CN102250925B - Glutamine synthetase gene, protein coded by same and cloning method therefore - Google Patents
Glutamine synthetase gene, protein coded by same and cloning method therefore Download PDFInfo
- Publication number
- CN102250925B CN102250925B CN2011101484380A CN201110148438A CN102250925B CN 102250925 B CN102250925 B CN 102250925B CN 2011101484380 A CN2011101484380 A CN 2011101484380A CN 201110148438 A CN201110148438 A CN 201110148438A CN 102250925 B CN102250925 B CN 102250925B
- Authority
- CN
- China
- Prior art keywords
- gene
- glutamine synthetase
- gly
- ala
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention relates to a glutamine synthetase gene, a protein coded by same and a cloning method thereof. The gene is from dunaliella, the base sequence of the gene is represented by SEQ ID No.1, and the gene has a function of synthesizing glutamine synthetase and is proved to be capable of obviously improve the glutamine synthesizing capacity of Escherichia coli which is a model organism. Thus, it is indicated that the disclosed full-length gene and the amino acid sequence of the gene have application values in plant gene engineering aspects of improving nitrogen utilization rate of organisms and increasing nitrogen content in organisms.
Description
Technical field
The present invention relates to a kind of glutamine synthetase gene, its proteins encoded and cloning process thereof.
Background technology
Nitrogen is one of essential nutrient of biology growing, comprises NO
3 -, NH
4 +And their some assimilation products, be used for the required amino acid of synthesising biological body and Nucleotide and multiple different nitrogenous compound.The nitrogen element also is the mineral element that plant has the call, and available nitrogenous source is fewer in the soil, and nature will become one of topmost factor of restriction crop yield.Because the nitrogen of external source is very effective to the raising of crop yield, therefore in the concentrated agrosystem, the interim inorganic nitrogenous fertilizer that applies becomes the important channel of improving crop yield.Why many varieties of crops are selected by cultivator, and a part of reason is exactly that they have higher usability to the nitrogenous fertilizer of using.Yet the application of inorganic nitrogenous fertilizer can cause a series of environmental problems, the eutrophy of especially fresh water, and then rivers and seawater.Just because of this, nowadays need reduce the usage quantity of nitrogenous fertilizer, and seek the higher plant gene type of nitrogen use efficiency.Higher nitrogen use efficiency can guarantee under the prerequisite that output does not descend, and reduces the usage quantity of nitrogenous fertilizer.
For the g and D of organism, the assimilation of nitrogen is crucial physiological process, inorganic nitrogen must the assimilate into Stimulina and organonitrogen such as l-asparagine could be absorbed and utilize by organism.Glutamine synthetase (glutamine synthetase; GS) be key enzyme on the path of nitrogen assimilation; It and NADPH-linked glutamate synthase combined action; The assimilation of catalysis ammonia, in vivo in the biosynthesizing of itrogenous organic substance as nitrogen donor, and extraneous inorganic nitrogen element also gets into biological intravital whole Nitrogen Cycling through this approach.Therefore glutamine synthetase has played crucial effect in organism nitrogen assimilation process.
The salt algae (
Dunaliella viridis) belong to volvocales, crinosity algae section, Dunaliella salina genus biology, be the strongest eucaryon unicellular algae of salt tolerance.Not only can also can stand salt concn 0.5-5.5 mol/L acute variation in the external environment in growths such as high sea brine and salt lakes.Under the environment of the high salt in the external world, the NO of lower concentration (mmole) in the salt algae environment capable of using
3 -And NH
4 +The nitrogen of research salt algae utilizes approach, helps making clear that plant nitrogen utilizes approach and regulatory mechanism under the hypersaline environment.Therefore, salt algae glutamine synthetase two gene
DvGS2Clone and Function Identification the proterties of farm crop has important meaning under the adverse circumstance for improving.
Summary of the invention
One of the object of the invention is to provide a kind of glutamine synthetase gene.
Two of the object of the invention is to provide the proteins encoded of this gene.
For reaching above purpose, the present invention realizes through following proposal:
A kind of glutamine synthetase gene, the sequence that it is characterized in that this gene are the base sequence shown in the SEQ ID NO 1.
A kind of proteins encoded of above-mentioned gene is characterized in that this proteic sequence is the aminoacid sequence shown in the SEQ ID NO 2.
A kind of recombinant expression vector, this recombinant vectors contains above-mentioned gene.
