CN101298616A - Promoter for expressing phosphor deficiency speciality induction in root and use thereof - Google Patents
Promoter for expressing phosphor deficiency speciality induction in root and use thereof Download PDFInfo
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- CN101298616A CN101298616A CNA2008101153013A CN200810115301A CN101298616A CN 101298616 A CN101298616 A CN 101298616A CN A2008101153013 A CNA2008101153013 A CN A2008101153013A CN 200810115301 A CN200810115301 A CN 200810115301A CN 101298616 A CN101298616 A CN 101298616A
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- 230000007812 deficiency Effects 0.000 title abstract description 10
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- FIXLYHHVMHXSCP-UHFFFAOYSA-H azane;dihydroxy(dioxo)molybdenum;trioxomolybdenum;tetrahydrate Chemical compound N.N.N.N.N.N.O.O.O.O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O[Mo](O)(=O)=O.O[Mo](O)(=O)=O.O[Mo](O)(=O)=O FIXLYHHVMHXSCP-UHFFFAOYSA-H 0.000 description 1
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- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
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Images
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a phosphorus deficiency specific inducement root-expression promoter and applications thereof. The promoter can be a DNA molecule as the following 1), 2) or 3): (1) the ribonucleotide sequence thereof is a DNA molecule in the 1st-serial in a sequence list; (2) a DNA molecule which can be hybridized with the DNA molecule of the 1st-serial in the sequence list under a strict condition and can drive targeted genes to be expressed specially in root in case of phosphorus deficiency; (3) a DNA molecule which has the homeology with the DNA by more than 90 percent and can drive targeted genes to be expressed specially in root in case of phosphorus deficiency. The promoter of the invention can be applied to the construction of carriers with economic value and the constructed carriers can be used for breeding transgenic plants with economic value.
Description
Technical field
The present invention relates to phosphor deficiency speciality induction expression promoter and application thereof in root.
Background technology
Promotor is the most important factor in the gene expression regulation factor, and it determines substantially whether a gene is expressed, when expressed and where express.By the mode of action and function, promotor can be divided into constitutive promoter, specificity promoter and inducible promoter three major types substantially.The feature of inducible promoter, for the gene of the type promotor control is not expressed under the condition that does not have inducible factor to exist or had only low-down expression, but in case be subjected to inducing of inducible factor, the expression of gene amount rapidly and increase considerably.For example: hormone induction promotor, chemically inducible promoter, photoinduction promoter etc.The characteristics of constitutive promoter are to be subjected to the expression of the structure gene of its control to have persistence, but not have the space-time specificity; RNA is relative with the protein expression amount constant, is not subjected to inducing of extraneous factor.For example: the Actinl promotor of corn Ubiqultin promotor and paddy rice.The feature of organ or tissue's specificity promoter is to be subjected to the genetic expression of its control or adjusting to have temporal and spatial, and often shows the characteristic of growing adjusting.For example, seed specific promoters, fruit-specific promoter, mesophyll cell specificity promoter, root-specific promoter.
Phosphorus is one of necessary for plant growth macronutrient, and it is not only ATP in the vegetable cell, the important composition composition of Nucleotide and phosphatide, and in energy transfer, albumen activation and carbon nitrogen metabolism, play very important regulating effect.Absolute phosphorus content very high usually (＞1000 μ M) in the soil, but phosphorus is very easily fixed with organic and inorganic form in soil, thereby the concentration of available phosphorus is very low, greatly about 2-10 μ M, the phosphorus that is diffused into the root surface is lower, is difficult to satisfy the needs of growth and development of plants.
Summary of the invention
The purpose of this invention is to provide phosphor deficiency speciality induction expression promoter and application thereof in root.
Promotor provided by the present invention derives from wheat, and called after TaPHT1.2 is following 1) or 2) or 3) dna molecular:
1) its nucleotide sequence is the dna molecular of sequence 1 in the sequence table;
2) under stringent condition with sequence table in sequence 1 dna molecule hybridize and can be at the dna molecular that lacks driving purposes gene specifically expressing in root under the phosphorus condition;
3) with 1) dna sequence dna that limits have 90% or more homology and can be under scarce phosphorus condition the dna molecular of driving purposes gene specifically expressing in root.
