CN107267535A - Mevalonate kinase TaMVK gene mutation bodies and its method for creating - Google Patents

Mevalonate kinase TaMVK gene mutation bodies and its method for creating Download PDF

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CN107267535A
CN107267535A CN201710720385.2A CN201710720385A CN107267535A CN 107267535 A CN107267535 A CN 107267535A CN 201710720385 A CN201710720385 A CN 201710720385A CN 107267535 A CN107267535 A CN 107267535A
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seq
sequences
tamvk
mevalonate kinase
codon
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楚秀生
褚蔚
李永波
崔德周
樊庆琦
隋新霞
黄承彦
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CROP Research Institute of Shandong Academy of Agricultural Sciences
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    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
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    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/01036—Mevalonate kinase (2.7.1.36)

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Abstract

The invention provides Mevalonate kinase TaMVK gene mutation bodies and its method for creating.The present invention is by changing Mevalonate kinase geneTaMVKMiddle specific amino acid codons are come the catalytic activity that improves or reduce its codase.From the wheat breed of clone, " the wild-type mevalonate kinase gene of Jimai 22 ", has formulated Mevalonate kinase geneTaMVK13 gene mutation bodies, and obtain relevant enzyme to substrate MVA（Mevalonic acid）With ATP enzyme kinetics parameter, it was demonstrated that by the change to Mevalonate kinase gene specific amino acid codons, can significantly improve or reduce the catalytic activity of enzyme.

Description

Mevalonate kinase TaMVK gene mutation bodies and its method for creating
Technical field
The invention belongs to field of plant molecular biology, and in particular to Mevalonate kinase TaMVK gene mutation bodies And its method for creating.
Background technology
Rite-directed mutagenesis refers to introduce specific base-pair in the specified site of target DNA fragment, so as to change its coding The technology of amino acid sequence.It is the strong instrument for studying the complex relationship between protein structure and function, is also real Test the means of the conventional genetic modification in room.Particular bases to some known gene carry out fixed point transformation, missing or inserted Enter, thus it is possible to vary orresponding amino acid sequence and the 26S Proteasome Structure and Function feature of protein, help to understand protein structure and function Relation.
Isoprenoid material is present in all organisms, and content is especially enriched in plant, is to maintain plant growth Important organic substance necessary to development, photosynthesis etc., can such as be used as growth substance and plant hormone（It is the basic element of cell division, de- Fall acid, gibberellin and brassinosteroid）, photosynthetic pigments（Such as chlorophyll, carotenoid）, membrane structure a part such as sitosterol, Electron transmission acceptor such as plastoquinone, reacted as glucosyl in glucose acceptor such as dolichol, and can regulating cell Growth such as prenyl protein.In addition, many plant isoprenoids also have important commercial value such as rubber, food fragrant Material, beverage, vitamin A. D. E, natural insecticide such as pyrethrin etc..The biosynthesis of isoprenoid material is main by first hydroxyl A series of enzyme enzyme' s catalysis in valeric acid approach, mevalonate kinase（MVK,MK;ATP:mevalonate-5- phosphotransferase;EC2.7.1.36）Be that three continuous ATP rely in enzymes in the metabolic pathway first, it will A phosphate group on ATP γ positions is transferred to formation mevalonic acid -5- phosphoric acid on the hydroxyl of mevalonic acid the 5th, and adjoint ADP release, is to control one of rate-limiting enzyme of whole metabolic pathway.At present respectively from arabidopsis（Arabidopsis thaliana）, paddy rice（Oryza sativa）, corn（Zea mays）, sorghum（Sorghum bicolor）, rubber（Hevea brasiliensis）, castor-oil plant（Ricinus communis）Mevalonate kinase gene is cloned Deng separation in plant, in this hair Before bright application, also do not disclose or delivered on initiative Mevalonate kinase TaMVK gene mutation bodies, gene is dashed forward Variant carries out Prokaryotic gene expression, zymoprotein and isolated and purified and its data such as enzymatic activity vitro detection and enzyme kinetics parameter study Information.
The content of the invention
For the defect of prior art, the invention provides Mevalonate kinase TaMVK gene mutation bodies and its wound Method processed.
It is an object of the invention to provide Mevalonate kinase TaMVK gene mutation bodies and its method for creating, Ke Yigao Effect obtains Mevalonate kinase TaMVK gene mutation bodies, is easy to improve the economical character of the crops such as wheat, improves yield, And the basic research of molecular biology can be carried out.
Technical scheme is as follows：
The invention provides Mevalonate kinase TaMVK gene mutation body method for creating, rite-directed mutagenesis by the following method Form：The corresponding DNA sequence dna of amino acid sequence different from other plants in Mevalonate kinase TaMVK conserved regions is determined Point mutation.
It is preferred that, Mevalonate kinase TaMVK gene mutation body method for creating, Mevalonate kinase TaMVK 14A, 24A, 35Q, 135V, 139G, 142M, 144A, 153S, 165L and 332N position amino acid in conserved region, are mutated respectively For 14S, 24T, 35N, 35R, 135E, 139D, 142L, 142F, 142Y, 144S, 153C, 165M and 332S.
It is preferred that, Mevalonate kinase TaMVK gene mutation body method for creating, Mevalonate kinase TaMVK The upstream and downstream primer of mutant is respectively：
A14S:F sequences are SEQ ID NO.6, A14S:R sequences are SEQ ID NO.7, and codon sports TCC by GCC；
A24T:F sequences are SEQ ID NO.8, A24T:R sequences are SEQ ID NO.9, and codon sports ACC by GCC；
Q35N:F sequences are SEQ ID NO.10, Q35N:R sequences are SEQ ID NO.11, and codon sports AAC by CAG；
FQ35R:F sequences are SEQ ID NO.12, FQ35R:R sequences are SEQ ID NO.13, and codon sports CGG by CAG；
V135E:F sequences are SEQ ID NO.14, V135E:R sequences are SEQ ID NO.15, and codon sports GAG by GTG；
G139D:F sequences are SEQ ID NO.16, G139D:R sequences are SEQ ID NO.17, and codon sports GAC by GGC；
M142L:F sequences are SEQ ID NO.18, M142L:R sequences are SEQ ID NO.19, and codon sports CTG by ATG；
M142F:F sequences are SEQ ID NO.20, M142F:R sequences are SEQ ID NO.21, and codon sports TTT by ATG；
F142Y:F sequences are SEQ ID NO.22, F142Y:R sequences are SEQ ID NO.23, and codon sports TAT by TTT；
A144S:F sequences are SEQ ID NO.24, A144S:R sequences are SEQ ID NO.25, and codon sports TCC by GCC；
S153C:F sequences are SEQ ID NO.26, S153C:R sequences are SEQ ID NO.27, and codon sports TGC by TCC；
L156M:F sequences are SEQ ID NO.28, L156M:R sequences are SEQ ID NO.29, and codon sports ATG by TTG；
N332S:F sequences are SEQ ID NO.30, N332S:R sequences are SEQ ID NO.31, and codon sports TCC by AAC.
