CN101583718A - Cytochrome P450 gene for increasing seed size or water stress resistance of plant - Google Patents
Cytochrome P450 gene for increasing seed size or water stress resistance of plant Download PDFInfo
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- CN101583718A CN101583718A CNA2008800026324A CN200880002632A CN101583718A CN 101583718 A CN101583718 A CN 101583718A CN A2008800026324 A CNA2008800026324 A CN A2008800026324A CN 200880002632 A CN200880002632 A CN 200880002632A CN 101583718 A CN101583718 A CN 101583718A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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
Abstract
The present invention relates to cytochrome P450 protein originating from Arabi dopsis thaliana which can be used for increasing seed size or storage protein content in seed or for increasing water stress resistance of plant, a gene encoding said protein, a recombinant plant expression vector comprising said gene, a method of increasing seed size or storage protein content in seed and a method of increasing water stress resis tance of plant by using said vector, plants produced by said method and transgenic see d of said plants. According to the present invention, by using cytochrome P450 geneof the present invention, seed size or storage protein content in seed can be increased or water stress resistance of plant can be increased.
Description
Technical field
The present invention relates to derive from the cytochrome p450 protein matter of Arabidopis thaliana (Arabidopsis thaliana), this cytochrome p450 protein matter can be used for increasing the size of seed or the content of seed storage protein, or is used to strengthen the resistance of plant to water stress.The invention still further relates to the described cytochrome p450 protein matter of coding gene, comprise the recombinant plant expression vector of described gene, and increase the method for the content of storage protein in the size of seed or the seed, and strengthen the method for plant to the resistance of water stress with described carrier.The invention still further relates to the plant of described method generation and the transgenic seed of described plant.
Background technology
The many enzymic catalytic reactions that relate to multiple substrate of Cytochrome P450 catalysis, i.e. oxidation, peroxidation and the reductive metabolism of endogenous substrate or xenobiotic substrate (xenobiotic substrate).Especially plant P450 has participated in various plants product synthetic bio-chemical pathway, comprise phenyl propyl (phenylpropanoids), alkaloid, terpene, lipid, cyanogenic glycoside and glucosinolate (glucosinolates) (Chapple, Annu.Rev.Plant Physiol.Plant Mol.Biol.1989,49:311-343).
Cytochrome P450 also is commonly referred to as P450 half mercaptan protein, and works as final oxydase in the polycomponent electron transport chain, and this electron transport chain is also referred to as the monooxygenase system that contains P450.As the specificity catalyzed reaction, this system comprises demethylation, hydroxylation, epoxidation, N-oxidation, sulfo-oxidation, N-, S-and O-dealkylation, deaminizatingization, desulfurization, and the reduction of nitrogen-containing group, nitryl group and N-oxide group.
The multiple effect of the P450 enzyme of tobacco plant relates to the transmission of various plants meta-bolites, for example phenyl propyl (phenylpropanoids), alkaloid, terpene, lipid, cyanogenic glycoside and glucosinolate (glucosinolates) and many other chemical bodies.In in the past 1 year, the composition that verified some P450 enzyme thanks to product to the plant mesophytic era works.
Based on above-mentioned prior art, the present invention finds when the function of research Cytochrome P450, the Cytochrome P450 that derives from Arabidopis thaliana can improve the content of storage protein in the size of plant seed or the plant, or strengthens the resistance of plant to water stress, has therefore finished the present invention.
Summary of the invention
The objective of the invention is to study the function of cytochrome p450 protein matter in plant that derives from Arabidopis thaliana.
To achieve these goals, the invention provides a kind of cytochrome p450 protein matter, this cytochrome p450 protein matter derives from Arabidopis thaliana and can be used for increasing the size of plant seed or the content of seed storage protein, or is used to strengthen the resistance of plant to water stress.
The present invention further provides the gene of the described cytochrome p450 protein matter of encoding.
The present invention further provides the recombinant plant expression vector that comprises described gene.
The present invention further provides the method that increases the content of storage protein in the size of seed or the seed with described carrier, and with the method for described carrier enhancing plant to the resistance of water stress.
The present invention also further provides with the plant of described method generation and the transgenic seed of described plant.
According to the present invention, use cytochrome P450 gene of the present invention can increase the content of storage protein in the size of seed or the seed, or strengthen the resistance of plant water stress.
Description of drawings
Fig. 1 has shown that the RT-PCR of the expression of AtCYP78A7 in different Arabidopis thaliana tissues analyzes.
Fig. 2 has shown the GUS tissue chemical analysis that AtCYP78A7 expresses.
Fig. 3 has shown that mistake is expressed size (A), the weight (B) of seed and the content (C) of seed storage protein (for example 12S sphaeroprotein and 2S albumin) that AtCYP78A7 has increased seed in Arabidopis thaliana.
Fig. 4 shows that ABA-responsive genes, arid/cold coerces the sxemiquantitative RT-PCR of genes involved.
Fig. 5 has shown the comparison of the drought stress response of wild-type and transgenic arabidopsis.Numeral cross each transgenic line express AtCYP78A7.Daws represents the fate behind the water stress; Fate after Darw represents to rewater.
Fig. 6 has shown the result (A) of the PCR that carries out for AtCYP78A7 expression of gene in the transgenosis rice plants that confirmed expression AtCYP78A7 and the result (B) of genomic hybridization reaction.The numeral that shows among the figure cross the number of each the transgenosis rice plants express AtCYP78A7.HW (Hwayoung) is contrast rice class group.
Fig. 7 has shown some transgenosis rice plants of producing among the present invention test result to the resistance of water stress.
Fig. 8 has shown the weighing results (for 100 seeds) of the seed of the transgenosis rice plants that produces among the present invention.The numeral that shows among the figure is crossed the number of each transgenosis rice plants of expressing AtCYP78A7.HW (Hwayoung) is contrast rice class group.
Embodiment
In order to realize the purpose of the invention described above, the invention provides a kind of cytochrome p450 protein matter, this cytochrome p450 protein matter derives from Arabidopis thaliana and has the aminoacid sequence shown in the SEQ ID NO:1, this protein can be used for increasing the size of plant seed or the content of seed storage protein, or is used to strengthen the resistance of plant to water stress.
The present invention relates to derive from the purposes of the cytochrome p450 protein matter of Arabidopis thaliana.Particularly, described protein can be used for increasing the size of plant seed or the content of seed storage protein, or is used to strengthen the resistance of plant to water stress.Storage protein can be the acid storage protein of 12S, the 12S alkalescence storage protein in the seed or the 2S storage protein in the seed in the seed in the described seed, but is not limited thereto.
Cytochrome p450 protein matter for according to one embodiment of the present invention can contain the aminoacid sequence shown in the SEQID NO:1.In addition, the variant of described aminoacid sequence is also included within the scope of the present invention.Described variant comprises that sequence changes but has the function similar to the aminoacid sequence shown in the SEQ ID NO:1 and the aminoacid sequence of immunity.Particularly, cytochrome p450 protein matter of the present invention can comprise with the aminoacid sequence shown in the SEQ ID NO:1 have at least 70%, preferred at least 80%, more preferably at least 90%, the more preferably aminoacid sequence of at least 95% homology also.
