CN102061300A - Streptomyces citrate synthase gene and encoding protein thereof - Google Patents
Streptomyces citrate synthase gene and encoding protein thereof Download PDFInfo
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Abstract
The invention discloses a streptomyces citrate synthase gene and an encoding protein thereof. The gene has a gene sequence shown in SEQ ID NO:1. The length of an overall ORF (Open Reading Frame) of the gene is 1,290bp. A polypeptide with 429 amino acids is encoded. An SdCS (amylase streptomyces citrate synthase gene) is connected with an expression vector pET-28b(+), E.coli Rosetta (DE3) competent cells are transformed, and induction expression and purification are carried out to obtain a soluble fusion protein His6-SdCS. The citrate synthase gene has a higher application value in a transgenic plant and can be used for genetic modification of the plant. By transformation of the SdCS gene, the output and the tolerance for stress environments of crops can be improved.
Description
1 technical field
The invention belongs to gene engineering technology field, relate to the albumen of streptomycete Oxalacetic transacetase gene, this genes encoding and the application of this gene.
2 background technologies
Oxalacetic transacetase (citrate synthase, CS, EC 4.1.3.7) almost is present in all organisms, is the crucial rate-limiting enzyme of multiple pathways metabolism in the cell and the marker enzyme of metabotic change.But the condensation reaction between CS catalysis oxaloacetic acid and the acetyl-CoA generates citric acid and coenzyme A.According to its location difference in eukaryotic cell, CS can be divided into plastosome CS (mCS), glyoxysome CS (gCS), peroxysome CS (pCS).CS has multiple isozyme, and these isozymes participate in various physiological processes, as plastosome energy metabolism, seed germination and degeneration-resistant etc.In recent years, because it is in using value agriculture, medically, the degree that CS is paid close attention to raises day by day.
The isozyme of CS is present in the different subcellular structures, participates in a plurality of important physical pathways metabolisms in the cell.Because citric acid plays an important role in plant metabolism, becomes focus gradually about the research that utilizes the CS gene to carry out transgenic plant in recent years.Existing studies show that plant can improve the utilizability of soil phosphorus by insoluble inorganic phosphorus in the secretion citric acid activating soil.The CS gene is imported in japonica rice and the long-grained nonglutinous rice, obtain the transgenic positive plant, make CS overexpression in rice plant,, can cultivate the transgenic rice plant of tolerant to low-phosphorus stress, be directly used in production to quicken the synthetic in a large number and secretion citric acid of root system by screening.In addition, organic acid such as CS can with Al
3+Complexing forms stabilized complex, and these mixtures are lower or even nontoxic to the toxicity of plant, so improve the expression activity of organic acid synthase gene, increases the synthetic and secretion of organic acid, helps strengthening the anti-aluminium poison patience of plant.Discover, overexpression CS gene in alfalfa, transgenic alfalfa obviously strengthens the tolerance of acid soil and aluminium poison, and the output of plant significantly improves.In addition, in the ripening process of fruit, the organic acid accumulation is relevant with the expression of CS.In the pineapple ripening process, the significant positive correlation of the active poling of the content of organic acid in the fruit (mainly being citric acid) and CS.This can be the raising fruit quality theoretical foundation is provided.The CS gene can significantly change plant growth state, improves the output of plant and coerces the tolerance of environment for some, therefore has higher using value.
Streptomycete (Streptomyces) is a kind of important prokaryotic organism, belongs to actinomycetales Gram-positive soil bacteria.It is dendritic that mycelia is branch, and good gas is saprophytic.Streptomycete has the good reputation of " the production of antibiotics person that the Nature is the most all-round ".At present, in the microbiotic of medical use, have 2/3rds to derive from streptomyces approximately.Simultaneously, the of a great variety and diverse in function of streptomycete secondary metabolites not only has antibacterium, effect such as antimycotic, antiviral and anticancer, and metabolite has the character of immunosuppressor, hypotensive and anti-hypercholesterolemia.Therefore, streptomycete is the important source of industrial acquisition secondary metabolite.In addition, but streptomycete can also produce multiple decomposing protein, xylogen, chitin and cellulosic ferment, decomposing nature is difficult for by the material of other microbiological degradation, as promote the decomposition of waste, can produce fertilizer, solve problem of environmental pollution and improve the value etc. of waste, industrial use is very extensive.In recent years, along with the structure of effective streptomycete expression system, the development of streptomycete molecular biology research causes that more and more people pay attention to rapidly.