The invention provides the wheat mevalonic acid of Mevalonate kinase TaMVK gene mutation bodies method for creating initiative Kinases TaMVK gene mutation bodies, Mevalonate kinase TaMVK gene mutation bodies TaMVK/A14S、TaMVK/A24T、 TaMVK/Q35N、TaMVK/Q35R、TaMVK/V135E、TaMVK/G139D、TaMVK/M142L、TaMVK/M142F、TaMVK/ F142Y、TaMVK/A144S、TaMVK/S153C、TaMVK/L165MWithTaMVK/N332S, respectively described wheat mevalonic acid 14A, 24A, 35Q, 135V, 139G, 142M, 144A, 153S, 165L and 332N position amino acid in kinases TaMVK conserved regions, 14S, 24T, 35N, 35R, 135E, 139D, 142L, 142F, 142Y, 144S, 153C, 165M and 332S are sported respectively.
It is preferred that, Mevalonate kinase TaMVK gene mutation bodies, described Mevalonate kinase TaMVK bases Because of mutant TaMVK/A14S、TaMVK/A24T、TaMVK/Q35N、TaMVK/Q35R、TaMVK/V135E、TaMVK/G139D、 TaMVK/M142L、TaMVK/M142F、TaMVK/F142Y、TaMVK/A144S、TaMVK/S153C、TaMVK/L165MWithTaMVK/N332S, upstream and downstream primer is respectively：
A14S:F sequences are SEQ ID NO.6, A14S:R sequences are SEQ ID NO.7, and codon sports TCC by GCC；
A24T:F sequences are SEQ ID NO.8, A24T:R sequences are SEQ ID NO.9, and codon sports ACC by GCC；
Q35N:F sequences are SEQ ID NO.10, Q35N:R sequences are SEQ ID NO.11, and codon sports AAC by CAG；
FQ35R:F sequences are SEQ ID NO.12, FQ35R:R sequences are SEQ ID NO.13, and codon sports CGG by CAG；
V135E:F sequences are SEQ ID NO.14, V135E:R sequences are SEQ ID NO.15, and codon sports GAG by GTG；
G139D:F sequences are SEQ ID NO.16, G139D:R sequences are SEQ ID NO.17, and codon sports GAC by GGC；
M142L:F sequences are SEQ ID NO.18, M142L:R sequences are SEQ ID NO.19, and codon sports CTG by ATG；
M142F:F sequences are SEQ ID NO.20, M142F:R sequences are SEQ ID NO.21, and codon sports TTT by ATG；
F142Y:F sequences are SEQ ID NO.22, F142Y:R sequences are SEQ ID NO.23, and codon sports TAT by TTT；
A144S:F sequences are SEQ ID NO.24, A144S:R sequences are SEQ ID NO.25, and codon sports TCC by GCC；
S153C:F sequences are SEQ ID NO.26, S153C:R sequences are SEQ ID NO.27, and codon sports TGC by TCC；
L156M:F sequences are SEQ ID NO.28, L156M:R sequences are SEQ ID NO.29, and codon sports ATG by TTG；
N332S:F sequences are SEQ ID NO.30, N332S:R sequences are SEQ ID NO.31, and codon sports TCC by AAC.
The application of Mevalonate kinase TaMVK gene mutation bodies, it is characterised in that：Mevalonate kinase TaMVK gene mutation bodies are expressed in change mevalonate kinase gene in the crops such as wheat and other plants, change secondary generation Thank to Product yields, improve seed size, the application of grain weight economical character；And to Mevalonate kinase gene introne, Extron and promoter structure research, research promoter function, exploitation about molecular labeling application.
The usefulness of this law is as follows：
The present invention is by changing Mevalonate kinase geneTaMVKMiddle specific amino acid codons are compiled to improve or reduce its The catalytic activity of code enzyme, is the further eukaryotic gene for carrying out genetic modification and modifying, build mevalonate kinase gene Expression vector simultaneously converts corresponding crop, and the plant of important commercial value is especially obtained by secondary metabolism, inquires into mevalonic acid and swashs Overexpression of the enzyme gene in recipient plant and secondary metabolite, crop kernel size and the grain Main Agronomic Characters such as again Relation, and then crop yield is improved, and mevalonate kinase gene intron, extron and promoter structure are cutd open Analysis, studies promoter function, develops the offer important technology deposit such as relevant molecular labeling.
From the wheat breed of clone, " the wild-type mevalonate kinase gene of Jimai 22 " has formulated wheat first hydroxyl to the present invention Kinase geneTaMVK13 gene mutation bodies, be respectivelyTaMVK/A14S、TaMVK/A24T、TaMVK/Q35N、 TaMVK/Q35R、TaMVK/V135E、TaMVK/G139D、TaMVK/M142L、TaMVK/M142F、TaMVK/F142Y、TaMVK/ A144S、TaMVK/S153C、TaMVK/L165MWithTaMVK/N332S, and relevant enzyme is obtained to substrate MVA（Mevalonic acid） With ATP enzyme kinetics parameter, it was demonstrated that, can by the change to Mevalonate kinase gene specific amino acid codons Significantly improve or reduce the catalytic activity of enzyme.
Brief description of the drawings
Fig. 1 is that Mevalonate kinase isolates and purifies (43kD) electrophoretogram, wherein 1 is albumen precipitation, 2 be on albumen Clearly, 3-7 is the albumen of elution, and Marker is protein standard.
Fig. 2 is that Mevalonate kinase gene TaMVK mutant proteins isolate and purify electrophoretogram, wherein 1 is A14S, 2 be A24T, and 3 be G139D, and 4 be S153C, and 5 be M142L, and 6 be M142F, and 6-1 is F142Y, and 7 be A144S, and 8 be Q35N, and 9 are Q35R, 10 be V135E, and 11 be L156M, and 12 be N332S, and MVK is wild-type protein.
Embodiment
The above of the present invention is described in further detail below by specific embodiment.But this should not be managed The scope solved as above-mentioned theme of the invention is only limitted to following instance.All technologies realized based on the above of the present invention are belonged to The technical scope of the present invention.
The Mevalonate kinase TaMVK gene mutation bodies of embodiment 1 and its method for creating
First, experimental method
Step 1 Mevalonate kinase geneTaMVKAmplification
Step 1.1 designs primer sequence：
mvkF（SEQ ID NO.1）:
5′GAGGATCGCATCATCACCACCATCACGAGATCCGCGCCCGCGCGCCCGGC 3′
mvkR（SEQ ID NO.2）:
5′CTGCAGAAGCTTTTATCCTTGGTGAATCTGAAGACCTTG3′
Step 1.2 wheatTaMVKFull length gene series amplification
PCR reaction systems are 20 μ l, and component is as follows：
mvkF 1μl
mvkR 1μl
5×FastPfu Buffer 4μl
2.5 mM dNTP 2μl
The μ l of 22 cDNA of Jimai 1
FastPfu DNA Polymerase 0.4μl
ddH2O 10.6μl
PCR programs are 94 DEG C of pre-degenerations 5 min, 94 DEG C of denaturation 30sec, 58 DEG C of anneal 30sec, 72 DEG C
Extend 1.5min, totally 34 circulations, 72 DEG C of extension 10min, 16 DEG C of insulations.
The recovery of step 1.3 amplified production
（1）In adsorption column CB2（Adsorption column is put into collecting pipe）Add 450 μ l equilibrium liquids BL, 13,000 rpm (~ 13,400 × g) 1 min of centrifugation, outwells the waste liquid in collecting pipe, adsorption column is placed back in collecting pipe.（Treated on the day of use Pillar）.
