CN101768213A - Protein related to plant tillering number and coding gene and application thereof - Google Patents
Protein related to plant tillering number and coding gene and application thereof Download PDFInfo
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- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8291—Hormone-influenced development
- C12N15/8298—Brassinosteroids
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Abstract
The invention discloses protein relevant to brassinolide signal conduction and a coding gene and the application thereof; the protein is the protein in the 1) or 2); 1) the protein is formed according to an amino acid sequence shown by a sequence 2 in a sequence table; 2) the amino acid residue sequence of the sequence 2 in a sequence table is substituted and/or lost and/or added by one or a plurality of the amino acid residues and is derived by 1) relevant to the plant tillering number. In the invention, DLT gene is cloned by utilizing a rice half-stuntedness little-tillering mutant, and molecule complementation verification is carried out; the result shows that DLT participates in the brassinolide signal conduction process to control the height, tillering and other phenotypes of the rice; after the DLT gene is transferred to the rice, the fact that the tillering number of the transgenosis rice with high DLT gene expression level is obviously increased; the invention has high potential application value on molecular breeding and improving crop production by utilizing genetic engineering measures to regulate plant hormone level.
Description
Technical field
The present invention relates to a kind of albumen relevant and encoding gene and application with plant tillering number.
Background technology
In the eighties of last century Green Revolution first time, the breeding expert cultivates the paddy rice and the wheat breed of semi-short-stalked, improved the lodging tolerance of crop greatly, successfully solved the contradiction of high yield and lodging, thereby world food output has been improved about 2.5 times, made huge contribution for solving the world food problem.Yet at that time to also unclear with the molecular nature of signal conduction gene SD1 and RHT1 with the synthetic of the gene-phytohormone gibberellin that causes this revolution.Today, we can influence the gene of crop yield by autotelic exploration discovery, thereby utilize genetic engineering means to be used, and make crop yield " revolutionary character " raising once more.
Brassinolide is the important plant hormone of a class, has participated in many processes of plant-growth and growth, and wherein the most tangible effect is height and the leaf angle of control plant.The height of plant is directly related with the plant lodging tolerance, and to catch the ability of Yanguan Pass closely related and the leaf angle is also with the dense planting ability of plant and plant.Compact plant can significantly reduce sheltering from heat or light mutually of plant leaf, thereby improves the capturing ability of each blade to light, improves the photosynthesis ability of colony, finally improves the crop yield of colony.Recently, the researchist finds that again brassinolide can also promote the generation and the transportation of photosynthetic assimilation product from the source to the storehouse of rice tillering, thereby promotes grouting solid (Wu, the C.Y. of rice paddy seed, Trieu, A., Radhakrishnan, P., Kwok, S.F., Harris, S., Zhang, K., Wang, J., Wan, J., Zhai, H., Takatsuto, S., Matsumoto, S., Fujioka, S., Feldmann, K.A.and Pennell, R.I. (2008) Brassinosteroids regulate grain filling in rice.Plant Cell, 20,2130-2145.).
Recent two decades comes in the model plant Arabidopis thaliana, synthetic and the signal transduction path of brassinolide is after deliberation more clearly (Gendron all, J.M.and Wang, Z.Y. (2007) Multiple mechanisms modulatebrassinosteroid signaling.Curr Opin Plant Biol, 10,436-441.; Li, J.and Jin, H. (2007) Regulation of brassinosteroid signaling.Trends Plant Sci, 12,37-41.), yet in important unifacial leaf model plant paddy rice, also know little about it.In route of synthesis, gene D2, D11, OsDWARF4 and BRD1 have been identified; In signal transduction path, mainly be to have found gene OsBRI and OsBZR1.In the limited gene that has identified, there are two expression of gene to suppress the output that plant is proved to be able to improve greatly paddy rice.First is mutant osdwarf4-1 (Sakamoto, T., the Morinaka of a paddy rice brassinolide synthetic gene OsDWARF4, Y., Ohnishi, T., Sunohara, H., Fujioka, S., Ueguchi-Tanaka, M., Mizutani, M., Sakata, K., Takatsuto, S., Yoshida, S., Tanaka, H., Kitano, H.and Matsuoka, M. (2006) Erect leaves caused by brassinosteroid deficiencyincrease biomass production and grain yield in rice.Nat Biotechnol, 24,105-109.).Because this mutant has significantly less leaf angle, therefore be particularly suitable for dense planting.Field test is found, apply same standard fertilizer, but under the high-density planting condition (44.4plants/m), the mutant plant can be improved about 32% output.Another example is to adopt genetic engineering means inhibited oil rape lactone acceptor encoding gene OsBRI1 and two the transgenic line BKD11 and the BKD22 (Morinaka that obtain, Y., Sakamoto, T., Inukai, Y., Agetsuma, M., Kitano, H., Ashikari, M.and Matsuoka, M. (2006) Morphologicalalteration caused by brassinosteroid insensitivity increases the biomass and grainproduction of rice.Plant Physiol, 141,924-931.).Same, rice yield about 10%～35% can improve owing to have axial blade in these two strain systems under the dense planting condition.Recently, thus another one study group had reported to cross expresses the example that a brassinolide synthetic gene improves rice yield.Though this transgenic paddy rice leaf angle of cross expressing becomes big, plant is slightly than wild-type height, because it has more tiller number, and plants grain and becomes big the change and weigh, so also can improve output (Wu, C.Y., Trieu, the A. of paddy rice greatly, Radhakrishnan, P., Kwok, S.F., Harris, S., Zhang, K., Wang, J., Wan, J., Zhai, H., Takatsuto, S., Matsumoto, S., Fujioka, S., Feldmann, K.A.and Pennell, R.I. (2008) Brassinosteroids regulate grain filling in rice.Plant Cell, 20,2130-2145.).These examples provide a new thinking to us: improve the output of paddy rice by flexible regulation and control brassinolide in the intravital level of plant.