Promotor in the described step 3) is with 1) promotor homology more than 95% is preferably arranged.
Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
Wherein, the sequence in the sequence table 1 is made up of 1305 deoxynucleotides.
TaPHT1.2 promotor of the present invention can be used for making up engineering carrier, can make up the expression cassette that contains the TaPHT1.2 promotor earlier, more described expression cassette is inserted into the multiple clone site of the carrier that sets out; Also can directly the TaPHT1.2 promotor be inserted into the multiple clone site of the carrier that sets out, in this carrier, insert goal gene again.
Wherein, described expression cassette is followed successively by described dna molecular, foreign gene and transcription terminator to the downstream from the upstream; The direction of described dna molecular is identical with the transcriptional orientation of foreign gene.
The transgenic cell line and the reorganization bacterium that contain the TaPHT1.2 promotor all belong to protection scope of the present invention.
Increase full length gene of the present invention or its any segmental primer to also belonging to protection scope of the present invention.
The present invention has cloned a promotor that is subjected to phosphor deficiency speciality induction specifically expressing in wheat root from wheat, and has proved the function of described promotor.Experimental result shows that TaPHT1.2 promotor of the present invention can be expressed by the driving purposes gene, mainly expresses in root hair cell and cells of superficial layer in the root system of wheat.TaPHT1.2 promotor of the present invention is lacking the expression that can induce goal gene under the phosphorus condition, can be used for driving reporter gene with indication and the plain nutritional status of the intravital phosphorus of diagnosis crop plant, perhaps be used for driving gene at Gen Mao and root system cells of superficial layer specifically expressing.TaPHT1.2 promotor of the present invention can be cultivation phosphorus efficiency new crop varieties theoretical foundation and genetic resources is provided.
Description of drawings
Fig. 1 is a pBI121-TaPHT1.2-GUS carrier synoptic diagram
Fig. 2 is for adding and subtracting the GUS coloration result after phosphorus is handled
Fig. 3 expresses for TaHT1.2 promoters driven GFP lacks in the cells of superficial layer of phosphorus root system maturation zone and the root hair cell
Fig. 4 is subjected to phosphorus starvation induced expression for the TaHT1.2 promoter-driven GUS
Fig. 5 lacks the activity influence of other nutritive element to GUS simultaneously for lacking phosphorus
Embodiment
The separation of embodiment 1, wheat phosphate radical transporter gene TaPHT1.2 promoter sequence
Extract wheat breed 54 genomic dnas of laying down for a short time, adopt the Universal Kit of Clontech company, wheat cdna group DNA is put down terminal Dra I, EcoR V, Pvu II and Stu I complete degestions with four, use T then
4DNA Ligase adds top connection (Adaptor) at the fragment two ends that enzyme is cut, according to the sequences Design on the joint 5 ' two primers, according to two nested-PCR primers of known TaPHT1.2 gene order design 3 ' end:
Joint sequence is as follows:
5′-GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTGGT-3′
3′-H2N-CCCGACCA-PO4-5′
5 ' end primer sequence is as follows:
Joint primer 1:5 '-GTA ATA CGA CTC ACT ATA GGG C-3 ';
The joint primer 2: 5 '-ACT ATA GGG CAC GCG TGG T-3 '.
3 ' end primer sequence is as follows: (gene specific primer)
Gene specific primer GSP1:5 '-GCCAGAAGCGGAAGAAACATAGCGTCC-3 ';
And GSP2:5 '-GAGCAGAGGACCATGAGGATGAGCGTGA-3 '.