（2）Single target DNA band is cut from Ago-Gel（Redundance is cut off as far as possible）It is put into clean In centrifuge tube, weight is weighed.
（3）Equimultiple bulk solution PC is added into blob of viscose（If gel weight is 0.1 g, its volume can be considered 100 μ l, Then add 100 μ l PC solution）, 50 DEG C of water-baths place 10 min or so, constantly centrifuge tube are leniently spun upside down therebetween, with true Blob of viscose is protected fully to dissolve.
（4）Previous step resulting solution is added in an adsorption column CB2（Adsorption column is put into collecting pipe）, 13,000 Rpm (~ 13,400 × g) centrifuges 1 min, outwells waste liquid in collecting pipe, adsorption column CB2 is put into collecting pipe.
（5）600 μ l rinsing liquids PW are added into adsorption column CB2（Absolute ethyl alcohol has been added using preceding first check whether）, 13,000 rpm (~ 13,400 × g) centrifuge 1 min, outwell the waste liquid in collecting pipe, adsorption column CB2 is put into collecting pipe.
（6）Repeat step 5.
（7）Adsorption column CB2 is put into collecting pipe, 13,000 rpm (~ 13,400 × g) centrifuge 2 min, remove as far as possible Remove rinsing liquid.Adsorption column is placed in into room temperature to place several minutes, thoroughly dried.
（8）Adsorption column CB2 is put into a clean centrifuge tube, appropriate wash vacantly is added dropwise to adsorbed film centre position De- buffer solution EB, room temperature places 2 min.13,000 rpm (~ 13,400 × g) centrifuge 2 min, collect DNA solution, -20 DEG C Save backup.
The connection and conversion of step 1.4 PCR primer
According to the concentration for reclaiming fragment, the ratio between product and pEASY- Blunt Simple Cloning Vector is determined Example.Connect step of converting as follows：
（1）4 μ l recovery DNA products are added in 200 μ l EP pipes and 1 μ l carriers are gently mixed.
（2）25 DEG C incubation 10min, after be placed on ice.
（3）Plus connection product is in 50 μ lE.coliIn DH5 α competent cells（When competent cell just thaws Add connection product）, flick mixing, ice bath 20-30 min.
（4）42 DEG C of heat shocks 90 seconds, are immediately placed on 2 min on ice.
（5）Plus the LB culture mediums of 1ml antibiotic-frees, 160rpm, 37 DEG C of incubation 1h.
（6）6000rpm centrifuges 3 min, discards part supernatant, retains 100-150 μ l, flicks suspension thalline, take whole Bacterium solution coated plate, 37 DEG C of overnight incubations.
The identification of step 1.5 positive clone molecule
The μ l of thalline PCR system 20 are prepared, component is as follows：
M13F 1μl
M13R 1μl
EASYTaq Mix 10μl
H2O 8μl
Edge clear, full 10 round and smooth colonies are chosen, are chosen in 1ml LB culture mediums, numbering is carried out, correspondence is clicked and entered In PCR systems.
PCR reaction conditions：94 DEG C of min of pre-degeneration 10（Cell lysis, inactivates nuclease）, 94 DEG C are denatured 30 sec, 55 DEG C 30 sec of annealing, 72 DEG C of extension 1min, 30 circulations extend 10 min, 16 DEG C of insulations after 72 DEG C.
Step 2 wheatTaMVKThe prokaryotic expression of full length gene ORFs
Step 2.1 design expression primer
PLM1F1（SEQ ID NO.3）:
5′CGCGCGGTACCAGGAGGAATTTAAAATGAGAGGATCGCATCATCACC3′
PLM1F2（SEQ ID NO.4）:
5′GAGGATCGCATCATCACCACCATCACGAGATCCGCGCCCGCGCGCCCGGC3′
PLM1R（SEQ ID NO.5）:
5′CTGCAGAAGCTTTTATCCTTGGTGAATCTGAAGACCTTG3′
Step 2.2TaMVKThe amplification of open reading frame sequence
Enter performing PCR as template using the full length sequence being connected on plasmid pEASY- Blunt Simple Cloning Vector to expand Increase, it is necessary to two-wheeled PCR.
First round PCR
20 μ l PCR systems, component is as follows：
PLM1F2 1μl
PLM1R 1μl
5×FastPfu Buffer 4μl
2.5 mM dNTP 2μl
The μ l of plasmid 1
FastPfu DNA Polymerase 0.4μl
ddH2O 10.6μl
PCR programs are 94 DEG C of pre-degeneration 5 min, 94 DEG C of denaturation 30sec, 60 DEG C of annealing 30sec, 72 DEG C of extension 1.5min, totally 10 Individual circulation, 72 DEG C of extension 10min, 16 DEG C of insulations.
Second wheel PCR
20 μ l PCR systems, component is as follows：
PLM1F1 0.8μl
PLM1R 0.8μl
5×FastPfu Buffer 4μl
2.5 mM dNTP 2μl
The μ l of first round PCR primer 1
FastPfu DNA Polymerase 0.4μl
ddH2O 11μl
PCR programs are 95 DEG C of pre-degeneration 5 min, 95 DEG C of denaturation 30sec, 60 DEG C of annealing 30sec, 72 DEG C of extension 1.5min, totally 35 Individual circulation, 72 DEG C of extension 10min, 16 DEG C of insulations.
Step 2.3 gel extraction purpose band, digestion recovery product and expression plasmid
Digestion system is as follows：
The μ l of expression plasmid pLM1 digestions system 15
Kpn1 0.75μl
Hind III 0.75μl
10×Green buffer 1.5μl
pLM1 10μl
ddH2O 2μl
TaMVKThe μ l of full length sequence pcr amplification product digestion system 20
Kpn1 1μl
Hind III 1μl
10×Green buffer 2μl
The μ l of PCR products 12
H2O 4μ
Step 2.4 connection construction of expression vector pLM 1-TaMVK
According to the concentration that fragment is reclaimed after digestion, the ratio between product and pLM1 carriers is determined, linked system is as follows：
pLM1 4μl
Reclaim the μ l of PCR digestion products 4
10×Buffer 1μl
T4 DNA Ligase 1μl
Stayed overnight in 16 DEG C of connections.
PLM1-TaMVK pairs of step 2.5E. coliThe conversion of DH5 α competent cells
Choose positive colony and extract plasmid and sequencing identification.
Step 2.6 pLM1-TaMVK recombinant plasmids pairE. coliBL21 expresses the conversion of competent cell
Choose the correct plasmid conversion of sequencingE. coliBL21 expresses competent cell.
Step 2.7 expression cellE. coliBL21 expansion culture and induction TaMVK expression
（1）It is transformed into pLM1-TaMVK recombinant plasmidsE. coliBL21 strains are inoculated into 20 ml LB culture mediums, and (Amp is dense Spend for 100mg/ml) in, 37 DEG C, 160rpm incubated overnights.
（2）The bacterium solution of 5ml incubated overnights is taken to be added in 500 ml LB nutrient solutions (Amp concentration 100mg/ml).37℃、 During 160rpm shaking table cultures 1.5h to thalline OD600=0.8, IPTG (final concentration of 1.5mM) is added.