Because brassinolide is subjected to complicated regulation and control from the metabolic balance in plant materials, therefore further make clear it signal transduction path and the signal conduction with synthetic between the relation of feedback regulation, serve extremely important in the intravital level of plant for molecular breeding thereby regulate it for final better utilised genetic engineering means.
Summary of the invention
The purpose of this invention is to provide a kind of albumen relevant and encoding gene thereof with plant tillering number.
The albumen relevant with plant tillering number provided by the present invention, name is called DLT, derive from Oryza paddy rice (Oryza sativa L.), is following 1) or 2) protein:
1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
2) with the aminoacid sequence of sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant tillering number by 1) deutero-protein.
The sequence of table 1. label
| Label | Residue | Sequence |
| ?Poly-Arg | 5-6 (being generally 5) | RRRRR |
| ?Poly-His | 2-10 (being generally 6) | HHHHHH |
| ?FLAG | ?8 | DYKDDDDK |
| ?Strep-tag?II | ?8 | WSHPQFEK |
| ?c-myc | ?10 | EQKLISEEDL |
Above-mentioned 2) but in the DLT synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Above-mentioned 2) encoding gene of the DLT in can be by the codon that lacks one or several amino-acid residue in the dna sequence dna shown in the 5 ' terminal 771-2624 bit base with sequence in the sequence table 1, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
Above-mentioned and the encoding gene plant tillering number associated protein also belongs to protection scope of the present invention.
Specifically can be following 1 with the encoding gene of plant tillering number associated protein)-4) in arbitrary described gene:
1) its encoding sequence be in the sequence table sequence 1 from 5 ' terminal 771-2624 position deoxyribonucleotide;
2) its nucleotide sequence is the sequence 1 in the sequence table;
3) the dna sequence dna hybridization that under stringent condition, can limit with sequence in the sequence table 1 and encode above-mentioned and the dna molecular plant tillering number associated protein;
4) with 1) gene have homology 90% or more, and encode above-mentioned and the dna molecular plant tillering number associated protein.
Gene in the described step 4) is with 1) gene homology more than 95% is preferably arranged.
Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Increase above-mentioned DLT full length gene or arbitrary segmental primer to also belonging to protection scope of the present invention.
Contain above-mentioned and recombinant expression vector, expression cassette, transgenic cell line and reorganization bacterium plant tillering number associated protein encoding gene and also belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of DLT gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pCAMBIA1302, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other plant expression vector of deriving.Conventional biological methods such as the plant expression vector that carries the present invention and brassinolide signal conduction associated protein encoding gene DLT can lead by Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity, agriculture bacillus mediated are transformed in vegetable cell or the tissue.By the plant transformed host is the plant that can tiller, as paddy rice, wheat etc.
When using the gene constructed recombinant plant expression vector of DLT, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) or paddy rice actin1 promotor etc., they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can in plant, express enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, antibiotic marker thing (gentamicin marker, kantlex marker or hygromix phosphotransferase marker etc.) or the anti-chemical reagent marker gene (as anti-weedkiller gene) etc. that can produce colour-change with resistance as adding.
Described recombinant expression vector specifically can be between the multiple clone site of pCAMBIA1302 the recombinant expression vector pCAMBIA1302-DLT that obtains from 5 ' terminal 771-2624 position deoxynucleotide that inserts sequence 1 in the sequence table.
Another object of the present invention provides a kind of raising plant tillering number purpose method.
Raising plant tillering number purpose method provided by the present invention is that above-mentioned encoding gene DLT with the plant tillering number associated protein is imported in the plant, obtains the transgenic plant that tillering number improves.
The plant of described plant for tillering is as paddy rice, wheat etc.
The 3rd purpose of the present invention provides a kind of method of cultivating transgenic plant.
The method of cultivation transgenic plant provided by the present invention is that above-mentioned encoding gene DLT with the plant tillering number associated protein is imported in the plant, obtains transgenic plant.The tillering number showed increased of these transgenic plant.
The plant of described plant for tillering is as paddy rice, wheat etc.
The present invention utilizes a paddy rice half to downgrade the mutant of tillering less, uses the map based cloning technology to clone the DLT gene and carried out the complementary element checking.The sensitivity tests of brassinolide and brassinolide Expression of Related Genes analysis revealed are being controlled the height of paddy rice, are being tillered and other phenotype thereby DLT has participated in the signal conductive process of brassinolide.The DLT gene changed in the paddy rice find, the transgenic paddy rice tillering number showed increased that the DLT gene expression dose raises.The present invention is utilizing genetic engineering means regulation and control hormone levels to carry out having the potential using value in the molecular breeding raising crop yield.
Description of drawings
Fig. 1 is that the phenotype of Mutant Rice and wild-type paddy rice compares.
Fig. 2 is PCR electrophoresis detection figure and the phenotypic map that changes the positive strain system of DLT gene over to.
Fig. 3 is a leaf angle sensitivity tests.
Fig. 4 is the expression of DLT gene in Different Organs and tissue.
Fig. 5 is subjected to the negative regulation of brassinolide for DLT expression of gene level.
Fig. 6 is a brassinolide Expression of Related Genes in the DLT mutant.
Fig. 7 is that DLT expression of gene level is identified and phenotype in the transgenic paddy rice.
Embodiment
Experimental technique described in the following embodiment if no special instructions, is ordinary method; Described reagent and biomaterial if no special instructions, all can obtain from commercial channels.
The rice varieties that relates among the following embodiment spends 11, bright extensive 63 and Shiokari in being, above rice varieties is all from Inst. of Genetics and Development Biology, CAS.
1, the acquisition of mutant and phenotype thereof
Find the mutant of a dwarfing from a rice mutant storehouse (from Inst. of Genetics and Development Biology, CAS), not only plant height half is short but also tillering number obviously reduces for this mutant, and the phenotype of this mutant as shown in Figure 1.Wherein, WT is the wild-type paddy rice, and dlt is the Mutant Rice that above-mentioned screening obtains.The plant height of this Mutant Rice be about spend in the wild-type paddy rice 11 60%, tillering number is about half of wild-type paddy rice, and plant type compactness, blade and fringe portion are upright, the plant fertility descends, the two stipes ratios of falling are obviously reduced; Microsection is observed and is found, the cell length of Mutant Rice diminishes, cell is arranged disorderly relatively.Above phenotype result shows that the Mutant Rice of above-mentioned acquisition is a more typical brassinolide defective mutant.
