CN100506990C - Malic dehydrogenase related to high photosynthetic capacity and resisting reversal of wheat, coded genes and method for breeding plants of resisting reversal - Google Patents
Malic dehydrogenase related to high photosynthetic capacity and resisting reversal of wheat, coded genes and method for breeding plants of resisting reversal Download PDFInfo
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Abstract
A malate dehydrogenase associated with the high optical effect and stress resistance of wheat, the gene coding it, the plant expression carrier containing said gene, and the method for culturing the transgenic plant with high optical effect and stress resistance are disclosed.
Description
Invention field
The present invention relates to the gene of the high light efficiency of wheat and degeneration-resistant relevant malate dehydrogenase (malic acid dehydrogenase), this enzyme of encoding and contain this expression carrier.
Background of invention
Malate dehydrogenase (malic acid dehydrogenase) is responsible for the mutual conversion between catalysis oxaloacetic acid and the oxysuccinic acid, and it is prevalent in various animals, plant and the most of bacterium, plays an important role in multiple metabolic process.In vegetable cell, can distinguish five type (Gietl usually according to the specificity of its coenzyme with in intracellular distribution, C., 1992, Biochim.Biophys.Acta, 1100:217-234): (1) depends on the chloroplast(id) MDH (EC 1.1.1.82) of NADP, the enzyme of this type is the oxysuccinic acid round-robin part of shuttling back and forth, and it plays an important role aspect the reduction equilibrium of forces between tenuigenin and the basal granule keeping.It also participates in enrichment CO in leaf sheath cell chloroplast(id) in the C4 plant
2(2) depend on the plastosome MDH of NAD, it is the part of tricarboxylic acid cycle, ubiquity in all eukaryotic cells, and it also participates in photorespiration and the C4 leaf sheath cell CO in plant
2Inrichment.(3) depend on the microbody MDH of NAD, it is present in glyoxysome and the peroxidase precursor, plays an important role in glyoxylate cycle and oxysuccinic acid-aspartic acid shuttle back and forth process, also participates in the photorespiration simultaneously.(4) depend on the chloroplast(id) MDH of NAD, it is less that this kind of enzyme is studied, only in a few plant as ice plant (Mesembryanthemumcryystallinum) (Winter, K. etc., 1982, Plant Physiol, 69:300-307), red autumnal leaves lamb's-quarters (Chenopodium rubrum) (Amino, S., 1992, Z Naturforsch, 47:545-552) and unicell green alga (Chlamydomonas reinhardtii) (Willeford, K.O.and Gibbs.M., 1989, Plant Physiol, 90:788-791) the middle existence of finding to have this kind enzymic activity, but find its definite evidence (Berkemeyer.M. etc. that exist at the cDNA of mouseearcress recently, 1998, J Biol Chem, 273:27927-27933).But its biological action it be unclear that.(5) depend on the tenuigenin MDH (cyMDH) of NAD.
To depending on the chloroplast(id) MDH of NADP, the plastosome MDH that depends on NAD has had extensive studies with the microbody MDH that depends on NAD, but the MDH of other types is but known little about it, and particularly depends on the tenuigenin MDH (cyMDH) of NAD in plant.CyMDH (EC 1.1.1.37) it has been generally acknowledged that in the various circulations of shuttling back and forth of carrying out relevant substrate and reducing power exchange between tenuigenin and organoid and plays a role.In having crassulacean acid metabolism round-robin Crassulaceae plant, utilize the acid of MDH synthesizing apple under the nice and cool at night and moistening environment of plant, store CO
2, and under arid by day and the pyritous situation, stomatal closure, CO
2Decarboxylation discharges from oxysuccinic acid, supplies with plant and carries out photosynthetic needs, thereby guaranteed that plant is to the adaptation of drought environment (Borland, A.M. is etc., 1998, Planta 205:342-351).MDH in studies show that in paddy rice, the salt tolerant adaptability of salt tolerant kind and its tenuigenin active relevant (RitambharaG. etc., 2000, Plant Science 156:23-34).The stimulation that the celery blade is subjected to high salt condition can obviously increase MDH activity in the tenuigenin (Aloni, B. etc., 1981, Plant Science Letters, 20:239-250).This show oxysuccinic acid metabolism in the tenuigenin and relevant malate dehydrogenase (malic acid dehydrogenase) thereof in the photosynthetic efficiency that improves plant, adapt in arid and the poor environment such as saline and alkaline and have vital role, thereby in the plant gene engineering technology field, have application potential widely.