（3）28 DEG C of incubated overnights.
（4）6000r/min centrifuges 10min, collects thalline 100ml deionized water rinsing thalline, 6000r/min centrifugations 10min, collects thalline.
Step 2.8 zymoprotein is isolated and purified
（1）Add 40 mL start buffers（Start buffer）Thalline is resuspended.
（2）Add 5mg lysozymes, 37 DEG C of water-bath lh.
（3）At least lh is placed in -80 DEG C.
（4）30 DEG C of water-baths are thawed.
（5）Sonicated cells 1h (broken 5s, be spaced 10s).
（6）9000r centrifuges 10min, and supernatant is used for protein purification.
（7）Ni-Trap posts are connected with 0.45 μm of filter, and syringe draws a drop in 5 ml aqua sterilisas injection post One drop ground excludes the ethanol in post.
（8）Take 3-5 ml 0.1M nickel sulfates to inject in post again, wash the nickel ion being not bound with off with 5 ml deionized waters, Then with 5 ml start buffer（Start buffer）Balance.
（9）Take in the supernatant injection post after 20 ml centrifugations.
（10）With 5 ml start buffers（Start buffer）Wash post.
（11）With 8ml dcq buffer liquid（Wash buffer）Wash post.
（12）With 5 ml elution buffer（Elution buffer）Destination protein is dissolved, and is connect with 1.5 ml centrifuge tubes Sample, is managed per 0.5ml mono-.
Step 2.9 SDS-PAGE electrophoresis detection protein
（1）Separation gel is prepared：
Deionized water 2.5ml
The ml of 30%Arc-Bis mother liquors 3.35
1.5M Tris-HCl（pH8.8） 2 ml
10% SDS 80μl
10% AP 80μl
TEMED 3.5 μl
（2）Glue is concentrated to prepare：
The ml of deionized water 1.7
The μ l of 30%Arc-Bis mother liquors 420
1M Tris-HCl（pH6.7） 320μl
10% SDS 25 μl
10% AP 25 μl
TEMED 3 μl
（3）Protein sample and 2 × SDS sample-loading buffers 1:1 mixing, 100 DEG C are boiled 5 min.Precipitation after ultrasound centrifugation, plus Enter the dissolving of 2ml 8M/L urea, add isometric sample-loading buffer, 100 DEG C are boiled 5 min.
（4）Separation gel 8ml is prepared, is added after TEMED with the mixing of 1ml pipettors（It is careful not to blow afloat bubble）, exist rapidly Separation gel is irrigated in the gap of two glass plates, then needed for perfusion concentration glue thereon plus 1-2ml deionized waters, is reserved Space, about 1.0 cm；After separation gel polymerization completely, coating liquid is poured out, gel top is washed with deionized for several times, with Remove unpolymerized acrylamide gel.
（5）Concentration glue 2.5ml is prepared, adds after TEMED and is well mixed, the perfusion concentration glue on separation gel, insertion is clean Comb；After polymerization completely, gel is fixed on electrophoretic apparatus, upper and lower groove respectively adds Tris- glycine running buffers, arranged Except the bubble between glass plate, comb is carefully pulled out.
（6）Ready sample is added in a predetermined order.80V constant pressures, during indicator to separation gel, voltage is adjusted to 120V, to be instructed dose stops electrophoresis when moving to bottom.
（7）Gel is taken out, fresh Coomassie brilliant blue dye liquor is added, gel is soaked with the dye liquor of about 5 times of volumes, is placed on gentle On the platform of shake, 2 h are dyed in room temperature.
（8）Remove and reclaim dye liquor for future use, it is gentle to shake 4-8 h by soak in destainer, therebetween should be more Change destainer 3-4 times.
The dialysis of step 2.10 zymoprotein
The zymoprotein of purifying is placed in bag filter, and bag filter two is clamped with sealing clip, appropriate albumen is added in large beaker saturating Analyse liquid（Ensure that bag filter can suspend）, rotor is put into, 4 DEG C of 20 h of dialysis on magnetic stirring apparatus, control rotor rotation are placed in Rotary speed should not be too fast, and dialyzate is changed 3 times in centre.The albumen after dialysis is collected, -80 DEG C of ultra low temperature freezers are put in standby.
Step 3 Mevalonate kinase geneTaMVKThe rite-directed mutagenesis of codon
The design of step 3.1 codon mutation primer
Found by amino acid alignment, 14A in Mevalonate kinase TaMVK conserved regions, 24A, 35Q, 135V, The position amino acid such as 139G, 142M, 144A, 153S, 165L and 332N is different from other plants, to inquire into these amino acid residues Influence to enzymatic activity, it is sported respectively 14S, 24T, 35N, 35R, 135E, 139D, 142L, 142F, 142Y, 144S, 153C, 165M and 332S.
The upstream and downstream primer of mutant is respectively：
A14S:F（SEQ ID NO.6）:GCAAGATCATCCTCTCCGGCGAGCACGCCGTCG;
A14S:R（SEQ ID NO.7）:CGACGGCGTGCTCGCCGGAGAGGATGATCTTGC；
Codon sports TCC by GCC.
A24T:F（SEQ ID NO.8）:CGTCCACGGCTCTACCGCCGTCGCCGCCGCC；
A24T:R（SEQ ID NO.9）:GGCGGCGGCGACGGCGGTAGAGCCGTGGACG；
Codon sports ACC by GCC.
Q35N:F（SEQ ID NO.10）:CGACCTCTACACGAACTCCTCCCTCCACCTG；
Q35N:R（SEQ ID NO.11）:CAGGTGGAGGGAGGAGTTCGTGTAGAGGTCG；
Codon sports AAC by CAG.
FQ35R:F（SEQ ID NO.12）:CGACCTCTACACGCGGTCCTCCCTCCACCTG；
FQ35R:R（SEQ ID NO.13）:CAGGTGGAGGGAGGACCGCGTGTAGAGGTCG；
Codon sports CGG by CAG.
V135E:F（SEQ ID NO.14）: GGCCTGGGAAGGTCGAGGTGAGCTCTGGCCTG；
V135E:R（SEQ ID NO.15）:CAGGCCAGAGCTCACCTCGACCTTCCCAGGCC；
Codon sports GAG by GTG.
G139D:F（SEQ ID NO.16）：GGTGAGCTCTGACCTGCCGATGGGC；
G139D:R（SEQ ID NO.17）：GCCCATCGGCAGGTCAGAGCTCACC；
Codon sports GAC by GGC.
M142L:F（SEQ ID NO.18）:GCTCTGGCCTGCCGCTGGGCGCCGGGCTTGGC；
M142L:R（SEQ ID NO.19）:GCCAAGCCCGGCGCCCAGCGGCAGGCCAGAGC；
Codon sports CTG by ATG.
M142F:F（SEQ ID NO.20）:GCTCTGGCCTGCCGTTTGGCGCCGGGCTTGGC；
M142F:R（SEQ ID NO.21）:GCCAAGCCCGGCGCCAAACGGCAGGCCAGAGC；
Codon sports TTT by ATG.