2, adopt the map based cloning technology to obtain the DLT gene
The Mutant Rice that above-mentioned steps 1 obtains is carried out genetic analysis, and the result shows that the phenotype of this Mutant Rice is to be controlled by the single-gene of a recessiveness.The Mutant Rice and bright extensive 63 that above-mentioned steps 1 obtains is hybridized, made up F
2Carry out map based cloning for colony.The zone of an about 1300Kbp of the assignment of genes gene mapping to the six chromosomal galianconism ends of Mutant Rice phenotype will be controlled.Because between do not have other molecule marker to use, calculate genetic distance and find, the genetic distance of about 0.5 cM of molecule marker of candidate gene distance.At the encoding gene (http://www.tigr.org/) that note of this area discover is the GRAS family protein, order-checking finds that this gene coding region has the disappearance of 62 bases, is DLT with this unnamed gene.The nucleotide sequence of DLT is shown in sequence in the sequence table 1, and its amino acid sequence coded is shown in sequence in the sequence table 2.
3, complementary confirmatory experiment
From a BAC clone OSJNBa0038F22 (available from Arizona Genomics Institute), utilize Restriction Enzyme BamHI and KpnI (available from Promega company) double digestion, scale off the fragment of 7577 bases, this fragment contains the complete sequence of above-mentioned DLT gene.Reclaim this fragment, be connected to binary vector pCAMBIA1300 (available from Cambia, Australia, http://www.cambia.org/daisy/cambia/home.html) on, and transform among the importing Agrobacterium AGL1 (available from ATCC), utilize its callus that infects the Mutant Rice that above-mentioned steps 1 screening obtains, concrete grammar is referring to following reference: easily rely on oneself Cao Shouyun, Wang Li, what strontium is clean, and storage is become a useful person, Tang Zuoshun, Zhou Piaohua, Tian Wenzhong. improve Agrobacterium-mediated Transformation paddy rice Frequency Study, Acta Genetica Sinica .2001,28 (4): 352-358.After the hygromycin resistance screening obtains regeneration plant, utilize the primer at deletion fragment two ends: forward primer 5 '-CATCAATCCATTGCAGGGACGAT-3 ' and reverse primer 5 '-CGTTGAGCGTGAAGTGCAGGAA-3 ' carries out pcr amplification to the genomic dna of regeneration plant, screens positive strain to be.The pcr amplification result is shown in Fig. 2 a.Wherein, dlt-C represents to screen the pcr amplification result that the positive strain that obtains is a paddy rice, and WT represents the pcr amplification result of wild-type paddy rice, and dlt represents the pcr amplification result of the mutant strain paddy rice that above-mentioned steps 1 screening obtains.The 324bp band has represented to lack the band of 62 bases, illustrates that this strain is that background is a Mutant Rice; The 386bp band is represented to have changed over to the DLT gene and is obtained positive plant.The phenotype that above-mentioned each strain is a paddy rice is shown in Fig. 2 b, and the result shows that the positive strain that screening obtains is that the phenotype of paddy rice recovers normal, illustrates that the DLT gene is the gene that causes the undesired phenotype of Mutant Rice.
The signal conduction of embodiment 2, DLT gene participation oil rape lactone
1, Mutant Rice and wild-type paddy rice are compared the susceptibility of brassinolide
According to document Hong, Z., Ueguchi-Tanaka, M., Umemura, K., Uozu, S., Fujioka, S., Takatsuto, S., Yoshida, S., Ashikari, M., Kitano, H.and Matsuoka, M. (2003) A ricebrassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of anew member of cytochrome P450.Plant Cell, 15, the method for 2900-2910. adopts micro drop method test wild-type paddy rice and the foregoing description 1 to screen the susceptibility of the leaf angle of the Mutant Rice that obtains to brassinolide.With the seed of the Mutant Rice of spending 1 screening of 11 (coming from Inst. of Genetics and Development Biology, CAS) and the foregoing description to obtain in the wild-type paddy rice after 30 ℃ of sproutings two days later, the planting seed of choosing the germinating unanimity is in water, in 30 ℃ of illumination boxs again through three days growth (10 hours illumination/14 hour dark), the ethanol that then 1ul is dissolved with 0ng, 10ng, 100ng and 1000ng brassinolide (E1641 is available from Sigma company) respectively drips to the top of second blade of paddy rice.Again through after the growth in three days, compare the angle of this blade and leaf sheath under the similarity condition.The result is shown in Fig. 3 a.Wherein, WT is the wild-type paddy rice, and dlt is the Mutant Rice that above-mentioned screening obtains.The result shows that the Mutant Rice that the foregoing description 1 screening obtains does not have reaction substantially to the brassinolide of different concns, and the wild-type paddy rice is relatively more responsive to brassinolide, and the brassinolide concentration of dropping is high more, and the angle of blade and leaf sheath is big more.
Higher method (the Wada of another kind of susceptibility, K., Marumo, S., Ikekawa, N., Morisaki, M.andMori, K. (1981) Brassinolide and homobrassinolide promotion of lamina inclination ofrice seedlings.Plant Cell Physiol., 22,323-325.) be Mutant Rice that 1 screening of wild-type paddy rice and the foregoing description is obtained seed 30 ℃ after sprouting two days later, the planting seed of choosing the germinating unanimity on the silk screen of immersion, under 30 ℃ of dark fully conditions through 8 days growth, get one section part that has 1 blade and 1 leaf sheath topmost, blade and leaf sheath all keep the length of 1cm, will float on pure water after last 24 hour under this section plant dark condition, place respectively the 2.5mmol/L maleic acid potassium solution that the final concentration that does not contain brassinolide and brassinolide is 5ng/ml, again through after 48 hours, the size of leaf angle relatively.The result is shown in Fig. 3 b.Wherein, WT is the wild-type paddy rice, and dlt is the Mutant Rice that above-mentioned screening obtains.-BL represents paddy rice is placed the maleic acid potassium solution that does not contain brassinolide, and+BL represents that it is the maleic acid potassium solution of 5ng/ml that paddy rice is placed the final concentration of brassinolide.The result shows that the Mutant Rice that the foregoing description 1 screening obtains is starkly lower than the wild-type paddy rice to the susceptibility of brassinolide.