A kind of host cell, this host cell contains above-mentioned recombinant vectors.
Of the present inventionComplementation test verified glutamine synthetase gene in the salt algae (
DvGS2) coded glutamine synthetase can bring into play the function of glutamine synthetase in intestinal bacteria; Prove that this gene can recover the ability of the synthetic Stimulina of intestinal bacteria two mutants, also indicated that it is in the using value of other biological nitrogen aspect efficiently utilizing simultaneously.
Description of drawings
Fig. 1 and Fig. 2 are glutamine synthetase gene encoded protein Subcellular Localization prognostic chart of the present invention, and it is very big to show that this proteins encoded is positioned at the possibility of chloroplast(id);
Fig. 3 is the evolutionary analysis figure of glutamine synthetase gene encoded protein of the present invention, shows that this proteins encoded belongs to glutamine synthetase family;
Fig. 4 is that glutamine synthetase gene of the present invention is at intestinal bacteria two mutants ET6017 (Genotype:F-, [araD139] B/r, Δ (argF-lac) 169; FlhD5301, Δ (fruK-yeiR) 725 (fruA25), relA1; RpsL150 (strR); Δ (glnG-glnA) 229, ha-10, the analysis that has complementary functions in deoC1).Add Stimulina in the substratum, the bacterial strain that changes DvGS2 over to and do not change DvGS2 over to all can normal growth;
Fig. 5 is that glutamine synthetase gene of the present invention is at intestinal bacteria two mutants ET6017 (Genotype:F-, [araD139] B/r, Δ (argF-lac) 169; FlhD5301, Δ (fruK-yeiR) 725 (fruA25), relA1; RpsL150 (strR); Δ (glnG-glnA) 229, ha-10, the analysis that has complementary functions in deoC1).Do not add Stimulina in the substratum, the bacterial strain that only changes DvGS2 over to can normal growth.
Specific embodiments
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these instances only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted concrete experiment condition in the following example, usually according to normal condition, like " molecular cloning (Molecular Cloning:A Laboratory Manual, 3rd ed.) ", or the condition of advising according to manufacturer.
Embodiment one:
DvGS2The clone of full length gene coding region and analysis:
This laboratory utilizes SMART
TMCDNA construction kit has made up 3 salt algae cDNA libraries, carries out large scale sequencing through the sequence to cDNA in the library, obtains corresponding EST library (data are not delivered).Through sequence alignment, be separated to a salt algae glutamine synthetase gene, called after
DvGS2Select the corresponding clone in cDNA library and check order, obtained to contain the cDNA sequence of this gene complete ORF.Sequencing result shows:
DvGS2CDNA total length 1440bp, contain the tailing signal TGTAA and poly (A) structure of green alga gene specific.The analytical results of Vector NTI9.1.0 software shows: one of this genes encoding contains 385 amino acid whose albumen, and the molecular weight of albumen size is about 42.2kDa, and iso-electric point is 5.78.Utilize 9.0 couples of the ProtComp Version of Euk-mPLoc 2.0 and the Softberry website of Cell-PLoc website
DvGS2The albumen of genes encoding carries out Subcellular Localization prediction, and it is very big that predicted data shows that this albumen is positioned at the possibility of chloroplast(id), and referring to Fig. 1 and 2, this conforms to the locating information of glutamine synthetase in other species.Evolutionary analysis shows that the albumen of genes encoding of the present invention belongs to glutamine synthetase family, referring to Fig. 3.
Embodiment two:
DvGS2Functional analysis in the intestinal bacteria two mutants:
Through the enzyme blanking method, restriction enzyme site does
SalI+
XbaI (these two restriction enzyme sites are positioned on the plasmid of storehouse) will
DvGS2Full-length cDNA from the plasmid of storehouse, downcut, obtain to comprise the gene fragment of complete ORF.
Then the purpose fragment of being cloned into is inserted among the intestinal bacteria constitutive expression carrier pEGFP-1, and changes intestinal bacteria two mutants ET6017 (Genotype:F-, [araD139] B/r over to; Δ (argF-lac) 169, flhD5301, Δ (fruK-yeiR) 725 (fruA25); RelA1, rpsL150 (strR), Δ (glnG-glnA) 229; Ha-10, deoC1) in.See also document: Merida, A., E. Flores, and F.J. Florencio,
Regulation of Anabaena sp. strain PCC 7120 glutamine synthetase activity in a Synechocystis sp. strain PCC 6803 derivative strain bearing the Anabaena glnA gene and a mutated host glnA gene.J Bacteriol, 1992.