To cyMDH in the plant the research work of gene level carry out less.Argentina scientist from pineapple leaf, be purified into a kind of protein, and prove it belong to cyMDH (Cuevas andPodesta, 2000, Physiol Plant, 108:240-248).Germany scientist is separated to the cDNA of a kind of cyMDH of coding from Crassulaceae plant ice plant (Mesembryanthemum crystallinum), but to its biochemical characteristic (Ocheretina and Scheibe that do not make a search, 1997, Gene, 199:145-148).The cDNA of the another one cyMDH that U.S. scientist is separated to from clover, and go out relevant protein at expression in escherichia coli, prove that it shows NAD
+High reducing activity and the oxidation activity high to NDAH (Miller, S.S. etc., 1998, Plant J, 15:173-184).So far, do not see the relevant report that malate dehydrogenase gene research is arranged at home and abroad as yet in wheat.
No matter in China still worldwide, wheat all is one of most important farm crop.It also is the widest crop of adaptability, and its output ranks first place in comprising the important farm crop of corn, paddy rice and potato.But wheat belongs to C3 plant, and its photosynthetic efficiency is lower than the C4 plant, as corn, simultaneously in production practice, because environmental stress is very huge as arid, the saline and alkaline loss that causes.Utilize the malate dehydrogenase gene of wheat, by genetic engineering means, the expression activity of regulatory gene improves the oxysuccinic acid metabolism, and the new strain of wheat that obtains high light efficiency and degeneration-resistant border has crucial meaning.
Summary of the invention
The present invention by being provided at a kind of plasmotype in the wheat malate dehydrogenase gene and by its encoded protein matter, reach the oxysuccinic acid metabolism of regulating in the wheat, the photosynthetic efficiency and the resistance of the step of going forward side by side joint wheat strengthen wheat and resist extraneous adverse circumstance as arid and saline and alkaline purpose.Another object of the present invention provides the expression plasmid that said gene can be translated into active protein.
An object of the present invention is to provide the malate dehydrogenase (malic acid dehydrogenase) of plasmotype in the grow wheat.
Another object of the present invention provides a kind of gene of malate dehydrogenase (malic acid dehydrogenase) of the wheat plasmotype of encoding.
A further object of the present invention provides and a kind ofly contains gene of the present invention and gene translation is become active protein expression plasmid, and plant expression vector that can transformed plant cells.
The invention provides a kind of dna sequence dna of malate dehydrogenase (malic acid dehydrogenase) of the wheat plasmotype of encoding, it is selected from down group:
(1), the dna sequence dna of the aminoacid sequence of coding SEQ ID NO:2 or
(2), can hybridize, and the dna sequence dna of the malate dehydrogenase (malic acid dehydrogenase) of can encoding with active plasmotype with the sequence of (1).
Described hybridization conditions is meant, with Hybond membrane place prehybridization solution (the 0.25mol/L sodium phosphate buffer, pH7.2,7%SDS) in, 65 ℃ of prehybridization 30min; Abandon prehybridization solution, add hybridization solution (0.25mol/L sodium phosphate buffer, pH7.2,7%SDS, isotope-labeled nucleotide fragments), 65 ℃ of hybridization 12hr; Abandon hybridization solution, (20mmol/L sodium phosphate buffer, pH7.2 5%SDS), wash film 2 times for 65 ℃, each 30min to add film washing liquid I; (20mmol/L sodium phosphate buffer, pH7.2 1%SDS), wash film 2 times for 65 ℃, each 30min to add film washing liquid II.
