CN101798342B - Rice glume development related protein TRI1 and encoding gene and application thereof - Google Patents

Abstract

The invention discloses a rice glume development related protein TRI1 and an encoding gene and application thereof. The rice glume development related protein (TRI1) is derived from oryza commonly cultivated rice (O. sativa L.) and is a protein described in (a) or (b): (a) a protein consisting of amino acid sequences as shown by a sequence 1 in a sequence table; and (b) a protein which is obtained by performing substitution and/or deletion and/or addition of one or a few amino acid residues on the amino acid sequence of the sequence 1, is correlative with the rice glume growth, and is derived from the sequence 1. The rice glume development related protein and the encoding gene thereof have important theoretical and practical meanings to the development of rice spikelets, in particular to molecular mechanism researches on the glume development and the breeding selection of rice grain forms and varieties, and provides an economical, quick and effective path for improving the grain form of crops. The rice glume development related protein TRI1 and the encoding gene thereof have extensive application spaces and market prospects in the agricultural field.

Description

Rice glume development related protein TRI 1 and encoding gene thereof and application

Technical field

The present invention relates to a kind of rice glume development related protein TRI 1 and encoding gene thereof and application.

Background technology

All kinds of floral organs of regulation and control form in the relevant flower development organ characteristic's gene cloning and functional analysis, be one of important breakthrough of in recent years molecular biology of plant development research, and formed comparatively ripe experimental model---the research work that the guidance of ABC model is relevant.The ABC model is by Coen (Coen ES and Meyerowitz EM.The warof the whorls:Genetic interactions controlling flower development.Nature, 1991,353:31-37) etc. the people carries out the homeotic gene that affects development of floral organs in model plant Arabidopis thaliana and the Common Snapdragon successively proposing on the basis of heredity and analysis of molecules, and this model has been described the phenomenon that the floral organ different sites is subjected to the different genes decision.According to phenotype and genetic analysis, the floral organ of four torus is to be controlled by the mutual of three genoids, and this three genoid is called A, B, C genoid, and wherein every genoid is responsible for the formation of the floral organ of adjacent two torus.Wherein the category-A gene is responsible for first, second torus, i.e. the formation of calyx, petal; The category-B gene is responsible for second, third torus, i.e. the formation of petal, stamen; The C genoid is responsible for the 3rd, fourth round ring, the i.e. formation of stamen, carpel.Studies show that by the mutant to some A, C genoid, the effect of A, C two genoids be mutual antagonism (Theissen G, and Saedler H.Plant biology.Floral quartets.Nature, 2001,409:469-471).

Paddy rice is as a class of gramineous crop, though its small ear, inflorescence and dicotyledons have very big-difference, its basic structure also can be regarded as by four torus and form.So the investigator has proposed a hypothesis, namely four of dicotyledons and paddy rice torus have corresponding relation, think that dicots ABC pattern is also applicable in gramineous crop.Poly-x female gene mutation body (spw1) (the Nagasawa N of paddy rice, Miyoshi M, Sano Y, Satoh H, Hirano H, SakaiH, Nagato, Y.SUPERWOMAN1 and DROOPING LEAF genes control floral organ identityin rice.Development, 2003,130:705-718), it shows as the organ molecular cloning that stamen and lodicule dystopy be converted into carpel and similar bran sheet and has also disclosed SPW1 genes encoding AP3---the albuminoid of paddy rice.The expressive site of SPW1 gene is mainly expressed in the initial former base of stamen and two allelotaxises of lodicule.Rice genome contains homology of two PI genes, OsMADS2 and OsMADS4, AP3 homology.Above these studies show that: the Type B gene is being guarded aspect the characteristic of specialization petal/lodicule and stamen.

The gene of regulation and control carpel specialization is the DL gene in the paddy rice, and the carpel of dl mutant has been replaced by stamen.The complete dystopy of mutant carpel that some dl defectives are serious converts stamen to, and the defective of floral organ only limits to occur in the fourth round ring.The molecular cloning of DL gene is disclosed the member of a YABBY protein family of this genes encoding.DL genetically deficient phenotype and genetic expression all show: the DL gene is the needed gene of carpel specialization.This gene also is the YABBY gene of found first control floral organ specialization, with many MADS---the function class of box gene is like (Yamaguchi T, Nagasawa N, Kawasaki S, Matsuoka, Nagato Y, Hirano HY.The YABBY gene DROOPINGLEAFregulates carpel specification and midrib development in Oryza sativa.ThePlant Cell, 2004,16:500-509).Studies show that: the characteristic of carpel is guarded in dicotyledons and gramineous crop paddy rice.

Lodicule, the peculiar floral organ of grass paddy rice.From anatomical research, with the petal homology of dicotyledons.Some nearest Molecular and Genetic Studies have also confirmed this point.In Arabidopis thaliana, the identity of petal is in conjunction with regulation and control by the function of category-A gene and category-B gene.Infer successively, lodicule can be replaced by other floral organs in some mutant of Gramineae category-B gene.In fact the spw1 mutant of paddy rice namely is so, and the SPW1 gene is expressed in the former base of the lodicule of wild-type plant.Other category-B genes, for example OsMADS2 and OsMADS4 gene are also expressed in the former base of lodicule, and category-A gene RAP1A (OsMADS15) also expresses in the former base of lodicule.In addition, the transposition expression of C genoid OsMADS3 can cause lodicule to change into stamen shape structure or change into the mosaic that is comprised of lodicule and stamen.Transposition is expressed the AG gene to cause in the transfer-gen plant petal to be converted into stamen shape petal similar in the variation of these phenotypes and the Arabidopis thaliana.The above fact shows: petal homology (the Bommert P of the lodicule of grass and dicotyledons, Satoh-Nagasawa N, Jackson D, and Hirano HY.Genetics and evolutionof infl orescence and fl ower development in grasses.Plant Cell Physiol., 2005,46:69-78).

The Problems of Identity of petal and inside and outside bran sheet is too controversial.Form comparative study shows, glumelle is a kind of organ of similar prophyll structure, and lemma is a kind of organ of similar bract.Lodicule dystopy in the spw1 mutant of paddy rice changes into inside/outside bran shape structure.Therefore Nagasawa etc. thinks that the second torus organ of spwl mutant forms the glumelle correspondence of can getting along well, can with the petal-shaped structural correspondence, and this petal-shaped structure can not be grown formation in spending normally, and this structure can only could produce in the mutant that the category-B gene activity is knocked.Yet, Luo (Luo Q, Zhou KD, Zhao XF, Zeng QC, Xia HG, Zhai WX, Xu JC, Wu XJ, Yang HS, Zhu LH.Identification and fine mapping of a mutant gene for pa1ea1ess spikelet in rice.Planta, 2005,221:222-230) etc. cause the glumelle disappearance to replace forming the foliation structure organ reaching a conclusion by the pal mutant: glumelle aspect the evolution with the sepal homology of dicotyledons.Therefore, the characteristic problem of inside and outside bran sheet remains a mystery.In order to address this problem, need to separate the mutant of some and the affected corresponding gene of inside and outside bran structure, in order to identify the characteristic of inside and outside bran.

Summary of the invention

The purpose of this invention is to provide a kind of rice glume development related protein TRI 1 and encoding gene thereof and application.

Rice glume development related protein provided by the invention (TRI1) derives from Oryza common cultivated rice (O.sativaL.) 93-11, is following (a) or protein (b):

(a) protein that is formed 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 relevant with the rice glume development protein that is derived by sequence 1.

In order to make the TRI1 in (a) be convenient to purifying, N-terminal or C-terminal that can the protein that the aminoacid sequence shown in the sequence 1 forms 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-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned (b) but in the TRI1 synthetic, also can synthesize first its encoding gene, carry out again biological expression and obtain.The encoding gene of TRI1 in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2, 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 gene (TRI1) of above-mentioned rice glume development related protein of encoding also belongs to protection scope of the present invention.

Described gene can be following 1) or 2) or 3) or 4) dna molecular:

1) dna molecular shown in the sequence 2 (cDNA) in the sequence table;

2) dna molecular shown in the sequence 3 (genomic dna) in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coding rice glume development related protein;

4) with 1) or 2) or 3) dna sequence dna that limits has 90% above homology, and the dna molecular of the rice glume development related protein of encoding.

Described stringent condition be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

The aminoacid sequence of TRI1 albumen is comprised of 248 amino-acid residues shown in the sequence 1 in the sequence table, and contains the protein structure domain DUF640 of a unknown function.The open reading frame of TRI1 gene is comprised of 747 Nucleotide shown in the sequence 2 of sequence table.The genomic gene of TRI1 gene is comprised of 2091 Nucleotide shown in the sequence 3 of sequence table, and its encoding sequence is from 5 ' end 593-1339 position Nucleotide.

