium after 4-5 weeks, induces transgenic plant.
10) transgenic plant aerial part, which is grown, moves back in 1/2SH9 culture medium root induction.
11) good to the root system development of transgenic plant, it is transplanted in the culture bottle equipped with sterile soil, is placed in group
Train room hardening.
12) the successful plantlet of transplant of hardening is in (25 ± 2 DEG C of phjytotron；18h illumination+6h is dark).
13) the DNA screening transgenic positive seedling of transgenic plant is extracted.
14) RNA for extracting positive seedling, analyzes its expression.Filter out high expression strain
15) positive seedling phenotype is observed, and the metabolite content of high expression strain is analyzed.
(3) measurement of anthocyanidin content
1) the M. truncatula fresh tissue sample under same growing environment is taken.
2) 50mg is accurately weighed after liquid nitrogen grinding being added, the methanol (salt for being 0.1% containing volume ratio of 5 times of volumes is added
Acid), ultrasonic 30min, 4 DEG C are overnight.
3) ultrasound 30min, 10000rmp are centrifuged 5min, take supernatant.
4) 750 μ L water and 750 μ L chloroforms are added, oscillation centrifugation takes supernatant.
5) it is measured at 530nm with ultraviolet specrophotometer.Using mole light absorption of Cyanidin -3-O- glucose as
The content of standard items estimation anthocyanidin.
In order to parse the mechanism of action of gene encoding for pyruvate kinase MtPK1 in M. truncatula, pass through fluorescence quantitative RT-RCR
Method is to MtPK1 in the seed that M. truncatula root, stem, leaf, flower and Post flowering 10d, 12d, 16d, 20d, 24d, 36d are harvested
Expression is studied.Result of study is shown in Fig. 8, expression of the MtPK1 gene in different tissues；MtPK1 is to acquire not
It include being extracted after seed material within 10 days, 12 days, 16 days, 20 days, 24 days, 36 days after root, stem, leaf, flower, beanpod and pollination with position
RNA and after being reversed to cDNA, using Mtactin2 as internal reference, carries out qRT-PCR.Fig. 8's the result shows that, in each of M. truncatula
Each period of position and seed development can detect the expression of MtPK1.Wherein, stem, beanpod and 10d, 12d, 16d,
Expression in 20d and 36d seed is higher, the expression highest in root and 24d seed.
Anthocyanidin in flavonoids is primarily present in the stem and leaf of M. truncatula, and the procyanidine in flavonoids is only being planted
It is accumulated in son, this is similar to the expression of MtPK1, illustrates that MtPK1 is related to the biosynthesis of anthocyanidin and procyanidine.
In order to further prove effect of the MtPK1 in anthocyanidin and procyanidine synthesis, the present invention is by tobacco mosaic disease
The gene encoding for pyruvate kinase MtPK1 of malicious CaMV 35S promoter driving is connected on carrier for expression of eukaryon pCXSN, by fusion
It is transferred in Agrobacterium EHA105.According to the method that the M. truncatula blade of mediated by agriculture bacillus converts, first lured under dark condition
Export callus is placed on the induction for starting regrowth under light.Firstly, being transformed into using 35S:pCXSN-MtPK1/GV3101 bacterial strain
In M. truncatula mutant NF0791, two resistance seedlings are obtained.MtPK1 gene is also transferred to M. truncatula wild type simultaneously
In R108, is identified through PCR and obtain 3 transgenic positive seedling OE-10, OE-26 and OE-30 (Fig. 9).The cauline leaf of overexpressing plants
Anthocyanin accumulation level is different, shows as most of transgenic line anthocyanidin contents and increases, stem and leaf base are purple.To mistake
The content of anthocyanidin is measured in expression strain, and the content of discovery OE-10 anthocyanidin is significantly higher than wild type and mutant (figure
10).The result shows that the higher material MtPK1 expression quantity of anthocyanidin content is high, the accumulation of stem's anthocyanidin is illustrated in these tables of data
It is positively correlated with the expression of MtPK1.
In summary, M. truncatula MtPK1 gene encodes a pyruvate kinase, the special substrate of MtPK1 proteins carry
Phosphoenolpyruvate forms pyruvic acid, and pH value will affect the catalytic efficiency of MtPK1, the acetone with known other plants
Acid kinase is different, and MtPK1 is a completely new plant pyruvate kinase.M. truncatula MtPK1 albumen is one and is positioned at cell
The albumen of matter can express in bacterium and plant, and the recombinant protein of expression is active, can in view of the overexpression of MtPK1
To improve the content of flavonoids, the content of anthocyanidin especially therein, therefore it can be used for microorganism or plant metabolism work
Journey production improves the yield of flavonoids.