Dna sequence dna of the present invention can have the sequence shown in the SEQ ID NO:1.This sequence is the full-length cDNA of the malate dehydrogenase gene (cyMDH) of coding wheat plasmotype, called after TaMDH.This sequence is altogether by 1340 based compositions, wherein, the untranslated district of 5 ' end comprises 44 bases, and the untranslated district of 3 ' end comprises 294 bases (comprising the PolyA of 51 based compositions), and the coding region is made up of 1002 bases (from 45 to 1046), wherein A accounts for 23.35% (234), C accounts for 22.55% (226), and G accounts for 27.54% (276), and T accounts for 26.55% (266), A+T accounts for 49.90% (500), and C+G accounts for 50.10% (502).Dna homolog analysis revealed in international geneseq database (GenBank EMBL DDBJ), the TaMDH gene is the new gene of not reported in the wheat, and it has homology with isolated cytoplasmic malate dehydrogenase gene from ice plant (Mesembryanthemum crystallinum) and clover (Medicago sativa).
The present invention also provides a kind of aminoacid sequence by above-mentioned dna sequence encoding.This aminoacid sequence preferably has the sequence shown in the SEQ ID NO:2.This protein sequence comprises 333 amino acid, shown in SEQ ID NO:2.In this protein sequence, hydrophobic amino acid accounts for 134, and hydrophilic amino acid accounts for 74, and basic aminoacids accounts for 31, and acidic amino acid accounts for 36, and this proteinic molecular weight is 35.5KD, and iso-electric point is 5.9.This protein is the new protein of not reported in the world.
The present invention also provides a kind of expression vector that contains above-mentioned dna sequence dna, and another plant expression vector.It can be the expression vector pET-28a that contains above-mentioned dna sequence dna, perhaps is used for the carrier that contains actin promotor and Bar selection genetic marker that vegetable cell transforms.The building process of these two kinds of expression vectors is as follows:
At TaMDH gene coding region both sides synthetic primer, and introduce BamHI and NotI restriction enzyme site respectively, its primer sequence is respectively:
5 '-primer: 5 '-CGGGATCCATGGCTGCGAAGGAACCGATG-3 ' 3 '-primer:
5’ATAAGAATGCGGCCGCTTACGCCAGGCATGAGTAGG-3’。
Go out the full length coding region of TaMDH gene through pcr amplification, under BamHI and NotI double digestion, be connected to (see figure 1) on the corresponding restriction enzyme site of expression vector pET-28a, cut integrity and the exactness that proves this expression vector with sequential analysis by enzyme.With expression vector transformed into escherichia coli 1477 bacterial strains, after process IPTG induces, e. coli protein through the SDS-PAGE gel electrophoresis analysis, is proved that the TaMDH gene has translated correct protein in intestinal bacteria.After above-mentioned coli strain induced through 37 ℃ of cultivations and IPTG, carry out cellular lysate, separating thallus protein, and this protein carried out the malate dehydrogenase enzyme activity assay, and used substrate is an oxysuccinic acid, obtaining its enzymic activity is 121.4 μ mol
-1Min
-1Mg protein
-1, prove that this protein has the catalytic activity of malate dehydrogenase (malic acid dehydrogenase).
The process pcr amplification goes out the full length coding region of TaMDH gene, under BamHI and NotI double digestion, is connected to the carrier (see figure 2) that contains Actin promotor and Bar selection genetic marker, proves the integrity and the exactness of this expression vector by restriction analysis.This plant expression vector can pass through particle bombardment or pollen tube channel transformed plant cells, thereby improves the oxysuccinic acid metabolism of plant, improves its photosynthetic efficiency and resistance.
Extract total RNA respectively from the root of wheat, stem, leaf texture, transfer on the nylon membrane with capillary tube technique through behind the electrophoresis, with the hybridization of wheat MDH gene probe, radioautograph detects results of hybridization, serves as that contrast makes the hybridization signal stdn with the rRNA probe.Detect of the expression of TaMDH gene with this method at the wheat different sites.The result who obtains shows that this gene all has expression in the tissues of nourishing and growing such as wheat root, stem and leaf.
