CN101603036A - A kind of rice pyruvate kinase and encoding gene thereof and application - Google Patents

Abstract

The invention discloses a kind of rice pyruvate kinase and encoding gene thereof and application.Rice pyruvate kinase disclosed by the invention is following (a) or protein (b): (a) protein of being made up of the aminoacid sequence shown in the sequence in the sequence table 1; (b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and to have a pyruvate kinase active by sequence 1 deutero-protein.The invention also discloses described proteic encoding gene and contain the recombinant expression vector of this gene and the bacterium of recombinating.By silence albumen disclosed by the invention, can cultivate short bar paddy rice.The present invention can promote the theoretical investigation that rice stem is grown, and helps to accelerate the plant height seed selection kind process of paddy rice or other monocot crops, thereby has important theory and practice significance.

Description

A kind of rice pyruvate kinase and encoding gene thereof and application

Technical field

The present invention relates to a kind of rice pyruvate kinase and encoding gene thereof and application.

Background technology

Paddy rice is an important crops, is that annual production in the world surpasses one of three generalized grain crops of 50,000,000 tons, is the main food of the whole world 1/3rd above populations, occupies critical role in national economy.The genome minimum of paddy rice is about 389Mbp in cereal crop, wherein is distributed with ten thousand genes of 3-4 to high-density.There are the collinearity relation in genome of paddy rice and the genome of other cereal crop, have become model plant (Goff, 1999 of genetics, molecular biology and genomics research at present; Goff et al., 2002; Yu et al., 2002).

The function overwhelming majority of ten thousand genes of 3-4 of above-mentioned paddy rice is unknown or supposition, therefore, need identify by experiment that it is at rice growth and to the function in the environment answering.To paddy gene, particularly the clone of the gene relevant with Main Agronomic Characters and functional study help to illustrate the inner link of crop gene type and phenotype, for rice breeding is provided fundamental basis, also can provide new breeding material for rice breeding simultaneously.Along with finishing of research of paddy rice structure gene group and paddy rice examining order in the world, the research of its functional genomics has become the emphasis of whole world research.

Plant height is the important economical characters of farm crop, and the paddy rice for the first time rise cause of the Green Revolution is planted rice varieties of short stem in large area, so current target of seeking the new resource of short stem person that becomes the breeding work again.The rice dwarf gene genetic mainly contains two types: a class is the qualitative character heredity by single-gene control, and another kind of is quantitative character heredity by controlled by multiple genes, mostly downgrades sudden change and is controlled by single recessive gene.

Japan paddy rice gene linkage group and NK (The Japanese Committee on Nomencla-ture and Linkage Groups of Rice Gene) are symbol with d with short bar gene and the unification of minority semi-short-stalked gene, according to the certified time sequence of short bar gene, it is numbered d1 to d61, wherein lack d8, d15, d16, d25, d34 and d36, have 55 short bar genes (Matsuo et al., 1997) with the d name; The semi-dwarf mutant gene is named with sd, as sd1-sd9 etc.

The endogenous hormones that influences the paddy rice plant height mainly contains two kinds: the plain class ester (BRs) of Plant hormones regulators,gibberellins (GA) and rape.The dwarfing of paddy rice or to increase sudden change be to be undergone mutation by the genes involved of GA or BRs route of synthesis or signal transduction path to cause mostly.The hormone-sensitive that the mutant of route of synthesis genes involved applies external source can recover the phenotype of plant height, and this class is downgraded sudden change and is called the responsive type sudden change; And the mutant of signal transduction path genes involved is insensitive to the hormone that external source applies, and this class sudden change is called the non-sensitive type sudden change.

Plant hormones regulators,gibberellins is the product of plant and secondary fungus metabolism diterpene approach, and (geranylgeranyl pyrophosphate GGPP) forms by precursor geranyl geranyl tetra-sodium.Intermediate GGPP is by isoprene tetra-sodium (isopentenyl pyrophophate, IPP) synthetic in plastid or kytoplasm respectively.Recent findings IPP in the plastid of plant is transformed by the X 5P that glyceraldehyde-3-phosphate and pyruvic acid are condensed into, rather than by the direct synthetic of mevalonic acid (Hedden and Proebsting, 1999).Usefulness stable isotopic tracer methods such as Schwender proof pyruvate salt (PYA) in plant is the precursor of synthetic GA precursor isopentenylpyrophosphate (IPP), is the initial precursor of GA synthetic (Schwender J 1996; Lichtenthaler HK 1997).

Pyruvate kinase is an important enzyme in the glycolytic pathway in the organism, catalytic reaction is to change the substrate phosphoenolpyruvic acid into pyruvic acid, produce ATP simultaneously, pyruvate kinase has a lot of important physical functions in plant, (P.K.AMBASHT, 2002) such as carbon skeletons are provided when providing ATP and lipid acid synthetic during as seed germination.Pyruvate kinase has influence on the synthetic of the initial precursor pyruvate salt of GA synthetic (PYA), because GA is the important endogenous hormones that influences paddy rice plant height proterties, therefore, pyruvate kinase finally has influence on the plant height proterties of paddy rice.

By genetic engineering means control crop growth is the well stimulation that cost is cheaper in the agriculture production, benefit is higher, in recent years, has cloned many and the closely-related gene of plant height.As from Arabidopis thaliana, being cloned into 5 biosynthetic key genes of GA: GA1, GA2, GA3, GA4, GA5.What the clone obtained has with GA signal conduction genes involved: the GAI of Arabidopis thaliana, RGA, the d8 of corn, the Rht of wheat, SLN1 of barley or the like.Thereby utilizing the biosynthesizing of these Gene Handling GA or BR or signal conduction to obtain suitable plant height, will be the important means that the future of agriculture is produced.

At present, the clone of paddy rice plant height character gene is undertaken by the mutant material, and there are many spontaneous mutation plant of short stem in nature, and the scientific research personnel has also obtained a lot of dwarfing mutant materials by various mutafacient system simultaneously.The T-DNA labeling acts is the method that a kind of more common reverse genetics is studied functional gene.In recent years, the technology of separation of T-DNA labeling acts and clone gene has obtained great progress (Krysan et al., 1999; Joen etal., 2000).Because T-DNA inserts at random, average each transformation plant only contains 1.4 copies, can genetic stability in transformation generation, and T-DNA itself not only can disturb expression of gene, it inserts the molecule marker that fragment can also become this gene of clone, therefore in the research of Arabidopis thaliana and rice genome, many successful application (Filleur, et al., 2001 have been obtained by agriculture bacillus mediated T-DNA labeling acts; Maria, etal., 2001; Shihshieh, et al., 2001).In the research of paddy rice functional genomics, there have been some laboratories to utilize the T-DNA labeling acts to make up the rice mutant storehouse both at home and abroad, and from the T of mutant strain system ₂Of short stem, leaf variegation, cape horn fever spot, leaf variation, flowering time have been obtained in generation in advance or various mutant phenotypes (Jeon, et al., 2000 such as postponement; Jeong, et al., 2002; Jung, et al., 2003; Chen, et al., 2003).An (2003) has analyzed the insertion site of T-DNA in rice genome in 3793 transformed plants, wherein has 1637 sites to drop in the gene order of NCBI note.