The present invention also provides coding to derive from the gene (AtCYP78A7) of the cytochrome p450 protein matter of Arabidopis thaliana, this cytochrome p450 protein matter has the aminoacid sequence shown in the SEQ ID NO:1, this protein can be used for increasing the size of plant seed or the content of seed storage protein, or is used to strengthen the resistance of plant to water stress.Preferably, described gene comprises the nucleotide sequence shown in the SEQ ID NO:2.In addition, the variant of described nucleotide sequence is also included within the scope of the present invention.Described variant comprises that sequence changes but has the function similar to the nucleotide sequence shown in the SEQ ID NO:2 and the nucleotide sequence of immunity.Particularly, the proteinic gene of Codocyte cytochrome p 450 can comprise with the nucleotide sequence shown in the SEQ ID NO:2 have at least 70%, preferred at least 80%, more preferably at least 90%, the more preferably nucleotide sequence of at least 95% homology also.
" sequence homology " for specific polynucleotide and polypeptide is to define by the corresponding zone of comparing best two sequences comparing.In such cases, for the optimum comparison of two sequences, polynucleotide in corresponding zone or polypeptide portion are compared with reference sequences (without any adding or disappearance), can contain and add or disappearance (being the room).
Described per-cent obtains by following method: the nucleotide base that two sequences determining to compare all exist or the number of sites of amino-acid residue, obtaining match bit thus counts, count divided by total number of sites in the zone of comparing with the match bit that obtains, what will obtain is on duty with 100 again, promptly obtains the percent value of sequence homology.Being used for this type of optimum comparison relatively can be by computer equipment with known tupe (Genetics Computer Group (GCG) for example, 575Science Dr., Madison, WI, GAP, BESTFIT, FASTA and the TFAST in the Wisconsin heredity software package or available from BlastN and the BlastX of NCBI (National Center for Biotechnology Information)) carry out or undertaken by mensuration.
The term that uses in this specification sheets " basically identical " or " similar substantially " refer to contain the polypeptide of sequence that can hybridize with the target polypeptide under stringency.Described stringency is that 2 * SSC solution and temperature are 65 ℃.
" similar substantially " polypeptide has described sequence, except the position of the different residues that can be changed by the conservative change of amino-acid residue.Conserved amino acid replaces and refers to that the amino-acid residue with similar side chain can replace mutually.For example, amino acid group with aliphatic lateral chain comprises glycine, L-Ala, Xie Ansuan, leucine and Isoleucine, amino acid group with aliphatic hydroxyl side chain comprises Serine and Threonine, comprise l-asparagine and glutamine with amino acid group with amide containing side chain, amino acid group with aromatic series side chain comprises phenylalanine, tryptophane and tyrosine, amino acid group with basic side chain comprises Methionin, arginine and Histidine, and the amino acid group with sulfur-containing side chain comprises halfcystine and methionine(Met).
The polynucleotide sequence of basically identical refer to comprise have at least 70%, preferred at least 80%, more preferably at least 90%, the more preferably polynucleotide of the sequence of at least 95% sequence identity also.According to another kind definition, when two nucleic acid molecules under stringency each other during specific hybrid, their nucleotide sequence relative to each other is a basically identical.The sequence-dependent characteristic of stringency may change under other different condition.Usually, when limiting ionic strength and pH value, the temperature that stringency is selected is hanged down about 10 ℃ than the melting temperature (Tm) (Tm value) of specificity sequence.The Tm value is 50% target sequence and the probe bonded temperature of mating fully (under ionic strength that limits and pH value condition).The Tm value of hybridization complex is determined by the length and the based composition of probe, and can be according to document (Sambrook, T. wait the people, (1989) Molecular Cloning-A Laboratory Manual (secondedition), molecular cloning-laboratory manual (second edition), 1-3 volume, Cold Spring HarborLaboratory, Cold Spring) in the information described calculate.The stringency of typical Southern engram analysis comprises with 0.2 * SSC solution washs under 65 ℃.For preferred oligonucleotide probe, typical wash conditions is for to wash under 42 ℃ with 6 * SSC solution.
According to an embodiment of the invention, the gene of Codocyte cytochrome p 450 can also comprise the promoter sequence of the encoding gene of Cytochrome P450, and this promoter sequence is made up of the nucleotide sequence shown in the SEQ ID NO:3.Gus gene and described promoter sequence are merged, and in plant, express after the gene that obtains subsequently, find that described gene is at dark cotyledon and the apical meristem of sprouting seedling/photogermination seedling, bud, flower, axillalry bud, or the expression among the developmental embryo is stronger.
In order to realize another object of the present invention, the invention provides comprising the recombinant plant expression vector of gene of the present invention.
Term " recombinant chou " refers to the proteinic cell that can duplicate allos Nucleotide or express described Nucleotide, peptide, heterologous peptides or allos nucleotide coding.The cell of recombinant chou can be expressed in non-existent gene or gene fragment in the native state cell with the form of justice or antisense.In addition, the cell of recombinant chou also can be expressed in the gene that exists in the native state cell, but this gene is modified and is incorporated in the cell by manual method.
Used term " carrier " refers to be delivered to one or more dna fragmentations and the nucleic acid molecule in the cell in this specification sheets.Described carrier can repetition DNA and can be independently duplicated in host cell.Term " transfer system " and " carrier " are used interchangeably usually.Term " expression vector " refers to comprise the recombinant DNA of following sequence: required encoding sequence; And in the specific host organism for other very important suitable nucleotide sequence of the expression of the described encoding sequence that can be operatively connected.As everyone knows, can use promotor, enhanser, termination signal and polyadenylation signal for eukaryotic cell.
The preferred embodiment of plant expression vector is the Ti-plasmids carrier, and when the Ti-plasmids carrier was present in the appropriate host (for example Agrobacterium tumefaciens), it can transmit the part of self, promptly so-called T zone to vegetable cell.The Ti-plasmids carrier of other type (seeing EP 0116718B1) is generally used for transmitting heterozygous genes (hybrid gene) in protoplastis, produce new plant by suitably inserting vegetable cell or inserting hybrid DNA to Plant Genome.The form of especially preferred Ti-plasmids carrier is at EP 0120516B1 and USP No.4, disclosed so-called binary vector in 940,838.Be used for introducing other carrier of DNA of the present invention, can be selected from the plant virus of double-stranded plant virus (for example CaMV), strand and derive from virus vector of Gemini virus or the like, for example incomplete plant viral vector to host plant.When plant host can not carry out suitable conversion, use described carrier especially favourable.
Described expression vector can comprise at least a selection markers.Described selection markers comprises dehydrofolic acid reductase enzyme (dehydrofolate reductase) gene or the neomycin resistance gene that is used for the eukaryotic cell cultivation.The described selection markers that is widely used in Plant Transformation is neomycin phosphotransferase II (nptII) gene, this gene isolation from Tn5 and the antagonism Totomycin (a kind of microbiotic) have resistance hygromycin phosphotransferase gene.