The PCR reaction selects for use Shanghai to give birth to the Pfu high-fidelity enzyme of worker company, and the PCR reaction system is for the first time: ultrapure water 40.5 μ l, 10 * PCR buffer, 5 μ l; The genomic fragment 1 μ l (200ng/ μ l) that adds top connection; Joint primer 1 (10 μ M) 1 μ l; Gene specific primer GSP1 (10 μ M) 1 μ l; Pfu enzyme (5u/ μ l) 0.5 μ l, dNTPs (10mM) 1 μ l.Reaction conditions is: earlier 94 ℃ 4 minutes; Then 94 ℃ 25 seconds, 72 ℃ 4 minutes, 7 circulations; Again 94 ℃ 25 seconds, 67 ℃ 4 minutes, 32 circulations; Last 67 ℃ 4 minutes.Get the template of 1 μ l as the PCR second time after then the PCR product being diluted 50 times, amplimer is respectively joint primer 2 and gene specific primer 2, and other component of PCR reaction system is with PCR is identical for the first time.For the second time the reaction conditions of PCR is: earlier 94 ℃ 4 minutes; Then 94 ℃ 25 seconds, 72 ℃ 4 minutes, 5 circulations; Again 94 ℃ 25 seconds, 67 ℃ 4 minutes, 20 circulations; Last 67 ℃ 4 minutes.
Amplified production reclaims test kit with QIAquick glue and carries out purifying by product description, is connected 8 hours with PUC18 T carrier under 16 ℃ then, makes up recombinant vectors PUC18 T-TaPHT1.2.Use the 2mm pole cup, 2500V is with recombinant vectors PUC18 T-TaPHT1.2 transformed into escherichia coli DH5 α, conversion product is grown containing on the LB plate culture medium of penbritin, and selected clone extracts plasmid, plasmid is checked order, AbI PRISM3700 DNA analysis instrument (Perkin-Elmer/Applied Biosystem) is used in order-checking, and sequencing result shows that the segmental nucleotide sequence that increases is shown in sequence in the sequence table 1, with this fragment called after TaPHT1.2, total length 1305bp.
1) promoter expression specificity analysis
Downcut the TaPHT1.2 promoter fragment with restriction endonuclease Hind III and BamH I from PUC18 T-TaPHT1.2, and the 35S promoter part of downcutting carrier pBI121 with these two enzymes, the TaPHT1.2 promotor is inserted between the Hind III and BamH I site of the pBI121 after enzyme is cut, make up pBI121-TaPHT1.2-GUS recombinant expression vector (Fig. 1), change the recombinant expression vector of the pBI121-TaPHT1.2-GUS that builds over to Agrobacterium GV3101.
With EGFP (Cat.#4999-100, Clontech, Palo Alto, CA) pJIT163 (Guerineau et al. is cloned in the coding region, Plant Molecular Biology, 1992,18:815-818) the 35S promoter downstream of carrier (Inst. of Genetics and Development Biology, CAS) obtains the pJIT163-GFP carrier.Downcut the TaPHT1.2 promoter fragment with restriction endonuclease Kpn I and Sal I from PUC18 T-TaPHT1.2, and the 35S promoter part of downcutting carrier pJIT163-GFP with these two enzymes, the TaPHT1.2 promotor inserted between the Kpn I of the pJIT163-GFP after enzyme is cut and Sal I site obtain the pJIT163-TaPHT1.2-GFP carrier.Then with the TaPHT1.2-GFP in Sac I and the XhoI cutting-out pJIT163-TaPHT1.2-GFP carrier, be connected into pBINPLUS (van Engelen FA again, Transgenic Research.1995, (SalI and XhoI are isocaudarners) makes up the pBINPLUS-TaPHT1.2-GFP recombinant expression vector between SacI 4:288-290) and the SalI site, changes the recombinant expression vector of the pBINPLUS-TaPHT1.2-GFP that builds over to Agrobacterium GV3101.
By dipping in colored dip method arabidopsis thaliana transformation (Colombia's type), results Arabidopis thaliana seed has obtained serial transformation generation respectively for pBI121-TaPHT1.2-GUS that makes up and pBINPLUS-TaPHT1.2-GFP recombinant expression vector.