Operation technique in the present embodiment without specified otherwise is the prior art, therefore excessive explanation herein.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Institute of Animal Sciences, Chinese Academy of Agricultural Sciences
<120>a kind of gene encoding for pyruvate kinase and its application
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<170> SIPOSequenceListing 1.0
<210> 1
<211> 1494
<212> DNA
<213>MtPK1 gene (Medicago truncatula)
<400> 1
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tctgttccaa tggtggagaa gcttctacaa gcaggcatga atgtcgctcg tttcaacttc 120
tctcatggtt cctatgaata tcatcaggaa acgcttgata atcttagaac cgctatgcaa 180
aataccggta ttctctgcgc cgtcatgctt gacactaagg ggccggagat tcgaaccgga 240
tttctcaagg atggaaagcc tgtccaactg aaacaaggtc aggaaataac catttcaacc 300
gactatgaca taaaaggaga tgagaatatg atctgtatga gctacaaaaa gttggcttat 360
gacgtgaagc ccggaagcat tgtattatgc gcagatggca ccatatcatt taaagtttta 420
tcatgtgaca aaaaagctgg tttggttcga tgctgctgtg aaaactctgc catgcttggc 480
gaaagaaaga atgttaatct tcctggagtc atagtggatc tcccaacatt gactgagaaa 540
gacaaggaag atatcatggt atggggagtt cctaataata ttgacatgat tgcactttct 600
tttgttcgaa aaggttctga tctggtggaa gttcgcaagt tgttgggaaa acatgctaag 660
aacatacttc tcatgtcaaa ggttgaaaac caagaaggag ttgcaaattt tgatgaaatc 720
cttacaaatt cagatgcatt tatggtggca cgtggcgacc ttggaatgga aattccaata 780
gagaagatat ttctagcaca aaaagtgatg atttataagt gtaatatcca aggaaagccg 840
gttgtcactg caacgcagat gttggagtca atgatcaaat cacctaggcc aaccagagct 900
gaagctactg atgtcgcgaa tgcagttctg gatggcacag attgtgtcat gcttagtggt 960
gaaactgctg ctggagctta tccagaactt gctgttcgaa ctatggctaa aatttgtgtt 1020
gaagctgaga gtaccatcaa ctatggagat gtatttaaaa ggataatgga gcactcacca 1080
gtaccgatgg gcccattgga gagtctagct tcttctgcgg ttaaaatggc aaactcagct 1140
aaagcagcac ttatattggt tttaactaga ggagggagta ctgcaaaatt agtggctaaa 1200
tatagggcag gcatgccaat tctttctgtt gtcgttcctg agattaagac cgataccttt 1260
gattggtcct gcagtgatga ggtccctgcc agacatagct tgatattccg aggattgatt 1320
ccagtactga gtgcgggttc tgctagagct tctcatgcag aaacaacaga agaggcacta 1380
gacttcgcca ttcagtatgc caaaacaaaa ggtctttgca ataacgggga ttctgtggtg 1440
gctctgcatc gtgtaggtgt cgcatcaatc atcaaaatct tgactgtgaa atga 1494
<210> 2
<211> 497
<212> PRT
<213>MtPK1 albumen (Medicago truncatula)
<400> 2
Met Met Ala Glu Lys Lys Pro Lys Thr Lys Ile Val Cys Thr Leu Gly
1 5 10 15
Pro Ala Ser Arg Ser Val Pro Met Val Glu Lys Leu Leu Gln Ala Gly
20 25 30
Met Asn Val Ala Arg Phe Asn Phe Ser His Gly Ser Tyr Glu Tyr His
35 40 45
Gln Glu Thr Leu Asp Asn Leu Arg Thr Ala Met Gln Asn Thr Gly Ile
50 55 60
Leu Cys Ala Val Met Leu Asp Thr Lys Gly Pro Glu Ile Arg Thr Gly
65 70 75 80
Phe Leu Lys Asp Gly Lys Pro Val Gln Leu Lys Gln Gly Gln Glu Ile
85 90 95
Thr Ile Ser Thr Asp Tyr Asp Ile Lys Gly Asp Glu Asn Met Ile Cys
100 105 110
Met Ser Tyr Lys Lys Leu Ala Tyr Asp Val Lys Pro Gly Ser Ile Val
115 120 125
Leu Cys Ala Asp Gly Thr Ile Ser Phe Lys Val Leu Ser Cys Asp Lys
130 135 140
Lys Ala Gly Leu Val Arg Cys Cys Cys Glu Asn Ser Ala Met Leu Gly
145 150 155 160
Glu Arg Lys Asn Val Asn Leu Pro Gly Val Ile Val Asp Leu Pro Thr
165 170 175
Leu Thr Glu Lys Asp Lys Glu Asp Ile Met Val Trp Gly Val Pro Asn
180 185 190
Asn Ile Asp Met Ile Ala Leu Ser Phe Val Arg Lys Gly Ser Asp Leu
195 200 205
Val Glu Val Arg Lys Leu Leu Gly Lys His Ala Lys Asn Ile Leu Leu
210 215 220
Met Ser Lys Val Glu Asn Gln Glu Gly Val Ala Asn Phe Asp Glu Ile
225 230 235 240
Leu Thr Asn Ser Asp Ala Phe Met Val Ala Arg Gly Asp Leu Gly Met