F142Y:F（SEQ ID NO.22）: GCTCTGGCCTGCCGTATGGCGCCGGGCTTGGC；
F142Y:R（SEQ ID NO.23）:GCCAAGCCCGGCGCCATACGGCAGGCCAGAGC；
Codon sports TAT by TTT.
A144S:F（SEQ ID NO.24）:CCTGCCGATGGGCTCCGGGCTTGGCTCGTCG；
A144S:R（SEQ ID NO.25）:CGACGAGCCAAGCCCGGAGCCCATCGGCAGG；
Codon sports TCC by GCC.
S153C:F（SEQ ID NO.26）：CGGCTGCGTTCTGCGCGTCGTTG；
S153C:R（SEQ ID NO.27）：CAACGACGCGCAGAACGCAGCCG；
Codon sports TGC by TCC.
L156M:F（SEQ ID NO.28）: CGTTCTCCGTGTCGATGTCGGGCGCGCTGC；
L156M:R（SEQ ID NO.29）:GCAGCGCGCCCGACATCGACACGGAGAACG；
Codon sports ATG by TTG.
N332S:F（SEQ ID NO.30）:CGACATTGAAGTATTCCTTGGTCTCGAAGCTC；
N332S:R（SEQ ID NO.31）:GAGCTTCGAGACCAAGGAATACTTCAATGTCG；
Codon sports TCC by AAC.
The PCR amplifications of step 3.2 gene mutation body
With the Mevalonate kinase gene of structureTaMVKVector plasmid pLM1-TaMVK is DNA profiling, utilizes each gene Mutant primer, enters performing PCR amplification, obtains upstream and downstream PCR primer respectively in two steps.
PCR reaction systems：PCR reaction systems：
PLM1-TaMVK 1μL PLM1-TaMVK 1μL
PLM1-F1 1μL PLM1-R 1μL
The μ L of 1 μ L variant primers upstream of mutant primer downstream 1
5×FastPfu Buffer 4μL 5×FastPfu Buffer 4μL
2.5 mM dNTP 2μL 2.5 mM dNTP 2μL
FastPfu DNA Polymerase 0.4μL FastPfu DNA Polymerase 0.4μL
ddH2O 10.6μL ddH2O 10.6μL
PCR reaction conditions：
94℃ 5min
94 ℃ 30S
60℃ 30S
72℃ 1min
72℃ 10min
30 circulations of reaction, 16 DEG C of insulations.
Obtain upstream and downstream product, gel extraction.Then, then enter performing PCR amplification, reaction system and course of reaction are as follows.
PCR reaction systems：
The μ L of upstream PCR primer 1
The μ L of downstream PCR product 1
5×FastPfu Buffer 4μL
2.5 mM dNTP 2μL
FastPfu DNA Polymerase 0.6μL
ddH2O 8.6μL
PCR reaction conditions：
94℃ 5min
94 ℃ 30S
47℃ 30S
72℃ 1.5min
5 circulations of reaction.
Then each 1 μ L of downstream mutant primer, plus the μ L of FastPfu 0.2 are added, enters performing PCR amplification.
PCR reaction conditions：
94℃ 5min
94 ℃ 30S
58℃ 30S
72℃ 1.5min
72℃ 10min
34 circulations of reaction, in 16 DEG C, insulation.
Step 3.3 obtains mutant product, carries out digestion connection.
With step 2.4.
Step 3.4 is converted and positive clone identification
With step 2.6.
The expression of step 3.5 mutant protein, purifying, electrophoresis detection and dialysis
With step 2.7,2.8,2.9,2.10.
Step 4 Mevalonate kinase TaMVK and its mutant enzyme kinetics parametric measurement
The measure of Mevalonate kinase kinetic parameter, using pyruvate kinase and the method for lactic dehydrogenase coupling reaction Carry out, it is specific as follows.
Following reagent is sequentially added in disposable Germany's Brand plastic cuvettes, makes reaction final volume 1mL.
0.1M kaliumphosphate buffers（pH 7.6）,
0.16mM NADH,
5mM MgCl2,
4mM ATP,
3mM mevalonic acids,
0.5mM PEPs,
10U pyruvate kinases,
10U lactic dehydrogenases,
Add a certain amount of mevalonate kinase and start reaction,
Volume 1mL is supplemented to H2O,
Determine NADH under 340nm wavelength and be oxidized time Change of absorption, obtain reaction slope.Background oxidation 2min is determined first, 50sec is determined immediately after adding enzyme.If enzymatic activity is stronger, absorbance value decrease speed is very fast under 340nm wavelength, conversely, Then enzymatic activity is weaker.Using mevalonate concentrations as gradient change, enzymatic activity is determined, dynamics of the enzyme to substrate mevalonic acid is obtained Parameter；Using ATP concentration as gradient change, enzymatic activity is determined, kinetic parameter of the enzyme to substrate A TP is obtained.
2nd, experimental result
（One）Mevalonate kinaseTaMVKCoding sequence
Using the cDNA of wheat breed Jimai 22 as template amplification, the Mevalonate kinase that length is 1167bp is obtainedTaMVKThe coding region sequence of gene, 388 amino acid residues of codified.
Wild-type wheatTaMVKGene coding region DNA sequence dna（SEQ ID NO.32）：
ATGGAGATCCGCGCCCGCGCGCCCGGCAAGATCATCCTCGCCGGCGAGCACGCCGTCGTCCACGGCTCTGCCG CCGTCGCCGCCGCCATCGACCTCTACACGCAGTCCTCCCTCCACCTGCCCCCCTCAGGTGAGGGCGGCGCCGGCGAA GTGGAGGTTGACCTGACGGACTCGGGCCTCGCCTTCTCCTGGCCATGCTCGCGCCTCCTCGAGGCGCTGGGGGAGAC CTCCCGCAAGGCGGAGCTGCAGGCCCCGAGGCCCTGCTCCCCGGAGGAGCTGGCCGCCATTGCCAGGCTCGTGGAGC TGCACGAGATACCCGAGGCCAAGATCTGGCTCTCCGCCGGCCTCTCCGCGTTCCTCTACCTCTACACCTCCATCCTG GGGTGTAGGCCTGGGAAGGTCGTGGTGAGCTCTGGCCTGCCGATGGGCGCCGGGCTTGGCTCGTCGGCTGCGTTCTC CGTGTCGTTGTCGGGCGCGCTGCTGACAGCGGCAGGTGTGGTTTCTGCTGAGGGCGCCGTTGGCGGAACGGAGTGGC AGTTGTTGGGGAAGGATCATCTTGAGCTGGTTAACACGTGGGCGTTCCAGGGGGAAAAGATTATTCATGGCAAGCCT TCTGGCATTGATAACGCTGTCAGCACTTTTGGAAGCATGATCAAATTCAAGAAGGGAGAATTGACGAACCTCAAATC TGGCAATCCAGTCAAAATGCTCATTACTGATACAAGGGTTGGTAGGAACACCAAGGCTCTGGTTGCTGGTGTGTCTG AAAGAGCATCTAGGCATCCCGATGCTATGGCTTCCGTCTTCcATGCAGTCAACACTATTAGTGAAGAGCTTTCCAGC ATTGTTGAGTTAGCTGCTACTGATGAGATAGCCATGACCTCAAAGGAAGAAAAGCTAGCAGAACTCATGGAGATGAA CCAAGGTTTGCTCCAGTGCATGGGAGTCAGTCATGCTTCTATAGAAACCGTGCTGCGCTCGACATTGAAGTATAACT TGGTCTCGAAGCTCACCGGAGCTGGTGGTGGAGGCTGTGTTTTGACGTTGATACCAACTCTATTGTCCAAGTTAGTT TTGGAGAAGGTCACCACGGAGCTAGAATCGCATGGTTTCCGCTGCTTCAAAGTCGAGGTCGGTGGACAAGGTCTTCA GATTCACCAAGGATAA