174(2): p. 650-4..This two mutants is glutamine synthetase gene because disappearance is encoded, so can't normal growth in the substratum that does not add Stimulina.This bacterial strain buy in
E. coliGenetic Stock Center, Yale University.By Fig. 4 and Fig. 5 the pipe amide synthetase disappearance phenotype that DvGS2 can complementary intestinal bacteria two mutants ET6017 is described.
DvGS2Expression, make intestinal bacteria two mutants ET6017 under the culture condition that does not add Stimulina, grow.This complementation test has been verified gene in intestinal bacteria
DvGS2Coded glutamine synthetase can be brought into play the function of glutamine synthetase in intestinal bacteria; Prove that this gene can recover the ability of the synthetic Stimulina of intestinal bacteria two mutants, also indicated that it is in the using value of other biological nitrogen aspect efficiently utilizing simultaneously.
Although the invention describes concrete example, having a bit is significantly to those skilled in the art, promptly can do various variations and change to the present invention under the premise without departing from the spirit and scope of the present invention.Therefore, accompanying claims has covered all these changes within the scope of the present invention.
Sequence table
< 110>Shanghai University
< 120>glutamine synthetase gene, its proteins encoded and cloning process thereof
<160> 2
<210> 1
<211> 1440
<212> DNA
<213>The salt algae (
Dunaliella viridis)
<400> 1
AATCGGCACG AGGATCACCA TGGCCACGAT GATGATGATG AAGCCTGCAC AGCTGGCGCA 60
GCGAGGCGCT TTTGGCCGGG CGGCAGCCCC TGCAGCGGGA GGCCGTGGCC TGTCTGTCAG 120
GGCCAATGCC GCCCCCAAGC TGGTGCAGAA GGCTGAGTAC ATCTGGACCG ATGGGCAGGA 180
GGGTGAGCCC CACAAGGGTC TGCTGTTCAA TGAGCTGAGG TCTAAGACCA AGACATTCGC 240
CAAGGAGAAC GGCCTGGACG CCTCTGAGTA CCCTGACTGG AGCTACGATG GCAGCAGCAC 300
CAACCAGGCT GAGGGTGACA ACTCTGACTG CATCATCAGG CCTGTGCGCG TGGTGCCTGA 360
CCCCATCCGT GGCGCTCCCC ACGTGCTGGT CATGTGCGAG GTCTTCGGCC CTGATGGCCA 420
ACCTCACCCA AGCAACACTC GTGCCAAGCT GCGTGAGCAG CTGACCCCAA AGGCTCTGGA 480
GCAGGACTGC TGGTTTGGCT TCGAGCAGGA ATACACCATG CTGGCAAGGA GCGGCAGGCC 540
CTACGGCTGG CCTGAGAACG GCTTCCCTGC TCCCCAGGGC CCTTACTACT GCGCCGTGGG 600
CTCTGAGTCT GTCTACGGCC GCCCCCTCGT GGAGGCCCAC CTGGAGGCCT GCATGGCTGC 660
CGGCATCAAC ATTAGCGGTT GCAACGCTGA GGTCATGCCT GGCCAGTGGG AGTTCCAGGT 720
TGGCCCCTCC GGACCTCTTG ACGTCGGAGA TGAGGTGCAC CTGGCCCGCT ACCTGCTGCA 780
CCGCCTGGGT GAGGACTTTG GCATTGTGGT GACCTTCGAG CCCAAGCCCA TGGTGGACTG 840
GAACGGCGCG GGCGCGCACA CCAACTTCTC CACCAAGGAC ATGCGCCAGC CCGGCGGCAT 900
GGACGCCATC CTGGCTTCCA TTGAGAAGCT GAGCAAGACC CACGTGGAGC ACATCAGCCA 960
GTACGGCCTG AACAACGACC AGCGCCTGAC CGGCAAGTTC GAGACCAGCG ACATCGACAC 1020
CTTCAAGTTT GGCATTGCCG ACCGCGGCAG CTCCATCCGT ATCCCTCTGC CTGTGCAGCT 1080