2, the expression of DLT gene in different tissues
Get Mutant Rice Different Organs or tissue that the foregoing description 1 screening obtains, extract its RNA respectively, employing is carried out reverse transcription available from the MMLV reversed transcriptive enzyme of Promega company and with reference to corresponding using method, utilize the real-time fluorescence quantitative PCR technology then, the using method that provides according to producer (Bio-Rad company), in the PCR system, add SYBR green I fluorescence dye, go up the DLT expression of gene situation that detects at quantitative real time PCR Instrument (Bio-Rad company); Be confidential reference items with paddy rice ACTIN1 gene simultaneously.The sequence that detects the forward primer of DLT gene is: 5 '-TGCGGATACTCAACGCCATCA-3 ', and the sequence of reverse primer is: 5 '-ACTCGCCGACTCCGGTGATC-3 '; The sequence that detects the forward primer of ACTIN1 gene is: 5 '-AGCAACTGGGATGATATGGA-3 ', the sequence of reverse primer is: 5 '-CAGGGCGATGTAGGAAAGC-3 '.Three repetition are established in experiment, the expression of DLT gene in Different Organs and tissue as shown in Figure 4, the longitudinal axis represent each organ and organize in the ratio of DLT gene and ACTIN1 expression of gene amount.Wherein, Fig. 4 a be the DLT gene at root, stem, leaf with the expression in spending, Fig. 4 b is the expression of DLT gene in different stipes.
The result shows, the DLT gene spend, stem is relative higher with the expression amount in the root, the expression amount in blade is lower; Find that in different stipes the DLT gene is falling expression amount in two joints than all low at the expression amount of other joint.This expression and its special shortening to fall two joint ratios corresponding, also with expression pattern (Yamamuro, C., the Ihara of the OsBRI that has reported, Y., Wu, X., Noguchi, T., Fujioka, S., Takatsuto, S., Ashikari, M., Kitano, H.and Matsuoka, M. (2000) Loss of function of a rice brassinosteroidinsensitivel homolog prevents internode elongation and bending of the lamina joint.Plant Cell, 12,1591-1606.) consistent.Illustrate that the DLT gene has participated in the signal transduction path of brassinolide.
3, the DLT gene is subjected to the negative adjusting of brassinolide
1umol/L brassinolide (E1641 is available from Sigma company) is sprayed onto on the wild-type paddy rice Shiokari seedling that sprouts a week, and the different time point water intaking rice in spray back is extracted its RNA, utilizes quantitative PCR technique to detect the DLT expression of gene after the reverse transcription.Three repetition are established in experiment, will not have that DLT expression of gene level set is 1 in the preceding paddy rice of sprinkling oil rape lactone, and different time points DLT expression of gene situation is shown in Fig. 5 a behind the sprinkling oil rape lactone.The result shows that behind the sprinkling oil rape lactone, DLT expression of gene level reduces gradually, arrives minimumly after 12 hours, is about 40% before handling.
And then adopt above-mentioned same procedure to detect at brassinolide synthesis mutant d2-1 and d11-2 (Hong, Z., Ueguchi-Tanaka, M., Umemura, K., Uozu, S., Fujioka, S., Takatsuto, S., Yoshida, S., Ashikari, M., Kitano, H.and Matsuoka, M. (2003) A rice brassinosteroid-deficientmutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochromeP450.Plant Cell, 15,2900-2910.Tanabe, S., Ashikari, M., Fujioka, S., Takatsuto, S., Yoshida, S., Yano, M., Yoshimura, A., Kitano, H., Matsuoka, M., Fujisawa, Y., Kato, H.and Iwasaki, Y. (2005) A novel cytochrome P450 is implicated in brassinosteroidbiosynthesis via the characterization of a rice dwarf mutant, dwarf11, with reduced seedlength.Plant Cell, 17,776-790.) middle DLT expression of gene situation.Three repetition are established in experiment, and the expression level of DLT gene in wild-type paddy rice Shiokari is set at 1, calculating DLT gene in these two synthesis mutants with wild-type in the ratio of expression level.The expression of DLT gene in wild-type paddy rice Shiokari, brassinolide synthesis mutant d2-1 and d11-2 is shown in Fig. 5 b.The result shows that the expression amount of DLT gene in brassinolide synthesis mutant d2-1 and d11-2 obviously raises than the expression amount in wild-type paddy rice Shiokari.
Above presentation of results, the DLT expression of gene is subjected to the negative regulation of brassinolide.
4, in the Mutant Rice with the brassinolide Expression of Related Genes
Adopt fluorescence quantifying PCR method to detect the foregoing description 1 and screen several known brassinolide synthetic gene D2 in the Mutant Rice that obtains, D11, OsCPD, OsBR6ox and two expression levels that may be subjected to its inductive gene OsXTR1, OsBLE2 in brassinolide signal conduction downstream.Amplification gene D2 wherein, the primer sequence of D11 and the OsCPD document Shimada that sees reference, A., Ueguchi-Tanaka, M., Sakamoto, T., Fujioka, S., Takatsuto, S., Yoshida, S., Sazuka, T., Ashikari, M.and Matsuoka, M. (2006) The rice SPINDL Y gene functions as a negative regulator of gibberellin signaling bycontrolling the suppressive function of the DELLA protein, SLR1, and modulatingbrassinosteroid synthesis.Plant J, 48, the forward sequence of the primer of 390-402. amplification gene OsBR6ox is: 5 '-CAGGTACGGGAGCGTGTT-3 ', reverse sequence are 5 '-TGAAGCCTTGGTAGTAGTTGGT-3 '.The primer sequence of the amplification gene OsXTR1 document Duan that sees reference, K., Li, L., Hu, P., Xu, S.P., Xu, Z.H.and Xue, H.W. (2006) Abrassinolide-suppressed rice MADS-box transcription factor, OsMDP1, has a negativeregulatory role in BR signaling.Plant J, 47,519-531.The primer sequence of the amplification gene OsBLE2 document Yang that sees reference, G., Matsuoka, M., Iwasaki, Y.and Komatsu, S. (2003) A novelbrassinolide-enhanced gene identified by cDNA microarray is involved in the growth ofrice.Plant Mol Biol, 52,843-854.Above-mentioned each expression of gene level set is 1 in 11 with spending in the wild-type paddy rice, calculates the ratio of above-mentioned each gene expression level in Mutant Rice and in the wild-type paddy rice.Three repetitions are established in experiment, and the result as shown in Figure 6.
The result shows that several known brassinolide synthetic gene D2, D11, OsCPD, the expression of OsBR6ox in Mutant Rice all have tangible accumulation, illustrates because the defective of signal conduction has caused the feedback regulation to the upstream synthetic gene; And may find by the detected result of the expression level of its inductive gene OsXTR1, OsBLE2 in brassinolide signal conduction downstream for two, these expression of gene downward modulations illustrate that the disappearance owing to the DLT gene causes the reduction of signal conduction to cause the derivative strength reduction of downstream gene.
The rice tillering number of embodiment 3, commentaries on classics DLT gene increases
1, the structure that contains the DLT expression carrier
Utilize PCR method, with 5 '-CCATGGATGTTGGCGGGTTGCTCGTTCTCGT-3 ' is forward primer, with 5 '-AGATCTGATGTTGGCGGGTTGCTCGTTCTCGT-3 ' is reverse primer, from oryza sativa genomic dna, amplify the open reading frame sequence of DLT gene, promptly in the sequence table sequence 1 from 5 ' terminal 771-2621 bit base.Add the restriction enzyme site of Restriction Enzyme NcoI and BglII respectively at forward and reverse primer end.To connect in the pMD18-T carrier (available from TAKARA company) after the recovery of PCR product, after guaranteeing correctly, order-checking utilize NcoI and BglII to carry out double digestion, enzyme is cut product and is connected into binary expression vector pCAMBIA1302 (available from Cambia, Australia, http://www.cambia.org/daisy/cambia/home.html) in, promptly obtains containing the recombinant expression vector pCAMBIA1302-DLT of DLT gene.
2, change acquisition, screening and the evaluation of the paddy rice of DLT gene
The recombinant expression vector 1302-35S-DLT that above-mentioned steps 1 is made up imports to by Agrobacterium AGL1 (available from ATCC) and spends in the rice varieties in 11 the callus, concrete transformation and selection method is referring to following document: easily rely on oneself Cao Shouyun, Wang Li, what strontium is clean, storage is become a useful person, Tang Zuoshun, Zhou Piaohua, Tian Wenzhong. improve Agrobacterium-mediated Transformation paddy rice Frequency Study, Acta Genetica Sinica, 2001,28 (4): 352-358.Obtain 10 strain T through resistance screening
0For the transgenic positive plant.
3, the evaluation of DLT gene expression dose and corresponding phenotype
Extract this 10 strain T respectively
0For the RNA of transgenic positive plant, adopt fluorescent quantitative PCR technique to detect DLT expression of gene level, concrete detection method is with embodiment 2.DLT expression of gene level set is 1 in 11 with spending in the wild-type paddy rice, calculates the ratio of DLT gene expression level in transgenic line and wild-type paddy rice.Three repetitions are established in experiment, and DLT expression of gene situation is shown in Fig. 7 a.7 strain T wherein
0DLT gene expression dose for the transgenic positive plant raises greatly.To this 7 strain T
0Carry out phenotype for the transgenic positive plant and observe, the result shows, wherein the DLT gene expression dose raises greater than 100 times 5 strain T
0All show the phenotype that leaf roll, leaf angle increase, tiller and increase, downgrade a little for the transgenic positive plant, as No. 5 plant among Fig. 7 b; And other two strain T
0In the transgenic positive plant, the rising of DLT gene expression dose is less than 100 times of wild-types, shows that tangible tillering number increases, plant uprises slightly, as No. 10 plant among Fig. 7 b.
Above presentation of results, the increase of DLT gene expression dose can cause the rice plant generation more to be tillered.
Sequence table
＜110〉Inst. of Genetics and Development Biology, CAS
＜120〉a kind of albumen relevant and encoding gene and application with plant tillering number
<130>CGGNARZ82117
<160>2
<210>1
<211>3084
<212>DNA
＜213〉paddy rice (Oryza sativa L.)
<400>1
gagtgagagt?gagagagtga?gagcagagac?caccaccacc?ggagaggtta?gtgagagagg???60
agtggtaatg?gtgaggcaac?aagagtaggt?tccatttcat?atcatcacta?ggatagcgta??120
gtttgtaggc?tgcatctcca?tctccatcgc?cattgattcg?cattgcatcc?atcattttag??180
gatgttctac?tagggttctt?gatttttctt?ttggtttgtt?gttttgacga?atggaggtat??240
tgttgggatt?cgccgcctgc?tgctcgtcgt?cgtcgtcgcc?gatgaggagg?ccgtgcgggc??300
tctgccccgg?catgtccgat?cgttcgtgat?ttgttttttc?tacatgtttt?agggcccatt??360
tgttcttgat?cctattcttt?gattcttttg?tactaagcat?tctaaggcga?agccacccat??420
tctttcctgc?atatatactt?acaaacacat?agcccccatc?tgatctcaca?aacattattt??480
ctctctcttt?ttttctcagt?tttttctttg?ttgatttact?gaccaaattc?tttggaagaa?????540
caacaagatc?atctggtttt?tatctgctca?ttcttttgta?catcgaatca?tatacatttc?????600
cattccacca?aagccttagc?cagataccac?agagagagtg?tgagagaaat?cagagtgaga?????660
aacagaggag?gaagaagaag?aagaagacga?ggaggaggag?gaggaggagc?aggaggagga?????720
ggaggtctct?tcttggcacg?tcgcgttccg?gcgagtgacg?tgtctccggg?atgttggcgg?????780
gttgctcgtt?ctcgtcgtcg?aggcatcaga?tgagcaccgc?gcagcgtttc?gacatcctcc?????840
cctgcggctt?ctccaagcgc?ggcagccgcg?gcgacggcgc?cgccccgcgg?gtcgccggcg?????900
acgccaggag?cggcgccacc?acctgctcct?tccggacgca?ccccgcgccg?ccggtcaccc?????960
agtccgtgtc?ctggggcgcc?aagccggagc?ccggcggcaa?tggcaatggc?gcccaccgcg????1020
ccgttaagcg?ggcgcatgac?gaggacgcgg?tcgaggagta?tggccccatt?gttcgcgcca????1080
agcggacgcg?gatgggcggc?gacggcgatg?aggtatggtt?ccatcaatcc?attgcaggga????1140
cgatgcaagc?gacggcggcg?ggagaaggag?aggaggcgga?ggaggagaag?gtcttcttgg????1200
tgccgagcgc?ggcggcgttc?ccgcacggca?tggccgccgc?ggggccatcg?ctggccgcgg????1260
ccaagaagga?ggagtacagc?aagtcgccgt?ccgactcgtc?gtcctcgtcg?ggcacggacg????1320
gcggctcgtc?ggcgatgatg?ccgccgccgc?agccgcccga?gttcgacgcg?aggaacggcg????1380
tgccggcgcc?ggggcaggcg?gagcgggagg?cgctggagct?ggtgcgcgcg?ctcaccgcgt????1440
gcgccgactc?cctctccgcc?ggcaaccacg?aggccgccaa?ctactacctg?gcccggctcg????1500
gcgagatggc?ctcgccggcg?gggcccacgc?cgatgcaccg?cgtggccgcc?tacttcaccg????1560
aggcgctcgc?gctccgcgtc?gtgcgcatgt?ggccgcacat?gttcgacatc?ggcccgccgc????1620
gggagctcac?cgacgacgcc?ttcggcggcg?gcgacgacga?cgccatggcg?ctgcggatac????1680
tcaacgccat?cacgcccatc?ccgaggttcc?tgcacttcac?gctcaacgag?cgcctcctcc????1740
gcgagttcga?ggggcacgag?cgcgtccacg?tcatcgactt?cgacatcaag?caggggctcc????1800
aatggccggg?cttgctccag?agcctggccg?cgcgggcggt?gcctccggcg?cacgtgcgga????1860
tcaccggagt?cggcgagtcg?aggcaggagc?tgcaggagac?gggagcgcgg?ctggcgcgcg????1920
tcgccgccgc?gctcggcctg?gcgttcgagt?tccacgccgt?ggtcgaccgg?ctcgaggacg????1980
tccgcctgtg?gatgctccac?gtcaagcgcg?gcgagtgcgt?ggccgtgaac?tgcgtcctcg????2040
ccatgcaccg?cctgctccgc?gacgacgccg?cgctgaccga?cttcctgggg?ctagcgcgca????2100
gcacgggcgc?caccatcctc?ctcctcggcg?agcacgaggg?cggcggcctc?aactcgggga????2160
ggtgggaggc?gcggttcgcg?cgcgcgctgc?ggtactacgc?cgcggcgttc?gacgcggtgg????2220
acgcggcggg?gctgccggag?gcgagccccg?cgagggccaa?ggcggaggag?atgttcgcgc????2280
gggagatccg?caacgcggtg?gcgttcgagg?gccccgagcg?gttcgagcgc?cacgagagct????2340
tcgccgggtg?gcggcggcgc?atggaggacg?gcggcgggtt?caagaacgcc?ggcatcggcg????2400
agcgcgaggc?gatgcagggg?cgcatgatcg?cgaggatgtt?cgggccggac?aagtacaccg????2460
tgcaggcgca?cggcggcggc?ggcagcggcg?gcggcgaggc?gctcacgctc?cggtggctgg????2520
accagccgct?gtacaccgtg?acggcgtgga?cgccggcggg?cgacggcgcg?ggaggcagca????2580
ccgtgtcggc?gtccacaaca?gcatcacatt?ctcagcaaag?ctaagctgac?gatgaatggt????2640
gattaggtga?agagaaagaa?agaacaaagc?ctttttttac?agtgcttctt?ttgttaatga????2700
tgattagttc?atacagtatg?acaattcttt?tatacattca?gagaaaagaa?agaagaaaga????2760
aaggtgtagt?tttttgtttt?atagattgat?aggtggaaag?attcaattaa?atcaaattca????2820
attcaatttt?tagattgtaa?ttctttataa?atattctttt?ggctgttgag?agagagtccc????2880
ctgcaaaatg?tagctgcatg?tagaagaaag?aaagcaaaga?agcagtagat?agattagcag????2940
gggcagcatc?tctcacagtc?actattagtg?tctccggctg?ttattataca?acattattat????3000
tacaatcaaa?ttctttcatc?attcattcta?catgtaatct?ctgttcagaa?tcagaatgaa????3060
atgaaacatg?tgttatattt?ctcc???????????????????????????????????????????3084
<210>2
<211>617
<212>PRT
＜213〉paddy rice (Oryza sativa L.)