Wild-type wheat TaMVK amino acid sequence（SEQ ID NO.33）：
MEIRARAPGKIILAGEHAVVHGSAAVAAAIDLYTQSSLHLPPSGEGGAGEVEVDLTDSGLAFSWPCSRLLEAL GETSRKAELQAPRPCSPEELAAIARLVELHEIPEAKIWLSAGLSAFLYLYTSILGCRPGKVVVSSGLPMGAGLGSSA AFSVSLSGALLTAAGVVSAEGAVGGTEWQLLGKDHLELVNTWAFQGEKIIHGKPSGIDNAVSTFGSMIKFKKGELTN LKSGNPVKMLITDTRVGRNTKALVAGVSERASRHPDAMASVFHAVNTISEELSSIVELAATDEIAMTSKEEKLAELM EMNQGLLQCMGVSHASIETVLRSTLKYNLVSKLTGAGGGGCVLTLIPTLLSKLVLEKVTTELESHGFRCFKVEVGGQ GLQIHQG
（Two）Mevalonate kinaseTaMVKThe initiative of gene mutation body and protein expression purifying
According to the Mevalonate kinase of designTaMVKGene mutation body primer, obtains TaMVK/A14S, TaMVK/ A24T、TaMVK/Q35N、TaMVK/Q35R、TaMVK/V135E、TaMVK/G139D、TaMVK/M142L、TaMVK/M142F、 13 gene mutation bodies such as TaMVK/F142Y, TaMVK/A144S, TaMVK/S153C, TaMVK/L165M and TaMVK/N332S. Protein expression, purification result show that the gene mutation body protein obtained is solvable, and purity is higher（Fig. 1, Fig. 2）.
（Three）The enzyme kinetics parameter of Mevalonate kinase wild type and mutant
Experiment obtains wheat breed, and " mevalonate kinase of Jimai 22 " wild type and 13 gene mutation body zymoproteins are to substrate Mevalonic acid（MVA）With ATP enzyme kinetics parameter（Table 1）.As a result show, compiled by changing Mevalonate kinase gene The codon of code amino acid, can significantly improve or reduce the catalytic activity of Mevalonate kinase.For substrate MVA, Mutant TaMVK/A14S, TaMVK/V135E and TaMVK/G139D maximum reaction rate are higher, are improved respectively than wild type 1.39,1.14 and 1.07 times, and TaMVK/F142Y maximum reaction rate is minimum, 0.57 times is reduced than wild type； TaMVK/A14S KMValue is maximum, and 1.91 times are improved than wild type, and TaMVK/Q35R KMValue is minimum, than wild type reduction 1.46 times.For substrate A TP, mutant TaMVK/A14S maximum reaction rate is also highest, is improved than wild type 1.50 times, and TaMVK/F142Y maximum reaction rate is also minimum, 0.36 times is reduced than wild type；TaMVK/ F142Y and TaMVK/M142F KMValue is larger, improves 2.15 and 2.12 times than wild type respectively, and TaMVK/Q35N KMValue Minimum, 0.61 times is reduced than wild type.
The wheat TaMVK wild types of table 1 and its mutant enzyme kinetic parameter
Described above is only the preferred embodiment of this patent, it is noted that come for those skilled in the art Say, on the premise of the art of this patent principle is not departed from, some improvement and replacement can also be made, these, which improve and replaced, also should It is considered as the protection domain of this patent.
SEQUENCE LISTING
<110>Crop Inst. of shandong Prov. Agriculture science Academy
<120>Mevalonate kinase TaMVK gene mutation bodies and its method for creating
<130> 2017
<160> 33
<170> PatentIn version 3.5
<210> 1
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<212> DNA
<213>Artificial sequence
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gaggatcgca tcatcaccac catcacgaga tccgcgcccg cgcgcccggc 50
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<211> 39
<212> DNA
<213>Artificial sequence
<400> 2
ctgcagaagc ttttatcctt ggtgaatctg aagaccttg 39
<210> 3
<211> 47
<212> DNA
<213>Artificial sequence
<400> 3
cgcgcggtac caggaggaat ttaaaatgag aggatcgcat catcacc 47
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<211> 50
<212> DNA
<213>Artificial sequence
<400> 4
gaggatcgca tcatcaccac catcacgaga tccgcgcccg cgcgcccggc 50
<210> 5
<211> 39
<212> DNA
<213>Artificial sequence
<400> 5
ctgcagaagc ttttatcctt ggtgaatctg aagaccttg 39
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<211> 33
<212> DNA
<213>Artificial sequence
<400> 6
gcaagatcat cctctccggc gagcacgccg tcg 33
<210> 7
<211> 33
<212> DNA
<213>Artificial sequence
<400> 7
cgacggcgtg ctcgccggag aggatgatct tgc 33
<210> 8
<211> 31
<212> DNA
<213>Artificial sequence
<400> 8
cgtccacggc tctaccgccg tcgccgccgc c 31
<210> 9
<211> 31
<212> DNA
<213>Artificial sequence
<400> 9
ggcggcggcg acggcggtag agccgtggac g 31
<210> 10
<211> 31
<212> DNA
<213>Artificial sequence
<400> 10
cgacctctac acgaactcct ccctccacct g 31
<210> 11
<211> 31
<212> DNA
<213>Artificial sequence
<400> 11
caggtggagg gaggagttcg tgtagaggtc g 31
<210> 12
<211> 31
<212> DNA
<213>Artificial sequence
<400> 12
cgacctctac acgcggtcct ccctccacct g 31
<210> 13
<211> 31
<212> DNA
<213>Artificial sequence
<400> 13
caggtggagg gaggaccgcg tgtagaggtc g 31
<210> 14
<211> 32
<212> DNA
<213>Artificial sequence
<400> 14
ggcctgggaa ggtcgaggtg agctctggcc tg 32
<210> 15
<211> 32
<212> DNA
<213>Artificial sequence
<400> 15
caggccagag ctcacctcga ccttcccagg cc 32
<210> 16
<211> 25
<212> DNA
<213>Artificial sequence
<400> 16
ggtgagctct ggcctgccga tgggc 25
<210> 17
<211> 25
<212> DNA
<213>Artificial sequence
<400> 17
gcccatcggc aggccagagc tcacc 25
<210> 18
<211> 32
<212> DNA
<213>Artificial sequence
<400> 18
