CN101659944A - Tea tree cytoplasmic malate dehydrogenase gene and encoding protein thereof - Google Patents
Tea tree cytoplasmic malate dehydrogenase gene and encoding protein thereof Download PDFInfo
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- CN101659944A CN101659944A CN200910145097A CN200910145097A CN101659944A CN 101659944 A CN101659944 A CN 101659944A CN 200910145097 A CN200910145097 A CN 200910145097A CN 200910145097 A CN200910145097 A CN 200910145097A CN 101659944 A CN101659944 A CN 101659944A
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- 102000013460 Malate Dehydrogenase Human genes 0.000 claims abstract description 18
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- 101710088194 Dehydrogenase Proteins 0.000 claims description 15
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a tea tree cytoplasmic malate dehydrogenase gene and an encoding protein thereof, wherein the enzyme has an amino acid sequence shown by SEQ ID NO: 1. Compared with the prior art, the malate dehydrogenase containing a Cam-cyMDH gene can be widely used for separating D, L-malic acid and be applied to the tea tree F1 hybrid.
Description
Technical field
The present invention relates to tea tree cytoplasmic malate dehydrogenase gene and proteins encoded thereof.
Background technology
Malate dehydrogenase (malic acid dehydrogenase) extensively distributes in vivo, the pMDH, the chMDH of chloroplast(id), the cytoplasmic cyMDH that are present in the gMDH of oxoethanoic acid body, mitochondrial mMDH, peroxysome, though the function difference of above-mentioned malate dehydrogenase (malic acid dehydrogenase), tissue exist difference, the kind difference of location difference, expression in the cell, but the MDH isozyme all belonged to.The MDH isozyme is widely used in the researchs such as the analysis of biomolecules systematics, genetic diversity and variation, ontogeny and species hybridization at present.Isozyme is with in a kind of or the genus, by different genes site or allelotrope encoded polypeptides chain monomer, is pure aggressiveness or heteromers, and their primary structure, physico-chemical property and physiological function are different, but the identical polymorphism enzyme of catalysis.Isozyme extensively is present in the organic sphere, and now existing more than 60 kind of intravital isozyme of animal and plant is studied report.The characteristics of isozyme are: having the specificity of tangible kind, tissue and etap, both can be used as physical signs, is again reliable genetic marker.
Malate dehydrogenase (malic acid dehydrogenase) (malate dehydrogenase, MDH) be one of the key enzyme of tricarboxylic acid cycle (TCA), but it is the inverse conversion between catalysis oxysuccinic acid and the oxaloacetic acid in tricarboxylic acid cycle, wherein oxaloacetic acid is an important intermediate product of biochemical reaction in the organism, connecting many important pathways metabolisms, is that amino acid forms one of main source of necessary carbon skeleton.In addition, malate dehydrogenase (malic acid dehydrogenase) also has important effect in the multiple physiological activity of cell, as relates to mitochondrial energy metabolism, oxysuccinic acid one aspartic acid shuttle system, the disease resistance of plant and the active oxygen metabolism in the plant disease-resistant process etc.
Tea tree [Camellia sinensis (L.) O.Kuntze] belongs to the Theaceae Camellia, is the perennial evergreen xylophyta.Tea tree has great economic worth, is a kind of worldwide drink that is widely current, and its secondary metabolite has great medical value especially.Although malate dehydrogenase (malic acid dehydrogenase) has the important physical biochemical function, up to now, in tea tree, do not see complete malate dehydrogenase gene clone's report as yet.
Summary of the invention
The 1st technical problem to be solved by this invention provides a kind of new tea tree cytoplasmic malate dehydrogenase gene.
It is said gene encoded protein matter that the 2nd technical problem to be solved by this invention provides.
The 3rd the above-mentioned proteinic application of technical problem to be solved by this invention.
The present invention obtains the Cam-cyMDH gene from tea tree, the aminoacid sequence of its coding shown in SEQ ID NO:1 is connected the Cam-cyMDH gene with the pGEX-4T-1 plasmid, make up recombinant expression plasmid pGEX-MDH; This recombinant plasmid transformed competent escherichia coli cell makes up recombinant protein GST-MDH.
Malate dehydrogenase (malic acid dehydrogenase) of the present invention is particularly separating D, the application of L MALIC ACID at the preparation chiral drug.