Arabidopis thaliana T1 carries out GUS to 9 days Arabidopis thaliana plant of germination and growth and dyes, the expression of detection gus gene for seed vertical cultivations 9 days in scarce phosphorus solid medium and normal solid medium respectively.The inorganic salt (mmol/L) composed as follows of normal solid medium: KH
2PO
4, 0.2000mmol/L; Ca (NO
3)
2.4H
2O, 2.0000mmol/L; FeEDTA, 0.1000mmol/L; MgSO
4.7H
2O, 0.5000mmol/L; KCl, 1.5000mmol/L; CaCl
2, 1.5000mmol/L; H
3BO
3, 0.0010mmol/L; (NH
4)
6Mo
7O
24.4H
2O, 0.0001mmol/L; CuSO
4.5H
2O, 0.0005mmol/L; ZnSO
4.7H
2O, 0.0010mmol/L; MnSO
4.H
2O, 0.0010mmol/L.Replace the KH of normal substratum with the KCl of 0.2mmol/L
2PO
4Be and lack the phosphorus solid medium.Normal solid medium and scarce phosphorus solid medium all contain 1% sucrose and 1% agar powder.
Arabidopis thaliana T1 changes normal water planting substratum over to after sprouting in normal solid medium for seed, in normal water planting substratum, cultivated for 3 weeks, change over to lack in the phosphorus water planting substratum again and grow a week, the Arabidopis thaliana plant of getting above-mentioned processing carries out GUS dyeing, detects the expression of gus gene.Normal water planting substratum is identical with the essentially consist of normal solid medium, and difference only is not contain agar and sucrose in the normal water planting substratum; It is identical with the essentially consist of scarce phosphorus solid medium to lack phosphorus water planting substratum, and difference only is to lack in the phosphorus water planting substratum and does not contain agar and sucrose.With the Arabidopis thaliana plant of in normal water planting substratum, cultivating for 4 weeks in contrast.
The GUS coloration result of Arabidopis thaliana plant in the solid medium as shown in Figure 3A, show change the Arabidopis thaliana of pB1121-TaPHT1.2-GUS plant in scarce phosphorus solid plate substratum germination and growth 9d after GUS dyeing, only detect the expression of gus gene, and portion does not detect the GUS expression on the ground at the root system that lacks the phosphorus plant; 9 days Arabidopis thaliana seedling of germination and growth does not detect the expression of gus gene in normal solid medium;
The GUS coloration result of Arabidopis thaliana plant in the water planting substratum is shown in Fig. 3 B, show and change gus gene expression in the root system that lacks the Arabidopis thaliana plant of the commentaries on classics pBI121-TaPHT1.2-GUS in one week of growth in the phosphorus water planting substratum over to, partly do not have gus gene to express on the ground, and the Arabidopis thaliana plant over-ground part of the commentaries on classics pBI121-TaPHT1.2-GUS in 4 weeks of growth still is the expression that root does not all have gus gene in normal water planting substratum.
The Arabidopis thaliana of the commentaries on classics pBI121-TaPHT1.2-GUS that "+P " representative is grown in normal substratum among Fig. 3, the Arabidopis thaliana of the commentaries on classics pBI121-TaPHT1.2-GUS that " P " representative is grown in lacking the phosphorus substratum.
The similar gus gene of GFP detected result lacks the expression that the commentaries on classics pBINPLUS-TaPHT1.2-GFP Arabidopis thaliana of growing 5 days in the phosphorus substratum can detect the GFP reporter gene, and 2 weeks still can detect GFP fluorescence later.
2) TaHT1.2 promoters driven GFP expresses in cells of superficial layer that lacks phosphorus root system maturation zone and root hair cell
TaHT1.2 promoters driven GFP reporter gene is mainly expressed at root system, and is just to express after scarce phosphorus is handled.Fluorescence is presented at root cap, meristematic zone and elongation zone and does not express; Express in the maturation zone, the cell of expression is root hair cell and cells of superficial layer (root hair cell and cells of superficial layer are root system absorbs nutrient from soil the main undertakers), and the result as shown in Figure 4.Illustrate that the function that the expressive site of TaHT1.2 promoters driven GFP and it are born is coincide.
