CN109337884B9

CN109337884B9 - Pyruvate kinase gene and application thereof

Info

Publication number: CN109337884B9
Application number: CN201811573141.7A
Authority: CN
Inventors: 庞永珍; 范存莹; 狄少康
Original assignee: Institute of Animal Science of CAAS
Current assignee: Institute of Animal Science of CAAS
Priority date: 2018-12-21
Filing date: 2018-12-21
Publication date: 2021-10-29
Anticipated expiration: 2038-12-21
Also published as: CN109337884B; CN109337884A

Abstract

The invention discloses a pyruvate kinase gene, the gene sequence of which is shown in SEQ ID NO. 1. The coded amino acid sequence of the pyruvate kinase gene is shown as SEQ ID NO. 2. The gene sequence of the pyruvate kinase is derived from medicago truncatula. The invention clones pyruvate kinase gene from medicago truncatula and systematically identifies the function of the pyruvate kinase gene, and finds that the gene not only can catalyze phosphoenolpyruvate to form pyruvate in vitro, but also can participate in the biosynthesis of flavonoid.

Description

Pyruvate kinase gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a pyruvate kinase gene and application thereof.

Background

Pyruvate Kinase (PK), one of the key regulatory enzymes in the glycolysis process. PK transfers the phosphate group of phosphoenolpyruvate (PEP) to ADP by transphosphorylation, forming one molecule of ATP and one molecule of pyruvate. Pyruvate kinase is essential for the control of metabolic flows through the glycolytic pathway, and more importantly, its substrate phosphoenolpyruvate and product pyruvate are involved in a variety of metabolic processes. Such as: pyruvate enters the tricarboxylic acid cycle of mitochondria, while phosphoenolpyruvate acts as an intermediate in the synthetic conversion of glycolysis products to proteins and fatty acids, in amounts that affect multiple metabolic processes in the plant. Phosphoenolpyruvate is involved twice in the process of producing shikimic acid, the first time is the process of using phosphoenolpyruvate as a substrate to synthesize DAHP (3-Deoxy-D-arabinoheptaulonate 7-phosphate) under the action of DAHP synthetase, and the other is the process of using phosphoenolpyruvate and shikimic acid-3-phosphate (Erythrosite-4-phosphate) to produce EPSP (5-Enolpyruvylshikimate3-phosphate) under the action of EPSP synthetase. In conclusion, phosphoenolpyruvate is an important bridge intermediate connecting two pathways, and pyruvate kinase is a key enzyme influencing the content of phosphoenolpyruvate.

Pyruvate kinase exists in all organisms, and non-plant-derived pyruvate kinase has been studied in large numbers, but the study of pyruvate kinase in plants is still very deficient, and pyruvate kinase exists in many copies in plants, and this evolutionary expansion has largely increased the diversity of pyruvate kinase gene expression and protein activity, thereby making plants more flexible to adapt to various complex and varied environmental conditions. The pyruvate kinase plays multiple functions in the plant development and metabolic process, and the research on the function of the pyruvate kinase has important significance for understanding the complex metabolism and the development process in the plant body. However, the function of pyruvate kinase gene in plants, especially the leguminous plant Tribulus terrestris is still unknown at present, and in view of the multiple functions of pyruvate kinase, the role and potential application value of pyruvate kinase in plant metabolism, the cloning and identification of plant pyruvate kinase gene with function, specificity and biological activity has important theoretical and application value.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a pyruvate kinase gene and application thereof, and the purpose is realized by cloning the pyruvate kinase gene with catalytic activity from the medicago truncatula belonging to the leguminous plant, identifying the function of the pyruvate kinase gene to generate active pyruvate, and simultaneously participating in flavonoid metabolism by the pyruvate kinase gene, so that important gene resources and technical methods are provided for regulating the biosynthesis of the flavonoid by using biotechnology in other organisms.

In order to achieve the purpose, the invention provides the technical scheme that the invention provides a pyruvate kinase gene, which is characterized in that the gene sequence of the pyruvate kinase is shown as SEQ ID NO. 1.