The application of malate dehydrogenase (malic acid dehydrogenase) of the present invention in the tea tree cross-breeding.
The present invention compared with prior art, but the malate dehydrogenase (malic acid dehydrogenase) widespread use that contains the Cam-cyMDH gene separates D, L MALIC ACID and the application in tea tree F1 cross-fertilize seed.
Description of drawings
The synoptic diagram of the pcr amplification of Fig. 1 Cam-cyMDH gene
M1:Marker DL2000; 11: the pcr amplification product of gene
The synoptic diagram of the amplification of Fig. 2 Cam-cyMDH gene protein coding region
M2:Marker DL2000; 21: the pcr amplification product in protein coding zone
The synoptic diagram of the double digestion of Fig. 3 recombinant plasmid.
M3:Marker DL2000; 31: the recombinant plasmid pGEX-MDH of double digestion; 32: the cyMDH behind the double digestion; 33: the pGEX-4T-1 plasmid behind the double digestion.
The synoptic diagram of the protein electrophorese of Fig. 4 pGEX-MDH.
M4: the recombinant protein that protein standard molecular weight 41 is expressed
The proteic hydrophobicity profile of Fig. 5 Cam-cyMDH.
Embodiment
Below in conjunction with embodiment the present invention is done detailed explanation.
Embodiment 1:
1, the acquisition of the extraction of the total RNA of tealeaves and cDNA.
Adopt the total RNA purification kit of plant (MACHEREY-NAGEL company) to extract the total RNA of tea tree (Dragon Well tea 43), adopt Reverse Transcript ion System test kit (Promega company) to obtain total cDNA the total RNA of the tea tree that extracts, and with total cDNA in-20 ℃ of preservations.
2, the acquisition of malate dehydrogenase (malic acid dehydrogenase) complete sequence.
Design upstream and downstream primer cyMDH-5 ' and cyMDH-3 '
CyMDH-5 ' sequence: GAAAAGGTTTTCAACGCACTCTCT,
Cy-MDH3 ' sequence: TTGGACAAAAACTGTTACCAAGTAG
With the total cDNA of tea tree is template, pcr amplification, and the PCR response procedures is: 95 ℃, the pre-sex change of 3min; 95 ℃ of 30s, 55 ℃ of 30s, 35 circulations of 72 ℃ of 1min30s reactions; 72 ℃ are extended 10min; 4 ℃ of preservations.1% agarose gel electrophoresis separates PCR product (Fig. 1), the AxyPrep dna gel reclaims test kit and reclaims the PCR product, be connected on the pMD19-T carrier (TaKaRa company), 16 ℃ of connections are spent the night, transfer in the intestinal bacteria E.coli DH5 α competent cell connecting liquid, the solid medium of falling LB (containing Amp) and on its surface even coating 40 μ l5-bromo-4-chloro-3-indoles-β-D-galactoside and 7 μ l isopropyl-s, after 12h cultivates, the picking hickie, hickie is placed in LB (the containing Amp) liquid nutrient medium cultivates, with the primer M13-47/RV-M in the pMD19-T support agent, carry out bacterium liquid PCR (bacterium colony PCR condition is: 95 ℃, the pre-sex change of 3min; 95 ℃ of 30s, 55 ℃ of 45s, 35 circulations of 72 ℃ of 1min reactions; 72 ℃ are extended 10min; 4 ℃ of preservations) the checking positive colony obtains the Cam-cyMDH gene, delivers the order-checking of Shanghai Ying Jun Bioisystech Co., Ltd.
Sequential analysis:
This full length gene 1235bp comprises the polyA tail of an ATG initiator codon and TAG terminator codon and 22bp, the 3 ' non-coding region that 5 ' non-coding region that 62bp is long and 174bp are long, the protein that contains 332 amino residue of encoding.