3) phosphor deficiency speciality induction analysis
Except lacking phosphorus, detected GFP expression of gene in the Arabidopis thaliana plant of vertical cultivation in nitrogen stress, iron deficiency and magnesium deficiency substratum.The composition of nitrogen stress, iron deficiency and magnesium deficiency substratum is respectively the normal solid medium that removes nitrogenous source, molysite and magnesium salts.
GFP test experience result only shows that the GFP of TaHT1.2 promoters driven just can be by abduction delivering under the condition that lacks phosphorus as shown in Figure 5.In addition, temperature (low temperature was handled 1 day for 4 ℃) and salt (1%NaCL) etc. do not have direct relation with the GFP expression of gene.
4) compound nutritional deficiency is to the influence of GUS expression
Because the mutual relationship in the soil between the various ions also affects the absorption of plant to them, and usually compound fertilising more can reach the purpose of volume increase in the production practice.Lack respectively under the situations such as nitrogen, iron element or magnesium simultaneously at scarce phosphorus, detected the expression of gus gene, the composition of nitrogen stress, iron deficiency and magnesium deficiency substratum is with 3) in.
Experimental result as shown in Figure 6, show and lack the expression that phosphorus while nitrogen stress then suppresses gus gene, lacking other nutritive element does not simultaneously then have tangible influence, and on behalf of scarce phosphorus, 2,1 represent scarce phosphorus and nitrogen stress, the scarce phosphorus of 3 representatives and iron deficiency and 4 to represent scarce phosphorus and magnesium deficiency among Fig. 6.
Sequence table
＜110〉Inst. of Genetics and Development Biology, CAS
＜120〉phosphor deficiency speciality induction expression promoter and application thereof in root
<130>CGGNARW81395
<160>1
<210>1
<211>1305
<212>DNA
<400>1
agtacggcat?acggatttca?atgctgccct?gtctgcttgc?tgcgaaatct?gcatatgttt 60
agtagtttgt?taaaaaaaaa?cacacttagt?agtttttatt?tcatatttca?gattaccaat 120
gttctaaacg?tatggttgct?gcatgctatt?ttgttcagaa?ccttatctaa?attctgaagt 180
tgggaattct?cctctgtttc?tgtgactact?atgttatgca?cctaatcctt?caagaatatt 240
tcctcatttt?ttctgtgtta?caatcaataa?aaaaatagca?cgttatagcg?ttctgagcag 300
tcttgctaaa?tgaaatcaat?gtacaattgt?tcgctatagt?gtggagatag?cgttgcctta 360
gcgcgatata?gcttcgctat?agctgatttt?aagggttgcg?ctattatgtc?atagcacgct 420
attttttgca?ttggttacaa?ccaaacaacc?ccccctcccc?cccttgttcc?tacatttcta 480
gaacaatgtg?aaccaagcat?ggccttttta?tgtaacagat?gaccaatgtt?ctgaacatat 540
agcccttgca?tatgtatttt?gttcataaac?tgtctgaagt?atgaaattag?attttagcaa 600
gctagcaaca?acaattgaac?taattatatt?tgtatgccta?tgtctgcaag?acctagcatt 660
gcatccattt?atcgtcgaaa?ttctgttttt?ttgttgttgc?ttctgatttg?atgaaatcgg 720
aggagtgcct?accttcttcc?tgccatacca?tgtactagtt?atagagagaa?caaattaact 780
ccatctctga?agaaaggtca?ccaaatccat?gccggtagtg?agaacataag?atacaactca 840
ccgtataatc?ttgacctttt?ctcgcaaagc?ttgcttgtgt?ctgtacaata?tgtttctttc 900
aggtatattc?ccagcataac?catgatctgg?cttcattatt?ttttatttcc?acaattctat 960
agtactaaat?ttgaaagtca?aacctgataa?acttaagttt?agcattcctt?aatccttcta 1020
ataataacag?aaatgtagcc?cgacatagct?gtagctctca?aggcaagaca?attgactccg 1080
cgtatatttt?tgtaagaact?atcttgacaa?tgtacaatcc?gttgttttac?aagcgatgcc 1140
gtacatatga?tattatatac?gtcgttgcaa?ctagcacaat?atgtaccttc?accttgcatt 1200
atttacgcct?atataatagt?atatatacgt?actagaagca?tcgaacaaag?cacacaacaa 1260
gagagcagca?gacgaagttt?agttgagaga?tcgccggcgg?ccatg 1305
Claims (7)
1, a kind of promotor is following 1) or 2) dna molecular:
1) its nucleotide sequence is the dna molecular of sequence 1 in the sequence table;
2) under stringent condition with sequence table in sequence 1 dna molecule hybridize and can be at the dna molecular that lacks driving purposes gene specifically expressing in root under the phosphorus condition;
3) with 1) dna sequence dna that limits have 90% or more homology and can be under scarce phosphorus condition the dna molecular of driving purposes gene specifically expressing in root.