Furthermore, the coded amino acid sequence of the pyruvate kinase gene is shown as SEQ ID NO. 2.

Furthermore, the gene sequence of the pyruvate kinase is derived from medicago truncatula.

The invention also provides a plasmid containing the pyruvate kinase gene.

The invention also provides application of the pyruvate kinase gene in improving flavonoid content.

The invention also provides application of the plasmid in improving flavonoid content.

In the technical scheme, the pyruvate kinase gene is cloned from medicago truncatula, the function of the pyruvate kinase gene is systematically identified, and the gene is found to be capable of generating active recombinant protein in bacteria and participating in the biosynthesis of flavonoids.

Drawings

FIG. 1 is a multiple alignment analysis of MtPK1 with other plant PK amino acid sequences;

FIG. 2 is a tree analysis of the evolution of plant pyruvate kinase;

FIG. 3 is the subcellular localization of MtPK 1;

FIG. 4 is an SDS-PAGE gel of the recombinant protein MtPK 1;

FIG. 5 is a graph of the effect of pH on the reaction rate of recombinant MtPK1 protein;

FIG. 6 is a graph of the effect of PEP concentration on the rate of reaction of recombinant MtPK1 protein;

FIG. 7 is a graph of the effect of ADP concentration on the rate of recombinant MtPK1 protein reaction;

FIG. 8 is a tissue expression profile of MtPK 1;

FIG. 9 shows PCR identification of a transgenic line in which MtPK1 is overexpressed in Medicago truncatula;

FIG. 10 is a graph of the relative content of anthocyanins in R108, NF0791 and over-expression strains.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

Example 1

The invention discloses a pyruvate kinase gene MtPK1, the gene sequence of which is shown in SEQ ID NO.1, wherein ATG in the sequence shown in SEQ ID NO.1 is an initiation codon, and TGA is a termination codon. The amino acid sequence of the protein coded by the gene is shown in SEQ ID NO. 2. The purpose is to heterologously express pyruvic acid in other organisms, and the excessive expression in the medicago truncatula can produce flavonoid.

The method for identifying the pyruvate kinase gene comprises the following steps:

(1) cloning of full-length fragment of MtPK1 gene

The PCR reactions were performed according to Phusion high fidelity DNA polymerase instructions supplied by Saimer Feishale science Inc. (Table 1, Table 2).

TABLE 1Phusion enzyme reaction System

Wherein the Forward primer has the sequence: ATGATGGCAGAGAAGAAACC, respectively; the sequence of Reverse primer is: TCATTTCACAGTCAAGATTTTG

TABLE 2 PCR procedure

(2) Glue recovery of destination strip

The reaction of adding A to the target fragment was carried out according to the instructions provided by Takara biomedical technology (Beijing) Co. The reaction system was prepared according to the following table (Table 3), and reacted at 72 ℃ for 15 min. After the reaction with A was completed, the desired band was recovered.

The target band was gel recovered according to the instructions of agarose gel DNA recovery kit provided by Beijing kang as a century Biotechnology Co. Weighing the weight of the centrifuge tube, cutting DNA of a single-purpose strip from the agarose gel, putting the DNA into the centrifuge tube, weighing and calculating the net weight of the gel.

1) Add 100. mu.L of BufferPG/100mg of gel.

2) Incubate in a 50 ℃ water bath while gently inverting the tube upside down until the gel mass is completely dissolved.

3) To the adsorption column, 200. mu.l of BufferPS was added to activate the column. Centrifuge at 13000g for 1min at room temperature, and pour off the waste liquid in the collection tube.

4) Cooling the gel solution to room temperature, adding into adsorption column, and standing for 2 min. Centrifuge at 13000g for 1min at room temperature, and pour off the waste liquid in the collection tube.

5) Mu.l of BufferPW was added to the adsorption column and allowed to stand for 5 min. Centrifuge at 13000g for 1min at room temperature. And (4) pouring the waste liquid in the collecting pipe.

6) Centrifuge at 13000g for 1min at room temperature. The adsorption column was placed in a new centrifuge tube, 50. mu.l of Buffer EB was suspended and dropped onto the middle of the adsorption membrane, and left at room temperature for 2 min. Centrifuge at 13000g for 1min at room temperature. Agarose gel electrophoresis determined the size of the bands of DNA collected and the concentration was determined using a NanoDrop 2000, stored at-20 ℃.

TABLE 3 recovery of fragment plus A System

(3) Ligation of pCXSN to the DNA fragment of interest

The target recovered fragment was ligated with the pCXSN vector recovered after Xcm I enzyme digestion according to the instruction provided by Takara biomedical technology (Beijing) Co. The reaction system was prepared according to the following table (table 4), gently mixed, and metal-bath at 16 ℃ overnight.

TABLE 4T4DNA ligase System

(4) Small extraction of plasmids

1) 50. mu.L of the resulting suspension was inoculated into 5mL of LB liquid medium containing Carb and cultured with shaking overnight at 37 ℃.

2) The overnight cultured broth was added to a 2mL sterile centrifuge tube, centrifuged at 12000g for 1min, and the supernatant was decanted.

3) Sucking 250 mu L P1 (confirming that RNAse I is added) and adding the mixture into the thallus precipitate, and blowing and beating the thallus by using a gun head to fully mix the thallus.

4) Aspirate 250 μ L P2 into the centrifuge tube and gently invert 7 times to lyse the bacteria thoroughly.

5) Aspirate 350 μ L N3 into the centrifuge tube and mix thoroughly by gently inverting 10 times immediately.

6) 12000g at room temperature, and 5min by centrifugation.

7) The supernatant was pipetted into an adsorption column and centrifuged at 12000g for 1min at room temperature.

8) The waste liquid in the collection tube was decanted and 150. mu.L PB was added to the adsorption column.

9) Centrifuge at 12000g for 1min at room temperature.

10) 400 μ L PW was added to the column and centrifuged at 12000g for 1min at room temperature.

11) Placing the adsorption column into a new 1.5mL centrifuge tube, sucking 50 μ L EB, suspending, adding into the middle part of the adsorption membrane, and standing at room temperature for 5 min. The plasmid was stored by centrifugation at 12000g for 1min at-20 ℃.

And carrying out double enzyme digestion identification on the obtained transformant extracted plasmid. Transformants identified as positive are cloned, expanded and then sent to be sequenced.

(5) Transformation of competent cells of E.coli (DH 5. alpha. and M15)

1) Coli competent cells were thawed on ice.

2) Add 5. mu.L plasmid, mix well and put on ice for 30 min.

3) Heat shock at 42 ℃ for 1min, take out quickly and place immediately and gently on ice for 2 min.

4) Add 500. mu.L LB liquid medium and mix well. The cells were cultured at 37 ℃ for 1 hour with shaking.

5) And (3) coating the bacterial liquid on an LB solid culture medium containing Carb by using a sterilized coating rod, airing on an ultra-clean bench, and sealing.

6) The cells were cultured at 37 ℃ overnight with standing.

7) And (4) picking a monoclonal antibody by using a sterile gun head to perform PCR identification on the bacterial liquid.

The MtPK1 gene is cloned and sequenced to find that the MtPK1 has the full length of 2629bp, contains 3 exons and 2 introns, encodes a protein sequence containing 497 amino acids, and the predicted isoelectric point (pI) of the protein is 8.14, and the relative molecular mass is about 55.68 kD.

Example 2

Full-length sequences of 13, 10 and 12 pyruvate kinases were obtained from arabidopsis, rice and medicago truncatula, respectively, for sequence alignment and evolutionary analysis, according to the annotations in the NCBIgenbank and KEGG SSDB databases.

Performing amino acid sequence homology alignment analysis by using DNAMAN, and analyzing a conserved structural domain contained in the DNAMAN according to a Pfam 31.0 database; the phylogenetic tree was constructed according to the Neighbor-join method using Mega7 software.

The invention carries out bioinformatics analysis on the amino acid sequence encoded by MtPK 1. Carrying out multiple sequence alignment analysis on the amino acid sequence of MtPK1 and the amino acid sequence of homologous protein thereof, wherein the alignment analysis result is shown in figure 1, and the beta barrel-shaped domain and the alpha/beta domain of pyruvate kinase in figure 1 are respectively at amino acid positions 8-350 and 367-493; the solid line represents the pyruvate kinase conserved domain active site. Consensus indicates amino acids with perfect sequence identity, and the conserved amino groups are highlighted in black. From the results of FIG. 1, it can be seen that all pyruvate kinases contain a conserved pyruvate kinase active site: [ LIVAC ] -x- [ LIVM ] - [ LIVM ] - [ SAPCV ] -K- [ LIV ] -E- [ NKRST ] -x- [ DEQHS ] - [ GSTA ] - [ LIVM ]. This site is rich in lysine and may play a major catalytic role. More importantly, analysis of the domains contained in these sequences in the Pfam 31.0 database revealed, in addition to a conserved pyruvate kinase active site, a β barrel domain (PF00224.14) at the N-terminus and an α/β domain (PF02887.9) at the C-terminus of these pyruvate kinases, further suggesting that they belong to the pyruvate kinase family.

According to the evolutionary tree shown in fig. 2, these pyruvate kinases apparently cluster into two major clusters: cytoplasmic pyruvate kinase (cytopalic PK) and plastid pyruvate kinase (Plastidial PK), the gene At3g49160 encoding pyruvate-like kinase forms a unique cluster. The PKc isozymes consistently aggregate into two small clusters (Cytosolic-1 and Cytosolic-2), and the PKp isozyme also aggregates into two small clusters (Plastidial-alpha and Plastidial-beta). The cytoplasmic pyruvate kinase MtPK1, MtPK2 and MtPK4 are clustered, the relativity of the cytoplasmic pyruvate kinase MtPK1, the MtPK2 and the MtPK4 is the most similar, and the homology of the amino acid sequences is as high as 99 percent. The 4 cytoplasmic pyruvate kinases (MtPK5, MtPK6, MtPK9 and MtPK10) distributed in the Cytosalic-2 have a sequence homology of up to 68%. OsPK1(Os11g0148500) which is also a cytoplasmic pyruvate kinase shares 68% homology with MtPK 1.

Example 3

The coding sequence of MtPK1 was cloned by primers MtPK1SalF and MtPK1 BamH with restriction sites, and the PCR product was digested with SalI and BamHI and ligated into the same restriction endonuclease digested vector pJIT163-hGFP to construct the fusion gene MtPK 1-hGFP. The correctly sequenced construct was introduced into Arabidopsis leaf protoplasts. Incubation is carried out for 16h at 25 ℃, fluorescence is detected by a Leica laser scanning confocal microscope by taking pJIT163-hGFP as a positive control.

In order to further determine that MtPK1 belongs to cytoplasmic pyruvate kinase, the invention constructs a transient expression vector of MtPK1-hGFP fusion expression driven by a CAMV 35S promoter, converts the transient expression vector into an arabidopsis mesophyll cell protoplast, and observes the positioning condition of fluorescent protein by using a Leica laser confocal microscope. The green fluorescence signal of GFP is clearly visible in the cytoplasmic region, whereas the autofluorescence region of chloroplasts has little green fluorescence. In FIG. 3, the top layer is the one that transiently expresses GFP in Arabidopsis protoplast cells, the bottom layer is the one that transiently expresses MtPK1-GFP fusion protein, and the localization of green fluorescent protein is observed by a Leica laser confocal microscope. GFP and Bright field indicate green fluorescence and Bright field, respectively. The results of fig. 3 show that: MtPK1 is a cytoplasmic-localized pyruvate kinase, consistent with cytoplasmic localization results predicted from the previous evolutionary tree analyses constructed using arabidopsis, rice and tribulus lucerne pyruvate kinase.

Example 4

The functional identification method of pyruvate kinase gene coding protein comprises the following steps:

steps (1) and (2) were the same as Steps (1) and (2) of example 1

(1) And (3) connecting the pQE30 vector and the target DNA fragment.

The target band and the vector after the recovery by the digestion were ligated according to the reaction system shown in the following Table (Table 5).

TABLE 5T4DNA ligase System

(2) Induction and purification of recombinant protein and determination of enzyme activity

1) 20 μ L of the resulting suspension was put into 2mL of LB liquid medium containing Carb and cultured overnight with shaking at 37 ℃.

2) Adding the overnight cultured bacterial liquid into 300mL LB liquid medium containing 100mg/L Carb and 50% glucose sterilized by high temperature and high pressure, and shake culturing at 37 deg.C to OD₆₀₀The value was 0.5-0.6, and 1mL of the bacterial solution was collected as a control.

3) To 300mL of the bacterial suspension was added 300. mu.L of 1M IPTG to a final concentration of 0.3mM, and the mixture was cultured at 16 ℃ for 24 hours.

4) Centrifugation was carried out at 9000g for 5min at 4 ℃ to collect the cells.

5) 10 volumes of lysis buffer and 1mg/mL of lysozyme were added and allowed to stand on ice for 30 min.

6) The cells were disrupted for 10min with an ultrasonicator to release the protein.

7) The supernatant was collected by centrifugation at 9000g for 30min at 4 ℃.

8) Adding the sample into a Washbuffer activated nickel column with 8 times of column volume, flushing the hybrid protein by using Washbuffer with 20 times of column volume after the sample flows through the affinity column filler, and finally eluting the target protein by using elutebufter with 5 times of column volume. The protein was collected in a Millpore (30kDa) tube and concentrated by centrifugation at 4000g for 15min at 4 ℃.

9) The salt and imidazole were washed off by adding 1mL of 100mM Tris-HCl, centrifugation at 4 ℃ and 4000g for 15 min.

10) And (2) adding the following components in a ratio of 500: 20 in a ratio of Coomassie brilliant blue, lambda_595nmDetermining the concentration of the recombinant protein; meanwhile, SDS-PAGE electrophoresis is carried out to confirm the size and purity of the recombinant protein.

11) Determination of pyruvate kinase Activity: 500 μ L of enzyme activity reaction system: 50mM HEPES-KOH (pH 6.4), 25mM KCl, 12mM MgCl₂2mM PEP, 1mM ADP, 1mM DTT, 5% (w/v) PEG8000, 0.15mM NADH and 2units/ml lactate dehydrogenase. Reacting at 28 deg.C for 5min at lambda_340nmAnd measuring the light absorption value.

12) Preparing an NADH standard curve: NADH solutions were prepared at various concentrations of 0, 12.5, 25, 50, 75, 100 and 150nmol at lambda_340nmThe change in absorbance was measured. And (5) preparing a standard curve according to the light absorption value and the concentration of NADH. Pyruvate kinase activity was calculated from a standard curve of NADH.

In order to detect the pyruvate kinase activity of MtPK1 protein, the MtPK1 gene is constructed on a pQE30 vector, and a prokaryotic expression system is used for expressing fusion protein. The fusion protein was separated and purified by using a nickel ion affinity resin, and the purity of the target protein was confirmed by SDS-PAGE electrophoresis (FIG. 4). In the presence of ADP, with K⁺And Mg²⁺As a cofactor, pyruvate kinase catalyzes the conversion of PEP to pyruvate, which is converted to lactate by Lactate Dehydrogenase (LDH) in the presence of reduced coenzyme I (NADH). NADH has strong light absorption at 340nm, and the activity of pyruvate kinase is calculated according to the change of the light absorption value. The results showed that the purified His-tagged recombinant protein showed a broad pH profile when both the substrate and the cofactor were in saturation, and reached the optimum pH around pH 6.5 (FIG. 5). Furthermore, at the optimum pH, the kinetic constants Km values for MtPK1 for PEP (FIG. 6) and ADP (FIG. 7) were 149. mu.M and 106. mu.M, respectively. In conclusion, MtPK1 is conserved and has active catalytic activityThe cytosolic pyruvate kinase of (1).

Example 5

(1) Transformation of Agrobacterium-infected competent cells

1) Freezing and thawing the agrobacterium tumefaciens competent cells stored in a refrigerator at the temperature of minus 80 ℃ on ice.

2) Add 2. mu.L of plasmid to the competence, gently flick and mix, place on ice for 15 min.

3) Placing in liquid nitrogen, and quickly freezing for 1 min; heat shock was performed at 37 ℃ for 5 min.

4) To the above liquid was added 500. mu.L of LB liquid medium without antibiotic, and shake-cultured at 28 ℃ for 3 hours.

5) And (3) coating the bacterial liquid on an LB solid culture medium added with corresponding antibiotics by using a sterilized coating rod, sealing, and standing and culturing at 28 ℃.

6) The single clone was picked for colony PCR, and after confirming the positive of the cultured colony, the strain was stored at-80 ℃.

(2) Transformation of transgenic plants

1) Activation of the strain: the pCXSN-MtPK1/EHA105 strain stored at-80 ℃ was freeze-thawed, 20. mu.L of the resulting strain was inoculated into 2mL of LB medium containing antibiotics, and cultured with shaking overnight at 28 ℃.

1) And (3) amplification culture: inoculating 1mL of activated bacteria into 100mL of LB medium containing antibiotics, and performing shaking culture at 28 deg.C to OD₆₀₀＝0.6。

2) Adding 1 drop of Tween-20 (surfactant) into tender leaves of herba Medicaginis for 5-8 weeks, and washing with tap water until no foam is formed.

3) Adding 30mL of 10% sodium hypochlorite for disinfection for 15 min; meanwhile, the well shaken strains are collected by 6000g centrifugation for 5min and are resuspended by sterile SH3a liquid for standby.

4) The sterilized leaves were rinsed 3-4 times with sterile water until no sodium hypochlorite remained.

5) Cutting the leaves into wounds by using a scalpel, mixing the cut leaves with the bacterial liquid uniformly, and infecting for 1 h.

6) The cells were plated on SH3a solid medium and co-cultured in the dark for 2 d.

7) Transferring to SH3a culture medium added with antibiotic to induce callus.

8) After 4-5 weeks, the calli were transferred to SH9 solid medium to induce transgenic plants.

10) After the overground part of the transgenic plant grows out, the transgenic plant is moved to 1/2SH9 culture medium to induce rooting.

11) And transplanting the transgenic plant into a culture bottle filled with sterilized soil when the root system of the transgenic plant grows well, and putting the transgenic plant into a tissue culture room for hardening seedlings.

12) Transplanting the successfully acclimated plants into a phytotron (25 +/-2 ℃; 18h light +6h dark).

13) Extracting DNA of the transgenic plant to screen transgenic positive seedlings.

14) And extracting RNA of the positive seedlings, and analyzing the expression level of the RNA. Screening out high expression strain

15) Positive seedling phenotype was observed and the metabolite content of the high expression lines was analyzed.

(3) Determination of anthocyanin content

1) Taking a fresh tissue sample of medicago truncatula under the same growth environment.

2) Adding liquid nitrogen, grinding, accurately weighing 50mg, adding 5 times volume of methanol (containing 0.1% hydrochloric acid), ultrasonic treating for 30min, and standing at 4 deg.C overnight.

3) Ultrasonic treating for 30min, centrifuging at 10000rmp for 5min, and collecting supernatant.

4) 750. mu.L of water and 750. mu.L of chloroform were added, and centrifuged with shaking to obtain the supernatant.

5) Measured at 530nm with an ultraviolet spectrophotometer. The content of anthocyanin is estimated by taking the molar light absorption of cyanidin-3-O-glucose as a standard substance.

In order to analyze the action mechanism of pyruvate kinase gene MtPK1 in the medicago truncatula, the expression condition of MtPK1 in the medicago truncatula roots, stems, leaves, flowers and seeds harvested 10d, 12d, 16d, 20d, 24d and 36d after flowering is researched by a fluorescent quantitative RT-PCR method. The research result is shown in figure 8, and the expression condition of the MtPK1 gene in different tissues is shown; MtPK1 is used for collecting seed materials of different parts including roots, stems, leaves, flowers and pods 10 days, 12 days, 16 days, 20 days, 24 days and 36 days after pollination, extracting RNA and reversing the RNA into cDNA, and then carrying out qRT-PCR by using Mtactin2 as an internal reference. The results in FIG. 8 show that MtPK1 expression was detected in all parts of Medicago truncatula and at all stages of seed development. Among them, the expression level was higher in stem, pod and 10d, 12d, 16d, 20d and 36d seeds, and the highest in root and 24d seeds.

Anthocyanin in flavonoid is mainly present in stems and leaves of medicago truncatula, and procyanidine in flavonoid is only accumulated in seeds, which is similar to the expression condition of MtPK1, and the result shows that MtPK1 is related to biosynthesis of anthocyanin and procyanidine.

In order to further prove the function of MtPK1 in anthocyanin and procyanidine synthesis, the pyruvate kinase gene MtPK1 driven by a tobacco mosaic virus CaMV 35S promoter is connected to a eukaryotic expression vector pCXSN, and the fusion gene is transferred into agrobacterium EHA 105. According to the agrobacterium-mediated method for transforming the leaves of the medicago truncatula, the callus is induced under dark conditions and then placed under light to start the induction of regeneration seedlings. First, two resistant seedlings were co-obtained by transformation into the Medicago truncatula mutant NF0791 using the 35S strain pCXSN-MtPK1/GV 3101. Meanwhile, the MtPK1 gene is transferred into wild R108 of the medicago truncatula, and 3 transgenic positive seedlings OE-10, OE-26 and OE-30 are obtained through PCR identification (figure 9). The accumulation levels of anthocyanin in stems and leaves of over-expressed plants are different, and the result shows that the anthocyanin content of most transgenic lines is increased, and the bases of the stems and leaves are purple. The content of anthocyanin in the over-expressed strain was determined, and the content of OE-10 anthocyanin was found to be significantly higher than that of the wild type and the mutant (FIG. 10). The result shows that the material MtPK1 with higher anthocyanin content has high expression level, and the data indicate that the accumulation of the stem anthocyanin is in positive correlation with the expression level of MtPK 1.

In conclusion, the MtPK1 gene of the medicago truncatula encodes pyruvate kinase, MtPK1 protein catalyzes a specific substrate, namely phosphoenolpyruvate, to form pyruvic acid, the pH value can influence the catalytic efficiency of MtPK1, and the MtPK1 is a brand-new plant pyruvate kinase different from the known pyruvate kinases of other plants. The alfalfa MtPK1 protein is a protein located in cytoplasm, can be expressed in bacteria and plants, and the expressed recombinant protein has activity, and can be used for improving the yield of flavonoid compounds in the production of microorganisms or plant metabolic engineering due to the fact that the overexpression of MtPK1 can improve the content of flavonoid compounds, particularly the content of anthocyanidin.

The operation methods not specifically described in the present embodiment are conventional techniques, and thus are not explained in detail herein.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Beijing animal husbandry and veterinary institute of Chinese academy of agricultural sciences

<120> pyruvate kinase gene and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1494

<212> DNA

<213> MtPK1 gene (Medicago truncatula)

<400> 1

atgatggcag agaagaaacc caaaacaaag atcgtgtgca cgctgggacc tgcatcgagg 60

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

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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

1. The application of over-expression of pyruvate kinase MtPK1 in improving the anthocyanin content of tribulus lucerne is characterized in that the amino acid sequence of pyruvate kinase MtPK1 is shown in SEQ ID NO. 2.

2. The use according to claim 1, wherein the nucleotide sequence of the pyruvate kinase MtPK1 gene is as shown in SEQ ID No. 1.

3. Use of a plasmid comprising the pyruvate kinase gene MtPK1 as defined in claim 2 for increasing the anthocyanin content of tribulus lucerne.