GAAGGGCTAC GGCTACCTGG AGGACCGCCG CCCGTCAGCT AATGTGGATC CCTACAATGT 1140
GGCCAGGCTG CTGATCAAGT CCACCATCAA CATGTAAAAA GTGATCCTGG GGACGATTTG 1200
TGAGGCCATC ATCTGACAGC TTTGACTTGA CAAGACTAGG ACATTAGGAG AGCTCCACGG 1260
AGCGCACACC TGCTTTCATA AATAACATGA ACAGAACAAG CATGCCTTGG AGCCTGCCTG 1320
TCTTGTTTGC CCGTGAGGGA GAACAAGGTG ATAGGGTATG CATTTGCACT GAATTCAACC 1380
TTAGCTTGTT GTGTAAGTCT GTGTTTCTCA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAC 1440
<210> 2
<211> 385
<212> PRT
<213>The salt algae (
Dunaliella viridis)
<400> 2
Met Ala Thr Met Met Met Met Lys Pro Ala Gln Leu Ala Gln Arg Gly 16
1 5 10 15
Ala Phe Gly Arg Ala Ala Ala Pro Ala Ala Gly Gly Arg Gly Leu Ser 32
20 25 30
Val Arg Ala Asn Ala Ala Pro Lys Leu Val Gln Lys Ala Glu Tyr Ile 48
35 40 45
Trp Thr Asp Gly Gln Glu Gly Glu Pro His Lys Gly Leu Leu Phe Asn 64
50 55 60
Glu Leu Arg Ser Lys Thr Lys Thr Phe Ala Lys Glu Asn Gly Leu Asp 80
65 70 75 80
Ala Ser Glu Tyr Pro Asp Trp Ser Tyr Asp Gly Ser Ser Thr Asn Gln 96
85 90 95
Ala Glu Gly Asp Asn Ser Asp Cys Ile Ile Arg Pro Val Arg Val Val 112
100 105 110
Pro Asp Pro Ile Arg Gly Ala Pro His Val Leu Val Met Cys Glu Val 128
115 120 125
Phe Gly Pro Asp Gly Gln Pro His Pro Ser Asn Thr Arg Ala Lys Leu 144
130 135 140
Arg Glu Gln Leu Thr Pro Lys Ala Leu Glu Gln Asp Cys Trp Phe Gly 160
145 150 155 160
Phe Glu Gln Glu Tyr Thr Met Leu Ala Arg Ser Gly Arg Pro Tyr Gly 176
165 170 175
Trp Pro Glu Asn Gly Phe Pro Ala Pro Gln Gly Pro Tyr Tyr Cys Ala 192
180 185 190
Val Gly Ser Glu Ser Val Tyr Gly Arg Pro Leu Val Glu Ala His Leu 208
195 200 205
Glu Ala Cys Met Ala Ala Gly Ile Asn Ile Ser Gly Cys Asn Ala Glu 224
210 215 220
Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Gly Pro Leu 240
225 230 235 240
Asp Val Gly Asp Glu Val His Leu Ala Arg Tyr Leu Leu His Arg Leu 256
245 250 255
Gly Glu Asp Phe Gly Ile Val Val Thr Phe Glu Pro Lys Pro Met Val 272
260 265 270
Asp Trp Asn Gly Ala Gly Ala His Thr Asn Phe Ser Thr Lys Asp Met 288
275 280 285
Arg Gln Pro Gly Gly Met Asp Ala Ile Leu Ala Ser Ile Glu Lys Leu 304
290 295 300
Ser Lys Thr His Val Glu His Ile Ser Gln Tyr Gly Leu Asn Asn Asp 320
305 310 315 320
Gln Arg Leu Thr Gly Lys Phe Glu Thr Ser Asp Ile Asp Thr Phe Lys 336
325 330 335
Phe Gly Ile Ala Asp Arg Gly Ser Ser Ile Arg Ile Pro Leu Pro Val 352
340 345 350
Gln Leu Lys Gly Tyr Gly Tyr Leu Glu Asp Arg Arg Pro Ser Ala Asn 368
355 360 365
Val Asp Pro Tyr Asn Val Ala Arg Leu Leu Ile Lys Ser Thr Ile Asn 384
370 375 380
385。
Claims (4)
1. glutamine synthetase gene, the sequence that it is characterized in that this gene is the base sequence shown in the SEQ ID NO 1.
2. the albumen of a genes encoding according to claim 1 is characterized in that this proteic sequence is the aminoacid sequence shown in the SEQ ID NO 2.
3. recombinant expression vector, this recombinant vectors contains gene according to claim 1.