With Protparam (
Http:// au.expasy.org/tools/protparam.html) the proteic physico-chemical property of prediction tea tree cy-MDH, infer that this protein molecular formula is C
1579H
2540N
426O
472S
16, relative molecular mass is 35.557kD, iso-electric point PI is 5.91; The theoretical transformation period of deriving is approximately 30h, and unstable parameter is 33.93, belongs to stabilize proteins (surpass 40 think protein instability).The many amino acid of content is (30 of Ala (38,11.4%), Val (37,11.1%), Leu in this albumen, 9.0%), the fewer amino acid of content is (6 of Phe (6,1.8%), His in this albumen, 1.8%) Trp (5,1.5%).Total electronegative residue (Asp+Glu) is 34, and positively charged residue (Arg+Lys) is 30.Fat index (Aliphatic index) is 100.15.Wetting ability mean number (GRAVY) is 0.133, predicts that this protein is hydrophobic protein, contains more Ala, Val relevant with Leu (Fig. 5) with it.
Embodiment 2:
1, pGEX-MDH construction of recombinant plasmid
The design primer:
cyMDH-F1：GAGATCGC
GGATCCATGGCGAAAGAACCAGTTC；
CyMDH-F2:GTACGCCG
CTCGAGAGAAAGGCAAGAGTATGCCAGA, underscore are respectively BamHI and Xho I restriction enzyme site.Classify template as with Cam-cyMDH gene total order among the embodiment 1, pcr amplification protein coding zone, reaction conditions is: 95 ℃, the pre-sex change of 3min; 95 ℃ of 30s, 55 ℃ of 30s, 35 circulations of 72 ℃ of 1min10s reactions; 72 ℃ are extended 10min; 4 ℃ of preservations.Amplified production (Fig. 2) adopted Nde I and BamHI double digestion 4 as a child with carrier pGEX-4T-1 (Pharmacia Corp), connected, and was converted into E.coli DH5 α competent cell, the flat board of falling LB, and screening positive clone obtains expression plasmid pGEX-MDH.Adopt Nde I and BamHI double digestion to identify back (Fig. 3), deliver the order-checking of Shanghai Ying Jun Bioisystech Co., Ltd, sequence alignment is found embodiment 1 unanimity as a result, and the sequence of amplification really is a tea tree cyMDH encoding gene.
2, malate dehydrogenase (malic acid dehydrogenase) induction expression of protein
To E.coli Rosetta (DE3) competent cell, the picking mono-clonal is in the test tube that contains 5ml LB liquid nutrient medium with the pGEX-MDH recombinant plasmid transformed, and 37 ℃, 225rpm cultivated 12 hours, obtained bacterium liquid.
Get enlarged culturing in the LB liquid nutrient medium that 1ml bacterium liquid is inoculated in 50ml, 37 ℃, 225rpm, shaking culture is to OD
600During for 0.4-1.0 (best 0.6); Add 0.5mM isopropyl-(IPTG), 20 ℃ of abduction delivering 16h;
The bacterium liquid that abduction delivering is good is in 4 ℃, and 5,000rpm, centrifugal 5min collects thalline, and ultrasonic disruption is found recombinant protein GST-MDH (Fig. 4) in supernatant.
Because the D-oxysuccinic acid is a kind of non-natural organic acid, be the acid of a kind of extremely important 4-carbon organic chiral, be mainly used in fields such as hand-type medicine, chiral additives, chiral auxiliary(reagent), malate dehydrogenase (malic acid dehydrogenase) of the present invention can be used to split D, L MALIC ACID obtains the D-oxysuccinic acid.
In tea tree F1 cross-fertilize seed, the difference of utilizing electrophoretic technique to detect between malate dehydrogenase (malic acid dehydrogenase) of the present invention and other MDH isozyme zymogram is identified the purity of cross-fertilize seed, help identifying, select the cross-fertilize seed of high yield, disease-resistant, degeneration-resistant advantage, the economic benefit that this is bigger for agriculture production brings.