In a large amount of dwarf genes of paddy rice, Dwarf1 and d-61 are successively cloned and are studied (Ashikari, etal., 1999; Yamamura, et al., 2000), Dwarf1 is the encoding gene that conducts the d subunit of relevant gtp binding protein with the Plant hormones regulators,gibberellins signal, the BR receptor kinase of d-61 genes encoding is the genes involved of brassinosteroid signal conduction, and the sudden change of these genes causes the plant height growth to be suppressed.Zhi etc. have cloned paddy rice D2 gene in 2003, by discovering to this gene, newcomer in the D2 genes encoding Cytochrome P450 family, belong to the CYP90D family similar to BR synthetic enzyme height, dwarf gene in paddy rice has then lacked this encoding function, make and cause the dwarfing of rice plant by the BR biosynthesis block.

Monna etc. (2002) utilize the method for map based cloning to separate and obtain Green Revolution genes involved sd-1, sd-1 site and the GA20 oxydase mutator gene of separating in Arabidopis thaliana (Ds20ox2) are chain, thereby the GA20 oxidase activity weakens and causes the plant dwarfing in the semi-dwarf mutant rice varieties of sd-1 control.Sasaki etc. have cloned the gene gid2 of a paddy rice GA signal transduction path in 2003, the product of its coding is a F-box albumen, are the acceptors of a kind of GA.Miyako etc. cloned paddy rice GID1 gene in 2005, and coded product is the GA receptor protein of solubility.Itoh etc. (2004) utilize the homologous clone method to clone 5 KO-like genes (OsKOL1-5), the KOs height homology of the albumen of these genes encodings and the Arabidopis thaliana of having cloned and summer squash, sequential analysis and complementary assay confirm, wherein the OsKOL2 gene is corresponding with D35, and coded product is the kaurene oxydase of GA route of synthesis.

Summary of the invention

The invention provides a kind of rice pyruvate kinase and encoding gene thereof and application.

Rice pyruvate kinase provided by the invention is following (a) or protein (b):

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and to have a pyruvate kinase active by sequence 1 deutero-protein.

Sequence 1 in the sequence table is made up of 527 amino-acid residues, is pyruvate kinase, called after OsPK1.From the N-terminal 30-379 of sequence 1 amino acids residue is the PK structural domain, and 394-524 amino acids residue is the PK_C structural domain.

The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant and replace outside the above-mentioned PK structural domain of sequence 1 and PK_C structural domain and/or lack and/or add.

In order to make the OsPK1 in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1 label

Label	Residue	Sequence
			??Poly-Arg	5-6 (being generally 5)	??RRRRR
??Poly-His	2-10 (being generally 6)	??HHHHHH
			??FLAG	??8	??DYKDDDDK
??Strep-tagII	??8	??WSHPQFEK
			??c-myc	??10	??EQKLISEEDL

Above-mentioned (b) but in the OsPK1 synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of OsPK1 in above-mentioned (b) can be by the codon with sequence in the sequence table 2 one or several amino-acid residue of disappearance in the dna sequence dna shown in 5 ' end the 1st to 1584 bit base, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The encoding gene of above-mentioned pyruvate kinase also belongs to protection scope of the present invention.

The encoding gene of described pyruvate kinase is following 1) or 2) or 3) dna molecular:

1) its nucleotide sequence is the dna molecular shown in the sequence 2 in the sequence table;

2) under stringent condition with 1) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;

3) with sequence table in the dna sequence dna that limits of sequence 2 have 90% above homology, and the identical function protein DNA molecule of encoding.

Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

Sequence 2 in the sequence table is made up of 1584 deoxyribonucleotides.

Contain OsPK1 expression of gene box or recombinant expression vector and also belong to protection scope of the present invention.

Available existing expression vector establishment contains the recombinant expression vector of OsPK1 gene.

When using OsPK1 to make up recombinant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be used in combination with other promotor; In addition, when using gene constructed expression carrier of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of the transgenosis bacterial strain being identified and being screened, can process used expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in bacterial strain, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene etc.

Described recombinant expression vector specifically can be pET-23b-OsPK1; Described pET-23b-OsPK1 obtains the multiple clone site that described gene inserts pET-23b; Described pET-23b-OsPK1 specifically can insert described gene between the XhoI and BamHI site of described pET-23b.

The transgenic cell line and the reorganization bacterium that contain the OsPK1 gene all belong to protection scope of the present invention.

Described reorganization bacterium specifically can be DH10B/pET-23b-OsPK1; Described DH10B/pET-23b-OsPK1 imports DH10B with pET-23b-OsPK1 and obtains.

The present invention also provides the described OsPK1 of a kind of expression proteic method, is by cultivating described reorganization bacterium, obtaining described OsPK1 albumen.

Described OsPK1 albumen or described OsPK1 gene can be applicable to cultivate short bar paddy rice.

The present invention also provides a kind of method of cultivating short bar paddy rice, is to cultivate short bar paddy rice by the OsPK1 gene in the reticent paddy rice.

The present invention has created T-DNA and has inserted the rice mutant storehouse that produces, and by the screening mutant library, has found that plant half is downgraded, the mutant A846 of sheath Bao Sui.Genetic analysis shows that the phenotype of mutant A846 is that (un-translated region UTR) weakens that its function causes because T-DNA is inserted in 5 of certain gene ' end untranslated region.The coding region of this gene is 1584bp, encodes one to contain 527 amino acid whose protein, has very high homology with the pyruvate kinase family gene of Arabidopis thaliana, contains typical PK-PK_C structural domain, with its called after OsPK1.Find that by mutant A846 being carried out anatomic observation it mainly is owing to first segment under the fringe of paddy rice obviously shortens that plant height is downgraded.Use microscope and scanning electron microscopic observation and find that the cell quantity of first segment does not have considerable change, but the length of cell obviously shortens.Because GA can promote the elongation of cell, mutant cells shortens may be relevant with GA.Can make mutant plant height recovery in seedling stage by applying GA3, prove that the A846 mutant is the sudden change of GA responsive type, and the sudden change of the plant height of responsive type be all relevant with some enzymes of GA or BRs route of synthesis.

For further verifying the function of OsPK1 gene, the present invention passes through at vivoexpression pyruvate kinase OsPK1 albumen, detect the proteic kinase activity of OsPK1 of expressing with Velemine Tanaka method, proved that the OsPK1 gene is the functional gene on the rice genome.OsPK1 genes encoding pyruvate kinase is first pyruvate kinase family gene of being identified function in the paddy rice.This gene plays an important role in the GA of rice stem building-up process, influences the formation of the initial precursor pyruvate salt of GA synthetic, and the elongation of rice stem first segment is obviously shortened, and then influences the height of plant.Illustrating of this gene function can promote the theoretical investigation that rice stem is grown, and accelerates the plant height seed selection kind process of paddy rice or other monocot crops, thereby has important theory and practice significance.