gctctggcct gccgctgggc gccgggcttg gc 32
<210> 19
<211> 32
<212> DNA
<213>Artificial sequence
<400> 19
gccaagcccg gcgcccagcg gcaggccaga gc 32
<210> 20
<211> 32
<212> DNA
<213>Artificial sequence
<400> 20
gctctggcct gccgtttggc gccgggcttg gc 32
<210> 21
<211> 32
<212> DNA
<213>Artificial sequence
<400> 21
gccaagcccg gcgccaaacg gcaggccaga gc 32
<210> 22
<211> 32
<212> DNA
<213>Artificial sequence
<400> 22
gctctggcct gccgtatggc gccgggcttg gc 32
<210> 23
<211> 32
<212> DNA
<213>Artificial sequence
<400> 23
gccaagcccg gcgccatacg gcaggccaga gc 32
<210> 24
<211> 31
<212> DNA
<213>Artificial sequence
<400> 24
cctgccgatg ggctccgggc ttggctcgtc g 31
<210> 25
<211> 31
<212> DNA
<213>Artificial sequence
<400> 25
cgacgagcca agcccggagc ccatcggcag g 31
<210> 26
<211> 23
<212> DNA
<213>Artificial sequence
<400> 26
cggctgcgtt ctccgcgtcg ttg 23
<210> 27
<211> 23
<212> DNA
<213>Artificial sequence
<400> 27
caacgacgcg gagaacgcag ccg 23
<210> 28
<211> 30
<212> DNA
<213>Artificial sequence
<400> 28
cgttctccgt gtcgatgtcg ggcgcgctgc 30
<210> 29
<211> 30
<212> DNA
<213>Artificial sequence
<400> 29
gcagcgcgcc cgacatcgac acggagaacg 30
<210> 30
<211> 32
<212> DNA
<213>Artificial sequence
<400> 30
cgacattgaa gtattccttg gtctcgaagc tc 32
<210> 31
<211> 32
<212> DNA
<213>Artificial sequence
<400> 31
gagcttcgag accaaggaat acttcaatgt cg 32
<210> 32
<211> 1167
<212> DNA
<213>Wheat（Triticum aestivum L.）
<400> 32
atggagatcc gcgcccgcgc gcccggcaag atcatcctcg ccggcgagca cgccgtcgtc 60
cacggctctg ccgccgtcgc cgccgccatc gacctctaca cgcagtcctc cctccacctg 120
cccccctcag gtgagggcgg cgccggcgaa gtggaggttg acctgacgga ctcgggcctc 180
gccttctcct ggccatgctc gcgcctcctc gaggcgctgg gggagacctc ccgcaaggcg 240
gagctgcagg ccccgaggcc ctgctccccg gaggagctgg ccgccattgc caggctcgtg 300
gagctgcacg agatacccga ggccaagatc tggctctccg ccggcctctc cgcgttcctc 360
tacctctaca cctccatcct ggggtgtagg cctgggaagg tcgtggtgag ctctggcctg 420
ccgatgggcg ccgggcttgg ctcgtcggct gcgttctccg tgtcgttgtc gggcgcgctg 480
ctgacagcgg caggtgtggt ttctgctgag ggcgccgttg gcggaacgga gtggcagttg 540
ttggggaagg atcatcttga gctggttaac acgtgggcgt tccaggggga aaagattatt 600
catggcaagc cttctggcat tgataacgct gtcagcactt ttggaagcat gatcaaattc 660
aagaagggag aattgacgaa cctcaaatct ggcaatccag tcaaaatgct cattactgat 720
acaagggttg gtaggaacac caaggctctg gttgctggtg tgtctgaaag agcatctagg 780
catcccgatg ctatggcttc cgtcttccat gcagtcaaca ctattagtga agagctttcc 840
agcattgttg agttagctgc tactgatgag atagccatga cctcaaagga agaaaagcta 900
gcagaactca tggagatgaa ccaaggtttg ctccagtgca tgggagtcag tcatgcttct 960
atagaaaccg tgctgcgctc gacattgaag tataacttgg tctcgaagct caccggagct 1020
ggtggtggag gctgtgtttt gacgttgata ccaactctat tgtccaagtt agttttggag 1080
aaggtcacca cggagctaga atcgcatggt ttccgctgct tcaaagtcga ggtcggtgga 1140
caaggtcttc agattcacca aggataa 1167
<210> 33
<211> 388
<212> PRT
<213>Wheat（Triticum aestivum L.）
<400> 33
Met Glu Ile Arg Ala Arg Ala Pro Gly Lys Ile Ile Leu Ala Gly Glu
1 5 10 15
His Ala Val Val His Gly Ser Ala Ala Val Ala Ala Ala Ile Asp Leu
20 25 30
Tyr Thr Gln Ser Ser Leu His Leu Pro Pro Ser Gly Glu Gly Gly Ala
35 40 45
Gly Glu Val Glu Val Asp Leu Thr Asp Ser Gly Leu Ala Phe Ser Trp
50 55 60
Pro Cys Ser Arg Leu Leu Glu Ala Leu Gly Glu Thr Ser Arg Lys Ala
65 70 75 80
Glu Leu Gln Ala Pro Arg Pro Cys Ser Pro Glu Glu Leu Ala Ala Ile
85 90 95
Ala Arg Leu Val Glu Leu His Glu Ile Pro Glu Ala Lys Ile Trp Leu
100 105 110
Ser Ala Gly Leu Ser Ala Phe Leu Tyr Leu Tyr Thr Ser Ile Leu Gly
115 120 125
Cys Arg Pro Gly Lys Val Val Val Ser Ser Gly Leu Pro Met Gly Ala
130 135 140
Gly Leu Gly Ser Ser Ala Ala Phe Ser Val Ser Leu Ser Gly Ala Leu
145 150 155 160
Leu Thr Ala Ala Gly Val Val Ser Ala Glu Gly Ala Val Gly Gly Thr
165 170 175
Glu Trp Gln Leu Leu Gly Lys Asp His Leu Glu Leu Val Asn Thr Trp
180 185 190
Ala Phe Gln Gly Glu Lys Ile Ile His Gly Lys Pro Ser Gly Ile Asp
195 200 205
Asn Ala Val Ser Thr Phe Gly Ser Met Ile Lys Phe Lys Lys Gly Glu
210 215 220
Leu Thr Asn Leu Lys Ser Gly Asn Pro Val Lys Met Leu Ile Thr Asp
225 230 235 240
Thr Arg Val Gly Arg Asn Thr Lys Ala Leu Val Ala Gly Val Ser Glu
245 250 255
Arg Ala Ser Arg His Pro Asp Ala Met Ala Ser Val Phe His Ala Val
260 265 270
Asn Thr Ile Ser Glu Glu Leu Ser Ser Ile Val Glu Leu Ala Ala Thr
275 280 285
Asp Glu Ile Ala Met Thr Ser Lys Glu Glu Lys Leu Ala Glu Leu Met
290 295 300
Glu Met Asn Gln Gly Leu Leu Gln Cys Met Gly Val Ser His Ala Ser
305 310 315 320
Ile Glu Thr Val Leu Arg Ser Thr Leu Lys Tyr Asn Leu Val Ser Lys
325 330 335
Leu Thr Gly Ala Gly Gly Gly Gly Cys Val Leu Thr Leu Ile Pro Thr
340 345 350
Leu Leu Ser Lys Leu Val Leu Glu Lys Val Thr Thr Glu Leu Glu Ser
355 360 365
His Gly Phe Arg Cys Phe Lys Val Glu Val Gly Gly Gln Gly Leu Gln
370 375 380
Ile His Gln Gly
385

Claims (6)

1. Mevalonate kinase TaMVK gene mutation body method for creating, it is characterised in that rite-directed mutagenesis by the following method Form：The corresponding DNA sequence dna of amino acid sequence different from other plants in Mevalonate kinase TaMVK conserved regions is determined Point mutation.