Sequence table
＜110〉pacify little normal university
＜120〉tea tree cytoplasmic malate dehydrogenase gene and proteins encoded thereof
<130>1
<160>1
<170>PatentIn?version?3.3
<210>1
<211>1235
<212>DNA
<213>Camellia?sinensis(L.)O.Kuntze
<400>1
gaaaaggttt?tcaacgcact?ctctcatttc?gcatcgtctc?ttcggtcaga?tctctctcta 60
caatggcgaa?agaaccagtt?cgcgtcctcg?tcactggtgc?tgcaggacaa?attggatatg 120
ctcttgttcc?tatgattgcc?agaggagtta?tgttgggacc?agaccagccc?gtgatccttc 180
acatgcttga?tattccacct?gctgctgagg?cattgaatgg?agtgaaaatg?gaattggtgg 240
atgctgcgtt?ccctcttctt?aaaggtgttg?ttgctaccac?tgatgttgtt?gaggcatgca 300
ctggcgtcag?cattgcagtc?atggtcggtg?ggttccctag?gaaagaaggc?atggaaagaa 360
aagatgtgat?gtcaaagaat?gttgccattt?acaaatctca?agcttctgct?ctggagagtt 420
atgctgctgc?aaactgcaag?gttttggttg?ttgctaaccc?tgccaacacc?aatgcgttga 480
ttctaaagga?atttgcacca?tcaatttccg?agaaaaacat?tacttgtttg?acaaggttgg 540
accacaacag?ggcccttggt?caagtttcag?agagattgaa?tgttccagta?tctgatgtga 600
aaaatgttat?tatttggggt?aatcattcct?caactcagta?tcctgatgtc?aaccatgcaa 660
ctgtcaagac?accagctgga?gagaagcctg?tccgtgagct?tgttgctaat?gatgaatggt 720
tgcatggaga?atttataaca?accgtccaac?agcgtggagc?tgctattatc?aaggctagaa 780
agttgtctag?cgcattgtct?gctgctagct?ctgcttgtga?tcacattcgt?gattgggtcc 840
ttggaactcc?agagggaact?tgggtttcta?tgggtgtata?ctttgatggc?tcatacaatg 900
tgccagctgg?gcttatttat?tctttccctg?ttacttgttg?caatggagta?tggacaattg 960
ttcaaggtct?cccaattgat?gatctttcaa?ggaaaaagtt?ggacttgacc?gccgaagagc 1020
ttactgagga?aaaggctctg?gcatactctt?gcctttcttg?aatttgctcc?taacctcctt 1080
tatgctgcag?taggtctcac?gcttttgtgt?gtttttggaa?gaaacagcta?cggttttgaa 1140
taatgccatt?gttatgtgaa?aacttaatgg?aaggatagat?ataaactccc?ctacttggta 1200
acagtttttg?tccaaaaaaa?aaaaaaaaaa?aaaaa 1235
Claims (4)
1, tea tree cytoplasmic malate dehydrogenase is characterized in that: it has SEQ ID NO:1, shown aminoacid sequence.
2, a kind of gene of the described malate dehydrogenase (malic acid dehydrogenase) of claim 1 of encoding.
3, the application of the described malate dehydrogenase (malic acid dehydrogenase) of claim 1 in the preparation chiral drug.
4, the application of the described malate dehydrogenase (malic acid dehydrogenase) of claim 1 in the tea tree cross-breeding.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910145097A CN101659944A (en) | 2009-09-27 | 2009-09-27 | Tea tree cytoplasmic malate dehydrogenase gene and encoding protein thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910145097A CN101659944A (en) | 2009-09-27 | 2009-09-27 | Tea tree cytoplasmic malate dehydrogenase gene and encoding protein thereof |
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| CN101659944A true CN101659944A (en) | 2010-03-03 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525825A (en) * | 2013-07-11 | 2014-01-22 | 华南农业大学 | Clone of plant manganese poison-resistant important gene ShMDH1 and application thereof |
| CN115927453A (en) * | 2023-01-31 | 2023-04-07 | 昆明理工大学 | Application of malic acid dehydrogenase gene in improving absorption and metabolism capacity of plant formaldehyde |
-
2009
- 2009-09-27 CN CN200910145097A patent/CN101659944A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103525825A (en) * | 2013-07-11 | 2014-01-22 | 华南农业大学 | Clone of plant manganese poison-resistant important gene ShMDH1 and application thereof |
| CN103525825B (en) * | 2013-07-11 | 2015-11-18 | 华南农业大学 | The clone of the resistance to manganese poisoning important gene ShMDH1 of one kind of plant and application thereof |
| CN115927453A (en) * | 2023-01-31 | 2023-04-07 | 昆明理工大学 | Application of malic acid dehydrogenase gene in improving absorption and metabolism capacity of plant formaldehyde |
| CN115927453B (en) * | 2023-01-31 | 2023-11-24 | 昆明理工大学 | Application of malate dehydrogenase gene in improving formaldehyde absorption and metabolism capability of plants |
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