Rice mutant A846 provided by the invention is that a plant height half is downgraded, the rice mutant of sheath Bao Sui.The evaluation of the establishment of mutant A846 and OsPK1 gene function, result of study can not only be improved the route of synthesis of the important endogenous hormones GA of plant, disclose the molecular mechanism of pyruvate kinase effect gene paddy rice plant height, and for the rice field breeding new dwarfing quality resource is provided, for the breeding of paddy gene engineering provides the functional gene resource, on paddy rice conventional breeding and genetic engineering breeding, have a wide range of applications.

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.

Description of drawings

Fig. 1 is the fine phenotype of mutant A846 and Japan

Fig. 2 detects figure for Southern blot

Fig. 3 inserts the site synoptic diagram for T-DNA

Fig. 4 is divided into from evaluation figure for mutant

Fig. 5 is mutant plant height anatomical features figure

Fig. 6 is the microscope anatomic observation figure of intercalary meristem of first segment under the mutant fringe

Fig. 7 is the stereoscan photograph of first segment internode maturation zone under the mutant fringe

Fig. 8 handles the seedling growing way of mutant for GA3

Fig. 9 is OsPK1 protein electrophoresis figure

Figure 10 detects the pyruvate kinase activity of expressing for surveying with Velemine Tanaka method

Embodiment

Substratum in following examples is composed as follows:

LB substratum: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl, 15g/L agar powder (solid medium need add); PH7.0.

NB ₀Substratum: macroelement (28.3g/L KNO ₃, 4g/L KH ₂PO ₄, 4.63g/L (NH ₄) ₂SO ₄, 1.85g/LMgSO ₄, 1.66g/L CaCl ₂), trace element (0.7578g/L MnSO ₄, 0.2g/L ZnSO ₄, 0.3g/LH ₃BO ₃, 0.0025g/L Na ₂MoO ₄, 0.0025g/L CuSO ₄, 0.0025g/L CoCl ₂) and organic composition (1g/L hydrochloric acid plug amine/VitB1,0.1g/L vitamin B6 hydrochloride/aldehyde/alcohol, 0.1g/L nicotinic acid, 10g/L inositol), 300mg/L casein hydrolysate, 500mg/L proline(Pro), 30g/L sucrose, 3g/L plant gel (Phytagel); PH5.8.

The ABC substratum:

A liquid: 20 * phosphoric acid buffer (60g/L K ₂HPO ₄, 20g/L NaH ₂PO ₄);

B liquid: 20 * salts solution (20g/L NH ₄Cl, 6g/L MgSO ₄7H ₂O, 3g/L KCl, 0.2g/L CaCl ₂, 0.05g/L FeSO ₄7H ₂O; PH7.0);

C liquid: 5g sucrose/900ml water;

Before facing usefulness, by 5: 5: 90 mixed.

AAM substratum: macroelement (29.4g/L KCl, 1.7g/L KH ₂PO ₄, 3.7g/L MgSO ₄, 4.4g/LCaCl ₂), trace element (1.69g/L MnSO ₄, 0.86g/L ZnSO ₄, 0.62g/L H ₃BO ₃, 0.0025g/LNa ₂MoO ₄,, 0.0025g/L CuSO ₄, 0.0025g/L CoCl ₂) and organic composition (1g/L hydrochloric acid plug amine/VitB1,0.1g/L vitamin B6 hydrochloride/aldehyde/alcohol, 0.1g/L nicotinic acid, the 10g/L inositol), the VITAMIN of MS minimum medium, 0.5g/L casein hydrolysate, 68.5g/L sucrose, 36g/L glucose, 100 μ M Syringylethanones; PH5.2.

2N6-AS substratum: macroelement (28.3g/L KNO ₃, 4g/L KH ₂PO ₄, 4.63g/L (NH ₄) ₂SO ₄, 1.85g/L MgSO ₄, 1.66g/L CaCl ₂), trace element (0.7578g/L MnSO ₄, 0.2g/L ZnSO ₄, 0.3g/L H ₃BO ₃, 0.0025g/L Na ₂MoO ₄, CuSO ₄, 0.0025g/L CoCl ₂) and the VITAMIN of N6 substratum, 2mg/L 2, and this fluoroacetic acid of 4-dichloro (2,4-D), 1g/L casein hydrolysate, 30g/L sucrose, 10g/L glucose, 2.0g/L Phytagel, 100 μ M Syringylethanones; PH5.2.

1/2MS substratum: 1/2MS inorganic salt and VITAMIN, 30g/L sucrose, 3.0g/L Phytagel; PH5.8.

Experimental technique among the following embodiment if no special instructions, is ordinary method.

The acquisition that embodiment 1, Japanese fine T-DNA insert mutant A846

Rice mutant A846 is that the method by Agrobacterium tumefaciens mediated conversion obtains, and concrete steps are as follows:

1, the rice callus tissue induces

Get the fine rice paddy seed shelling of sophisticated Japan, with 70% Ethanol Treatment 1-2min, 0.1% mercury chloride is handled 15-20min, the deionized water rinsing of sterilization 4-6 time.After aseptic filter paper blots surface-moisture, be inoculated into contain 2mg/ L 2,4 these fluoroacetic acid of dichloro (2, the 4-D) NB of (Fluka company) ₀On the substratum, the parafilm film seals, and secretly cultivates induced embryonic callus in 25 ℃ of incubators.After 7-10 days, peel off the callus that is derived by scultellum, succeeding transfer culture is on identical substratum.The dark cultivation after about 20 days in 25 ℃ of incubators obtains the embryo callus of the vigorous growth that can be used for transforming.

2, cultivation of Agrobacterium and pre-treatment

PCAMBIA 1301 (CAMBIA company) is imported the agrobacterium tumefaciens EHA105 bacterium EHA105/pCAMBIA 1301 that obtains recombinating.

EHA105/pCAMBIA 1301 is rule 28 ℃ of dark cultivations 2 days on the LB solid medium that contains 50mg/L kantlex (Amresco company) and 10mg/L Rifampin (Sigma company).Picking list bacterium colony is to containing in the identical antibiotic LB liquid nutrient medium, and 28 ℃, about 12 hours of 200rpm wave and culture to bacterial concentration reaches OD ₅₉₅≈ 0.5.Transfer in containing identical antibiotic ABC substratum with 1% inoculum size, 28 ℃, about 14 hours of 200rpm wave and culture to bacterial concentration reaches OD again ₅₉₅≈ 0.8-1.0.Bacterium liquid is poured in the 50mL centrifuge tube of sterilization, the centrifugal 30min of 3800rpm under the eppendorf 5810R whizzer room temperature collects thalline, outwells supernatant, with the resuspended thalline of isopyknic AAM substratum once, behind the recentrifuge, uses the resuspended thalline of AAM substratum to OD ₅₉₅≈ 0.3, is equipped with to transform to use.