4. host cell, this host cell contains recombinant vectors according to claim 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101484380A CN102250925B (en) | 2011-06-03 | 2011-06-03 | Glutamine synthetase gene, protein coded by same and cloning method therefore |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101484380A CN102250925B (en) | 2011-06-03 | 2011-06-03 | Glutamine synthetase gene, protein coded by same and cloning method therefore |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102250925A CN102250925A (en) | 2011-11-23 |
| CN102250925B true CN102250925B (en) | 2012-10-31 |
Family
ID=44978493
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101484380A Active CN102250925B (en) | 2011-06-03 | 2011-06-03 | Glutamine synthetase gene, protein coded by same and cloning method therefore |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102250925B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966240A (en) * | 2013-04-15 | 2014-08-06 | 上海大学 | Glutamine synthetase gene of acinetobacter as well as encoding protein and cloning method of glutamine synthetase gene |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103205441A (en) * | 2013-04-16 | 2013-07-17 | 上海大学 | Galactococcus glutamine synthetase gene, and encoding protein and clone method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101407824A (en) * | 2008-11-24 | 2009-04-15 | 昆明理工大学 | Construction and use of plant expression vector of Arabidopsis thaliana cytoplasm type glutamine synthetase gene |
-
2011
- 2011-06-03 CN CN2011101484380A patent/CN102250925B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101407824A (en) * | 2008-11-24 | 2009-04-15 | 昆明理工大学 | Construction and use of plant expression vector of Arabidopsis thaliana cytoplasm type glutamine synthetase gene |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103966240A (en) * | 2013-04-15 | 2014-08-06 | 上海大学 | Glutamine synthetase gene of acinetobacter as well as encoding protein and cloning method of glutamine synthetase gene |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102250925A (en) | 2011-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107164347A (en) | Control Culm of Rice rugosity, tiller number, grain number per spike, mass of 1000 kernel and the ideotype gene NPT1 of yield and its application | |
| CN107326032A (en) | A kind of gene and application for being used for cadmium pollution soil repair and improving cadmium-resistant vegetable | |
| CN102757969A (en) | Phosphorus transportprotein gene GmPT5 related to phosphorus transport of soybean nodulation and application thereof | |
| CN103289972A (en) | Rice long-grain related gene and application for same | |
| CN102112613A (en) | Genes, proteins and vectors for increaseing tolerance of plants and microbes to abiotic stresses and use thereof | |
| CN109628466A (en) | A kind of alfalfa salt tolerance and drought resistance gene MsCKX and its application | |
| CN110078804A (en) | A kind of protein and its gene improving plant and the resistance to Low nitrogen stress ability of microorganism | |
| CN101824079A (en) | Buckwheat Na+/H+ antiporter FtNHX and coding gene and application thereof | |
| CN102250925B (en) | Glutamine synthetase gene, protein coded by same and cloning method therefore | |
| CN106636028A (en) | Rice protein with imidazolinone herbicide resistance activity and coding gene and application of rice protein | |
| CN104480120A (en) | Plant salt-tolerant related gene PpSIG2 as well as encoding protein and application thereof | |
| CN102121018A (en) | Gene with asparagine synthetase function, encoding protein and application thereof | |
| CN108864264B (en) | Corn OXS2a gene, and encoding protein and application thereof | |
| CN103194462A (en) | Bacillus subtilis glutamine synthetase gene as well as encoding protein and cloning method thereof | |
| CN105037516B (en) | Corn OXS2 gene family, its coding albumen and application | |
| CN104109192A (en) | Wheat draught-resistant gene and use thereof | |
| CN104371991B (en) | Atriplex canescens cysteine protease gene clone and its application | |
| CN113046375A (en) | SpCPK33 gene and application of encoding protein thereof in regulation and control of tomato drought tolerance | |
| CN112899293A (en) | Carotenoid cracking dioxygenase related gene in mulberry and prokaryotic expression thereof | |
| CN101671683B (en) | Synthetase gene relative to carotene synthesis, coded protein and application thereof | |
| CN102250926B (en) | Gene with function of glutamine synthelase, and encoded protein and cloning method thereof | |
| CN112481278A (en) | Biosensor based on AIP induction and application thereof | |
| CN111893134B (en) | Application of OXS2 gene and encoding protein thereof in improving salt stress resistance of plants | |
| CN103966241A (en) | Micrococcus glutamine synthetase gene as well as encoding protein and cloning method thereof | |
| CN103205441A (en) | Galactococcus glutamine synthetase gene, and encoding protein and clone method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |