CN101230354B - Clone and use of rice phosphatidylinositol-3kinase gene - Google Patents
Clone and use of rice phosphatidylinositol-3kinase gene Download PDFInfo
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- CN101230354B CN101230354B CN2007100513500A CN200710051350A CN101230354B CN 101230354 B CN101230354 B CN 101230354B CN 2007100513500 A CN2007100513500 A CN 2007100513500A CN 200710051350 A CN200710051350 A CN 200710051350A CN 101230354 B CN101230354 B CN 101230354B
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Abstract
The invention discloses a cloning and application of OsIP3K. By the RT-PCR technology, the total length of nucleic acid sequences of OsIP3K can be achieved by cloning form rice. The OsIP3K can be induced by IPTG in E. coli to obtain a protein with high expression. The analysis of HPLC shows that the protein can phosphorylate IP3 into OsIP4 at third and sixth bits, and has double enzyme activity. In addition, the invention also reports the application of the protein in vegetative growth, reproductive growth, hormone response and yeast salt-resistant.
Description
Technical field
The present invention relates to the clone of rice phosphatidylinositol--3 kinase gene (OsIP3K), more particularly utilize RT-PCR from paddy rice, to clone the total length nucleotide sequence of OsIP3K.The invention still further relates to aminoacid sequence, prokaryotic expression, enzyme activity determination and the application of this gene in the salt adverse circumstance of OsIP3K gene.
Background technology
Phosphatidylinositol--3 kinase gene (IP3K) has been proved extensively and has been present in the animal, and brings into play the effect of wanting emphatically.Mammals IP3K participates in having regulated many kinds of biological procedureses, comprise (Xia such as brain development, memory formation and learning process, H.J., Yang, G. (2005) Inositol 1,4,5-trisphosphate3-kinasesL:functions and regulations.Cell Res.15:83-91).The IP3K of overexpression fruit bat can improve fruit bat to H
2O
2Oxidation tolerance (Monnier, V., Girardot, F., Audin, W.Tricoire, H (2002) .Control of oxidative stress resistance by IP
3Kinase in Drosophila melanogaster.Free Radic Biol Med.33:1250-1259), show that thus the degeneration-resistant reaction of this gene and animal is relevant.
And the phosphatidylinositol--3 kinase gene in plant (Arabidopis thaliana) and the yeast also shows the characteristic of polyphosphoinositide kinase gene.The phosphatidylinositol--3 kinase gene of Arabidopis thaliana (IP3K) is an important kinase gene of regulating phosphoinositide metabolism in the vegetable cell, and its coded albumen can be with InsP3 (IP
3) phosphoric acid turns to tetra phosphoinositide (IP
4) and five phosphoinositide (IP
5) (Jill Stevenson-Paulik, Audrey R.Odom, and John D.York. (2003) .Molecular and Biochemical Characterization ofTwo Plant Inositol Polyphosphate 6-/3-/5-Kinases.J.Biol.Chem.277:42711-42718.Xia H-J, Brearley C, Elge S, et al. (2003) .Arabidopsisinositol polyphosphate 6-/3-kinase is a nuclear protein that complementsa yeast mutant lacking a functional ArgR-Mcm1transcription complex.Plant Cell.15:449-463.).IP
3With IP
4Be to regulate cell Ca
2+The important messenger molecule of equilibrated was so phosphatidylinositol--3 kinase gene ought to participate in regulating vegetable cell Ca indirectly
2+Signal (Xia H-J, Brearley C, Elge S, et al. (2003) .Arabidopsis inositol polyphosphate6-/3-kinase is a nuclear protein that complements a yeast mutant lackinga functional ArgR-Mcm1 transcription complex.Plant Cell; 15:449-463).The first phosphatidylinositol--3 kinase gene of plant is cloned (Jill Stevenson-Paulik from Arabidopis thaliana, Audrey R.Odom, and John D.York. (2002) .Molecular and BiochemicalCharacterization of Two Plant Inositol Polyphosphate 6-/3-/5-Kinases.J.Biol.Chem.277:42711-42718), its functional study is just in being reported by the main declarer of this patent in recent years, find that the Arabidopis thaliana phosphatidylinositol--3 kinase gene has transcriptional regulation, change Arabidopis thaliana polyphosphoinositide kinase gene over to yeast IP3K (ARG82/IPK2) mutant, can compensate phenotype (the Xia H-J that this mutant can not be grown when the arginine defective, Brearley C, Elge S, et al. (2003) Arabidopsisinositol polyphosphate 6-/3-kinase is a nuclear protein that complementsa yeast mutant lacking a functional Arg-Mcm1transcription complex.plantcell.15:449-463).Confirmed also that recently the Arabidopis thaliana phosphatidylinositol--3 kinase gene can be by the approach adjusting pollen tube relevant with calcium ion and growth (the Xu J of root, Brearley CA, Lin WH, et al (2005) .Arole of Arabidopsis inositol polyphosphate kinase AtIpk2 α, in pollengermination and root growth.Plant Physiol.137:94-103.).
Paddy rice has extremely important strategic position as important crops in national economy.Simultaneously, paddy rice can provide the important basic knowledge of biology for plant research as important model plant.In conjunction with the known function of phosphatidylinositol--3 kinase gene, especially its effect in adverse circumstance, the applicant thinks that rice phosphatidylinositol--3 kinase gene (OsIP3K) has crucial value for improvement paddy rice and other farm crop.The content of the application's patent is the first research to phosphatidylinositol--3 kinase gene clone and function thereof in the paddy rice.
Summary of the invention
The object of the present invention is to provide expression, hormone and the adverse circumstance inductive of a kind of clone of rice phosphatidylinositol--3 kinase gene, kinase whose activity, tissue to express.This gene provides a technological approaches for the improvement rice quality.
The invention still further relates to the application of a kind of rice phosphatidylinositol--3 kinase gene sequence in phosphoinositide metabolism.
The invention still further relates to the application of a kind of rice phosphatidylinositol--3 kinase gene sequence in the hormone signal approach.
The invention still further relates to of the application of a kind of rice phosphatidylinositol--3 kinase gene sequence at rice nutrition growth and reproductive growth.
The invention still further relates to the application of a kind of rice phosphatidylinositol--3 kinase gene sequence in the improvement rice varieties.
The invention still further relates to the application of a kind of rice phosphatidylinositol--3 kinase gene sequence in improving the anti-salt of yeast.
To achieve these goals, the present invention has adopted following technical measures:
(1) clone of paddy rice triphosphoric acid kinase gene (OsIP3K): utilize Arabidopis thaliana IP3K gene order (GenBank accession no:AJ245521), with rice genome sequence alignment among the GENBANK (http://www.ncbi.nlm.nih.gov/), obtain 1 the highest sequence of homology.According to the sequences Design primer that is found, clone the cDNA segment that obtains I in Rice P3K by the RT-RCR method, this fragment of analysis revealed promptly is the goal gene that we need, it has nucleotide sequence shown in the SEQ ID NO.1.This gene is made up of 888bp, 295 amino acid of encoding, and it has aminoacid sequence shown in the SEQ ID NO.2.This albumen is 32kDa, is positioned on the second karyomit(e) of paddy rice, does not contain intron.This goal gene is connected on the carrier pGEM-T (purchase of Promega company), the product called after pT-OsIP3K of gained, it has the nucleotide sequence shown in the SEQ ID NO.1.I in Rice P3K aminoacid sequence and Arabidopis thaliana, nematode and yeast IP3K aminoacid sequence have 48%, 27% and 25% homology respectively.Sequential analysis shows: rice Os IP3K contains the conserved domain that exists: PXXXDXKXG in animal and Arabidopis thaliana IP3K; But IP3K is the same with Arabidopis thaliana, and OsIP3K lacks the calmodulin binding domains of N-end, and this structural domain ubiquity in animal IP3K.OsIP3K also has peculiar 4 conservative regions among the Arabidopis thaliana IP3K in addition.Above result has supported that further the gene that is obtained is the phosphatidylinositol--3 kinase gene of paddy rice.
(2) construction of prokaryotic expression vector: with EcoRI and PstI (restriction enzyme) digested plasmid pT-OsIP3K (making up among the present invention), be connected on pMAL-c2 (NEB company) carrier of cutting with same enzyme, obtain expression vector pMAL-c2-OsIP3K, it has nucleotide sequence shown in the SEQ ID NO.1.PMAL-c2-OsIP3K expression in intestinal bacteria [XL1-Blue (NEB company)] can be obtained this albumen (Fig. 3), be used for further analysis.
(3) enzyme is lived and analyzed: (it is through inducing to induce the intestinal bacteria XL1-Blue that carries plasmid pMAL-c2-OsIP3K, can express aminoacid sequence shown in the SEQ ID NO.2), and get its bacterium liquid and carry out enzymatic activity experiment and high performance liquid chromatography experiment, detect its reaction substrate and product.Find that OsIP3K can catalysis 1,4, the 5-InsP3 generates tetra phosphoinositide, has 3 and 6 phosphorylation activities.(Fig. 4) explanation OsIP3K has dual phosphoinositide kinase activity.
(4) tissue expression analysis: utilization RT-PCR technology for detection the expression of OsIP3K in rice root, stem, leaf, bud, inflorescence, find that rice phosphatidylinositol--3 kinase gene all has expression (Fig. 5) in above tissue.Illustrate that this gene is a constitutive expression gene, hint that it may have latent effect in vegetation growth of plant and reproductive growth.
(5) abduction delivering analysis: at methyl jasmonate (MeJA), glucose (Glucose), dormin (ABA), Plant hormones regulators,gibberellins (GA), salt (NaCl) is handled down, find the rice seedling expression amount under above hormone and adverse circumstance processing, the expression amount of OsIP3K all improves (Fig. 6), illustrates that OsIP3K may participate in hormone signal approach and adverse circumstance reaction process.
(6) structure of transgene carrier: with pT-OsIP3K is template, and (KpnI, BamHI) primer of (restriction enzyme) increases, and obtains the segment of one section about 900bp with containing.This segment and pCAMBIA1301 (Australian taxi driver brother Lun Biya (CAMBIA) company) are used KpnI and BamHI double digestion respectively, reclaim product, connection obtains P1301-OsIP3K, and it has nucleotide sequence shown in the SEQ ID NO.1.And change the P1301-OsIP3K that successfully constructs over to Agrobacterium EHA105 (Australian Colombia (CAMBIA) company).
(7) inducing and genetic transformation of rice callus tissue: mature seed shells, and places 70% ethanol 30 seconds, transfers to 0.1%HgCl
2 Middle sterilization 10 minutes, aseptic washing 5 times.Mature embryo after the sterilization is inoculated on the inducing culture, secretly cultivated for 3 weeks (22-28 ℃).Downcut callus, change on the pre-culture medium, secretly cultivated 3 days.The Agrobacterium EHA105 that picking contains P1301-OsIP3K (this patent obtains, and it has nucleotide sequence shown in the SEQ ID NO.1) puts LB liquid nutrient medium (containing kantlex (kan) 50mg/L), and 28 ℃, 200 rev/mins (rpm) shook 12 hours.Take out 10-15mL and put in the LB substratum of new preparation, shake to OD
600=0.5-0.8, centrifugal 10 minutes of 6000rpm, precipitation was placed 20 minutes with suspending in isopyknic suspension culture base.From pre-culture medium, take out callus, put into dip-dyeing solution 30 minutes.Take out callus, inhale with sterilization filter paper and remove surperficial dip-dyeing solution, change common culture medium over to, secretly cultivated three days.Callus is put into sterilized water (containing Rifampin (cef) 300-500mg/L) washing 30 minutes, again with the sterilized water washing that does not contain cef 5-6 time.Screening: callus is put into screening culture medium, and 2-3 is after week, and the callus that cutting-out newly grows is placed on the screening culture medium after screening culture medium 2-3 week again, changes division culture medium over to after 2 weeks.Differentiation under illumination through about 45 days, callus is divided into green plant, then immature plant is forwarded on the root media,, form complete plant through 15-20 days induce, obtain transfer-gen plant, these transfer-gen plants detect through PCR, prove its positive transfer-gen plant, forward the experimental plot continued growth to, obtain sophisticated seed at last, this is that T1 is for seed.
(8) yeast compensation experiment: make up pYES2-OsIP3K (making up among the present invention) Yeast expression carrier, change in the yeast ARG82/IPK2 mutant.The salt resistance of discovery transgenic yeast under the salt adverse circumstance improves 20-30%, sees Figure 11.
Advantage of the present invention: to the clone of rice phosphatidylinositol--3 kinase gene (OsIP3K), help the research of plant phosphoinositide signal transduction path and mechanism, simultaneously because phosphatidylinositol--3 kinase gene has showed the critical function at aspects such as adverse circumstance and growths in fruit bat, Arabidopis thaliana, therefore can utilize rice phosphatidylinositol--3 kinase gene to improve the resistivity of paddy rice, be expected to improve crop quality environment stress.
Description of drawings
Fig. 1: bacterium colony PCR detects.M is marker (DL2000), and 1-8 is respectively 8 resistance clones of picking.
Fig. 2: enzyme is cut detection.M is marker (DL2000), three clones of corresponding numbering in 2,4,6 corresponding diagram 1.
Fig. 3: 10% polyacrylamide gel electrophoresis figure.M is the standard protein molecular weight, 1,2 for carrying the electrophoresis band of XL1-Blue bacterial strain product of empty carrier pMAL-c2, and 3-6 is the electrophoresis band that carries the XL1-Blue bacterial strain product of carrier pMAL-c2-OsIP3K, and wherein 1,3,5 is electrophoresis band without IPTG inductive bacterial strain product; 2,4,6 is the electrophoresis band of IPTG inductive bacterial strain product.
Fig. 4: high performance liquid chromatography (HPLC) detects the enzymic activity of OsIP3K.A is that reversed-phase liquid chromatography detects enzyme reaction product alive; B is the phosphorylation site that the anionresin liquid chromatography further detects this enzyme.
Fig. 5: RT-PCR detects the expression of OsIP3K gene in rice tissue.M is marker (DL2000), P: inflorescence, B: bud, L: leaf, S: stem, R: root.
Fig. 6: RT-PCR detects the expression of OsIP3K gene under hormone and adverse circumstance processing.Wherein C4 and C24 are the expression amount of 0 hour and 24 hours materials that are untreated.4,8,12,24 are respectively hormone or adverse circumstance handled 4 hours, and 8 hours, 12 hours, the expression amount of 24 hours seedling.
Fig. 7: enzyme is cut and is detected P1301-OsIP3K.1 is KpnI and BamHI double digestion, and 2 are the detection of SalI single endonuclease digestion.
Fig. 8: rice conversion schema.From left to right, be respectively callus and post generation, screening is broken up and is taken root.
Fig. 9: transgenic paddy rice PCR identifies.-negative contrast ,+positive contrast, 1-15 is respectively 15 transgenic lines.
Figure 10: enzyme is cut and is detected pYES2-OsIP3K.M is marker (DL2000), and pYES2-OsIP3K is for making up the figure that plasmid is cut with KpnI and BamHI enzyme.
Figure 11: the anti-salt drop experiment of yeast.CK is the contrast of handling without salt, WT is the yeast wild type strain, ipk2 is a yeast ARG82/IPK2 mutant, ipk2+Vector is for having transformed the yeast ARG82/IPK2 mutant of pYES2 empty carrier, and ipk2+pYES2-OsIP3K is for having transformed the yeast ARG82/IPK2 mutant of pYES2-OsIP3K carrier.
Embodiment
The clone of embodiment 1.OsIP3K gene
In order to obtain the full length sequence of OsIP3K gene, we utilize BLAST (
Http:// www.ncbi.nlm.nih.gov/BLAST/) search software compares the sequence and the rice genome sequence of Arabidopis thaliana phosphatidylinositol--3 kinase gene (GenBank accession no:AJ245521), find out a sequence the highest, infer that this sequence is the rice phosphatidylinositol--3 kinase gene sequence with its homology.According to this gene design special primer, [forward primer: (5 '-GATCGAATTCATGGCCTGCGACCTGCGCCCG-3 '), reverse primer: (5 '-GTACTCTAGATCAAGAATGATCTGAAGACGCCT-3 ')], and be material with the blade of spending 11 (preservations of academy of agricultural sciences, Hubei Province) in the japonica rice, obtain the cDNA segment by RT-PCR, PCR gained target gene fragment is connected on the carrier pGEM-T (purchase of Promega company), the product called after pT-OsIP3K of gained, it has the nucleotide sequence shown in the SEQ ID NO.1.
This assignment of genes gene mapping is made up of 888bp in No. 2 karyomit(e) of paddy rice, 295 amino acid of encoding, and it has the aminoacid sequence shown in the SEQ ID NO.2, does not contain intron.I in Rice P3K aminoacid sequence and Arabidopis thaliana, nematode and yeast IP3K aminoacid sequence have 48%, 27% and 25% homology respectively.With the clustalw program (
Http:// bioinfo.biosino.org:6000/) carry out amino acid sequence analysis and find that OsIP3K contains the conserved domain that exists: PXXXDXKXG in animal and Arabidopis thaliana IP3K; But IP3K is the same with Arabidopis thaliana, lacks the terminal calmodulin binding domains of N-, and this site ubiquity in animal IP3K.OsIP3K albumen also has peculiar 4 conservative regions in Arabidopis thaliana IP3K in addition.
At first use EcoRI and PstI digested plasmid pT-OsIP3K (obtaining among the embodiment 1), the fragment that obtains is connected on pMAL-c2 (NEB company) carrier of cutting with same enzyme, obtain expression vector pMAL-c2-OsIP3K, and transformed into escherichia coli XL1-Blue (NEB company), bacterium liquid is applied to LB (the peptone 10g/L that contains the ammonia benzyl, yeast extract 5g/L, NaCl 5g/L) dull and stereotyped last 37 ℃ of grow overnight, select 8 single bacterium colonies.With OsPr:5 '-TCAAGAATGATCTGAAGACG-3 ', OsPm:5 '-GGCGCCCGGACCGGTCGG-3 ' makes bacterium colony PCR for primer and detects, and finds that the 1-7 bacterium colony all expands the band (Fig. 1) that about 400bp.2,4, No. 6 bacterium colonies choosing wherein extract plasmid, cut the band (Fig. 2) that obtains to be about 700bp with the SalI enzyme, and the result is consistent with the purpose clip size.Enzyme is cut the result and is shown the success of pMAL-c2-OsIP3K (it has the nucleotide sequence shown in the SEQ ID NO.1) construction of recombinant plasmid.
The expression of embodiment 3.Maltose Binding Protein-OsIP3K (MBP-OsIP3K) fusion rotein in intestinal bacteria
Choose among the embodiment 2 No. 2 and No. 4 two positive colonies of verifying are inoculated in the LB substratum activation and spend the night, be transferred in the fresh LB liquid nutrient medium OD to be cultured to then respectively
600Be about at 0.5 o'clock, will do not contain IPTG (contrast) and add IPTG inductive bacterium liquid co-cultivation is got the 1ml contrast respectively and induced processing after 4 hours bacterium liquid, centrifugal collecting cell.After cell is cleaved, polyacrylamide gel electrophoresis with 10% separates target protein, with coomassie brilliant blue staining (Fig. 3), wherein pMAL-c2 empty carrier itself has the nucleotide sequence of label protein maltose binding protein, through inducing the maltose binding protein (MBP) that can obtain about 42KDa, the intestinal bacteria XL1-Blue that contains pMAL-c2-OsIP3K is through inducing, but expressed fusion protein MBP-OsIP3K, be about 74KDa, consistent with this gene Fusion molecular weight of albumen size of prediction.
The kinase whose enzyme activity determination of embodiment 4.OsIP3K
To contain the XL1-Blue bacterial strain (among the embodiment 2 obtain) of pMAL-c2-OsIP3K and 0.4 μ M ATP respectively with Ins (1,4, the 5) P of 40 μ M
3(△), Ins (3,4,6) P
3(●), (two kinds of InsP3s) Ins (3,4,5,6) P
4(zero) (a kind of tetra phosphoinositide) is 30 ℃ of warm down baths 2 hours.Utilize reverse resin isolation technical point from each component.(Fig. 4-A), its corresponding product is IP can to obtain a peak by radioassay
4With above catalysis Ins (1,4,5) P
3Reaction gained IP
4Product, with [
3H] mark [
3H] Ins (1,3,4,5) P
4And Ins (1,4,5,6) P
4Isomer [
3H] Ins (3,4,5,6) P
4As the wash-out contrast, pass through anion-exchange column together.According to elution time, determine its phosphorylation site.Product of catalyzed reaction with [
3H] Ins (1,3,4,5) P
4Reach peak value (53 minutes) at one time, and another product with [
3H] Ins (3,4,5,6) P
4Reached peak value at 55.5 minutes, visible catalyzed reaction can obtain two products, is respectively Ins (1,3,4,5) P
4And Ins (1,4,5,6) P
4(Fig. 4-B).The OsIP3K gene that can show our resulting separation thus has the P with Ins (1,4,5)
3Catalysis is to Ins (1,3,4,5) P
4And Ins (1,4,5,6) P
4Activity, that is to say that this enzyme has 3,6 phosphorylation activities.This experimental results show that the OsIP3K gene rice phosphatidylinositol--3 kinase of encoding really, and this enzyme has dual enzymic activity.
The tissue expression of embodiment 5.OsIP3K gene
Choose the material of rice root, stem, leaf, bud, five tissues of inflorescence respectively, extract RNA, method by RT-PCR detects OsIP3K expression of gene situation, find that the OsIP3K gene all has expression in root, stem, leaf, bud, inflorescence, hint OsIP3K may have latent effect (Fig. 5) in vegetation growth of plant and reproductive growth.
The relation of embodiment 6.OsIP3K gene and adverse circumstance and hormone
The paddy rice that sprouted for two weeks is used 100 μ M methyl jasmonates (MeJA) respectively, 40mM glucose (Glucose), 50 μ M dormins (ABA), 100 μ M Plant hormones regulators,gibberellins (GA) and 250mM salt (NaCl) are handled, and choose and have handled 4,8,12,24 hours seedling is extracted RNA, by the method detection OsIP3K expression of gene amount of RT-PCR.Discovery is under above hormone and adverse circumstance processing, and the expression amount of OsIP3K is improved (Fig. 6), illustrates that the OsIP3K gene may participate in hormone signal approach and adverse circumstance reaction.
The structure of embodiment 7. rice phosphatidylinositol--3 kinase gene sense expression vectors
With PT-OsIP3K (embodiment 1 obtains) is template, use forward primer: (5 '-GGGTACCATGGCCTCCGACCTGCGCCCG-3 '), reverse primer: (5 '-CGCGGATCCGCGAGAATGATCTGAAGACGCCT-3 ') increase (restriction enzyme site that this primer contains KpnI and BamHI) obtains the segment of one section about 900bp.This segment and pCAMBIA1301 are used KpnI and BamHI double digestion respectively, reclaim product, connection obtains P1301-OsIP3K, and it has the nucleotide sequence shown in the SEQ ID NO.1.Connect product and use KpnI and BamHI double digestion and SalI single endonuclease digestion to identify (Fig. 7), obtain the band of about 900bp and 700bp respectively, prove to successfully construct.
And utilize freeze-thaw method to transform Agrobacterium EHA105 (Holsters M, Depicker A, Depicker A, (1978) .Transfection and transformation of Agrobacteriumtumefaciens.Molecular and General Genetics, 183:181-187.)
The acquisition of embodiment 8. rice conversion and transfer-gen plant
1) sterilization of seed: mature seed shells, and places 70% ethanol 30 seconds, changes 0.1%HgCl sterilization 10 minutes over to, aseptic washing 5 times
2) inoculation: the mature embryo after will sterilizing is inoculated on the inducing culture, secretly cultivates for 3 weeks (22-28 ℃).
3) the pre-cultivation: downcut callus, change on the pre-culture medium, secretly cultivated 3 days.
4) dip-dyeing solution preparation: picking mono-clonal Agrobacterium EHA105 (P1301-OsIP3K, it has the nucleotide sequence shown in the SEQ IDNO.1).Be inoculated in LB liquid nutrient medium (containing kan 50mg/L), 28 ℃, 200rpm shook 12 hours.Take out 10-15mL and put in the LB substratum of new preparation, shake to OD
600=0.5-0.8, centrifugal 10 minutes of 6000rpm will precipitate suspension, place 20 minutes.
5) contaminate: from pre-culture medium, take out callus, put into dip-dyeing solution 30 minutes.Take out callus, remove surperficial dip-dyeing solution, change common culture medium over to, secretly cultivated three days with inhaling on the sterilization filter paper.
6) washing: callus is put into sterilized water (containing cef 300-500mg/L) washing 30 minutes, again with the sterilized water washing that does not contain cef 5-6 time.
7) screening: callus is placed into screening culture medium, and 2-3 downcut the callus that newly grows and, is placed on the new screening culture medium after week to new screening culture medium 2-3 again after week, after 2 weeks, then put into screening culture medium screening 2-3 week.
8) differentiation: the differentiation under illumination through about 45 days, callus is divided into green plant
9) take root: then immature plant is forwarded on the root media,, form complete plant, obtain transfer-gen plant through 15-20 days induce.Fig. 8
| Inducing culture | N6 substratum+2,4-D (2mg/L)+sucrose (30g/L)+casamino acid (300mg/L), pH5.8+agar (8g/L), packing sterilization. |
| Pre-culture medium | N6 substratum+2,4-D (2mg/L)+sucrose (30g/L)+glucose (10 g/L), pH 5.2 ,+agar (8g/L).Sterilization back+AS (20mg/L, 100% EtOH dissolving) |
| Be total to culture medium | N6 substratum+2,4-D (2mg/L)+sucrose (30g/L)+glucose (10 g/L), pH 5.2 ,+agar (8g/L).Sterilization back+AS (20mg/L, 100% EtOH dissolving) |
| Screening culture medium | N6 substratum+2,4-D (2mg/L)+sucrose (30g/L)+casami no acid (300mg/L), pH 5.8+agar (8g/L), Hym (20mg/ml) |
| Root media | MS+casamino acid (500mg/L)+6-BA (1.2mg/L)+NAA (0.2 mg/L), pH 5.8.+sorbitol (5g/L). |
The evaluation of embodiment 9. transfer-gen plants
The transgenic seedling that obtains is transplanted to soil, grew one month, individual plant is collected blade, extract leaf DNA, use forward primer: (5 '-GCCGTGCACTTGTTTGTCGGG-3 ') and reverse primer: (5 '-GTACTCTAGATCAAGAATGATGTGAAGACGCCT-3 ') carries out PCR and detects, the positive transfer-gen plant of the pairing plant of product of band can be amplified, and the aminoacid sequence (Fig. 9) shown in the SEQ ID NO.2 can be expressed.
The structure of embodiment 10. Yeast expression carriers
At first use plasmid pT-OsIP3K (obtaining among the embodiment 1) to be template, use forward primer: (3 '-GGTCGGTACCATGGCCTCCGACCTGCGCC-3 ') and reverse primer: (5 '-CGGGATCCCGTCAAGAATGATCTGAAGACGCCT-3 ') carries out pcr amplification. with the segment of PCR gained with restriction enzyme KpnI and BamHI double digestion, and be connected on the carrier pYES2 (purchase of Invitrogen company) that same enzyme enzyme cuts, the product called after pYES2-OsIP3K of gained, it has the nucleotide sequence shown in the SEQ ID NO.1, expresses the aminoacid sequence shown in the SEQ ID NO.1.And it is changed among the intestinal bacteria XL1-Blue (NEB company).Extract plasmid, cut evaluation (Figure 10) with KpnI and BamHI enzyme.Can obtain a band that is about 900bp, prove to successfully construct.Then pYES2 and pYES2-OsIP3K are transformed in the yeast ARG82/IPK2 mutant [buy in EUROSCARF (
Http:// www.rz.uni-frankfurt.de/FB/fb16/mikro/euroscarf)].
The four primary yeast bacterium [wild type strain (purchase) that picking embodiment 10 mentions in EUROSCARF, yeast ARG82/IPK2 mutant, and the conversion that obtains among the embodiment 10 mutant of pYES2 empty carrier and pYES2-OsIP3K], be inoculated in complete minimal substratum (the Jill Stevenson-Paulik that contains 2% semi-lactosi, activation is 2 days among the Audrey R.Odom, and John D.York. (2002) Molecular and Biochemical Characterization of Two Plant InositolPolyphosphate 6-/3-/5-Kinases.J.Biol.Chem.277:42711-42718).Adjust bacterial strain concentration to OD600=1.0.(concentration is respectively 10 with 10 times of serial dilutions of they difference
0, 10
-1, 10
-2, 10
-3, 10
-4).And respectively get 1ul respectively point sample in 0M (CK) is arranged, 0.6M and 0.8MNaCl complete minimal substratum (with 2% semi-lactosi as the charcoal source) on.The result shows that the yeast saline-alkaline tolerance that transforms the pYES2-OsIP3K carrier significantly is higher than mutant and the mutants which had 20-30% (seeing Figure 11) that contains empty carrier.
SEQUENCE?LISTING
＜110〉Wuhan University
＜120〉clone of rice phosphatidylinositol--3 kinase gene and application
＜130〉clone of rice phosphatidylinositol--3 kinase gene and application
<160>2
<170>PatentIn?version?3.1
<210>1
<211>888
<212>DNA
<213>Oryza?sativa
<400>1
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aagctgggcc?cgctcgtcga?cggcgagggg?ctcttctaca?agcccctcca?ggccggggag 120
cgcggggagc?acgaggccgc?cttctacgcc?gcgttcaccg?cgcacccggc?cgtcccgccc 180
cgggtccggg?gcgccttctt?cccgcgcttc?cacggcaccc?gcttcctccc?ggccccagcc 240
agccccggcg?gcgcgcccta?cccgcacatc?gtcctcgacg?acctcctcgc?gggcctcccg 300
tccccctgcg?tcgccgacgt?caagatcggc?gcctgcacgt?ggccgccgcg?atccccggac 360
ccctacgtcg?ccaagtgcct?cgccaaggac?cgcgagacca?ccagcgcgct?cctcggcttc 420
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tcggagctga?aggggatcga?cgccgccggg?gtccgccgcg?tgctccgccg?ctacgtgtcc 540
acgggcggcg?gcgacggcct?ggactgcgcg?ctcgccgccg?cggtgtacgg?aggggagggc 600
ggcgtcctgg?cgcagctgcg?ggagctcaag?gcgtggttcg?aggagcaaac?cctgtaccac 660
ttctactcgg?cgtcgattct?gttcggctac?gacgccaatg?cggcggcggc?ggctgctccc 720
ggaggtggaa?gcggcggtgt?aagggtgaag?ctggtggact?tcgcgcatgt?cgacgatggg 780
gacggggtga?ttgaccacaa?cttcttgggc?gggctctgct?cgctcatcaa?gttcatcggc 840
gacattgtcg?ccgaggttac?cgagaaggcg?tcttcagatc?attcttga 888
<210>2
<211>295
<212>PRT
<213>Oryza?sativa
<400>2
Met?Ala?Ser?Asp?Leu?Arg?Pro?Pro?Glu?His?Gln?Val?Ala?Gly?His?Arg
1 5 10 15
Ala?Ser?Ala?Asp?Lys?Leu?Gly?Pro?Leu?Val?Asp?Gly?Glu?Gly?Leu?Phe
20 25 30
Tyr?Lys?Pro?Leu?Gln?Ala?Gly?Glu?Arg?Gly?Glu?His?Glu?Ala?Ala?Phe
35 40 45
Tyr?Ala?Ala?Phe?Thr?Ala?His?Pro?Ala?Val?Pro?Pro?Arg?Val?Arg?Gly
50 55 60
Ala?Phe?Phe?Pro?Arg?Phe?His?Gly?Thr?Arg?Phe?Leu?Pro?Ala?Pro?Ala
65 70 75 80
Ser?Pro?Gly?Gly?Ala?Pro?Tyr?Pro?His?Ile?Val?Leu?Asp?Asp?Leu?Leu
85 90 95
Ala?Gly?Leu?Pro?Ser?Pro?Cys?Val?Ala?Asp?Val?Lys?Ile?Gly?Ala?Cys
100 105 110
Thr?Trp?Pro?Pro?Arg?Ser?Pro?Asp?Pro?Tyr?Val?Ala?Lys?Cys?Leu?Ala
115 120 125
Lys?Asp?Arg?Glu?Thr?Thr?Ser?Ala?Leu?Leu?Gly?Phe?Arg?Val?Ser?Gly
130 135 140
Val?Arg?Val?Val?Asp?Ala?Arg?Gly?Gly?Ala?Val?Trp?Arg?Pro?Asp?Arg
145 150 155 160
Ser?Glu?Leu?Lys?Gly?Ile?Asp?Ala?Ala?Gly?Val?Arg?Arg?Val?Leu?Arg
165 170 175
Arg?Tyr?Val?Ser?Thr?Gly?Gly?Gly?Asp?Gly?Leu?Asp?Cys?Ala?Leu?Ala
180 185 190
Ala?Ala?Val?Tyr?Gly?Gly?Glu?Gly?Gly?Val?Leu?Ala?Gln?Leu?Arg?Glu
195 200 205
Leu?Lys?Ala?Trp?Phe?Glu?Glu?Gln?Thr?Leu?Tyr?His?Phe?Tyr?Ser?Ala
210 215 220
Ser?Ile?Leu?Phe?Gly?Tyr?Asp?Ala?Asn?Ala?Ala?Ala?Ala?Ala?Ala?Pro
225 230 235 240
Gly?Gly?Gly?Ser?Gly?Gly?Val?Arg?Val?Lys?Leu?Val?Asp?Phe?Ala?His
245 250 255
Val?Asp?Asp?Gly?Asp?Gly?Val?Ile?Asp?His?Asn?Phe?Leu?Gly?Gly?Leu
260 265 270
Cys?Ser?Leu?Ile?Lys?Phe?Ile?Gly?Asp?Ile?Val?Ala?Glu?Val?Thr?Glu
275 280 285
Lys?Ala?Ser?Ser?Asp?His?Ser
290 295
Claims (1)
1. the application of rice phosphatidylinositol--3 kinase gene in the anti-salt of yeast, the nucleotide sequence of described gene is shown in SEQ ID No.1.
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| CN2007100513500A CN101230354B (en) | 2007-01-24 | 2007-01-24 | Clone and use of rice phosphatidylinositol-3kinase gene |
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| CN2007100513500A CN101230354B (en) | 2007-01-24 | 2007-01-24 | Clone and use of rice phosphatidylinositol-3kinase gene |
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| CN101230354B true CN101230354B (en) | 2011-06-08 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434691A (en) * | 2016-09-22 | 2017-02-22 | 天津师范大学 | Application of OsFTL12 gene in control over paddy rice reproductive growth transition and plant type establishment |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102094008B (en) * | 2010-11-30 | 2012-12-05 | 深圳华大基因科技有限公司 | BGIos1010 gene and application thereof |
| CN114891811B (en) * | 2022-05-12 | 2023-05-26 | 华南农业大学 | Application of rice inositol triphosphate kinase gene |
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2007
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Non-Patent Citations (4)
| Title |
|---|
| NM_001053521.GENBANK.2006, |
| NM_001053521.GENBANK.2006,;夏惠君等.拟南芥三磷酸肌醇激酶的新功能-在酵母细胞中参与抗逆反应.中国细胞生物学学会第八界会员代表大会暨学术大会论文摘要集.2003,第"7-23"页,共1页. * |
| 周婷等.水稻三磷酸肌醇激酶的分离和鉴定.中国植物生理学会第九次全国会议论文摘要汇编.2004,第181页. * |
| 夏惠君等.拟南芥三磷酸肌醇激酶的新功能－在酵母细胞中参与抗逆反应.中国细胞生物学学会第八界会员代表大会暨学术大会论文摘要集.2003,第"7-23"页，共1页. |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434691A (en) * | 2016-09-22 | 2017-02-22 | 天津师范大学 | Application of OsFTL12 gene in control over paddy rice reproductive growth transition and plant type establishment |
| CN106434691B (en) * | 2016-09-22 | 2019-08-16 | 天津师范大学 | OsFTL12 gene builds up the application of aspect in the transformation of control rice reproductive growth and plant type |
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| CN101230354A (en) | 2008-07-30 |
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