3 summary of the invention
First problem to be solved by this invention provides streptomycete---a streptomyces diastaticus Oxalacetic transacetase gene (SdCS) of not finishing genome sequencing as yet at present.It has the gene order shown in sequence table SEQ ID NO:1.
Second problem to be solved by this invention provides coded protein s dCS of said gene and preparation method thereof, and it has the aminoacid sequence shown in sequence table SEQ ID NO:2.
The 3rd problem to be solved by this invention provides recombinant plasmid and the construction process thereof that contains streptomyces diastaticus SdCS gene.
The 4th problem to be solved by this invention provides the application of streptomyces diastaticus SdCS gene in transgenic plant.
4 description of drawings
Fig. 1 streptomyces diastaticus genome clone PCR first time is electrophorogram as a result.
M:DL-2000Marker; 1,2:PCR amplified production, 1563bp.
Fig. 2 SdCS gene ORF conservative fragments electrophorogram.
M:DL-2000Marker; The 1:PCR amplified production, 1290bp.
Fig. 3 His
6-SdCS fusion rotein SDS-PAGE electrophoresis and Western blot analysis chart
(A) SDS-PAGE electrophoresis (12%gel).M:protein molecular mass marker; Lane 1, the SdCS fusion rotein of purifying; Lane 2, pET-SdCS recombinant plasmid transformed Rosetta (DE3) bacterium, and the last white protein behind the IPTG abduction delivering, the molecular weight of SdCS own is about 45kDa; Lane 3, and pET-SdCS recombinant plasmid transformed Rosetta (DE3) bacterium is without the last white protein of IPTG abduction delivering; Lane 4, and the unloaded plasmid of pET-28b (+) transforms Rosetta (DE3) bacterium, the last white protein behind the abduction delivering.
(B) Western-blot analyzes.Lane 1, the SdCS fusion rotein of purifying; Lane 2, negative control, and the unloaded plasmid of pET-28b (+) transforms Rosetta (DE3) bacterium, the last white protein behind the IPTG abduction delivering.The residing position of the protein protomer of SdCS shown in arrow band.
5 embodiments
5.1 the extraction of streptomycete DNA, homology primer design and pcr amplification
With
Genomic DNA Purification Kit (Promega company) extracting streptomycete M1033 (Shandong Province's food fermentation industry research institute designing institute) genomic dna.According to the homology between the streptomyces CS gene, design a pair of primer: P1 (5 '-AAGTGGGGGATGGTAGAGACAGT-3 ') and P2 (5 '-CCTCACAGACGCCCGGCCGGATC-3 '), with the streptomyces diastaticus genomic dna is template, and P1 and P2 are primer, uses high-fidelity Prime
The HS archaeal dna polymerase carries out pcr amplification, amplification reaction condition: 94 ℃ of pre-sex change 3min; 94 ℃ of 30sec, 60 ℃ of 45sec, 72 ℃ of 2min, 30 circulations; 72 ℃ are extended 5min.
Primer P1/P2 amplified production detects through 1% agarose gel electrophoresis, the results are shown in Figure 1.Use DNA GelExtraction Kit (Axygen) to reclaim the PCR product.This dna fragmentation is connected to pMD19-T carrier (TaKaRa), and positive colony is delivered to Shanghai living worker bio-engineering corporation and is checked order, and obtains the dna fragmentation of a 1563bp.
5.2 the acquisition of total length SdCS gene fragment
The sequence of gained is carried out the comparison of Nucleotide BLAST homology find that comprise a complete ORF in this fragment, total length 1290bp is shown in sequence table SEQ ID NO:1.429 amino acid whose polypeptide of this ORF coding are shown in sequence table SEQ ID NO:2.The nucleotide sequence homology that gained ORF and this belong to known Oxalacetic transacetase gene can reach more than 90%; Amino acid sequence homology is also more than 90%.Therefore, this ORF that comprises in the primer P1/P2 amplified fragments should be the full gene of coding streptomyces diastaticus CS.
According to two gene-specific primer: the P3 of SdCS gene conservative fragments sequences Design that obtained (5 '-CTCCTGC
CATATGAGCGACAACTCTGTAGTACTGC-3 ', underline position are restriction enzyme Nde I restriction enzyme site) and P4 (5 '-ATTAGTAT
GCGGCCGCTCAGCGCTCCTCGACGGGCACG-3 ', underline position are Not I restriction enzyme site), the complete ORF of pcr amplification streptomyces diastaticus CS, amplification reaction condition: 94 ℃ of pre-sex change 3min; 94 ℃ of 30sec, 56 ℃ of 45sec, 72 ℃ of 1.5min, 30 circulations; 72 ℃ are extended 5min.The pcr amplification product of primer P3/P4 is seen Fig. 2 through 1% agarose gel electrophoresis detected result.
5.3pET-SdCS the abduction delivering of construction of prokaryotic expression vector and fusion rotein
The pcr amplification product of primer P3/P4 was connected as a child with Not I double digestion 4 through Nde I respectively with carrier pET-28b (+), obtained prokaryotic expression plasmid pET-SdCS.To E.coli Rosetta (DE3) competent cell, the picking mono-clonal is in 5ml LB liquid nutrient medium with the pET-SdCS recombinant plasmid transformed, and 37 ℃, 225rpm cultivates 12h.Get enlarged culturing in the LB liquid nutrient medium that 1ml bacterium liquid is inoculated in 50ml, 37 ℃, 225rpm, shaking culture is to OD
600≈ 0.6-0.8 adds IPTG to final concentration 0.5mM, 20 ℃ of abduction delivering 18h.
With the bacterium liquid after inducing in 4 ℃, 5,000rpm, centrifugal 5min collects thalline, SDS-PAGE detects behind the ultrasonic disruption, finds recombinant protein His
6-SdCS is present in the last white protein of solubility (Fig. 3 A).Co
2+Ion affinity chromatography (
Purification Kit, Clontech) the 12%SDS-PAGE isolation identification of the protein behind the purifying, there is a specific band (as Fig. 3 A) position that is slightly larger than 45kDa at molecular weight, with molecular weight size (47.8kDa) basically identical of the SdCS that infers by aminoacid sequence; In addition, Western blot result shows, also a band that specificity is very strong arranged being slightly larger than 45kDa place, illustrates that the protein of purifying collection has His exactly
6The SdCS of-tag (Fig. 3 B).The Oxalacetic transacetase gene has higher using value in transgenic plant, can be used for the improvement of genes of plant.By changing the SdCS gene over to, can improve the output of farm crop and coerce the tolerance of environment for some.
SEQUENCE?LISTING
＜110〉Anhui Normal University
＜120〉a kind of streptomycete Oxalacetic transacetase gene and proteins encoded thereof
<130>1
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Ser?Thr?Ile?Arg?Leu?Leu?Ala?Lys?Leu?Pro?Thr?Ile?Ala?Ala?Tyr?Ala
165 170 175
Tyr?Lys?Lys?Ser?Val?Gly?His?Pro?Phe?Val?Tyr?Pro?Arg?Asn?Asp?Leu
180 185 190
Gly?Tyr?Val?Glu?Asn?Phe?Leu?Arg?Met?Thr?Phe?Ser?Val?Pro?Ala?Gln
195 200 205
Glu?Tyr?Asp?Leu?Asp?Pro?Val?Val?Val?Ser?Ala?Leu?His?Lys?Leu?Leu
210 215 220
Ile?Leu?His?Ala?Asp?His?Glu?Gln?Asn?Cys?Ser?Thr?Ser?Thr?Val?Arg
225 230 235 240
Leu?Val?Gly?Ser?Ser?Gln?Ala?Asn?Met?Phe?Ala?Ser?Ile?Ser?Ala?Gly
245 250 255
Ile?Asn?Ala?Leu?Trp?Gly?Pro?Leu?His?Gly?Gly?Ala?Asn?Gln?Ser?Val
260 265 270
Leu?Glu?Met?Leu?Glu?Gly?Ile?Arg?Asp?Asn?Gly?Gly?Asp?Val?Asp?Ala
275 280 285
Phe?Ile?Arg?Lys?Val?Lys?Asn?Lys?Glu?Asp?Gly?Val?Arg?Leu?Met?Gly
290 295 300
Phe?Gly?His?Arg?Val?Tyr?Lys?Asn?Phe?Asp?Pro?Arg?Ala?Lys?Ile?Ile
305 310 315 320
Lys?Ala?Ala?Ala?His?Asp?Val?Leu?Ser?Ala?Leu?Gly?Lys?Ser?Asp?Glu
325 330 335
Leu?Leu?Asp?Ile?Ala?Leu?Lys?Leu?Glu?Glu?His?Ala?Leu?Ser?Asp?Asp
340 345 350
Tyr?Phe?Val?Ser?Arg?Ser?Leu?Tyr?Pro?Asn?Val?Asp?Phe?Tyr?Thr?Gly
355 360 365
Leu?Ile?Tyr?Arg?Ala?Met?Gly?Phe?Pro?Thr?Glu?Met?Phe?Thr?Val?Leu
370 375 380
Phe?Ala?Leu?Gly?Arg?Leu?Pro?Gly?Trp?Ile?Ala?Gln?Trp?His?Glu?Met
385 390 395 400
Ile?Lys?Glu?Pro?Gly?Ser?Arg?Ile?Gly?Arg?Pro?Arg?Gln?Ile?Tyr?Thr
405 410 415
Gly?Val?Val?Glu?Arg?Asp?Phe?Val?Pro?Val?Glu?Glu?Arg
420 425
Claims (4)
1. a streptomycete Oxalacetic transacetase gene is characterized in that, has the gene order shown in the SEQ ID NO:1.
2. the albumen by the described genes encoding of claim 1 is characterized in that: have the aminoacid sequence shown in the SEQ ID NO:2.
3. a recombinant plasmid and construction process thereof that contains the described gene of claim 1.
4. the application of the described streptomycete Oxalacetic transacetase of claim 1 gene in plant genetic engineering.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105626738A CN102061300A (en) | 2010-11-27 | 2010-11-27 | Streptomyces citrate synthase gene and encoding protein thereof |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010105626738A CN102061300A (en) | 2010-11-27 | 2010-11-27 | Streptomyces citrate synthase gene and encoding protein thereof |
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| Publication Number | Publication Date |
|---|---|
| CN102061300A true CN102061300A (en) | 2011-05-18 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010105626738A Pending CN102061300A (en) | 2010-11-27 | 2010-11-27 | Streptomyces citrate synthase gene and encoding protein thereof |
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| Country | Link |
|---|---|
| CN (1) | CN102061300A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543260A (en) * | 2016-02-06 | 2016-05-04 | 中国热带农业科学院橡胶研究所 | Application of HbCS4 gene in improvement of growth rate of prokaryotic expression bacteria and study of latex producing ability of rubber tree |
| CN109266665A (en) * | 2018-10-17 | 2019-01-25 | 安徽师范大学 | A kind of gene of heat-resisting citrate synthase, engineering bacteria and its expression containing the gene |
-
2010
- 2010-11-27 CN CN2010105626738A patent/CN102061300A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543260A (en) * | 2016-02-06 | 2016-05-04 | 中国热带农业科学院橡胶研究所 | Application of HbCS4 gene in improvement of growth rate of prokaryotic expression bacteria and study of latex producing ability of rubber tree |
| CN105543260B (en) * | 2016-02-06 | 2019-03-19 | 中国热带农业科学院橡胶研究所 | HbCS4 gene is improving prokaryotic expression bacterium growth rate, is studying the application in rubber tree production glue ability |
| CN109266665A (en) * | 2018-10-17 | 2019-01-25 | 安徽师范大学 | A kind of gene of heat-resisting citrate synthase, engineering bacteria and its expression containing the gene |
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Application publication date: 20110518 |