For the plant expression vector according to an embodiment of the invention, promotor can be CaMV 35S, Actin muscle, ubiquitin, pEMU, MAS or Histidine promotor arbitrarily, but is not limited thereto.Term " promotor " refer to RNA polymerase for initial transcribing the bonded dna molecular, corresponding to the DNA zone of structure gene upstream.Term " plant promoter " refer to can be in vegetable cell initial promotor of transcribing.Term " formation type promotor " refers in promoters active all under most envrionment conditionss and under developmental condition or the cytodifferentiation state.Because transformant can screen in different steps by number of mechanisms, so the preferred formation type promotor of the present invention.Therefore, be not limited to select formation type promotor herein.
Terminator can be rouge alkali synthetase (NOS) or rice αDian Fenmei RAmyl A terminator, but is not limited thereto.About the necessity of terminator, it has been generally acknowledged that this zone can strengthen reliability and validity that vegetable cell is transcribed.Therefore, highly preferably use terminator within the scope of the invention.
In order to realize another object of the present invention, the invention provides the method for the content of storage protein in the size that increases seed or the seed, and strengthening the method for plant to the resistance of water stress, this method comprises with recombinant plant expression vector transformed plant cells of the present invention with overexpressing cell cytochrome p 450 gene.Preferably, described plant is selected from the group of being made up of Arabidopis thaliana, rice, Semen Brassicae campestris, wheat, barley, corn, soybean, potato, red bean, oat and millet, but is not limited thereto.
Term " plant tissue " can be differentiated tissues or undifferentiated tissue, what comprise root, stem, leaf, pollen, seed, cancerous tissue and have a multiple shape is used for cultured cells (being individual cells), protoplastis, bud and callus, but is not limited thereto.Plant tissue can be in the plant, or is in the state of organ culture, tissue culture or cell cultures.
Term " vegetable cell " comprises the vegetable cell of (planta) in the plant, also comprises the vegetable cell and the protoplasma of cultivation conditions.
Plant Transformation refers to DNA to be delivered to any means in the plant.This kind method for transformation not necessarily has regeneration period and/or tissue culture phase.The conversion of plant species is very common at present, not only is used for dicotyledons, also is used for monocotyledons.In principle, can using arbitrarily, method for transformation is incorporated into hybrid DNA of the present invention in the suitable progenitor cell (progenitor cell).Described method suitably be selected from calcium/polyoxyethylene glycol method of being used for protoplastis (Krens, people such as F.A., 1982, Nature 296,72-74; NegrutiuI. wait the people, June 1987, Plant Mol.Biol.8,363-373), electroporation method (the people such as Shillito R.D. who is used for protoplastis, 1985Bio/Technol.3,1099-1102), micro-injection method (the people such as Crossway A. who is used for plant component, 1986, Mol.Gen.Genet.202,179-185), particle bombardment method (DNA or RNA by embedding) (the Klein T.M.et al. that is used for the various plants component, 1987, Nature 327,70), or (not exclusively) virus infection method (EP 0301316) that in agrobacterium tumefaciens (Agrobacterium tumefaciens), shifts by the intrusion of full ripe pollen or sporule plant or the mediated gene that is converted or the like.The preferred method of the present invention comprises that agrobacterium-mediated DNA shifts.Particularly preferably use EP A 120516 and USP No.4 in the present invention, disclosed so-called binary vector technology in 940,838.
In order to realize another object of the present invention, the content that the invention provides the size of seed or storage protein increases or to the resistance enhanced plant of water stress.By with method transformed plant cells of the present invention, can overexpressing cell cytochrome p 450 gene, the result increases the content of storage protein in the size of seed or the seed, or plant is strengthened the resistance of water stress.Preferably, described plant can be selected from the group of being made up of Arabidopis thaliana, rice, Semen Brassicae campestris, wheat, barley, corn, soybean, potato, red bean, oat and millet, but is not limited thereto.
In order to realize another object of the present invention, the invention provides transgenic seed with recombinant vectors plant transformed of the present invention.Preferably, described plant can be selected from the group of being made up of Arabidopis thaliana, rice, Semen Brassicae campestris, wheat, barley, corn, soybean, potato, red bean, oat and millet, but is not limited thereto.
Describe the present invention in detail below with reference to following embodiment.Yet this only illustrates the present invention for particular examples, rather than limits the scope of the invention by these embodiment.
Material and method
Vegetable material and growth conditions
Use the environmental Ws-2 of Arabidopis thaliana as the wild-type that is used to transform.Seed is carried out surperficial degerming, freezing 2 days at 4 ℃, make it contain 1x Murashige and Skoog salt (Murashige T then, S koogF (1962) Physiol Plant 15:473-497) and be added with on the 0.8% agar solid medium of 1% sucrose (transferring pH5.8), and under the photoperiod condition of illumination in 16 hours (22-24 ℃)/8 hours dark (18-20 ℃) germinate and growth with KOH.The plant of cultivating in soil also grows under identical photoperiod condition.
The formation type of AtCYP78A7 is expressed in Arabidopis thaliana
The genomic fragment that contains the AtCYP78A7 coding region increases with a pair of primer by Pwo polysaccharase (Roche, Mannheim, Germany) as template with arabidopsis thaliana genomic dna.For clone PCR products easily, in primer, introduce KpnI and XbaI enzyme cutting site: 78A7KpF, 5 '-GG in the following manner
GGTACCCATCAACCCAAAATAATGGAGTTGATG-3 ' (SEQ ID NO:4), 78A7XbR, 5 '-GC
TCTAGACATTCTGCAATTCATACCTCTCGACAA-3 ' (SEQ ID NO:5).With the SmaI site of PCR product cloning to the pUC19 carrier.Measure the perfect kernel nucleotide sequence of PCR product, to check the mistake of PCR.With the KpnI/XbaI endonuclease bamhi subclone of PCR product to (Gleave AP (1992) Plant Mol Biol 20:1203-1207) between the CaMV 35S promoter and ocs3 ' of pART7.To contain the NotI endonuclease bamhi subclone (Gleave AP (1992) PlantMol Biol 20:1203-1207) in binary vector pART27 of crossing expression cassette from pART7.The expression cassette of crossing among the pART27 is transformed among the edaphic bacillus GV3101 by electroporation, and is incorporated into (Clough SJ, BentAF (1998) Plant J 16:735-743) in the environmental Ws-2 plant with the plant infestation method.On the MS flat board that contains kantlex (40 μ g/mL), screen transgenic plant.
Going down to posterity and the GUS staining procedure of promoter construct
The genomic fragment (length is about 2.5kb) that contains the AtCYP78A7 promoter region obtains from the BAC clone (MYH9) with SalI/BamHI digestion.Promoter fragment comprises the part opening code-reading frame (ORF) of this gene, so that the translation of itself and gus gene is merged.With SalI/BamHI fragment subclone in pB I101 binary vector.Described promoter construct is transformed among the edaphic bacillus GV3101 by electroporation, and uses the plant infestation method to be incorporated into (Clough SJ, BentAF (1998) Plant J 16:735-743) in the environmental Ws-2 plant.On the MS flat board that contains kantlex (40 μ g/mL), screen transgenic plant.The transgenic line that isozygotys (homozygous transgenic lines) that contains promoter construct from T3 for screening.According to Stomp method (Stomp A-M (1992) In S.R.Gallagher ed, GUS protocols:Using the GUS gene as a reporter of geneexpression, Academic Press, San Diego, CA pp.103-113) carries out the GUS dyeing of plant and plant tissue.The painted tissue of GUS is by the ethanol dehydration of series.
Reverse transcription-polymerase chain reaction
With TRIzol reagent (Invitrogen, Carlsbad, CA) total RNA of purifying plant tissue.Using described total RNA (5 μ g) to carry out the first chain cDNA by MMLV ThermoScript II (Invitrogen) synthesizes.The condition of pcr amplification is as follows: 96 ℃ of initial sex change 5 minutes, and 15 seconds, 55 ℃ annealing of 94 ℃ of sex change were extended 1 minute (totally 27 circulations) in 30 seconds and 72 ℃ then, and last 72 ℃ were extended 5 minutes.The transcript of amplification coding tubulin-2 is as positive control.The primer sequence that is used for RT-PCR is listed at table 1.
Table 1 is used for the primer sequence of RT-PCR
Proteinic extraction and SDS-PAGE
500 ripe exsiccant seeds are extracted damping fluid [125mM Tris-HCl (pH8.8), 1%SDS, 10% glycerine, 50mM S-WAT] with 400 μ l carry out homogenate with pestle and mortar.After centrifugal, get each extract 5 μ l and be used for SDS-PAGE (Laemmli UK (1970) Nature 227:680-685).With Bio-Rad protein test kit is the standard test protein content with BSA.
Processed
Wild-type that 5 weeks are big and transgenic arabidopsis plant are used for drought stress and handle.Make the plant suction 12 hours that soil cultivates, remove superfluous water, carried out desiccation stress in 18 days by stopping to irrigate then.In order to check that whether plant can recover, and waters again after desiccation stress is carried out 18 days from desiccation stress.
RNA extracts and microarray hybridization
Extract total RNA of 12 days seedling with TRIzol reagent (Invitrogen).Isolating total RNA is further purified with the small-sized test kit of RNeasy plant (Qiagen, Germany).The total RNA of 15 μ g that gets every sample prepares cDNAs with SuperscriptII ThermoScript II (Invitrogen), with the specification sheets (Genisphere of Genisphere3DNA Array 900DNA labelling kit according to manufacturers, Montvale NJ) prepares micro probe array with Cy3 and Cy5 mark by described cDNA.The arabidopsis oligonucleotide microarray of this cDNA probe and 29000 elements is hybridized, and this microarray makes with Qiagen-Operon Arabidopsis Genome Array Ready Oligo Set (AROS) Version 3.0 (http://www.ag.arizona.edu/microarray/) in Arizona university.In brief, hybridization is undertaken by following two steps: 1) with the oligomer hybridisation of point sample on cDNA and the slide glass, and 2) by in the building-up process of first chain, mixing the acquisition sequence of cDNA, cDNAs and 3-DNA fluorescence dendritic polymerization body (dendrimers) are hybridized.Whole cDNA and fluorescence dye are hybridized in the SDS-alkali hybridization buffer that volume provides for the manufacturers of 35 μ L.The hybridization of described cDNA was carried out on MAUI crossing system and MAUI Mixer AO Hybridization Chamber Lids (BioMicro Systems, Salt Lake City), 60 ℃ of hybridization 18 hours.Clean slide glass according to experimental technique then, and carried out dry air in centrifugal 10 minutes.As mentioned above, 3-DNA hybridization was carried out 4 hours at 55 ℃, prevented the fluorescein oxidation except add 0.5mM dithiothreitol (DTT) (DTT) in twice cleaning of beginning, and other is all identical with aforesaid method.The step that comprises dyestuff exchange slide glass, 4 repetitions are carried out in each experiment, to eliminate the fluorescence deviation of dyestuff.Strain is #19 and #38 for transgenic arabidopsis, and we use 3 and 2 slide glasss respectively.
Scanning and data analysis
After the hybridization, (Axon Instruments, Union City CA) scan slide glass, and (Axon Instruments, Union City CA) carries out quantitatively spot to use General Pro4.0 then with General 4000B.The microarray results of scanning is input to sensibility analysis software 3.0 editions (Acuity analysissoftware 3.0 (Axon Instruments, Union City, CA)) in, and carry out homogenization with Global LOWESSNormalization (people such as Yang, (2002) Nucleic Acids Res 30:e15).Generate the data file of each experiment then, following screening conditions are satisfied in described experiment: [(intermediate value sum＞=100) and (Flags＞=0) and (F635%Sat＜3) and (F532%Sat＜3) and (RgnR2 (635/532)＞0.6) and (SNR635＞3) and (SNR532＞3)].Described screening has been got rid of by the data point of GenePix error flag or intermediate value sum and has been less than the data point of 100 (very weak) or the data point (may not be real spot) that pixel is lower than background.Analyzed the spot that meets these standards at least on 75% the used slide glass.In order to compare wild-type and transgenic line, calculated the mean value of the ratio intermediate value of the spot that meets these standards in each data set.2 data sets that obtain carry out cluster with the K-means clustering algorithm in the Acuity software (K-means clustering algorithm).From Arabidopis thaliana information resources net (TAIR, ftp: //ftp.arabidopsis.org/home/tair/home/tair/) go up to collect the note of the clone on the microarray and body (ontology) function of gene, and classify according to the classification that Gene Ontology Consortium (www.geneontology.org) provides.
The preparation of transgenosis rice plants
Obtain to contain the NotI endonuclease bamhi of 35S:AtCYP78A7:3 ' ocs from the pART27 binary vector.Then, form flat end with the Pfu archaeal dna polymerase.With after the SmaI digestion, with fragment cloning in the pCAMBIA1201 binary vector.The carrier that is used for producing the transgenosis rice plants of preparation like this is incorporated into agrobacterium tumefaciens (Agrobacterium tumefaciens) AGL24 bacterial strain with electric-shocking method.The process of described generation transgenosis rice plants realizes by the early stage transfection method that uses peltate tissue (scutellum tissue).Screening transgenosis rice plants on the MS solid medium that contains Totomycin (50 μ g/ml).
The genomic hybridization method
Based on CTAB method isolation of genomic DNA from rice plants.The genomic dna (10 μ g) of purifying is digested with SacI, carry out 1% agarose gel electrophoresis then.DNA with digestion transfers on the nylon membrane with the kapillary method, carries out hybridization with the AtCYP78A7DNA probe of [32P-dCTP]-mark.The condition of hybridization and washing is identical with the condition described in the prior art (people (2003) Journalof Plant Biology.46:263-270 such as Kim HB).
The transgenosis rice plants is to the analysis of the resistance of water stress
The rice plants seedling that will germinate in the MS solid medium and grow about 7 days is transferred in the soil, and growth is about 1 month in the greenhouse.Before water stress is handled 12 hours, that the basin of planting plant is under water fully.After removing water, no longer irrigated described plant in 10 days, so that described plant is carried out water stress.In order to determine whether described plant can overcome described water stress, after 10 days again to described plant watering.
Embodiment 1: the RT-PCR of the express spectra of dissimilar tissues analyzes
Measure the express spectra of dissimilar tissues in the transgenic plant of the present invention with RT-PCR.Found that the AtCYP78A7 gene almost expresses in a organized way in institute, especially the very strong (see figure 1) of expression in bud, flower, angle fruit (silique) and plant seedlings.
Embodiment 2: to using promotor:: the analysis of the expression of GUS
Measure the express spectra of dissimilar tissues in the described transgenic plant with gus reporter gene.Found that the AtCYP78A7 gene is in dark cotyledon and the strong (see figure 2) of the expression among apical meristem and bud, flower, axillalry bud or the developmental embryo of sprouting seedling/photogermination seedling.
Embodiment 3: cross the transgenic arabidopsis of expressing the AtCYP78A7 gene
1) content of the seed size of transgenic plant, seed weight and storage protein
In crossing the transgenic plant of expressing, as Fig. 3-A as seen, the size of seed is bigger than wild-type (Ws-2).As Fig. 3-B as seen, the weight ratio wild-type (Ws-2) of seed nearly weighs 50%.In addition, as Fig. 3-C as seen, the content of storage protein in the seed (for example 12S sphaeroprotein and 2S albumin etc.) has also increased with respect to wild-type (Ws-2).Wild-type (Ws-2) and transgenic seed (#9 and #19) with equal amount are measured seed weight.From the wild-type (Ws-2) of equal amount and transgenic seed (#9 and #19), extract total storage protein respectively.
2) microarray analysis of transgenic plant
Carry out microarray analysis with the oligonucleotide chip that comprises all arabidopsis gene group sequences.From the plant seedlings of growing 12 days, separate total RNA, carry out microarray analysis independently separately then 5 times.Found that in transgenic plant of the present invention the storage protein of the seed of 12S and 2S increases, and the expression of gene of the gene (a kind of plant hormone) of response ABA or response low temperature/drought stress increases (seeing Table 2).
Table 2. microarray analysis has disclosed is expressing in the transgenic arabidopsis of AtCYP78A7 gene excessively, and the gene of coding seed storage protein, the gene of ABA-response protein matter or arid/cold are coerced relevant proteinic gene and raised.
Whether increase difference isolation of RNA s from wild-type (Ws-2) and transgenosis kind of plant (#9 of 35S:AtCYP78A7, #19 and #38 strain system) in order to determine that said gene is expressed in the transgenic plant of the present invention.After carrying out RT-PCR, find that their expression has all increased (see figure 4) in transgenic plant of the present invention.
3) transgenic plant of the present invention are to the resistance of water stress
Study the resistance of transgenic plant of the present invention based on the result who obtains from above-mentioned microarray analysis to water stress.For wild-type, find that plant begins to wither after water stress is handled 12 days, dead fully at the 18th day then.On the other hand,, find that plant begins to wither after water stress is handled 18 days, in case water once more then can from water stress, recover fully for transgenic plant of the present invention (#9 of 35S:AtCYP78A7, #19 and #38 strain system).And wild-type plant can not recover (see figure 5).
Embodiment 4: cross the preparation of the transgenosis rice plants of expressing the AtCYP78A7 gene and be used to detect the test of described plant to the resistance of water stress
Prepare 18 independent mistakes and express the strain system of the transgenosis rice plants of AtCYP78A7.In the transgenosis rice plants strain of all preparations system, confirm that the expression of selection markers gene (hyg) (is seen Fig. 6-A).Except that the #14 strain system, all transgenosis rice plants strains systems confirm that all the AtCYP78A7 expression of gene is arranged and (see Fig. 6-A).From the transgenosis rice plants of such preparation, extract genomic dna, carry out the genomic hybridization reaction as probe with the AtCYP78A7 gene.Results verification be incorporated in the genome that AtCYP78A7 gene in the plant successfully is incorporated into rice plants and (see Fig. 6-B).For some like this transgenosis rice plants of preparation measure test to the resistance of water stress.Found that wild-type plant (Hwayoung) is handled at water stress began to wither in back 7 days, and transgenic plant of the present invention can not wither (seeing Fig. 7 left side plate).After water stress is handled 10 days, again to plant watering.Wild-type plant can not recover, but all transgenosis rice plants of the present invention can well return to their standard state, except that #5 strain system (seeing Fig. 7 right side plate).
Express the transgenosis rice plants of AtCYP78A7 for 18 independent mistakes, measure the weight of 100 seeds respectively.Found that the seed weight heavy 10-33% (see figure 8) of the seed weight of #5, #10 and #18 strain system than non-transgenic plant.
Sequence table
＜110〉Myongji University Industry And School Cooperation
＜120〉be used to increase the size of seed or plant cytochrome P450 gene to the resistance of water stress
<130>PCT07005
<160>29
<170>KopatentIn?1.71
<210>1
<211>536
<212>PRT
＜213〉Arabidopis thaliana
<400>1
Met?Glu?Leu?Met?Asn?Leu?Ala?Ser?Lys?Glu?Thr?Ser?Tyr?Trp?Met?Ile
1 5 10 15
Ala?Leu?Pro?Ala?Gly?Phe?Gly?Ser?Gln?Asn?Leu?His?Asp?Val?Ser?Thr
20 25 30
Leu?Gly?Tyr?Leu?Phe?Leu?Ala?Val?Val?Phe?Leu?Ser?Ile?Val?Thr?Trp
35 40 45
Ala?Leu?Ala?Gly?Gly?Gly?Gly?Val?Ala?Trp?Lys?Asn?Gly?Arg?Asn?Arg
50 55 60
Leu?Gly?Arg?Val?Ala?Ile?Pro?Gly?Pro?Arg?Gly?Ile?Pro?Val?Phe?Gly
65 70 75 80
Ser?Leu?Phe?Thr?Leu?Ser?Arg?Gly?Leu?Ala?His?Arg?Thr?Leu?Ala?Ala
85 90 95
Met?Ala?Trp?Ser?Arg?Ala?Asn?Thr?Glu?Ile?Met?Ala?Phe?Ser?Leu?Gly
100 105 110
Ser?Thr?Pro?Val?Ile?Val?Ala?Ser?Glu?Pro?Asn?Ile?Ala?Arg?Glu?Ile
115 120 125
Leu?Met?Ser?Pro?His?Phe?Ala?Asp?Arg?Pro?Val?Lys?Gln?Ser?Ala?Lys
130 135 140
Ser?Leu?Met?Phe?Ser?Arg?Ala?Ile?Gly?Phe?Ala?Pro?Asn?Gly?Thr?Tyr
145 150 155 160
Trp?Arg?Met?Leu?Arg?Arg?Ile?Ala?Ser?Thr?His?Leu?Phe?Ala?Pro?Arg
165 170 175
Arg?Ile?Leu?Ala?His?Glu?Ala?Gly?Arg?Gln?Leu?Asp?Cys?Ala?Glu?Met
180 185 190
Val?Lys?Ala?Val?Ser?Val?Glu?Gln?Asn?Gly?Ala?Gly?Ser?Val?Val?Leu
195 200 205
Arg?Lys?His?Leu?Gln?Leu?Ala?Ala?Leu?Asn?Asn?Ile?Met?Gly?Ser?Val
210 215 220
Phe?Gly?Arg?Arg?Tyr?Asp?Pro?Leu?Ala?Gln?Lys?Glu?Asp?Leu?Asp?Glu
225 230 235 240
Leu?Thr?Ser?Met?Val?Arg?Glu?Gly?Phe?Glu?Leu?Leu?Gly?Ala?Phe?Asn
245 250 255
Trp?Ser?Asp?Tyr?Leu?Pro?Trp?Leu?Gly?Tyr?Phe?Tyr?Asp?Ser?Ile?Arg
260 265 270
Leu?Asn?Gln?Arg?Cys?Ser?Asp?Leu?Val?Pro?Arg?Ile?Arg?Thr?Leu?Val
275 280 285
Lys?Lys?Ile?Ile?Asp?Glu?His?Arg?Val?Ser?Asn?Ser?Glu?Lys?Lys?Arg
290 295 300
Asp?Ile?Gly?Asp?Phe?Val?Asp?Val?Leu?Leu?Ser?Leu?Asp?Gly?Asp?Glu
305 310 315 320
Lys?Leu?Gln?Glu?Asp?Asp?Met?Ile?Ala?Val?Leu?Trp?Glu?Met?Ile?Phe
325 330 335
Arg?Gly?Thr?Asp?Thr?Thr?Ala?Leu?Leu?Thr?Glu?Trp?Thr?Met?Ala?Glu
340 345 350
Leu?Val?Leu?Asn?Pro?Asn?Val?Gln?Thr?Lys?Leu?Arg?Asp?Glu?Ile?Leu
355 360 365
Thr?Ala?Val?Gly?Asp?Gly?Ala?Asp?Gly?Asp?Val?Ala?Asp?Ala?Asp?Leu
370 375 380
Ala?Lys?Leu?Pro?Tyr?Leu?Asn?Ala?Val?Val?Lys?Glu?Thr?Leu?Arg?Leu
385 390 395 400
His?Pro?Pro?Gly?Pro?Leu?Leu?Ser?Trp?Ala?Arg?Leu?Ser?Thr?Ser?Asp
405 410 415
Val?Gln?Leu?Ser?Asn?Gly?Met?Val?Ile?Pro?Lys?Gly?Thr?Thr?Ala?Met
420 425 430
Val?Asn?Met?Trp?Ala?Ile?Thr?His?Asp?Gln?Thr?Val?Trp?Ser?Asp?Pro
435 440 445
Leu?Lys?Phe?Asp?Pro?Glu?Arg?Phe?Thr?Gly?Asn?Ala?Asp?Met?Asp?Ile
450 455 460
Arg?Gly?Gly?Asp?Leu?Arg?Leu?Ala?Pro?Phe?Gly?Ala?Gly?Arg?Arg?Val
465 470 475 480
Cys?Pro?Gly?Lys?Asn?Met?Gly?Leu?Ala?Thr?Val?Thr?Arg?Trp?Val?Ala
485 490 495
Glu?Leu?Val?Arg?Arg?Phe?Glu?Trp?Gly?Gln?Asp?Gln?Thr?Glu?Pro?Val
500 505 510
Asp?Leu?Gly?Glu?Val?Leu?Lys?Leu?Ser?Cys?Glu?Met?Glu?His?Pro?Leu
515 520 525
Arg?Ala?Val?Val?Thr?Glu?Ile?Phe
530 535
<210>2
<211>1611
<212>DNA
＜213〉Arabidopis thaliana
<400>2
atggagttga?tgaatttggc?ttcaaaagaa?acaagctatt?ggatgattgc?actgcctgcc 60
ggttttggat?cccaaaacct?acatgatgtt?tccaccctag?gctatctatt?ccttgccgtc 120
gtttttctct?ctatagtcac?gtgggctctc?gccggaggcg?gtggtgtcgc?ttggaagaac 180
ggccgtaacc?ggttgggtcg?tgtcgcgatc?cctggtcctc?gtggcatacc?agtattcggc 240
agtcttttca?ctctcagccg?aggcttggct?catcggacgt?tagcagccat?ggcttggagc 300
cgagccaaca?ctgagattat?ggcttttagc?cttggttcaa?cgccggttat?cgtggcttct 360
gaaccaaaca?tagctcgtga?gattctgatg?tcgcctcact?tcgcggaccg?gccggttaag 420
cagtctgcta?agagcctcat?gttcagccga?gccataggtt?tcgccccaaa?cgggacttac 480
tggcgcatgt?taagaaggat?cgcatcgact?cacctatttg?ctcctcggcg?tatcttagca 540
cacgaagctg?ggcgccagct?agactgcgct?gaaatggtga?aagctgtgtc?agtggagcaa 600
aacggcgctg?gatcagtcgt?tttaaggaaa?cacttacaac?tagccgcctt?gaacaacatc 660
atgggaagtg?tttttgggag?aagatacgat?cctctggctc?agaaagagga?tcttgatgag 720
cttacatcaa?tggttaggga?agggttcgag?cttttgggtg?cttttaattg?gtctgattat 780
cttccatggc?tcggttattt?ctacgactca?attcgtttaa?accaacgttg?ctcagatctc 840
gtccctcgaa?ttagaaccct?cgtcaagaaa?atcatcgacg?aacatcgagt?tagtaactct 900
gagaagaaaa?gagacattgg?agattttgtt?gatgtcttat?tgtctttaga?cggtgatgag 960
aaacttcaag?aagatgacat?gatcgccgtt?ttatgggaga?tgatttttcg?agggacagat 1020
acaacggcgt?tattaacgga?gtggaccatg?gccgagctag?tactgaaccc?taacgtgcaa 1080
accaagttac?gagacgagat?tttaactgct?gtgggcgacg?gcgccgacgg?agacgtggca 1140
gatgctgacc?tggcaaaact?cccgtaccta?aacgcagtgg?tgaaggaaac?tctaaggctg 1200
catcctcctg?gaccactgct?ttcatgggct?cgtctttcca?cgtcagacgt?ccagctcagc 1260
aatggcatgg?tgattccaaa?gggaactaca?gcgatggtca?acatgtgggc?tataacccac 1320
gaccagactg?tatggtccga?cccgctaaag?tttgacccgg?agagattcac?tgggaatgct 1380
gacatggata?ttcgtggtgg?ggatctaagg?cttgcaccgt?ttggagccgg?taggagagtg 1440
tgtccgggga?agaacatggg?gctagctact?gtgactcggt?gggtggctga?gttggtacga 1500
cggttcgagt?ggggtcagga?tcagaccgag?ccagttgatc?ttggtgaggt?cttgaagctt 1560
tcttgtgaga?tggagcatcc?gttacgtgcc?gttgtaacgg?aaatatttta?a 1611
<210>3
<211>2416
<212>DNA
＜213〉Arabidopis thaliana
<400>3
gtcgacccgc?cccattccct?gccaaacctc?ccttcttcac?cattcacact?tttttctttt 60
ctttgaccaa?ttgaaaaatt?atttatatac?attaattaat?agttaaaagt?aaacaaaatg 120
agaagcacca?atctattttg?gttaattttc?catttagatg?taactttcat?gatagatgtt 180
gagattcaaa?cctattacct?aatatgttaa?gcatgaagaa?tggatgattt?ggtgcatccc 240
aacctcggcc?tctaagattc?cacaacggat?tcctctaact?ttcaccaatt?tgttaggctt 300
ttttattcaa?tattttcaca?agcattatct?tcaaagaaaa?cggtaaacga?cgagctagaa 360
atatgcaaca?tacaaattgc?atgagacgtt?tagttaactg?gttttctaat?gtgatttgat 420
tgtctcttgt?tcttattatt?aaagattata?tattctcgta?aatgaaaatt?gttttatgtg 480
aaacggcatt?tgcttaatct?aacgcctaag?taaaaatcta?gtgttctaca?tcatgtgttc 540
gaattagatc?tatgttcaaa?atgtgcgatg?tgtttttaca?tattattatt?gtatatacta 600
ctgactacag?tttgtcgtag?tatacgtctt?atagaaattt?tgattgagca?ttgaggagtt 660
cttttgagta?atttgtatgt?tgctatttag?attatgtatt?tgcaaattaa?attgttagat 720
ttgtacgtcg?taaacgatat?aaatatttta?aatatgataa?gtgataaata?ggaaaccatt 780
tccattactt?aacatcattg?atacaataca?aagatttgaa?tctcacgata?agaattggtt 840
gctttctcta?ttaggatcac?tataaaaact?tgtgatcaac?gagttttaat?gaataatatc 900
agtatttttt?gtaaaaaaaa?tagcgataaa?aatattatat?taatcgttga?ttaaatataa 960
tatattgtta?cgatcggata?aaaaatatta?atttattaat?gatcgatcga?taaatctatt 1020
atttaataaa?tagattatat?aagacaaata?aatagattat?ataagacaaa?aatggtcaaa 1080
cgatattaaa?tataaataaa?tacatacatc?gtgtttttcg?agttggatat?atgatcgtta 1140
attaaatata?ttgttacgat?catataaaaa?gatcatttct?tagtaatagt?tcgggatgga 1200
ttggtgttgt?tcctctacgc?ctaggacacc?agaatatcga?accatgaata?tcaatcgata 1260
aatatattat?ttaaataatc?gcttatatac?gacaaaaaaa?atcaaaatga?tattgaatta 1320
atatagataa?atacatacac?aaatctggat?gcgatagcaa?aaacctagaa?cgtctatttt 1380
tttttttgca?tttcctgaat?ttttttttaa?tcagccatat?actcataagt?tttacaaaaa 1440
tgtctccttt?atctcatcca?ctttaaaatt?acttattcat?caaattttaa?cagtagaaga 1500
cttaaaacta?cagtttctta?gcaaaaaata?aataatttac?atagtcccca?attatatact 1560
tcgaaaacta?tatattacta?aaagctcaaa?tataatgttc?aattatactc?tcatggaaaa 1620
ttataattct?gttctactac?aaactcaaaa?aatatgaaga?taattaatca?gggcaagtat 1680
ctcgctgcac?gtcacaacta?tctaaagcca?tcgatatcta?taaccgacta?atattctttc 1740
ttctctaaag?aacatattca?ttggtttgtc?ctttcgtatt?ctctaagccg?aaatatacgg 1800
actcattgaa?ttcttttttc?ggtttaacga?acctcattga?attctcacat?cgatgcaaag 1860
taacatcaca?acccaagaaa?aaaacggtgc?aaacataatt?aaatatagct?aattaagaaa 1920
aacaaaataa?aaatgccgaa?ttcattgtca?taacgccctc?tttcacacac?atatctttca 1980
tttcatccaa?caccaaacca?attcatgcga?cccacctcct?ccctccacgt?gaatctctcc 2040
caattttaac?cttgccaatt?ttcctatatt?ttacatcaac?attacttata?tataaacggg 2100
ataaattata?tagaatcatt?tcaaaaccga?cgtctttctt?cccatgcatt?gcgcatttaa 2160
ttaacgctgc?caaaacatac?atgtggatat?atatatagtt?tttttatata?tagatgtacc 2220
tttaccttca?acttctccat?tgttcttctc?caagtacttc?tctcttcaac?caaaaaaaaa 2280
gagtactttc?cttctcatag?gatttttttg?ctttctcttg?aaaaaccttt?tgttattgtt 2340
attggttcgt?acgttgttca?ttatttgaca?agaaacaaga?ttacaaaaaa?aaaaagtaca 2400
tcaacccaaa?ataatg 2416
<210>4
<211>35
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial
<400>4
ggggtaccca?tcaacccaaa?ataatggagt?tgatg 35
<210>5
<211>35
<212>DNA
＜213〉artificial sequence
<220>
＜223〉artificial
<400>5
gctctagaca?ttctgcaatt?catacctctc?gacaa 35
<210>6
<211>27
<212>DNA
＜213〉Arabidopis thaliana
<400>6
ggtacgacgg?ttcgagtggg?gtcagga 27
<210>7
<211>30
<212>DNA
＜213〉Arabidopis thaliana
<400>7
cattactcca?tttagatttt?agacccacaa 30
<210>8
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>8
ataaaattca?aagtgtaagc?aaaac 25
<210>9
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>9
attaattaga?aaagaagtcc?aaggt 25
<210>10
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>10
atcacgaaga?ctaataaaac?aaagt 25
<210>11
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>11
aacaaattaa?cacttaggaa?aattg 25
<210>12
<211>24
<212>DNA
＜213〉Arabidopis thaliana
<400>12
aaacaaaaga?ctacattgtt?gaga 24
<210>13
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>13
tacgtattta?aaatgtgcta?gtgag 25
<210>14
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>14
aaaaggtata?gcagaaaaga?ttaaa 25
<210>15
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>15
catcatatta?ttacaccaca?caaat 25
<210>16
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>16
aagaagatta?ggtatgtgaa?tagga 25
<210>17
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>17
aacatcttct?gttgtttgat?aagat 25
<210>18
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>18
aaaagttagt?ggagaggaga?agtat 25
<210>19
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>19
agatctatct?agtagctgaa?ccaca 25
<210>20
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>20
attctgttga?agaggctcca?aaatc 25
<210>21
<211>27
<212>DNA
＜213〉Arabidopis thaliana
<400>21
aatacatcaa?agacgtcaaa?caaaaca 27
<210>22
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>22
ttatatccaa?aggcatctct?tgagt 25
<210>23
<211>25
<212>DNA
＜213〉Arabidopis thaliana
<400>23
aaacgatcca?tagaacacac?aaact 25
<210>24
<211>27
<212>DNA
＜213〉Arabidopis thaliana
<400>24
gagccttaca?acgctactct?gtctgtc 27
<210>25
<211>27
<212>DNA
＜213〉Arabidopis thaliana
<400>25
acaccagaca?tagtagcaga?aatcaag 27
<210>26
<211>26
<212>DNA
＜213〉Arabidopis thaliana
<400>26
gatccggtcg?gcatctactc?tatttc 26
<210>27
<211>26
<212>DNA
＜213〉Arabidopis thaliana
<400>27
cttgacattg?gggagtttag?cgagag 26
<210>28
<211>24
<212>DNA
＜213〉Arabidopis thaliana
<400>28
tcatctaata?accagttcga?tttc 24
<210>29
<211>23
<212>DNA
＜213〉Arabidopis thaliana
<400>29
gactacaaca?tccagaagga?gtc 23
Claims (12)
1, a kind of cytochrome p450 protein matter, it is characterized in that, this cytochrome p450 protein matter is derived from Arabidopis thaliana and has the aminoacid sequence shown in the SEQ ID NO:1, and can be used in the content of storage protein in the size that increases seed or the seed, or be used to strengthen the resistance of plant to water stress.