<400>2
Met?Leu?Ala?Gly?Cys?Ser?Phe?Ser?Ser?Ser?Arg?His?Gln?Met?Ser?Thr
1???????????????5???????????????????10??????????????????15
Ala?Gln?Arg?Phe?Asp?Ile?Leu?Pro?Cys?Gly?Phe?Ser?Lys?Arg?Gly?Ser
20??????????????????25??????????????????30
Arg?Gly?Asp?Gly?Ala?Ala?Pro?Arg?Val?Ala?Gly?Asp?Ala?Arg?Ser?Gly
35??????????????????40??????????????????45
Ala?Thr?Thr?Cys?Ser?Phe?Arg?Thr?His?Pro?Ala?Pro?Pro?Val?Thr?Gln
50??????????????????55??????????????????60
Ser?Val?Ser?Trp?Gly?Ala?Lys?Pro?Glu?Pro?Gly?Gly?Asn?Gly?Asn?Gly
65??????????????????70??????????????????75??????????????????80
Ala?His?Arg?Ala?Val?Lys?Arg?Ala?His?Asp?Glu?Asp?Ala?Val?Glu?Glu
85??????????????????90??????????????????95
Tyr?Gly?Pro?Ile?Val?Arg?Ala?Lys?Arg?Thr?Arg?Met?Gly?Gly?Asp?Gly
100?????????????????105?????????????????110
Asp?Glu?Val?Trp?Phe?His?Gln?Ser?Ile?Ala?Gly?Thr?Met?Gln?Ala?Thr
115?????????????????120?????????????????125
Ala?Ala?Gly?Glu?Gly?Glu?Glu?Ala?Glu?Glu?Glu?Lys?Val?Phe?Leu?Val
130?????????????????135?????????????????140
Pro?Ser?Ala?Ala?Ala?Phe?Pro?His?Gly?Met?Ala?Ala?Ala?Gly?Pro?Ser
145?????????????????150?????????????????155?????????????????160
Leu?Ala?Ala?Ala?Lys?Lys?Glu?Glu?Tyr?Ser?Lys?Ser?Pro?Ser?Asp?Ser
165?????????????????170?????????????????175
Ser?Ser?Ser?Ser?Gly?Thr?Asp?Gly?Gly?Ser?Ser?Ala?Met?Met?Pro?Pro
180?????????????????185?????????????????190
Pro?Gln?Pro?Pro?Glu?Phe?Asp?Ala?Arg?Asn?Gly?Val?Pro?Ala?Pro?Gly
195?????????????????200?????????????????205
Gln?Ala?Glu?Arg?Glu?Ala?Leu?Glu?Leu?Val?Arg?Ala?Leu?Thr?Ala?Cys
210?????????????????215?????????????????220
Ala?Asp?Ser?Leu?Ser?Ala?Gly?Asn?His?Glu?Ala?Ala?Asn?Tyr?Tyr?Leu
225?????????????????230?????????????????235?????????????????240
Ala?Arg?Leu?Gly?Glu?Met?Ala?Ser?Pro?Ala?Gly?Pro?Thr?Pro?Met?His
245?????????????????250?????????????????255
Arg?Val?Ala?Ala?Tyr?Phe?Thr?Glu?Ala?Leu?Ala?Leu?Arg?Val?Val?Arg
260?????????????????265?????????????????270
Met?Trp?Pro?His?Met?Phe?Asp?Ile?Gly?Pro?Pro?Arg?Glu?Leu?Thr?Asp
275?????????????????280?????????????????285
Asp?Ala?Phe?Gly?Gly?Gly?Asp?Asp?Asp?Ala?Met?Ala?Leu?Arg?Ile?Leu
290?????????????????295?????????????????300
Asn?Ala?Ile?Thr?Pro?Ile?Pro?Arg?Phe?Leu?His?Phe?Thr?Leu?Asn?Glu
305?????????????????310?????????????????315?????????????????320
Arg?Leu?Leu?Arg?Glu?Phe?Glu?Gly?His?Glu?Arg?Val?His?Val?Ile?Asp
325?????????????????330?????????????????335
Phe?Asp?Ile?Lys?Gln?Gly?Leu?Gln?Trp?Pro?Gly?Leu?Leu?Gln?Ser?Leu
340?????????????????345?????????????????350
Ala?Ala?Arg?Ala?Val?Pro?Pro?Ala?His?Val?Arg?Ile?Thr?Gly?Val?Gly
355?????????????????360?????????????????365
Glu?Ser?Arg?Gln?Glu?Leu?Gln?Glu?Thr?Gly?Ala?Arg?Leu?Ala?Arg?Val
370?????????????????375?????????????????380
Ala?Ala?Ala?Leu?Gly?Leu?Ala?Phe?Glu?Phe?His?Ala?Val?Val?Asp?Arg
385?????????????????390?????????????????395?????????????????400
Leu?Glu?Asp?Val?Arg?Leu?Trp?Met?Leu?His?Val?Lys?Arg?Gly?Glu?Cys
405?????????????????410?????????????????415
Val?Ala?Val?Asn?Cys?Val?Leu?Ala?Met?His?Arg?Leu?Leu?Arg?Asp?Asp
420?????????????????425?????????????????430
Ala?Ala?Leu?Thr?Asp?Phe?Leu?Gly?Leu?Ala?Arg?Ser?Thr?Gly?Ala?Thr
435?????????????????440?????????????????445
Ile?Leu?Leu?Leu?Gly?Glu?His?Glu?Gly?Gly?Gly?Leu?Asn?Ser?Gly?Arg
450?????????????????455?????????????????460
Trp?Glu?Ala?Arg?Phe?Ala?Arg?Ala?Leu?Arg?Tyr?Tyr?Ala?Ala?Ala?Phe
465?????????????????470?????????????????475?????????????????480
Asp?Ala?Val?Asp?Ala?Ala?Gly?Leu?Pro?Glu?Ala?Ser?Pro?Ala?Arg?Ala
485?????????????????490?????????????????495
Lys?Ala?Glu?Glu?Met?Phe?Ala?Arg?Glu?Ile?Arg?Asn?Ala?Val?Ala?Phe
500?????????????????505?????????????????510
Glu?Gly?Pro?Glu?Arg?Phe?Glu?Arg?His?Glu?Ser?Phe?Ala?Gly?Trp?Arg
515?????????????????520?????????????????525
Arg?Arg?Met?Glu?Asp?Gly?Gly?Gly?Phe?Lys?Asn?Ala?Gly?Ile?Gly?Glu
530?????????????????535?????????????????540
Arg?Glu?Ala?Met?Gln?Gly?Arg?Met?Ile?Ala?Arg?Met?Phe?Gly?Pro?Asp
545?????????????????550?????????????????555?????????????????560
Lys?Tyr?Thr?Val?Gln?Ala?His?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Glu
565?????????????????570?????????????????575
Ala?Leu?Thr?Leu?Arg?Trp?Leu?Asp?Gln?Pro?Leu?Tyr?Thr?Val?Thr?Ala
580?????????????????585?????????????????590
Trp?Thr?Pro?Ala?Gly?Asp?Gly?Ala?Gly?Gly?Ser?Thr?Val?Ser?Ala?Ser
595?????????????????600?????????????????605
Thr?Thr?Ala?Ser?Hi?s?Ser?Gln?Gln?Ser
610?????????????????615
Claims (10)
1. an albumen is following 1) or 2) protein:
1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
2) with the amino acid residue sequence of sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant tillering number by 1) deutero-protein.