The invention provides and the high light efficiency of wheat and degeneration-resistant relevant malate dehydrogenase gene and relative protein.Should be noted that, those skilled in the art in the technology of the present invention field use TaMDH gene of the present invention or its TaMDH gene expression plasmid, by the genetically modified organism technology, make up plant expression vector, with the plant expression vector transformed plant cells that makes up with the plant transformed cell culture is become plant, conversion is by agriculture bacillus mediated, particle bombardment or pollen tube channel are to realize purpose of the present invention, can develop transgenic plant with high light efficiency and resistance, for example, cultivate with nourish and grow the oxysuccinic acid metabolism in the tissue of regulation and control wheat, thereby change the photosynthetic efficiency of wheat, and the transgenic wheat of extraneous poor environment ability or other resistant transgenic plant are resisted in increase.Indicated as a preferred embodiment of the present invention, utilize transgenosis to obtain new strain of wheat, it is synthetic to increase the oxysuccinic acid that wheat nourishes and grows in the tissue, and the ability of extraneous poor environment is resisted in improvement.
For this reason, a last free-revving engine of the present invention provides a kind of gene constructed plant expression plasmid of TaMDH of the present invention of using, and by the plant transgene biotechnology, cultivates the method with high light efficiency and degeneration-resistant relevant transfer-gen plant.
Accompanying drawing is described:
Fig. 1: expression TaMDH gene expression plasmid.
Fig. 2: the plant expression vector of expression TaMDH gene
Embodiment
The separation and the sequential analysis of embodiment one, wheat malate dehydrogenase gene probe
(Triticum aestivum L) plants in the greenhouse with wheat, normally waters and applies fertilizer, to growing into 2-3 internode, gather root, stem, leaf texture, with TRI reagent (Molecular Research Center, Inc, Cincinnati USA) extracts total RNA, uses PolyAT
(Promega company, Madison USA) separate Poly (A) to mRNA Isolation test kit
+RNA is with content and the purity of ultraviolet spectrophotometer analysis RNA.
The condition of the synthetic cDNA article one chain of reverse transcription is: Poly (A)
+RNA 1ug, 5 μ mol/L primer 5 '-GACTCGAGTCGACATCGA (T)
17-3 ', 0.5mmol/L dNTP, 50 RNasin of unit, 65 ℃ are incubated 10 minutes, after cooled on ice, add 200U SuperScript
TMIIRNase H-Reverse Transcriptase (Gibco), 42 ℃ are incubated 1 hour, add EDTA to final concentration 10mM, and in 95 ℃ of insulations 10 minutes, in cooled on ice.
The primer of first round PCR is M1:5 '-GACTCGAGTCGACATCG-3 ', M2:5 '-ATGAThGCnmGnGGnrT-3 ', and condition is: 3 μ L cDNA reaction solutions, 1 μ mol/L primer, 0.4mmol/L dNTP, 2.5U Taq archaeal dna polymerase (Gibco company, Grand Island, NY, USA), in 95 ℃ of sex change 5 minutes, 50 ℃ of renaturation 2 minutes, 72 ℃ were extended 40 minutes, carried out 40 circulations then (95 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 3 minutes), last 72 ℃ were extended 10 minutes.
Second to take turns the PCR primer be M3:5 '-ATGyTnGGnsCnrAyCArCC-3 ', M4:5 '-TCnykdATrTGrTCrCAnGC-3 ', and condition is: use 1 μ L first round PCR reaction solution, in 95 ℃ of sex change 5 minutes, other reacted the same first round.The PCR product adopts through 1.0% agarose gel electrophoresis
DNA Isolation test kit (Gibco company, Grand Island, NY, USA) purifying electrophoresis product.Be connected behind the purifying on the pGEM-T Easy carrier (Promega company, Madison, USA).Condition of contact be 16 ℃ 12 hours.The transformation receptor bacterium is E.coli DH5 α, and CaCl is adopted in the preparation of competent cell
2Treatment process (Sambrook, J. etc. (eds), Molecular cloning:A laboratory manual, 2
NdEd., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY, 1989).To connect product and join in the competent cell, ice bath 30 minutes, 42 ℃ of heat shocks 90 seconds add the LB liquid nutrient medium, and 37 ℃ of insulations 45 minutes are coated on the LB flat board that contains penbritin (100ug/ml) and X-Gal (20ug/ml) 37 ℃ of overnight incubation.White clone is inoculated on the LB liquid nutrient medium and (contains penbritin 100ug/ml), and 37 ℃ of overnight incubation are extracted plasmid, carry out restriction analysis after, carry out sequential analysis with ABI 377 dna sequence analysis instrument, determine the MDH probe of wheat.