2. Mevalonate kinase TaMVK gene mutation body method for creating according to claim 1, it is characterised in that： 14A, 24A, 35Q, 135V, 139G, 142M, 144A, 153S, 165L in the Mevalonate kinase TaMVK conserved regions With 332N positions amino acid, sport respectively 14S, 24T, 35N, 35R, 135E, 139D, 142L, 142F, 142Y, 144S, 153C, 165M and 332S.
3. Mevalonate kinase TaMVK gene mutation body method for creating according to claim 2, it is characterised in that： The upstream and downstream primer of the Mevalonate kinase TaMVK mutant is respectively：
A14S:F sequences are SEQ ID NO.6, A14S:R sequences are SEQ ID NO.7, and codon sports TCC by GCC；
A24T:F sequences are SEQ ID NO.8, A24T:R sequences are SEQ ID NO.9, and codon sports ACC by GCC；
Q35N:F sequences are SEQ ID NO.10, Q35N:R sequences are SEQ ID NO.11, and codon sports AAC by CAG；
FQ35R:F sequences are SEQ ID NO.12, FQ35R:R sequences are SEQ ID NO.13, and codon sports CGG by CAG；
V135E:F sequences are SEQ ID NO.14, V135E:R sequences are SEQ ID NO.15, and codon sports GAG by GTG；
G139D:F sequences are SEQ ID NO.16, G139D:R sequences are SEQ ID NO.17, and codon sports GAC by GGC；
M142L:F sequences are SEQ ID NO.18, M142L:R sequences are SEQ ID NO.19, and codon sports CTG by ATG；
M142F:F sequences are SEQ ID NO.20, M142F:R sequences are SEQ ID NO.21, and codon sports TTT by ATG；
F142Y:F sequences are SEQ ID NO.22, F142Y:R sequences are SEQ ID NO.23, and codon sports TAT by TTT；
A144S:F sequences are SEQ ID NO.24, A144S:R sequences are SEQ ID NO.25, and codon sports TCC by GCC；
S153C:F sequences are SEQ ID NO.26, S153C:R sequences are SEQ ID NO.27, and codon sports TGC by TCC；
L156M:F sequences are SEQ ID NO.28, L156M:R sequences are SEQ ID NO.29, and codon sports ATG by TTG；
N332S:F sequences are SEQ ID NO.30, N332S:R sequences are SEQ ID NO.31, and codon sports TCC by AAC.
4. the wheat first of Mevalonate kinase TaMVK gene mutation bodies method for creating initiative according to claim 2 Hydroxyl kinase TaMVK gene mutation bodies, it is characterised in that：Mevalonate kinase TaMVK gene mutation bodies TaMVK/ A14S、TaMVK/A24T、TaMVK/Q35N、TaMVK/Q35R、TaMVK/V135E、TaMVK/G139D、TaMVK/M142L、 TaMVK/M142F、TaMVK/F142Y、TaMVK/A144S、TaMVK/S153C、TaMVK/L165MWithTaMVK/N332S, respectively For 14A in the Mevalonate kinase TaMVK conserved regions, 24A, 35Q, 135V, 139G, 142M, 144A, 153S, 165L and 332N positions amino acid, sport respectively 14S, 24T, 35N, 35R, 135E, 139D, 142L, 142F, 142Y, 144S, 153C, 165M and 332S.
5. Mevalonate kinase TaMVK gene mutation bodies according to claim 4, it is characterised in that：Described is small Wheat mevalonate kinase TaMVK gene mutation bodies TaMVK/A14S、TaMVK/A24T、TaMVK/Q35N、TaMVK/Q35R、 TaMVK/V135E、TaMVK/G139D、TaMVK/M142L、TaMVK/M142F、TaMVK/F142Y、TaMVK/A144S、 TaMVK/S153C、TaMVK/L165MWithTaMVK/N332S, upstream and downstream primer is respectively：
A14S:F sequences are SEQ ID NO.6, A14S:R sequences are SEQ ID NO.7, and codon sports TCC by GCC；
A24T:F sequences are SEQ ID NO.8, A24T:R sequences are SEQ ID NO.9, and codon sports ACC by GCC；
Q35N:F sequences are SEQ ID NO.10, Q35N:R sequences are SEQ ID NO.11, and codon sports AAC by CAG；
FQ35R:F sequences are SEQ ID NO.12, FQ35R:R sequences are SEQ ID NO.13, and codon sports CGG by CAG；
V135E:F sequences are SEQ ID NO.14, V135E:R sequences are SEQ ID NO.15, and codon sports GAG by GTG；
G139D:F sequences are SEQ ID NO.16, G139D:R sequences are SEQ ID NO.17, and codon sports GAC by GGC；
M142L:F sequences are SEQ ID NO.18, M142L:R sequences are SEQ ID NO.19, and codon sports CTG by ATG；
M142F:F sequences are SEQ ID NO.20, M142F:R sequences are SEQ ID NO.21, and codon sports TTT by ATG；
F142Y:F sequences are SEQ ID NO.22, F142Y:R sequences are SEQ ID NO.23, and codon sports TAT by TTT；
A144S:F sequences are SEQ ID NO.24, A144S:R sequences are SEQ ID NO.25, and codon sports TCC by GCC；
S153C:F sequences are SEQ ID NO.26, S153C:R sequences are SEQ ID NO.27, and codon sports TGC by TCC；
L156M:F sequences are SEQ ID NO.28, L156M:R sequences are SEQ ID NO.29, and codon sports ATG by TTG；
N332S:F sequences are SEQ ID NO.30, N332S:R sequences are SEQ ID NO.31, and codon sports TCC by AAC.
6. the application of the Mevalonate kinase TaMVK gene mutation bodies according to claim 1-5, it is characterised in that： Mevalonate kinase TaMVK gene mutation bodies are changing mevalonate kinase gene in the crops such as wheat and other plants Expression, changes secondary metabolite yield, improves seed size, the application of grain weight economical character；And to wheat mevalonic acid Kinase gene introne, extron and promoter structure research, research promoter function, exploitation about molecular labeling application.
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CN105112430A (en) * 2015-09-11 2015-12-02 山东省农业科学院作物研究所 Wheat 3-hydroxy-3-methylglutaryl-coenzyme A reductase (TaHMGR) gene, isolation and cloning method thereof, site-specific mutagenesis method thereof and enzyme function detection method thereof

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CN102392032A (en) * 2011-11-28 2012-03-28 山东省农业科学院作物研究所 Triticum aestivum Mevalonate kinase gene TaMVK, and separation cloning and enzymatic activity determination method thereof
CN105112430A (en) * 2015-09-11 2015-12-02 山东省农业科学院作物研究所 Wheat 3-hydroxy-3-methylglutaryl-coenzyme A reductase (TaHMGR) gene, isolation and cloning method thereof, site-specific mutagenesis method thereof and enzyme function detection method thereof

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Application publication date: 20171020