2. the described proteic encoding gene of claim 1.
3. encoding gene according to claim 2 is characterized in that: described proteic encoding gene is following 1)-4) in arbitrary described gene:
1) its encoding sequence be in the sequence table sequence 1 from 5 ' terminal 771-2624 position deoxyribonucleotide;
2) its nucleotide sequence is the sequence 1 in the sequence table;
3) the dna sequence dna hybridization that under stringent condition, can limit with sequence in the sequence table 1 and encode above-mentioned and the dna molecular plant tillering number associated protein;
4) with 1) gene have homology 90% or more, and encode above-mentioned and the dna molecular plant tillering number associated protein.
4. the recombinant expression vector, expression cassette, transgenic cell line and the reorganization bacterium that contain claim 2 or 3 described genes.
5. total length or arbitrary segmental primer of amplification claim 2 or 3 described genes are right.
6. described albumen of claim 1 or claim 2 or the 3 described genes application in improving plant tillering number.
7. one kind is improved plant tillering number purpose method, is that claim 2 or 3 described encoding genes are changed in the plant, obtain that tillering number improves transgenic plant.
8. method according to claim 7 is characterized in that: described plant is paddy rice or wheat.
9. a method of cultivating transgenic plant is that claim 2 or 3 described encoding genes are changed in the plant, obtains transgenic plant.
10. method according to claim 9 is characterized in that: described plant is paddy rice or wheat.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102473663A CN101768213B (en) | 2008-12-30 | 2008-12-30 | Protein related to plant tillering number and coding gene and application thereof |
| PCT/IB2009/055988 WO2010076766A1 (en) | 2008-12-30 | 2009-12-29 | Genes associated with plant tiller number and uses thereof |
| ARP090105185 AR074961A1 (en) | 2008-12-30 | 2009-12-30 | GENES ASSOCIATED WITH THE NUMBER OF PLANET RETONS AND THEIR USES. |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102473663A CN101768213B (en) | 2008-12-30 | 2008-12-30 | Protein related to plant tillering number and coding gene and application thereof |
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| Publication Number | Publication Date |
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| CN101768213A true CN101768213A (en) | 2010-07-07 |
| CN101768213B CN101768213B (en) | 2012-05-30 |
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| Country | Link |
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| CN (1) | CN101768213B (en) |
| AR (1) | AR074961A1 (en) |
| WO (1) | WO2010076766A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127536A (en) * | 2010-11-30 | 2011-07-20 | 深圳华大基因科技有限公司 | BGIos286 gene and application thereof |
| CN103740750A (en) * | 2014-01-13 | 2014-04-23 | 华南农业大学 | Method for cultivating transgenic plants with dwarf, increased tiller number and improved drought resistance |
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| US10041085B2 (en) * | 2013-04-09 | 2018-08-07 | Cotton Research Institute, Chinese Academy of Agricultural Sciences | Plant type related protein, and coding gene and application thereof |
| CN103275975B (en) * | 2013-05-21 | 2015-05-27 | 四川农业大学 | Wheat new few-tillering QTL (quantitative trait locus), primer pair, molecular marker, molecular marking method and application |
| CN105123495B (en) * | 2015-07-16 | 2017-03-22 | 江苏沿海地区农业科学研究所 | Application of partially indica-type paddy rice dominant dwarf mutant |
| CN105462992B (en) * | 2016-01-06 | 2019-03-05 | 河南师范大学 | Rice Os RhoGAP2 gene is improving the application in rice effective tillering |
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| CN112852828A (en) * | 2019-11-27 | 2021-05-28 | 江苏师范大学 | Plant pollen tube vacuole fluorescence labeling construction method and application |
| CN112707956B (en) * | 2021-02-01 | 2022-04-19 | 中国农业科学院作物科学研究所 | Corn ear row number related protein and coding gene and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102127536A (en) * | 2010-11-30 | 2011-07-20 | 深圳华大基因科技有限公司 | BGIos286 gene and application thereof |
| CN102127536B (en) * | 2010-11-30 | 2012-08-22 | 深圳华大基因科技有限公司 | BGIos286 gene and application thereof |
| CN103740750A (en) * | 2014-01-13 | 2014-04-23 | 华南农业大学 | Method for cultivating transgenic plants with dwarf, increased tiller number and improved drought resistance |
| CN103740750B (en) * | 2014-01-13 | 2016-01-13 | 华南农业大学 | A kind of method of cultivating dwarfing, increasing the transgenic plant of drought resistance of tillering, improve |
Also Published As
| Publication number | Publication date |
|---|---|
| AR074961A1 (en) | 2011-02-23 |
| WO2010076766A1 (en) | 2010-07-08 |
| CN101768213B (en) | 2012-05-30 |
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