The result of sequential analysis shows that isolating probe has 683 Nucleotide, has homology with isolated cytoplasmic malate dehydrogenase gene in ice plant (Mesembryanthemum crystallinum) and the clover (Medicago sativa).And its encoded protein matter has the catalytic active center IWGNH of malate dehydrogenase (malic acid dehydrogenase).
The screening of embodiment two, wheat malate dehydrogenase gene and sequential analysis
With wheat stalk Poly (A)
+RNA is a template, and (CA USA) synthesizes cDNA, through external packing, is built into the cDNA library of wheat stalk for Stratagene company, LaJolla with ZAP vector.Get the 50ng dna probe, add 50uCi
32P-dCTP carries out mark, and 37 ℃ are incubated 1 hour, add EDTA to final concentration 10mM termination reaction.Probe is by behind the Sephadex G-50 post, boils 10 minutes in 100 ℃, immediately in cooled on ice, hybridizes.
Get 50000pfu and pave plate, and it is transferred on the nitrocellulose filter, add 1.5M NaCl sex change 2 minutes in 0.5MNaOH, 1.5M NaCl adds 0.5M Tris-HCl (pH8.0) renaturation 5 minutes, 0.2M Tris-HCl (pH7.5) adds 2 x SSC rinsings 30 seconds, film is clipped in the two-layer Whatman filter paper, and 80 ℃ of vacuum bakeouts 2 hours add hybridization solution (6 x SSC afterwards, 5 xDenhardt, 0.5% SDS, 100 μ g/mL milt DNA, 50% methane amide), 42 ℃ are incubated 2 hours, add the probe of mark, 42 ℃ of hybridization are spent the night, and add under the 0.1% SDS room temperature through 6 x SSC and wash twice, each 30 minutes, add 0.1% SDS with 0.1 x SSC again and wash twice in 65 ℃, each 20 minutes, afterwards in-80 ℃ of following radioautograph, after determining positive colony, plaque is taken off, and 4 ℃ of extractions are spent the night in SM solution, carry out the two-wheeled screening by same program again.
For the positive plaque that obtains, the method that provides by Stratagene company is translated into plasmid DNA, through behind the restriction analysis, adopts ABI 377 dna sequence analysis instrument to carry out sequential analysis.
The dna sequence dna that records is shown in SEQ ID NO 1, with its called after TaMDH.It is the malate dehydrogenase gene of plasmotype in the wheat, is the new gene of not reporting as yet so far.
The translation of embodiment three, wheat malate dehydrogenase gene coded protein
The a pair of primer of synthetic:
5 '-primer: 5 '-CGGGATCCATGGCTGCGAAGGAACCGATG-3 '
3 '-primer:
5’-ATAAGAATGCGGCCGCTTACGCCAGGCATGAGTAGG-3’
Introducing BamHI and NotI restriction enzyme site respectively at the primer two ends, is template with the TaMDH gene, carries out pcr amplification, amplification condition: 10ng DNA, 1 μ mol/L primer, 0.4mmol/LdNTP, 2.5U Taq archaeal dna polymerase (Gibco company, Grand Island, NY, USA).In 95 ℃ of sex change 5 minutes, carry out then 30 circulations (95 ℃ 1 minute, 55 ℃ 1 minute, 72 ℃ 1.5 minutes), last 72 ℃ were extended 10 minutes.
