CN105420208A - Rice acid phosphatase gene OsPAP10c and application thereof - Google Patents
Rice acid phosphatase gene OsPAP10c and application thereof Download PDFInfo
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Abstract
The invention discloses rice purple acid phosphatase protein gene encoded protein with the amino acid sequence shown in SEQ ID NO: 2. The invention further provides a rice purple acidi phosphatase OsPAP10c gene encoding the protein, and the nucleotide sequence of the rice purple acidi phosphatase OsPAP10c gene is shown in SEQ ID NO: 1. The invention further provides application of the rice purple acid phosphatase OsPAP10c gene. According to the application, genetically modified rice is built and improves the activity of acid phosphatase, and correspondingly, the phosphorus absorption and utilization efficiency is improved.
Description
Technical field
The invention belongs to plant genetic engineering field.Specifically, the present invention relates to a kind of by PCR cloning rice purple acid phosphatase OsPAP10c gene, and obtain overexpression vegetable material by transgenic technology.Also relate to and utilize this gene to improve crop activity of acid phosphatase, promote the efficiency raising that crop absorbs phosphorus and utilizes.
Background technology
Acid phosphatase (Acidphosphatase, E.C.3.1.3.2) is the nonspecific phosphohydrolase of a class, because thus the hydrolytic activity of its best gains the name at acidic conditions (pH4.0-7.6).Acid phosphatase is present in all floristics, is discharged by inorganic phosphate radical ion by cutting phosphatide key from various P contained compound.When plant is under scarce phosphorus condition, the activity of acid phosphatase in plant materials and around rhizosphere can raise (Bozzoetal., 2006).It is generally acknowledged under scarce phosphorus condition, intracellular acid phosphatase is used for the P contained compound in degradation of cell, to increase the phosphate anion in tenuigenin needed for various biochemical reaction.At aboveground vegetation part, first acid phosphatase in born of the same parents degrades the P contained compound in ageing leaves, the phosphorus element being reclaimed out is transported to young blade or reproductive organ, preferentially to meet the demand (Robinsonetal., 2012a) of phosphorus element in these tissues.In soil, plant root is secreted into external acid phosphatase and is considered to act on the organo phosphorous compounds around rhizosphere, its phosphorus-containing acid ion is discharged, thus greatly improves the content of inorganic phosphorus (Wangetal., 2014) of plant available utilization.Meanwhile, plant can also secreting acidic Phosphoric acid esterase in cell walls, organophosphorus in degradation of cell interstitial utilizes (Shaneetal., 2014) for plant-growth.
Phosphorus is one of necessary main nutrient elements of crop growth.In nature and farmland, although the total phosphorus content in soil is very abundant, belongs to insoluble phosphorus more or exist with the form of organophosphorus, can not be utilized by crop, therefore soil lacks the important factor that phosphorus has become restriction agriculture production.The deficiency of soil available phosphorus, forces modern agriculture to use a large amount of phosphate fertilizer to meet the needs of crop growth.But phosphorus ore is a kind of resource of irreproducibility, according to the Ministry of Land and Resources's statistics nineties, the existing 2,700,000,000 tons of signature phosphorus ore reserves of China only enough use about 70 years.Worldwide phosphorus ore is also on the verge of exhaustion, the phosphate fertilizer resource estimation of global low cost by 2060 by depleted (Gilbert, 2009).The main dependence on import of China's high-quality phosphate fertilizer, phosphate rock resource is exhausted will cause phosphate fertilizer price continuous rise, restriction China's agricultural development.
Research shows, the plant of some acid phosphatase enzyme mutant, and its phosphorous use efficiency is weakened greatly, and the phosphorus element recovery ability in Lao Ye also reduces, and finally causes poor growth (Hurleyetal., 2010 of mutant plant under scarce phosphorus condition; Robinsonetal., 2012b).On the contrary, by overexpression acid phosphatase, the utilising efficiency of plant to organophosphorus can be improved, promote growing and output of plant.The acid phosphatase AtPAP15 of process LAN Arabidopis thaliana in soybean, the growth of render transgenic plant in the substratum taking phytic acid as phosphorus source or soil can be better than non-transgenic plant (Wangetal., 2009).Overexpression acid phosphatase OsPAP10a in paddy rice, also can significantly improve the organophosphorus utilising efficiency (Tianetal., 2012) of transfer-gen plant.Therefore by biotechnology overexpression acid phosphatase, carrying out molecular breeding research has very good application prospect, but patent relevant is both at home and abroad few.The purple acid phosphatase of contriver in early stage to all 29 paddy rice has carried out systems analysis, has screened the purple acid phosphatase that some participate in phosphorus signal reaction.Except acid phosphatase OsPAP10a, other acid phosphatase function is not also all reported, the potentiality of these acid phosphatases in phosphorus efficiency molecular breeding are not all known yet.Compared with wild type control, overexpression rice Os PAP10a only improves about 1.5 times acid phosphatases, and in another research, overexpression rice bean PvPAP3 also only improves the acid phosphatase of 1.8 times.
Reference is as follows:
Bozzo, G.G., Dunn, E.L., andPlaxton, W.C. (2006) .Differentialsynthesisofphosphate-starvationinduciblepur pleacidphosphataseisozymesintomato (Lycopersiconesculentum) suspensioncellsandseedlings.PlantCell & Environ29,303-313 (Bozzo, G.G., Dunn, E.L., phosphorus starvation induced in andPlaxton, W.C.2006 tomato different acid phosphatase synthesis analysis.Vegetable cell and environment, 29,303-313).
Gilbert, N. (2009) .Environment:Thedisappearingnutrient.Nature461,716-718. (Gilbert, N.2009 environment: a kind of nutritive element of disappearance.Nature, 461,716-718.).
Hurley, B.A., Tran, H.T., Marty, N.J., Park, J., Snedden, W.A., Mullen, R.T., andPlaxton, W.C. (2010) .TheDual-targetedPurpleAcidPhosphataseIsozymeAtPAP26isEs sentialforEfficientAcclimationofArabidopsisthalianatoNut ritionalPhosphateDeprivation.PlantPhysiol.153, 1112-1122. (Hurley, B.A., Tran, H.T., Marty, N.J., Park, J., Snedden, W.A., Mullen, R.T., andPlaxton, the acid phosphatase AtPAP26 of the two-way location of W.C.2010 lacks in phosphorus adaptation reaction at Arabidopis thaliana and plays indispensable effect.Plant physiology, 153,1112-1122).
Robinson, W.D., Carson, I., Ying, S., Ellis, K., andPlaxton, W.C. (2012a) .EliminatingthepurpleacidphosphataseAtPAP26inArabidopsis thalianadelaysleafsenescenceandimpairsphosphorusremobili zation.NewPhytol196,1024-1029. (Robinson, W.D., Carson, I., Ying, S., Ellis, K., andPlaxton, W.C.2012a sudden change AtPAP26 postpones leaf senile and weakens the transhipment of phosphorus.New plant scholar, 196,1024-1029).
Robinson, W.D., Park, J., Tran, H.T., DelVecchio, H.A., Ying, S., Zins, J.L., Patel, K., McKnight, T.D., andPlaxton, W.C. (2012b) .ThesecretedpurpleacidphosphataseisozymesAtPAP12andAtPAP 26playapivotalroleinextracellularphosphate-scavengingbyA rabidopsisthaliana.JExpBot63, 6531-6542. (Robinson, W.D., Park, J., Tran, H.T., DelVecchio, H.A., Ying, S., Zins, J.L., Patel, K., McKnight, T.D., andPlaxton, the keying action of W.C.2012b secretor type AtPAP12 and AtPAP26 in environment phosphorus organophosphorus absorbs.Botany Experiment magazine, 63,6531-6542).
Shane, M.W., Stigter, K., Fedosejevs, E.T., andPlaxton, W.C. (2014) .Senescence-induciblecellwallandintracellularpurpleacidp hosphatases:implicationsforphosphorusremobilizationinHak eaprostrata (Proteaceae) andArabidopsisthaliana (Brassicaceae) .JExpBot65, 6097-6106. (Shane, M.W., Stigter, K., Fedosejevs, E.T., andPlaxton, W.C.2014 aging induction cell walls and cell in purple acid phosphatase: Hakea grass and Arabidopis thaliana in phosphorus transporter function.Phytology magazine, 65,6097-6106).
Tian, J., Wang, C., Zhang, Q., He, X., Whelan, J., andShou, H. (2012) .OverexpressionofOsPAP10a, ARoot-AssociatedAcidPhosphatase, IncreasedExtracellularOrganicPhosphorusUtilizationinRice .JIntegrPlantBiol54,631-639. (Tian, J., Wang, C., Zhang, Q., He, X., Whelan, J., andShou, H.2012 the acid phosphatase OsPAP10a of a kind of plant root table of overexpression improves the utilising efficiency of environment organophosphorus.Integrate phytology magazine, 54,631-639).
Wang, L., Lu, S., Zhang, Y., Li, Z., Du, X., andLiu, D. (2014) .ComparativegeneticanalysisofArabidopsispurpleacidphosph atasesAtPAP10, AtPAP12, andAtPAP26providesnewinsightsintotheirrolesinplantadapta tiontophosphatedeprivation.JIntegrPlantBiol56, 299-314. (Wang, L., Lu, S., Zhang, Y., Li, Z., Du, X., andLiu, D.2014AtPAP10, the comparative genomics analytical proof of AtPAP12 and AtPAP26 its lack function in phosphorus adaptability plant.Integrate phytology magazine, 56,299-314).
Wang, X., Wang, Y., Tian, J., Lim, B.L., Yan, X., andLiao, H. (2009) .OverexpressingAtPAP15enhancesphosphorusefficiencyinsoyb ean.PlantPhysiol151,233-240. (Wang, X., Wang, Y., Tian, J., Lim, B.L., Yan, X., andLiao, H.2009 overexpression AtPAP15 improves the phosphorus efficiency of phosphorus soybean.Plant physiology, 151,233-240).
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of protein and the gene thereof that significantly can improve transgenic paddy rice acid phosphatase, and thus obtained transgenic plant, and utilizes described gene pairs crop to carry out the method transformed.The method can improve transgenic paddy rice activity of acid phosphatase and reach 5-10 doubly.
In order to solve the problems of the technologies described above, the present invention realizes by following technical solution: the protein providing a kind of paddy rice purple acid phosphatase protein gene coding, and it is the aminoacid sequence shown in SEQIDNO:2.
The present invention also provides a kind of gene of above-mentioned protein of encoding, and it has the nucleotide sequence (described in underscore corresponding above-mentioned amino acid) shown in SEQIDNO:1.
The present invention also provides a kind of transgenic plant comprising above-mentioned nucleic acid.
The present invention also provides a kind of method transformed paddy rice purple acid phosphatase, comprises the gene transformation Rice Callus with having the nucleotide sequence shown in SEQIDNO:1, then the callus regeneration after transforming is cultivated into plant.
More specifically: the object of this invention is to provide a kind of new gene OsPAP10c from the fine middle clone of paddy rice wild-type variety Japan, the DNA sequence dna as shown in SEQIDNO:1 and Fig. 1.The protein sequence of its correspondence is as shown in SEQIDNO:2 and Fig. 2, and this protein belongs to purple acid phosphatase gene family.
Another object of the present invention is to provide a kind of method that the OsPAP10c of utilization gene carries out rice conversion, specifically, the invention provides the genophore with the sequence shown in SEQIDNO:1, wherein carrier as shown in Figure 3, containing OsPAP10c gene, this carrier can be expressed by above-mentioned nucleotide sequence coded protein.
Present invention also offers a kind of method utilizing plant expression vector conversion of plant to affect paddy rice acid phosphatase.
Realize concrete technological step of the present invention as follows:
1. cloning rice OsPAP10c gene
By round pcr, cloning rice OsPAP10c gene, is cut by enzyme and connects equimolecular biology techniques by gene constructed to rice conversion expression vector.See Fig. 3.
2. Transgenic Rice
Transfer-gen plant is obtained by utilizing the fast preparation method of agriculture bacillus mediated Transgenic Rice acceptor.
3. regulate the expression of OsPAP10c gene in paddy rice:
By the expression of transgenic technology overexpression OsPAP10c gene in paddy rice, obtain the transgenic paddy rice of overexpression, and detect transfer-gen plant by methods such as Semiquatitative RT-PCR assay and WesternBlot (Fig. 4).
4.OsPAP10c gene function preliminary evaluation
Transgenosis overexpression OsPAP10a, after the material of overexpression OsPAP10c and wild rice seed germination, be incubated in paddy rice liquid nutrient medium, the seedling growing to 10 days is normally, process two weeks under lacking phosphorus and supply ATP condition, measure enzymic activity and the phosphorus content of all plant.After overexpression OsPAP10a, transgenic line activity of acid phosphatase raise about 1.5 times (with report before consistent), and after overexpression OsPAP10c, the ground of transgenic line and the activity of acid phosphatase of subsurface all raise about 5 times (Fig. 5).Further analysis secretion property activity of acid phosphatase finds, the transgenic line of overexpression OsPAP10c improves the activity of acid phosphatase of about 10 times, and utilizes Westernblot to prove, OsPAP10c is present in (Fig. 6) in secretory protein.Meanwhile, the transgenic line ATP degradation capability of overexpression OsPAP10c significantly improves, and under ATP treatment condition, the transgenic line blade phosphorus content also corresponding raising of overexpression OsPAP10c, illustrates that this material use ATP ability improves (Fig. 7).
Rock Phosphate (72Min BPL) crop one does not open renewable resource, and phosphate fertilizer price goes up year by year in the world, becomes a kind of strategic resource gradually.Current China is the first in the world phosphate fertilizer importer, in order to tackle the phosphate fertilizer crisis of China and preserve the ecological environment, in the urgent need to improving phosphate fertilizer utilising efficiency and the Low phosphorus tolerance of crop.The present invention obtains paddy rice purple acid phosphatase gene OsPAP10c by clone technology, and obtains overexpression material by transgenosis and the preliminary evaluation function of this gene.Report before comparing, the present invention has found that a kind of secretion is to the purple acid phosphatase in environment first in paddy rice, this Phosphoric acid esterase can increase substantially the activity of acid phosphatase of transgenic line simultaneously, promote that transgenic line is to the utilising efficiency of organophosphorus, improves the field yield of transgenic paddy rice.
The purple acid phosphatase of contriver to all 29 paddy rice has carried out systems analysis, has screened the purple acid phosphatase that some participate in phosphorus signal reaction.Except acid phosphatase OsPAP10a, other acid phosphatase function is not also all reported, the potentiality of these acid phosphatases in phosphorus efficiency molecular breeding are not all known yet.In the present invention, overexpression OsPAP10c improves plant above ground and underground part acid phosphatase about 5 times, improves the acid phosphatase about 10 times of secretor type.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the DNA sequences encoding of OsPAP10c gene;
Fig. 2 is the protein sequence of OsPAP10c;
Fig. 3 is the overexpression vector figure of OsPAP10c;
Fig. 4 is OsPAP10c transgenic paddy rice Molecular Identification;
Fig. 5 is the enzyme assay of OsPAP10c transgenic paddy rice;
Fig. 6 is the secretory protein enzyme assay of OsPAP10c transgenic paddy rice and WesternBlot;
Fig. 7 is the analysis of OsPAP10c transgenic paddy rice organophosphorus Utilization ability.
Remarks illustrate: OsPAP10c-Oe1, OsPAP10c-Oe2, OsPAP10c-Oe3 in figure represent three independently transgenic events.
Embodiment
Embodiment 1, cloning rice OsPAP10c gene and structure overexpression vector:
Utilize primer to increase from rice root cDNA OsPAP10c total length encoder block (Fig. 1), upstream primer and downstream primer sequence are respectively 5 '-CGGGATCCATGGGGATGCTGCGGTGG-3 ' and 5 '-CCCCCGGGTTATACATCGTCGTTGGTGGG-3 '.Amplified production and binary vector pTF101.1-Ubi reclaim digestion products after utilizing BamH I and Sma I enzyme to cut respectively.Utilize DNA ligase to import in pTF101.1-Ubi carrier by OsPAP10c amplified production, obtain and transform whole carrier Ubi-OsPAP10c (Fig. 3).
Embodiment 2, Transgenic Rice:
1, the wild-type fine paddy rice of Japan (OryzasatvaL.sspjaponicacvNipponbare);
2, rice transgenic method
1) preparation of Mature Embryos of Rice callus
After Mature seed of rice shelling, select the full bright and clean seed without bacterial plaque and put into beaker, with 70% ethanol disinfection 1min.Remove ethanol, add 20% (v/v) NaClO solution (available chlorine about 1 ~ 1.2%) sterilization 90min.Remove NaClO solution, by sterile water wash 5 times, in sterilized water, soak 30min last 1 time.Remove sterilized water, seed is placed on aseptic filter paper and blots.With tweezers, seed is proceeded in inducing culture, 28 DEG C of light culture 10-14d.Culture dish opened by Bechtop, with tweezers, budlet and grain of rice shell is removed, leave embryo callus (faint yellow, fine and close irregular), insert in long-grained nonglutinous rice subculture medium, 28 DEG C of light culture 5-10d.
2) cultivation of Agrobacterium
By the agrobacterium strains stock solution preserved YEP (adding 50mg/LKan, 50mg/LStr), 28 DEG C of line activation 1-1.5d.With sterile toothpick picking Agrobacterium mono-clonal in 5mlYEP (containing 50mg/LKan and 50mg/LStr) nutrient solution, 28 DEG C, 250rpm shaking culture 12-36h is saturated to bacterium liquid OD600, therefrom draw Agrobacterium bacterium liquid 50 μ l in 30mlYEP (containing 50mg/LKan and 50mg/LStr) nutrient solution, 28 DEG C, 250rpm shaking culture 12-16h is to bacterium liquid OD6000.8-1.5.By cultivating complete agrobacterium liquid low-speed centrifugal (4000rpm, 10min), abandon supernatant, resuspended with appropriate AAM nutrient solution (containing 200 μMs of As), be diluted to the bacteria suspension of OD600=0.3-0.5.
3) screening of Dual culture and kanamycin-resistant callus tissue
The Rice Callus growing to a certain size is chosen, cuts into granular, put into Agrobacterium bacteria suspension, shaking culture 30 minutes.Callus is taken out, is placed on aseptic filter paper and drains 30-40min.Then callus is placed on Dual culture base.After 25 DEG C of light culture 2.5d, callus is taken out, by sterile water wash 5-6 time, need therebetween ceaselessly to vibrate.Again by the sterile water wash 1 ~ 2 time containing 250mg/L carbenicillin disodium.Finally be placed on aseptic filter paper and drain 2h.The callus of drying is proceeded on the Selective agar medium containing 250mg/L carbenicillin disodium and 50mg/L Totomycin and carry out first round selection, 28 DEG C, light culture 14d.The initial callus of growing up is forwarded to and takes turns selection, 28 DEG C, light culture containing 250mg/L carbenicillin disodium and 80mg/L Hygromycin selection media carry out second, until the resistant calli of graininess grows.If end in three weeks does not grow kanamycin-resistant callus tissue, the Selective agar medium that need forward identical component to carries out third round selection.
4) differentiation of kanamycin-resistant callus tissue and seedling
The kanamycin-resistant callus tissue 2-3 that picking obtains from same callus is placed in division culture medium, 28 DEG C of illumination cultivation [14h/10h (day/light) photoperiod, light intensity is 2000lx].After differentiation culture 20-50d, callus can differentiate seedling, when green bud grows to about 3-5cm, proceeds in root media, 28 DEG C of illumination cultivation [14h/10h (day/light) photoperiod, light intensity is 2000lx].
5) transplanting of transgenic seedling and Molecular Identification
After root culture 10-15d, shoot root portion and cauline leaf are broken up more complete plant is chosen, open sealed membrane, add appropriate distilled water or sterilized water, after culturing room hardening 2-3d, wash away agar, proceed in rice nutrition liquid and cultivate 2 weeks.Utilize weedicide grass fourth phosphorus to smear transgenic leaf, select the transgenic line of resistance.Extract RNA and the total protein of positive transgenic material, utilize RT-PCR and WesternBlot to carry out transgenic seedlings qualification.Finally the transgenic positive seedling obtained is moved into land for growing field crops or potted plant, sowing.
Embodiment 3:
Material and wild rice seed 1% nitric acid of transgenosis overexpression OsPAP10a, overexpression OsPAP10c break dormancy, and room temperature places 16 hours, and clear water rinses out nitric acid repeatedly, in 37 DEG C of incubator dark culturing two days to showing money or valuables one carries unintentionally.After vernalization completes, cultivate with rice nutrition liquid (table 1).Greenhouse periodicity of illumination be 12h daytime/12h night, light intensity 3000lux, day temperature 30 DEG C, night 22 DEG C.Grow to the seedling of 10 days in normal condition, process two weeks under lacking phosphorus and supply ATP condition, measure enzymic activity and the phosphorus content of all plant.
Table 1 water planting Rice under Condition nutrient solution prescription
* nutritive medium 2M salt acid for adjusting pH to 5.5.
Embodiment 4, transgenic paddy rice Molecular Identification:
1, Semi quantitative PCR analysis:
Get 50-100mg sample tissue liquid nitrogen fully to grind, add 1mlTrizol reagent, continue to be ground to complete powder.After placing a moment to dissolving, tissue homogenate is transferred in clean centrifuge tube.Add 200ml chloroform, concuss 15 seconds, in 4 DEG C, centrifugal 10 minutes of 12000g.Supernatant proceeds in a new centrifuge tube, again adds 200ml chloroform, concuss 15 seconds, in 4 DEG C, and the centrifugal 10min of 12000g.Get in supernatant to new centrifuge tube, add equal-volume Virahol, after putting upside down mixing, room temperature places 10min, in 4 DEG C, and the centrifugal 10min of 12000g.Abandon supernatant, fully wash twice with 75% ethanol (need configure with RNAseFree water).After drying at room temperature, by 20-40 μ lRNAseFree water dissolution.RNA sample is kept in-80 DEG C of Ultralow Temperature Freezers.
Process of reverse-transcription of the present invention all adopts the Revertase (M-MLVReverseTranscriptase, Promega) of Promega company, and the operation instructions that total serum IgE provides according to test kit carries out reverse transcription.After reverse transcription completes, sample is placed in 75 DEG C of process 10min, cooled on ice.Of short duration centrifugal after ,-20 DEG C preserve samples.
Semi quantitative PCR analysis is carried out again after first template being diluted 5-10 times.In semiquantitive PCR process, first carry out pcr analysis to OsACTIN, agarose gel electrophoresis detects PCR primer.By controlling the applied sample amount of PCR primer, all samples being regulated unanimously, then according to this level, target gene being analyzed.
2, Westernblot analyzes:
Get vegetable material and weigh fresh weight, with in the mortar of Liquid nitrogen precooler, tissue being ground.Add the protein extract (protein extract: 2%SDS, 60mMTris-HCl, pH8.5,2.5%glycerol, 0.13mMEDTA, 1Xproteaseinhibitorcocktail) of two volumes (2ml/g).Mixing, in 4 DEG C, centrifugal 15 minutes of 12000g, gets supernatant.Be kept in-80 DEG C of Ultralow Temperature Freezers after measuring protein concentration.
Protein sample 5 × SDS electrophoretic buffer is diluted to 10-15 μ g total protein concentration, and 100 DEG C are boiled 5 minutes, and by protein denaturation, 13000rpm, centrifugal 10 minutes of room temperature, gets supernatant liquor loading.After 60V electrophoresis 15 minutes (concentrated glue), 150V electrophoresis 90 minutes (separation gel).Gel (not dyeing) after SDS-PAGE is separated is placed in transferring film damping fluid and soaks 1 minute.Sponge, filter paper, gel, nitrocellulose, filter paper and sponge are overlayed on supporting rack in regular turn, between gap, can not bubble be had.Insert in transferring film groove, gel connects (-) pole, and pvdf membrane connects (+) pole, and strength of current is 0.75-1.0mA/cm2 filter membrane, transfer 1.5-2.0h.After (90V, 350mA, 1.5h) completes, TBST washes film 3 times, each 10min.
Pvdf membrane is placed in TBSTM, hatches 1h, washes film 3 times, each 10min with TBST.Add in TBST (or with 0.1%TBSTM) at 1%BSA, then add primary antibodie by 1/500-5000, hatch 1-2h.Film is washed 3 times, each 10min with TBST.Add in TBST at 1%BSA, then by 1/5000 add two resist, hatch 1-2h.TBST washes film 3 times, each 10min.BCIP/NBT/ nitrite ion=1:1:38 mixing, film is placed in one and carries out color reaction 3-10min, and the film after colour developing is placed in water and terminates reaction, result scanner record.
Embodiment 5, paddy rice activity of acid phosphatase are analyzed:
Get the reaction solution (10mMpNPP, 50mMsodiumacetate, pH5.5) that 10 μ g albumen are placed in 600 μ L preheatings, at 25 DEG C, react after 10 minutes, add NaOH (1M) termination reaction of 1.2ml, absorption value (Fig. 6,7) is measured in 410nm wavelength.
The foregoing is only several embodiments of the present invention, it should be pointed out that all distortion that those of ordinary skill in the art can directly be derived from content disclosed by the invention or be associated, all should protection scope of the present invention be thought.
Claims (4)
1. the protein of paddy rice purple acid phosphatase protein gene coding, is characterized in that: be the aminoacid sequence shown in SEQIDNO:2.
2. to encode the paddy rice purple acid phosphatase OsPAP10c gene of protein as defined in claim 1, it is characterized in that: be the nucleotide sequence shown in SEQIDNO:1.
3. the purposes of paddy rice purple acid phosphatase OsPAP10c gene as claimed in claim 1, it is characterized in that: for building transgenic paddy rice, described transgenic paddy rice improves activity of acid phosphatase, the corresponding efficiency that improve phosphorus absorption and utilization.
4. pair method that paddy rice purple acid phosphatase is transformed, is characterized in that: comprise the gene transformation Rice Callus with having the nucleotide sequence shown in SEQIDNO:1, then the callus regeneration after transforming is cultivated into plant.
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| CN108588116A (en) * | 2018-05-10 | 2018-09-28 | 华南农业大学 | The application of soybean purple acid phosphatase gene GmPAP35 |
| CN110923253A (en) * | 2019-12-19 | 2020-03-27 | 浙江大学 | Application of OsPTP1 in efficient plant phosphorus breeding |
| CN113667658A (en) * | 2020-12-29 | 2021-11-19 | 浙江大学 | Application of protein phosphatase OsPP74 in improving phosphorus absorption of rice |
| CN115976071A (en) * | 2022-10-27 | 2023-04-18 | 沈阳农业大学 | Application of PAP10a gene in regulation and control of rice blast resistance |
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| CN104946684A (en) * | 2015-06-17 | 2015-09-30 | 华南农业大学 | Function of purple acid phosphatase GmPAP33 gene for promoting reuse of phosphorus in soybean mycorrhiza |
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| CN104946684A (en) * | 2015-06-17 | 2015-09-30 | 华南农业大学 | Function of purple acid phosphatase GmPAP33 gene for promoting reuse of phosphorus in soybean mycorrhiza |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588116A (en) * | 2018-05-10 | 2018-09-28 | 华南农业大学 | The application of soybean purple acid phosphatase gene GmPAP35 |
| CN108588116B (en) * | 2018-05-10 | 2021-02-19 | 华南农业大学 | Application of soybean purple acid phosphatase gene GmPAP35 |
| CN110923253A (en) * | 2019-12-19 | 2020-03-27 | 浙江大学 | Application of OsPTP1 in efficient plant phosphorus breeding |
| CN113667658A (en) * | 2020-12-29 | 2021-11-19 | 浙江大学 | Application of protein phosphatase OsPP74 in improving phosphorus absorption of rice |
| CN115976071A (en) * | 2022-10-27 | 2023-04-18 | 沈阳农业大学 | Application of PAP10a gene in regulation and control of rice blast resistance |
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