245 250 255
Glu Ile Pro Ile Glu Lys Ile Phe Leu Ala Gln Lys Val Met Ile Tyr
260 265 270
Lys Cys Asn Ile Gln Gly Lys Pro Val Val Thr Ala Thr Gln Met Leu
275 280 285
Glu Ser Met Ile Lys Ser Pro Arg Pro Thr Arg Ala Glu Ala Thr Asp
290 295 300
Val Ala Asn Ala Val Leu Asp Gly Thr Asp Cys Val Met Leu Ser Gly
305 310 315 320
Glu Thr Ala Ala Gly Ala Tyr Pro Glu Leu Ala Val Arg Thr Met Ala
325 330 335
Lys Ile Cys Val Glu Ala Glu Ser Thr Ile Asn Tyr Gly Asp Val Phe
340 345 350
Lys Arg Ile Met Glu His Ser Pro Val Pro Met Gly Pro Leu Glu Ser
355 360 365
Leu Ala Ser Ser Ala Val Lys Met Ala Asn Ser Ala Lys Ala Ala Leu
370 375 380
Ile Leu Val Leu Thr Arg Gly Gly Ser Thr Ala Lys Leu Val Ala Lys
385 390 395 400
Tyr Arg Ala Gly Met Pro Ile Leu Ser Val Val Val Pro Glu Ile Lys
405 410 415
Thr Asp Thr Phe Asp Trp Ser Cys Ser Asp Glu Val Pro Ala Arg His
420 425 430
Ser Leu Ile Phe Arg Gly Leu Ile Pro Val Leu Ser Ala Gly Ser Ala
435 440 445
Arg Ala Ser His Ala Glu Thr Thr Glu Glu Ala Leu Asp Phe Ala Ile
450 455 460
Gln Tyr Ala Lys Thr Lys Gly Leu Cys Asn Asn Gly Asp Ser Val Val
465 470 475 480
Ala Leu His Arg Val Gly Val Ala Ser Ile Ile Lys Ile Leu Thr Val
485 490 495
Lys
Claims (6)
1. a kind of gene encoding for pyruvate kinase, which is characterized in that the gene order of the pyruvate kinase is as shown in SEQ ID NO.1.
2. gene encoding for pyruvate kinase according to claim 1, which is characterized in that the coding amino acid sequence of the gene encoding for pyruvate kinase
Column are as shown in SEQ ID NO.2.
3. gene encoding for pyruvate kinase according to claim 1, which is characterized in that the gene order of the pyruvate kinase derives from puncture
Chenopodiaceae clover.
4. including the plasmid of gene encoding for pyruvate kinase described in claim 1.
5. the described in any item gene encoding for pyruvate kinase of claim 1-3 are improving the application in Flavonoid Content.
6. plasmid as claimed in claim 4 is improving the application in Flavonoid Content.
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| CN201811573141.7A CN109337884B9 (en) | 2018-12-21 | 2018-12-21 | Pyruvate kinase gene and application thereof |
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| CN109337884A true CN109337884A (en) | 2019-02-15 |
| CN109337884B CN109337884B (en) | 2021-09-17 |
| CN109337884B9 CN109337884B9 (en) | 2021-10-29 |
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Cited By (2)
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| CN109486838A (en) * | 2018-12-21 | 2019-03-19 | 中国农业科学院北京畜牧兽医研究所 | A kind of transcription factor gene and application thereof of regulation plant flavonoids synthesis |
| CN116004650A (en) * | 2022-08-31 | 2023-04-25 | 四川农业大学 | Gene OsPK7 for regulating male sterility of rice, encoding protein and application thereof |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109486838A (en) * | 2018-12-21 | 2019-03-19 | 中国农业科学院北京畜牧兽医研究所 | A kind of transcription factor gene and application thereof of regulation plant flavonoids synthesis |
| CN116004650A (en) * | 2022-08-31 | 2023-04-25 | 四川农业大学 | Gene OsPK7 for regulating male sterility of rice, encoding protein and application thereof |
| CN116004650B (en) * | 2022-08-31 | 2024-02-23 | 四川农业大学 | Gene OsPK7 for regulating male sterility of rice, encoding protein and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109337884B9 (en) | 2021-10-29 |
| CN109337884B (en) | 2021-09-17 |
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