The PCR product adopts through 1.0% agarose gel electrophoresis
DNA Isolation test kit (Gibco company, Grand Island, NY, USA) purifying electrophoresis product behind BamHI and NotI double digestion, is connected carrier pET-28a (Novagen Company, USA; Cat No.69872-3) on, transformed into escherichia coli 1477 competent cells, transformant ice bath are after 30 minutes, and 42 ℃ of heat shocks 50 seconds add the LB liquid nutrient medium, and 37 ℃ of insulations 45 minutes are coated on the LB flat board that contains kantlex (50ug/ml).With resistance bacterium colony 37 ℃ of concussion overnight incubation in LB liquid nutrient medium (containing kantlex 50ug/ml), after carrying out 1/100 dilution again, 37 ℃ of concussions were cultivated 2 hours, add IPTG to final concentration 1mM, continue to cultivate 3 hours, centrifugal 10 minutes of 12000g collects thalline, and thalline adds 100ul lysate (50mM Tris-HCl pH6.8, the 100mM dithiothreitol (DTT), 2%SDS, 0.1% tetrabromophenol sulfonphthalein, 10% glycerine), 100 ℃ were boiled 5 minutes, tropina carries out SDS-PAGE electrophoresis (gel strength 12%) according to a conventional method, and the result is presented at wheat malate dehydrogenase (malic acid dehydrogenase) protein expression in the bacterium, and its structure is shown in SEQ ID NO2.
Embodiment four, the active mensuration of malate dehydrogenase (malic acid dehydrogenase)
Intestinal bacteria 50ml after IPTG induced centrifugal 10 minutes in 4000g, add 4ml cellular lysate liquid (20mMTris-HCl pH7.5,1mM PMSF, the 2mM dithiothreitol (DTT), the 100ug/ml N,O-Diacetylmuramidase), 30 ℃ are incubated 15 minutes, use ultrasonication 20 times, centrifugal 10 minutes of 4000g gets supernatant liquor and carries out enzyme activity determination.The enzyme activity determination reaction solution comprises 100mM glycine buffer (pH10), 2.5mM NAD, and the 85mM oxysuccinic acid, the 1ul protein solution, cumulative volume 500ul measures A down at 30 ℃
340Increase calculate the activity of malate dehydrogenase (malic acid dehydrogenase).Protein content is pressed method (Bradford, M.M, 1976, Anal.Biochem., 72:248-254) mensuration of Bio-Rad company.
The result shows, is substrate with the oxysuccinic acid, and its enzymic activity is 121.4 μ mol
-1Min
-1Mgprotein
-1, prove that this protein has the catalytic activity of malate dehydrogenase (malic acid dehydrogenase).This shows that the protein of TaMDH coded by said gene has the function of catalysis malate dehydrogenase, thereby can control the oxysuccinic acid metabolism in the wheat tissue.
The detection of TaMDH gene expression product in embodiment five, the wheat tissue
Adopt the TaMDH expression of gene product in the Northern blot hybridization detection wheat different tissues, isolating total RNA sample from different wheat tissues, in 1.4% agarose/denaturing formaldehyde gel electrophoresis, separate according to a conventional method, transfer to (12 hours transfer time) on the nylon membrane with 20 x SSC by capillary tube technique, nylon membrane is clipped in the two-layer Whatman filter paper, 80 ℃ of vacuum bakeouts 2 hours, film is placed in the hybridization bottle, add hybridization solution (6 x SSC afterwards, 5 x Denhardt, 0.5%SDS, 100 μ g/mL milt DNA, 50% methane amide) 10ml, 42 ℃ of prehybridizations 2 hours, add the malate dehydrogenase gene probe of mark, 42 ℃ of hybridization are spent the night, and add under the 0.1% SDS room temperature through 6 x SSC and wash twice, each 30 minutes, add 0.1% SDS with 0.1 x SSC again and wash twice in 65 ℃, each 10 minutes, in-80 ℃ of following one weeks of radioautograph.Film after the development adds 0.1 x SSC and adds 0.1% SDS and wash twice in 95 ℃, and each 15 minutes, add the hybridization solution that contains 18S rRNA probe afterwards, 42 ℃ of hybridization are spent the night.Film is taken out, wash film by identical program, in-80 ℃ of following radioautograph 1 hour.
To the signal of hybridization through Phosphor Image scanning, and with the rRNA signal standardization, obtain TaMDH relative expression quantity in the wheat different tissues, as shown in table 1:
TaMDH expression of gene amount in the table 1. wheat different tissues (is 100 with leaf)
| Tissue | Leaf | Stem | Root |
| Relative expression quantity | 100 | 112 | 45 |
The result shows that wheat malate dehydrogenase gene TaMDH expresses all obvious expression in the tissue of nourishing and growing.Expression amount in root is lower than the expression in stem and the leaf slightly.
Embodiment six, the catalysis characteristics of TaMDH albumen in the oxysuccinic acid metabolism
Malate dehydrogenase (malic acid dehydrogenase) can the catalysis oxaloacetic acid and oxysuccinic acid between mutual conversion, but the malate dehydrogenase (malic acid dehydrogenase) of difference in functionality is very different on the directivity of reaction, thereby, and further have influence on its biological function in the metabolism that has influenced oxysuccinic acid in varying degrees.The malate dehydrogenase (malic acid dehydrogenase) TaMDH of wheat at expression in escherichia coli, is induced intestinal bacteria 100ml bacterium liquid with 1mM IPTG, centrifugal 10 minutes in 4000g, add 10ml cellular lysate liquid (50mMTris-HCl pH8.5,500mM NaCl, 10% glycerine, 1% Triton X-100,1mMPMSF, 20mM dithiothreitol (DTT), the 100ug/ml N,O-Diacetylmuramidase), 30 ℃ are incubated 15 minutes, use ultrasonication 20 times, centrifugal 10 minutes of 4000g, get supernatant liquor, cross Ni-NTA His-
(Novagen USA), obtains the TaMDH albumen of purifying to the Resins affinity column, measures the activity of its catalysis oxaloacetic acid reduction and oxysuccinic acid oxidation respectively with this protein.The oxaloacetic acid reductive is measured and is adopted 100mM Tris damping fluid (pH7.5), 0.225mM NADH, and the 0.06-1.2mM oxaloacetic acid, the 1ul protein solution, cumulative volume 1000ul measures A down at 30 ℃
340Reduction calculate the activity of enzyme.The mensuration of oxysuccinic acid oxidation adopts 100mM glycine buffer (pH10), 2.5mM NAD, and the 1-50mM oxysuccinic acid, the 1ul protein solution, cumulative volume 500ul measures A down at 30 ℃
340Increase calculate the activity of enzyme.Adopt two counting backward technique calculating K m, Vmax, kcat, and enzyme kinetics constant such as kcat/KM, the result is as shown in table 2.
Table 2.TaMDH is to oxysuccinic acid oxidation and oxaloacetic acid reductive catalysis characteristics
The result shows that TaMDH catalysis oxaloacetic acid reductive activity is obviously greater than the oxysuccinic acid oxidation, and its ratio can reach 28.1 (oxidations of oxaloacetic acid reduction/oxysuccinic acid).Therefore this kind malate dehydrogenase (malic acid dehydrogenase) can enrichment CO
2Thereby, improve the photosynthetic efficiency of wheat, and resist in the extraneous poor environment (as arid) in increase and to have important effect.
Sequence table
＜110〉CAS Botanical Institute
＜120〉the high light efficiency of wheat and degeneration-resistant relevant malate dehydrogenase (malic acid dehydrogenase), its encoding gene and the method for cultivating adversity resistant plant
<130>
<160>2
<170>PatentIn?version?3.1
<210>1
<211>1340
<212>DNA
<213>wheat，Triticum?aestivum?L.
<400>1
<210>2
<211>333
<212>PRT
<213>wheat?Triticum?aestivum?L.
<400>2
Claims (5)
1, a kind of dna sequence dna of the malate dehydrogenase (malic acid dehydrogenase) of encoding, it is the sequence shown in the SEQ ID NO:1.
2. aminoacid sequence by the described dna sequence encoding of claim 1, it is the sequence shown in the SEQ ID NO:2.
3, a kind of expression vector that contains the described dna sequence dna of claim 1.
4, according to the described expression vector of claim 3, it is a kind of plant expression vector.
5, according to the described expression vector of claim 3, it is the expression vector pET-28a that contains the described dna sequence dna of claim 1.
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| CN1440458A (en) * | 2000-07-04 | 2003-09-03 | 德古萨股份公司 | Nucleotide sequences coding for MDHA gene |
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| 玉米苹果酸脱氢酶基因的分离与结构分析. 胡建广，赵相山，刘军，袁自强，杨金水.植物学报，第41卷第1期. 1999 * |
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