2, the expression cassette that contains the described dna molecular of claim 1, described expression cassette are followed successively by the described dna molecular of claim 1, goal gene and transcription terminator to the downstream from the upstream; The direction of the described dna molecular of claim 1 is identical with the transcriptional orientation of goal gene.
3, the application of the described promotor of claim 1 in making up engineering carrier.
4, the recombinant expression vector that contains described dna molecular of claim 1 or the described expression cassette of claim 2.
5, the transgenic cell line or the reorganization bacterium that contain described dna molecular of claim 1 or the described expression cassette of claim 2.
6, the amplification described dna molecular total length of claim 1 and any segmental primer thereof are right.
7, the application of the described promotor of claim 1 in cultivating transgenic plant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101153013A CN101298616B (en) | 2008-06-20 | 2008-06-20 | Promoter for expressing phosphor deficiency speciality induction in root and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008101153013A CN101298616B (en) | 2008-06-20 | 2008-06-20 | Promoter for expressing phosphor deficiency speciality induction in root and use thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101298616A true CN101298616A (en) | 2008-11-05 |
| CN101298616B CN101298616B (en) | 2011-04-13 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008101153013A Expired - Fee Related CN101298616B (en) | 2008-06-20 | 2008-06-20 | Promoter for expressing phosphor deficiency speciality induction in root and use thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101298616B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559673A (en) * | 2010-12-16 | 2012-07-11 | 华中农业大学 | Promoter specifically induced and expressed under condition of lack of phosphorus in cabbage type rape |
| CN103865929A (en) * | 2014-03-06 | 2014-06-18 | 中国科学院遗传与发育生物学研究所 | Promoter related to phosphorus absorption transfer and application thereof |
| CN108424912A (en) * | 2018-02-01 | 2018-08-21 | 山西省农业科学院作物科学研究所 | The promoter of corn low-phosphorus stress induced expression and its application |
-
2008
- 2008-06-20 CN CN2008101153013A patent/CN101298616B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559673A (en) * | 2010-12-16 | 2012-07-11 | 华中农业大学 | Promoter specifically induced and expressed under condition of lack of phosphorus in cabbage type rape |
| CN102559673B (en) * | 2010-12-16 | 2013-07-17 | 华中农业大学 | Promoter specifically induced and expressed under condition of lack of phosphorus in cabbage type rape |
| CN103865929A (en) * | 2014-03-06 | 2014-06-18 | 中国科学院遗传与发育生物学研究所 | Promoter related to phosphorus absorption transfer and application thereof |
| CN103865929B (en) * | 2014-03-06 | 2016-03-02 | 中国科学院遗传与发育生物学研究所 | A kind of promotor relevant to phosphorus absorption and transport and application thereof |
| CN108424912A (en) * | 2018-02-01 | 2018-08-21 | 山西省农业科学院作物科学研究所 | The promoter of corn low-phosphorus stress induced expression and its application |
| CN108424912B (en) * | 2018-02-01 | 2021-07-13 | 山西省农业科学院作物科学研究所 | Promoter for low-phosphorus stress induced expression of corn and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101298616B (en) | 2011-04-13 |
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