2, cytochrome p450 protein matter according to claim 1 is characterized in that, the aminoacid sequence shown in this cytochrome p450 protein matter and the SEQ ID NO:1 has at least 70% sequence homology.
3, the gene of the described cytochrome p450 protein matter of coding claim 1.
4, the proteinic gene of Codocyte cytochrome p 450 according to claim 3 is characterized in that, this gene has the nucleotide sequence shown in the SEQ ID NO:2.
5, the proteinic gene of Codocyte cytochrome p 450 according to claim 3 is characterized in that this gene also comprises the promoter sequence of the encoding gene of Cytochrome P450, and this promoter sequence is made up of the nucleotide sequence shown in the SEQ ID NO:3.
6, the proteinic gene of Codocyte cytochrome p 450 according to claim 3 is characterized in that, this gene is at dark cotyledon and the apical meristem of sprouting seedling/photogermination seedling, bud, flower, axillalry bud, or strongly expressed among the developmental embryo.
7, a kind of plant expression vector of reorganization, wherein, the plant expression vector of this reorganization comprises claim 3 or 4 described genes.
8, the content of storage protein or strengthen the method for plant in a kind of size that increases seed or the seed to the resistance of water stress, it is characterized in that this method comprises that the plant expression vector with the described reorganization of claim 7 comes transformed plant cells so that the step of the gene overexpression of Cytochrome P450.
9, method according to claim 8 is characterized in that, described plant is selected from the group of being made up of Arabidopis thaliana, rice, Semen Brassicae campestris, wheat, barley, corn, soybean, potato, red bean, oat and millet.
10, a kind of plant, wherein, the seed size of this plant or the content of storage protein increase or the resistance of water stress are strengthened, and this plant produces by the described method of claim 8.
11, plant according to claim 10 is characterized in that, this plant is selected from the group of being made up of Arabidopis thaliana, rice, Semen Brassicae campestris, wheat, barley, corn, soybean, potato, red bean, oat and millet.
12, the transgenic seed of claim 10 or 11 described plants.
Applications Claiming Priority (10)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR1020070005866 | 2007-01-19 | ||
| KR10-2007-0005864 | 2007-01-19 | ||
| KR1020070005864 | 2007-01-19 | ||
| KR1020070005864A KR100834378B1 (en) | 2007-01-19 | 2007-01-19 | Cytochrome p450 gene for strengthening apical dominance of plant |
| KR1020070005870 | 2007-01-19 | ||
| KR10-2007-0005866 | 2007-01-19 | ||
| KR1020070005870A KR100794395B1 (en) | 2007-01-19 | 2007-01-19 | Cytochrome p450 gene for increasing seed size of plant |
| KR10-2007-0005870 | 2007-01-19 | ||
| KR1020070005866A KR100834380B1 (en) | 2007-01-19 | 2007-01-19 | Cytochrome p450 gene for increasing water stress resistnace of plant |
| PCT/KR2008/000250 WO2008088161A1 (en) | 2007-01-19 | 2008-01-15 | Cytochrome p450 gene for increasing seed size or water stress resistance of plant |
Publications (2)
| Publication Number | Publication Date |
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| CN101583718A true CN101583718A (en) | 2009-11-18 |
| CN101583718B CN101583718B (en) | 2012-09-26 |
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| CN2008800026324A Expired - Fee Related CN101583718B (en) | 2007-01-19 | 2008-01-15 | Cytochrome p450 gene for increasing seed size or water stress resistance of plant |
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| Country | Link |
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| KR (1) | KR100794395B1 (en) |
| CN (1) | CN101583718B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174523A (en) * | 2011-01-18 | 2011-09-07 | 山东师范大学 | Genes for regulating size of seeds as well as protein coded therewith and application thereof |
| CN111304219A (en) * | 2020-03-26 | 2020-06-19 | 华中农业大学 | GL1 gene separated from rice WZ1 and application thereof in increasing rice grain length |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100877729B1 (en) | 2007-05-01 | 2009-01-08 | 명지대학교 산학협력단 | Two Cytochrome P450 Genes Regulating Fruit Size and Seed Productivity |
| KR101316597B1 (en) | 2010-12-20 | 2013-10-16 | 대한민국 | Method for controlling plant growth with gene derived from Oryza sativa |
| KR101815737B1 (en) * | 2015-09-02 | 2018-01-08 | 숙명여자대학교산학협력단 | Gene for promoting plant growth and a transgenic plant comprising the same |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1867674B (en) * | 2003-10-16 | 2011-11-09 | 美国无烟烟草有限责任公司 | Cloning of cytochrome P450 genes from nicotiana |
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2007
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102174523A (en) * | 2011-01-18 | 2011-09-07 | 山东师范大学 | Genes for regulating size of seeds as well as protein coded therewith and application thereof |
| CN102174523B (en) * | 2011-01-18 | 2012-05-23 | 山东师范大学 | Genes for regulating size of seeds as well as protein coded therewith and application thereof |
| CN111304219A (en) * | 2020-03-26 | 2020-06-19 | 华中农业大学 | GL1 gene separated from rice WZ1 and application thereof in increasing rice grain length |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101583718B (en) | 2012-09-26 |
| KR100794395B1 (en) | 2008-01-15 |
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