3, the common cultivation of EMBRYO IN RICE callus and Agrobacterium

The rice callus of the faint yellow vigorous growth that picking step 1 prepares is organized to sterile petri dish, and the Agrobacterium bacterium liquid that step 2 prepares is poured into culture dish, stirs mixing gently and rice callus is organized fully be immersed in the bacterium liquid.Behind the 15-20min, outwell bacterium liquid, callus is inoculated on the 2N6-AS substratum, the dark cultivation 2-3 days in 25 ℃ of incubators.Check the growth of Agrobacterium situation during this time, cover with the rice callus tissue surface with Agrobacterium and be advisable.

4, the regeneration of the screening of rice conversion body and plant

Callus after cultivating altogether is transferred in the triangular flask of sterilization, about 15 times of washed with de-ionized water with sterilization, till the liquid clarification, add the sterilization deionized water that contains 500mg/L cephamycin (Sigma company) and 200mg/L penbritin (Huabei Pharmaceutic Co., Ltd) again, 120rpm shakes and cleans 2hr under 25 ℃ of conditions.Outwell deionized water, and callus is transferred on the aseptic filter paper, the callus after drying is inoculated in and contains 2mg/L 2, the NB of 4-D, 250mg/L cephamycin, 200mg/L penbritin and 50mg/L hygromycin B (Roche company) ₀On the substratum, the parafilm film seals, the dark cultivation 20-30 days in 25 ℃ of incubators.Often observe callus state and growing state during this time, avoid living contaminants.Picking hygromycin B resistant calli succeeding transfer culture is in same medium, and dark again cultivation is about 15 days in 25 ℃ of incubators.

The hygromycin B resistant calli of vigorous growth is inoculated in contains 1.0mg/L 6-benzyl aminopurine (6-BA) (Sigma company), 2.0mg/L a-naphthylacetic acid (NAA) (Beijing chemical reagents corporation), the NB of 5.0mg/L dormin (ABA) (Sigma company) and 50mg/L hygromycin B ₀Presorting on the substratum, the dark cultivation after about 15 days in 25 ℃ of incubators, the fine and close resistant calli of picking white is inoculated in and contains 2.0mg/L 6-BA again, 1.0mg/L 3-indolyl acetic acid (IAA) (Beijing chemical reagents corporation), 1.0mg/L NAA, the NB of 1.0mg/L 6-chaff aminopurine (KT) (Sigma company) and 50mg/L hygromycin B ₀Break up about 20 days of illumination cultivation in 25 ℃ of incubators (2000lux, the dark 8hr of illumination 16hr/) on the substratum.The callus that differentiation is sprouted is inoculated on the 1/2MS solid medium that contains 0.5mg/L NAA, and 25 ℃ of illumination cultivation (4000lux, the dark 8hr of illumination 16hr/) after about 30 days, move to solarium's cultivation with healthy and strong plant.

Obtained the mutant strain of 132 strain systems altogether.

5, the phenotype of transfer-gen plant is observed

After treating that transfer-gen plant is solid, T1 is for seed in sowing.In process of growth, the variation of each organ morphology of the plant of each transgenic lines of observed and recorded, quantity etc.

Observe and find, mutant strain A846 plant is downgraded, and plant height has only 1/2 of wild-type, sees Fig. 1.Among Fig. 1, the left side is Japan's fine (wild-type), and the right is mutant strain A846.The T1 of A846 is in the isolates, and wildtype phenotype is 25: 8 with the ratio of downgrading phenotype, through x ²Check, P＞0.5 shows that segregation ratio meets 3: 1, infers that thus the short bar phenotype of mutant strain A846 may cause single gene mutation to cause because T-DNA inserts.

The genetic analysis of embodiment 2, mutant strain A846

The mutant phenotype of transgenic rice plant also not all is because T-DNA inserts (An et al., 2005) that cause, so must confirm the dependency that mutation type surface and T-DNA insert by genetic analysis.

One, the extraction of oryza sativa genomic dna is with quantitative

The method (Malmberg et al., 1985) of pressing Dellaporta etc. is extracted the T2 generation short bar plant and the fine genomic dna of Japan of A846 respectively, and concrete steps are as follows:

(1) get the fresh rice leaf of 3-5g, liquid nitrogen flash freezer is ground into powder in mortar fast, changes in the 50ml centrifuge tube;

(2) add 20ml immediately and be preheated to 65 ℃ extraction damping fluid (1M Tris-HCl (pH8.0) 100ml/L, 0.5M EDTA (pH8.0) 100ml/L, 5M NaCl 100ml/L, 10%SDS 125ml/L, beta-mercaptoethanol 1.5ml/L) in, 65 ℃ of water-bath 30min;

(3) add 7.5ml 5M Potassium ethanoate, put upside down mixing gently, place 20min on ice, the centrifugal 20min of eppendorf 5810R whizzer 4000rpm transfers to supernatant in another clean 50ml centrifuge tube;

(4) add the 15ml chloroform: primary isoamyl alcohol (24: 1), put upside down mixing gently, the centrifugal 20min of 4000rpm transfers to supernatant in another clean 50ml centrifuge tube;

(5) add the Virahol that 15ml ices precooling, put upside down mixing gently, place 30min for-20 ℃;

(6) the centrifugal 20min of 4000rpm abandons supernatant, washes once with 20ml 70% ethanol, is inverted centrifuge tube on clean filter paper, after room temperature is dried, precipitation is resuspended in 700 μ l ddH ₂Among the O;

(7) change in the clean 1.5ml centrifuge tube, add isopyknic chloroform: primary isoamyl alcohol (24: 1), put upside down mixing gently, the centrifugal 15min of 12000rpm, supernatant is changed in the clean 2ml centrifuge tube, add the dehydrated alcohol of 70 μ l3M sodium-acetates (pH5.2) and the precooling of 2 times of volume ice, put upside down mixing gently, place 30min for-20 ℃;

(8) the centrifugal 15min of 12000rpm abandons supernatant, and 1ml 70% ethanol is washed one time, and the centrifugal 10min of 12000rpm abandons supernatant, and room temperature is dried post precipitation, is dissolved in 100 μ l ddH ₂Among the O; Add 2 μ l RNase (10mg/ml).

(9) the quantitative of DNA determined with ultraviolet determination method or gel electrophoresis.

Two, Southern blotting analyzes

1, the preparation of probe

A hygromycin B resistant gene is arranged on the T-DNA of plant expression vector pCAMBIA 1301.Design a pair of probe primer Hyg-1 and Hyg-2 according to this gene order.Plasmid DNA with pCAMBIA 1301 is that template is carried out pcr amplification, and the fragment that to obtain a length be 858bp is the probe of preparation.

The sequence of primer Hyg-1 and Hyg-2 is as follows:

Hyg-1：5′-TGCGCCCAAGCTGCATCAT-3′，

Hyg-2：5′-TGAACTCACCGCGACGTCTGT-3′。

The extracting method of the plasmid DNA of pCAMBIA 1301 is seen " molecular cloning experiment guide " (Sambrook andRussell, 2001).

The reaction conditions of pcr amplification: 94 ℃ of 4min; 94 ℃ of 1min, 60 ℃ of 1min, 72 ℃ of 1min, 35 circulations; 72 ℃ of 8min.

The reaction system of pcr amplification: 1 * PCR reaction buffer, 1U Taq archaeal dna polymerase (Takara company), 20ng pCAMBIA 1301 plasmid DNA, 0.2mM dNTP, 0.2 μ M Hyg-1,0.2 μ M Hyg-2, ddH ₂O is supplemented to 20 μ l.

2, Southern blotting analyzes

Get the genomic dna of Japanese fine plant and A846 T2 generation short bar plant each 10 μ g of genomic dna, add 1 μ l HindIII (Takara company) and 2.5 μ l, 10 * M damping fluid, ddH ₂O is supplemented to 25 μ l, and 37 ℃ of enzymes are cut and spent the night, the DNA after cutting with 2V/cm voltage electrophoretic separation enzyme on 1% sepharose, about 6hr of time.

Commentaries on classics film, probe mark and the hybridization of DNA glue all carries out with reference to " molecular cloning experiment guide " (Sambrook andRussell, 2001), and concrete steps are as follows:

DNA glue is the about 15min of sex change in 0.25M HCl, and to tetrabromophenol sulfonphthalein indicator yellowing, the about 20min of sex change in 0.4M NaOH again becomes blueness again to the tetrabromophenol sulfonphthalein indicator; With the up capillary tube technique of 0.4M NaOH DNA is transferred on the Hybond-N+ nylon membrane (Amersham company).α- ³²Carry out Southern hybridization behind P-dCTP (Beijing Fu Rui bio-engineering corporation) and Prime-a-Gene Labeling System labelling kit (Promega company) label probe.

See Fig. 2 from the Southern results of hybridization.Among Fig. 2, P:pCAMBIA 1301 plasmids; NB: Japan is fine; A846: mutant strain.As seen from the figure, mutant strain has a hybrid belt, contrasts Japanese fine amixia band.This result shows that the T-DNA in the mutant A846 genome is that single copy inserts.

Embodiment 3, T-DNA insert the site and analyze

Determine the insertion site of T-DNA by staggered polymerase chain reaction (TAIL-PCR) method (Liu et al., 1995) of hot heterogeneity.

Three special primers in the TAIL-PCR reaction are respectively:

SP1：5′-GGTGACCAGCTCGAATTTCCC-3′，

SP2：5′-TGAATCCTGTTGCCGGTCTTG-3′，

SP3：5′-GCGCGCGGTGTCATCTATGT-3′。

Random primer is:

AD4：5′-TG(A/T)GNAG(A/T)ANCA(G/C)AGA-3′。

T2 with A846 is a template for the short bar plant genomic dna that isozygotys, and carries out pcr amplification with above-mentioned primer, amplification program reference literature (Liu et al., 1995).The 3rd step amplified production carries out agarose gel electrophoresis, reclaim test kit (Shen can lottery industry bio tech ltd) with gel and reclaim the specific amplified band, be connected to check order behind the pGEM-T easy carrier (Promega company) (the biological limited liability company of three rich polygala roots).

The sequence of TAIL-PCR amplified production is carried out blastn analyze in Genbank database (http://www.gramene.org/), find that T-DNA is inserted on the o.11 karyomit(e) of rice genome.Be cloned into T-DNA with PCR method and be inserted in opposite side sequence on the rice genome site, find because the insertion that T-DNA inserts, cause the disappearance of 29 bases on the rice genome by comparison.T-DNA is inserted in LOC_Os11g05110The promoter region of gene, T-DNA insert the site synoptic diagram and see Fig. 3.

The insertion of embodiment 4, mutant character and T-DNA isolating checking altogether

TAIL-PCR amplified production sequence comprises part T-DNA and inserts the oryza sativa genomic dna of site flank, therefore, rice genome sequence with T-DNA sequence and its insertion site flank respectively designs a primer composition combination of primers, mutant colony is divided into from checking have more obvious advantage.

The sequence of a pair of primer is as follows:

Primer sequence on the T-DNA: 5 '-TCCGAGGGCAAAGAAATA-3 ';

Sequence on the rice genome: 5 '-GCCGGCCTTGAGGCAGGAGGAG-3 '.

Extract the genomic dna of the T2 of Japanese warm and fine A846 respectively, carry out pcr amplification with above-mentioned primer for the short bar plant of isozygotying.The PCR detected result as shown in Figure 4, among Fig. 4, M:DL2000Marker; The T2 of 1-10:A846 is for isozygotying short bar plant; C: Japan fine (NB).As seen from the figure, short bar plant all has an amplified band, contrasts the Japanese fine amplified band that then do not have, and the insertion that proves short bar proterties and T-DNA is isolating altogether.

The functional analysis of embodiment 5, mutator gene

One, the information biology of mutator gene function prediction

LOC_Os11g05110One of long codes district coding contain 527 amino acid whose protein.Its sequence is carried out the functional domain analysis on http://www.sanger.ac.uk/Software/Pfam website, find that it has typical PK-PK_C structural domain, the pyruvate kinase of may encoding.At present, the pyruvate kinase family gene member who has identified in Arabidopis thaliana has 12.The aminoacid sequence compare of analysis is the result show, among the mutant strain A846 because T-DNA inserts a pyruvate kinase gene order homology (identity) of the protein of genes encoding of block function and Arabidopis thaliana up to (88%).Therefore, with this blocking gene called after OsPK1.

Search in the cDNA of japonica rice database (http://www.gramene.org/), find 1 with the OsPK1 gene on amino acid levels homology up to 96% sequence (numbering Os12g0145700), temporarily with its called after OsPK2.Although so high homology is arranged between the two, the afunction of OsPK1 gene can cause phenotype generation considerable change separately, illustrates that the OsPK2 gene function is different with OsPK1, can not form to each other and have complementary functions.

Two, the effect of OsPK1 in the growing of rice stem

1, anatomic observation

The T2 that dissects Japanese warm and fine A846 respectively observes relatively their anatomical features for isozygotying short bar plant, and anatomical features is seen Fig. 5.By dissect finding, the plant height of short bar plant have only Japan fine 1/2, downgrade and mainly occur in first segment under the fringe, the length variations of fringe and leaf sheath is not obvious, so the reason that mutant strain A846 plant height is downgraded mainly is obviously to shorten between first segment under the fringe,

2, electron microscopic observation

In order to verify that reason that internode shortens is that cell number reduces or cell length reduces and causes, use Quanta200 scanning electronic microscope (FEI Co.) and observe the isozygoty cell of the maturation zone between short bar plant and Japanese fine first segment of T2 generation of A846 relatively.Fig. 6 is the microscope anatomic observation figure of intercalary meristem of first segment under the mutant fringe.Among Fig. 6, NB: Japan is fine; A846: mutant strain.As seen from the figure, the number of variations of cell is not obvious.

The electron microscopic observation sample adopts desiccating method preparation (Jenks et al., 1992).The T2 of A846 is fine after 2 weeks of heading with Japan for the short bar plant of isozygotying, and gathers the cane of first segment, air drying 48-72hr under the room temperature.Get branch incision the suitable big or small cane from the position, middle and upper part of each material, two sections upwards sticks on the load sample platform with conductive resin, behind the metal spraying (30sec/ time, 5 times), can observe under scanning electron microscope.Electronic Speculum the results are shown in Figure 7.Among Fig. 7, NB: Japan is fine; A846: mutant strain; 2 top figure amplify 200 times photo, and 2 following figure amplify 500 times photo.

Electron microscope showed, the cell length of the first segment of mutant strain obviously shortens, and compares with Japan is fine, and in mutant strain, the length of cell has only about half of the fine middle cell length of Japan.Infer that thus the OsPK1 gene plays an important role in the elongation process of paddy rice internode, weaken the OsPK1 expression of gene, the length of cell is shortened, thereby produce plant dwarfing isophenous because T-DNA inserts.

Embodiment 6, apply the influence of external source Plant hormones regulators,gibberellins (GA) to the mutant growth of seedling

For checking OsPK1 mutant is GA responsive type or non-sensitive type sudden change, in the 1/2MS substratum, add the external source GA3 of different amounts respectively, the final concentration of GA3 is respectively 10 in each substratum ^-1, 10 ^-2, 10 ^-3, 10 ^-4Ppm.With T2 generation of above culture medium culturing fine plant of Japan and A846 short bar plant of isozygotying, after 20 days, measure the height of plant respectively.

The results are shown in Figure 8, as seen from the figure, at lower concentration (10 ^-4Ppm) under the condition, the plant height of mutant strain seedling can return to the height near wild-type, shows that the OsPK1 sudden change is the sudden change of GA responsive type.The sudden change of responsive type generally all is influenced the causing of function by the enzyme of the metabolic pathway of synthesizing of GA, illustrates that the OsPK1 sudden change influences the function of the initial precursor pyruvate salt of GA synthetic synthetic important enzyme-pyruvate kinase.

Embodiment 7, vivoexpression mutator gene albumen OsPK1 also detect its pyruvate kinase activity

One, the acquisition of OsPK1 encoding gene

As follows according to a pair of primer of the consequence devised of sequential analysis:

OsPK1-F：5’-GGGATCCATGCATTCGACGAATCTGCTG-3；

OsPK1-R：5’-CCTGCAGCTAATCGTCCAGCTCAATGATC-3’。

Full-length cDNA with the fine paddy rice of Japan is a template, carries out pcr amplification with above primer.The PCR product is carried out agarose gel electrophoresis detect, the result shows, adopts to have obtained the band that molecular weight is about 1584bp, conforms to expected results.With 4 ℃ of preservations of this PCR product, serve extra large Invitrogen Corporation order-checking, sequencing result shows that the nucleotide sequence that increases is seen the sequence 2 of sequence table, encoding amino acid sequence is the protein OsPK1 of the sequence 1 of sequence table.

Reclaim test kit (TIANGEN) with sepharose and reclaim this fragment.Should reclaim fragment and pMD-18T (Dalian TaKaRa; Article No.: D103A) connect, make up cloning vector.The pMD-18T carrier called after pMD-18T-OsPK1 from the OsPK1 gene of 5 ' terminal 1-1584 position deoxyribonucleotide that will contain sequence 2 in the ordered list.

Two, proteic expression of OsPK1 and purifying

1, the proteic expression of OsPK1

Cut pMD-18T-OsPK1 with XhoI enzyme and BamHI enzyme enzyme, obtain the OsPK1 gene, the OsPK1 gene that obtains is connected between the multiple clone site XhoI and BamHI site of expression vector pET-23b (+), itself and terminal 6 Histidines are in the same reading frame, obtain recombinant expression vector pET-23b-OsPK1.Recombinant expression vector pET-23b-OsPK1 is imported in the DH10B cell, cultivated 12 hours, induce certain hour with IPTG after, get cleer and peaceful precipitation respectively and carry out electrophoresis detection, electrophoresis result is seen Fig. 9 A.Among Fig. 9 A, M:marker; 1: do not induce supernatant; 2: induced precipitation not; 3: induce the 1h supernatant; 4: induce the 1h precipitation; 5: induce the 2h supernatant; 6: induce the 2h precipitation.

As seen from the figure, the OsPK1 gene protein almost all comes out with the inclusion body formal representation, and major part all is an inactive form.

2, the proteic purifying of OsPK1

The inclusion body OsPK1 albumen that obtains is carried out protein renaturation with the dialysis refolding method, and concrete steps are as follows:

(1) processing before dialysis tubing uses.

A) cut 4 sections dialysis tubings, in the dialysis tubing rinsing liquid, boil 30min, dialysis tubing will be opened fully therebetween, with fully well-done;

B) with distilled water with the thorough rinsing of dialysis tubing;

C) dialysis tubing is soaked in the water, boils 15min, cooling is placed on 4 ℃ of refrigerator overnight;

D) with 4 trumpeter's art sutures dialysis tubing one end ligation is sealed, boil the back room temperature preservation.

(2) concentration of adjustment ion exchange chromatography eluted protein is 0.1mg/ml, and the metaprotein solution after the dilution is respectively charged into the dialysis tubing of anticipating, and (molecular weight cut-off is 1.4 * 10 ³).

(3) dialysis tubing is put into a beaker that 400ml dialyzate I is housed, stirred dialysis 4h under 4 ℃ of conditions; The dialyzate I that more renews (2M urine is disorderly, 20mM Tris, 0.1mM GSSG, 0.9mM GSH) repeats this step twice.

(4) dialysis tubing is put into the beaker that 400ml dialyzate II is housed, stirred dialysis 4h under 4 ℃ of conditions; The dialyzate II that more renews (0.5M urea, 20mM Tris, 0.1mM GSSG, 0.9mM GSH) repeats this step twice.

(5) dialysis tubing is put into the beaker that 400ml 0.01M phosphate buffered saline buffer is housed, stirred dialysis 4h under 4 ℃ of conditions.

(6) dialysis tubing is put into the beaker that 400ml sterilization deionized water is housed, stirred companion's dialysis 4h under 4 ℃ of conditions.

(7) solution in the dialysis tubing is merged, put into freeze drier and carry out lyophilize ,-70 ℃ of preservations.

With the Ni ion column purifying of the albumen after the renaturation, the albumen behind the purifying carries out electrophoresis detection, sees Fig. 9 B.Among Fig. 9 B, M:marker; 1: the OsPK1 albumen behind the purifying.

Obtain the OsPK1 albumen of active condition.

Three, the proteic pyruvate kinase activity of OsPK1

Detect the proteic pyruvate kinase activity of expressing of OsPK1 with reference to Velentine Tanaka method, pyruvate kinase can catalysis phosphoenolpyruvic acid (PEPI) change pyruvic acid into, and pyruvic acid is reduced to lactic acid and makes DPNH (NADH) change oxidized form of nicotinamide-adenine dinucleotide (NAD into when serum lactic dehydrogenase exists ⁺).NADH has special absorption peak at 340nm wavelength place.The optical density(OD) of observing this wavelength changes, and can measure the pyruvate kinase activity.

As standard specimen, detect the proteic pyruvate kinase activity of OsPK1 of the purifying that step 2 obtains with the pyruvate kinase of the Sigma company that buys.The results are shown in Figure 10, among Figure 10, CK1:ddH ₂O (blank); CK2: pyruvate kinase+serum lactic dehydrogenase+NADH; CK3: phosphoenolpyruvic acid+serum lactic dehydrogenase+NADH; CK4: pyruvate kinase+phosphoenolpyruvic acid+serum lactic dehydrogenase; Standard specimen: pyruvate kinase+phosphoenolpyruvic acid+serum lactic dehydrogenase+NADH (positive control); Purifying protein: OsPK1 albumen+phosphoenolpyruvic acid+serum lactic dehydrogenase+NADH.

As seen from the figure, the OsPK1 protease activity is a standard specimen pyruvate kinase active 1/2.

Sequence table

＜110〉Shenzhen Graduate School of Tsinghua University

＜120〉a kind of rice pyruvate kinase and encoding gene thereof and application

<130>CGGNARY81381

<160>2

<210>1

<211>527

<212>PRT

＜213〉Japanese fine paddy rice (Oryza sativa L.japonica.cv.Nipponbare)

<400>1

Met?His?Ser?Thr?Asn?Leu?Leu?Leu?Glu?Glu?Pro?Ile?Arg?Met?Ala?Ser

1???????????????5???????????????????10??????????????????15

Ile?Leu?Glu?Pro?Ser?Lys?Pro?Ser?Phe?Phe?Pro?Ala?Met?Thr?Lys?Ile

20??????????????????25??????????????????30

Val?Gly?Thr?Leu?Gly?Pro?Lys?Ser?Arg?Ala?Val?Asp?Thr?Ile?Ser?Ser

35??????????????????40??????????????????45

Cys?Leu?Lys?Ala?Gly?Met?Ser?Val?Ala?Arg?Phe?Asp?Phe?Ser?Trp?Gly

50??????????????????55??????????????????60

Asp?Ala?Glu?Tyr?His?Gln?Glu?Thr?Leu?Glu?Asn?Leu?Lys?Leu?Ala?Ile

65??????????????????70??????????????????75??????????????????80

Lys?Ser?Thr?Lys?Lys?Leu?Cys?Ala?Val?Met?Leu?Asp?Thr?Val?Gly?Pro

85??????????????????90??????????????????95

Glu?Leu?Gln?Val?Val?Asn?Lys?Ser?Glu?Ala?Ala?Ile?Ser?Leu?Glu?Ala

100?????????????????105?????????????????110

Asn?Gly?Thr?Val?Val?Leu?Thr?Pro?Asp?Gln?Gly?Gln?Glu?Ala?Ser?Ser

115?????????????????120?????????????????125

Glu?Leu?Leu?Pro?Ile?Asn?Phe?Ser?Gly?Leu?Ala?Lys?Ala?Leu?Lys?Pro

130?????????????????135?????????????????140

Gly?Ala?Thr?Ile?Phe?Val?Gly?Gln?Tyr?Leu?Phe?Thr?Gly?Ser?Glu?Thr

145?????????????????150?????????????????155?????????????????160

Thr?Ser?Val?Trp?Leu?Glu?Val?Ser?Glu?Val?Lys?Gly?Asp?Asp?Val?Val

165?????????????????170?????????????????175

Cys?Val?Ile?Lys?Asn?Ser?Ala?Thr?Leu?Ala?Gly?Ser?Leu?Phe?Thr?Leu

180?????????????????185?????????????????190

His?Cys?Ser?Gln?Ile?His?Ile?Asp?Leu?Pro?Thr?Leu?Ser?Asp?Glu?Asp

195?????????????????200?????????????????205

Lys?Glu?Val?Ile?Arg?Arg?Trp?Gly?Ala?Pro?Asn?Lys?Ile?Asp?Phe?Leu

210?????????????????215?????????????????220

Ser?Leu?Ser?Tyr?Thr?Arg?His?Ala?Glu?Asp?Val?Arg?Gln?Ala?Arg?Glu

225?????????????????230?????????????????235?????????????????240

Phe?Leu?Ser?Lys?Leu?Gly?Asp?Leu?Ser?Gln?Thr?Gln?Ile?Phe?Ala?Lys

245?????????????????250?????????????????255

Ile?Glu?Asn?Val?Glu?Gly?Leu?Asn?His?Phe?Asp?Glu?Ile?Leu?Gln?Glu

260?????????????????265?????????????????270

Ala?Asp?Gly?Ile?Ile?Leu?Ser?Arg?Gly?Asn?Leu?Gly?Ile?Asp?Leu?Pro

275?????????????????280?????????????????285

Pro?Glu?Lys?Val?Phe?Leu?Phe?Gln?Lys?Ser?Ala?Leu?His?Lys?Cys?Asn

290?????????????????295?????????????????300

Met?Ala?Gly?Lys?Pro?Ala?Val?Val?Thr?Arg?Val?Val?Asp?Ser?Met?Thr

305?????????????????310?????????????????315?????????????????320

Asp?Asn?Leu?Arg?Pro?Thr?Arg?Ala?Glu?Ala?Thr?Asp?Val?Ala?Asn?Ala

325?????????????????330?????????????????335

Val?Leu?Asp?Gly?Ser?Asp?Ala?Ile?Leu?Leu?Gly?Ala?Glu?Thr?Leu?Arg

340?????????????????345?????????????????350

Gly?Leu?Tyr?Pro?Val?Glu?Thr?Ile?Ser?Ile?Val?Gly?Lys?Ile?Cys?Ala

355?????????????????360?????????????????365

Glu?Ala?Glu?Lys?Val?Phe?Asn?Gln?Asp?Leu?Tyr?Phe?Lys?Arg?Thr?Val

370?????????????????375?????????????????380

Lys?Tyr?Val?Gly?Glu?Pro?Met?Thr?His?Leu?Glu?Ser?Ile?Ala?Ser?Ser

385?????????????????390?????????????????395?????????????????400

Ala?Val?Arg?Ala?Ala?Ile?Lys?Val?Lys?Ala?Ser?Val?Ile?Ile?Cys?Phe

405?????????????????410?????????????????415

Thr?Ser?Ser?Gly?Arg?Ala?Ala?Arg?Leu?Ile?Ala?Lys?Tyr?Arg?Pro?Thr

420?????????????????425?????????????????430

Met?Pro?Val?Leu?Ser?Val?Val?Ile?Pro?Arg?Leu?Lys?Thr?Asn?Gln?Leu

435?????????????????440?????????????????445

Arg?Trp?Ser?Phe?Thr?Gly?Ala?Phe?Glu?Ala?Arg?Gln?Ser?Leu?Ile?Val

450?????????????????455?????????????????460

Arg?Gly?Leu?Phe?Pro?Met?Leu?Ala?Asp?Pro?Arg?His?Pro?Ala?Glu?Ser

465?????????????????470?????????????????475?????????????????480

Thr?Ser?Ala?Thr?Asn?Glu?Ser?Val?Leu?Lys?Val?Ala?Leu?Asp?His?Gly

485?????????????????490?????????????????495

Lys?Ala?Ser?Gly?Val?Ile?Lys?Ser?His?Asp?Arg?Val?Val?Val?Cys?Gln

500?????????????????505?????????????????510

Lys?Val?Gly?Asp?Ser?Ser?Val?Val?Lys?Ile?Ile?Glu?Leu?Asp?Asp

515?????????????????520?????????????????525

<210>2

<211>1584

<212>DNA

＜213〉Japanese fine paddy rice (Oryza sativa L.japonica.cv.Nipponbare)

<400>2

atgcattcga?cgaatctgct?gctggaggag?cccatcagga?tggcatccat?cctggagcca????60

tccaagccga?gctttttccc?ggcgatgacc?aagatcgtgg?ggacgctggg?tcccaagtcg?????120

cgcgccgtcg?acaccatctc?ctcctgcctc?aaggccggca?tgtcggttgc?caggttcgat?????180

ttctcgtggg?gagacgctga?gtaccaccag?gaaaccctcg?aaaacctcaa?gctcgccatc?????240

aagtccacca?agaagctctg?tgctgtcatg?ctcgacactg?tcggcccgga?gttgcaggtg?????300

gtgaacaaga?gtgaggctgc?aatctccctt?gaagcgaatg?gcactgttgt?tctcacccct?????360

gaccaggggc?aggaggcctc?ctccgaattg?ctgcccatca?acttctccgg?gcttgctaag?????420

gctctgaaac?caggtgctac?aatttttgtg?gggcaatact?tgttcactgg?cagtgagact?????480

acttcagttt?ggttagaggt?ttctgaagtt?aaaggagatg?atgtggtttg?tgtgataaaa?????540

aattcggcta?ctctggctgg?ttcactgttc?accctgcatt?gttctcagat?tcatatcgac?????600

ttacctacac?tgtctgatga?ggacaaggag?gttatcagaa?gatggggagc?tccaaacaag?????660

attgacttcc?tctctctgtc?ttatacaaga?cacgcggaag?atgtgcgaca?ggcacgtgag?????720

ttcctctcaa?agttaggcga?ccttagccaa?actcagattt?ttgccaaaat?tgagaatgtt?????780

gagggtttga?atcattttga?tgaaatcctg?caagaggcgg?atggtatcat?tctgtcaaga?????840

ggaaaccttg?gaattgatct?tccacctgaa?aaggtgtttt?tgtttcaaaa?gtctgctctg?????900

cacaagtgca?acatggctgg?aaagcctgct?gttgttactc?gtgttgtgga?cagtatgact?????960

gacaacctca?ggcctactcg?tgcggaggct?actgatgtgg?caaatgctgt?acttgacggg????1020

agtgatgcca?ttctccttgg?tgctgagact?ctccgtggtc?tgtacccagt?tgagactatc????1080

tcaattgtgg?gcaaaatttg?cgctgaggca?gagaaggtat?tcaaccaaga?tttatacttc????1140

aagcggactg?tgaaatatgt?tggggagccc?atgacccact?tggagtctat?cgcttcttct????1200

gcggtgcgag?ctgctatcaa?agtgaaggct?tctgtcatta?tttgcttcac?atcatctgga????1260

cgcgctgcaa?gactaattgc?caagtacagg?cccaccatgc?ctgttctatc?tgttgtcatt????1320

cctcgtctga?aaacaaacca?actgaggtgg?agtttcactg?gcgcatttga?agcaagacaa????1380

tcactcatag?ttagaggtct?ctttccgatg?cttgctgatc?ctcgccatcc?agctgaatcg????1440

accagcgcta?ccaatgagtc?agttttgaag?gttgctcttg?accatggcaa?agcttccggt????1500

gtgatcaagt?cgcatgatcg?cgttgttgtc?tgccagaaag?tgggtgattc?ctcggttgtg????1560

aagatcattg?agctggacga?ttag???????????????????????????????????????????1584

Claims (10)

1, a kind of albumen is following (a) or protein (b):

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and to have a pyruvate kinase active by sequence 1 deutero-protein.

2, the described proteic encoding gene of claim 1.

3, gene according to claim 2 is characterized in that: described protein coding gene is following 1) or 2) or 3) dna molecular:

1) its encoding sequence is the dna molecular shown in the sequence 2 in the sequence table;

2) under stringent condition with 1) the dna sequence dna hybridization that limits and the dna molecular of encoding said proteins;

3) with 1) or 2) dna sequence dna that limits has 90% above homology, and the identical function protein DNA molecule of encoding.

4, contain claim 2 or 3 described expression of gene box or recombinant expression vectors.

5, recombinant expression vector according to claim 4 is characterized in that: described recombinant expression vector is pET-23b-0sPK1; Described pET-23b-0sPK1 inserts the recombinant vectors that the multiple clone site of pET-23b obtains with claim 2 or 3 described genes.

6, the transgenic cell line or the reorganization bacterium that contain claim 2 or 3 described genes.

7, reorganization bacterium as claimed in claim 6 is characterized in that: described reorganization bacterium is DH10B/pET-23b-0sPK1; Described DH10B/pET-23b-0sPK1 imports DH10B with described pET-23b-0sPK1 and obtains.

8, the described proteic method of a kind of expression claim 1 is to cultivate claim 6 or 7 described reorganization bacterium, obtains the described albumen of claim 1.

9, described albumen of claim 1 or claim 2 or the 3 described genes application in cultivating short bar paddy rice.

10, a kind of method of cultivating short bar paddy rice is to cultivate short bar paddy rice by claim in the reticent paddy rice 2 or 3 described genes.

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