The present invention also protects the sequence 2 of sequence table from the dna fragmentation shown in the 1st to 355 Nucleotide of 5 ' end, and this dna fragmentation can be used for making up RNA interfering, thereby suppresses the expression of described gene.

The recombinant expression vector that contains described gene or described dna fragmentation also belongs to protection scope of the present invention.

Available existing plant expression vector construction contains the recombinant expression vector of described gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (such as kermes synthetic enzyme Nos gene), plant gene (storing protein gene such as soybean) 3 ' end to transcribe such as the Agrobacterium crown-gall nodule all has similar functions.When using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, such as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, they can use separately or be combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (such as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Available existing plant vector makes up the recombinant vectors (interference vector) that contains described dna fragmentation.Described recombinant expression vector is to insert respectively the recombinant plasmid that dna fragmentation A and dna fragmentation B obtain between the multiple clone site of pTCK303/JL1460-TRI1; The sequence 2 that described dna fragmentation A is sequence table is from the DNA shown in the 1st to 355 Nucleotide of 5 ' end; The sequence 2 that described dna fragmentation B is sequence table is from the reverse complemental DNA of the DNA shown in the 1st to 355 Nucleotide of 5 ' end.Described dna fragmentation A specifically inserts between the BamHI and KpnI restriction enzyme site of pTCK303/JL1460-TRI1, and described dna fragmentation B specifically inserts between the SpeI and SacI restriction enzyme site of pTCK303/JL1460-TRI 1.

The expression cassette, transgenic cell line and the recombinant bacterium that contain described gene or described dna fragmentation all belong to protection scope of the present invention.

The primer pair of described gene or described dna fragmentation of increasing also belongs to protection scope of the present invention.

Another object of the present invention provides a kind of method of cultivating transgenic paddy rice.

The method of cultivation transgenic paddy rice provided by the present invention, the gene of the described rice glume development related protein of coding can be imported in the purpose paddy rice (such as rice cell or tissue), obtain clever shell width (being the width at wide position of clever shell) and be higher than the transgenic paddy rice of described purpose paddy rice, thereby improve the output of paddy rice.Specifically, described recombinant expression vector can be imported in the purpose paddy rice, obtain the transgenic paddy rice that clever shell width is higher than described purpose paddy rice.Carry described gene expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated by using, and the plant tissue that transforms is cultivated into plant.

The method of cultivation transgenic paddy rice provided by the present invention can import described interference vector in the purpose paddy rice, obtains the transgenic paddy rice that clever shell width diminishes.Described purpose paddy rice specifically can be spends 17 in the rice varieties.

Described albumen, described gene, described dna fragmentation, described recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium all can be applicable to plant breeding, cultivate high yield plant or Model Plants.Described albumen, described gene, described dna fragmentation, described recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium all can be applicable to change the grain type of rice grain.

Oryza common cultivated rice (O.sativa L.) 93-11 has produced a mutant library that variation is abundant through EMS mutagenesis, pass through phenotypic screen, found a triangle grain husk shell mutant, and the method for using map based cloning has found the gene of controlling this mutant character, names to be TRI1.The sudden change of this gene can make the growth of clever shell morph, than normal wild-type grain husk shell narrowed width, and the length indifference, the most obvious feature is the variation that shows triangular shape at its tip, very beaky shape.As everyone knows, the clever shell of paddy rice is not only the part of floral organ, " storehouse " in " source, storehouse relation " in the grain forming process especially, and the capacity in " storehouse " can limit size and the weight of grain.The narrowed width of tri1 mutant, and deformity occurs, thus limited growth and the grouting of grain, form the grain of distortion.The grain of mutant is less than the wild-type in size, light than wild-type on weight.This gene not only having vital role aspect the research of rice glume development mechanism, has wide application and market outlook at agriculture field.

The invention provides albumen and the new gene of encoding gene thereof of a control rice glume development.This albumen and encoding gene thereof are for the Study on Molecular Mechanism of paddy rice Spikelet development, particularly glume development, and the seed selection of rice grain type kind has important theory and practical significance, and provide an economy, approach fast and effectively for the grain type of Crop Improvement.The present invention has wide application space and market outlook at agriculture field.

Description of drawings

Fig. 1 is the phenotype contrast of mutant tri1 and wild-type 93-11.

Fig. 2 is the map based cloning of TRI1.

Fig. 3 is for take the genomic dna of 10 rice varieties, 93-11 and tri1 as template, and (used Marker be the DNA MarkerIII of sky root biochemical technology company limited to the banding pattern of the pcr amplification product under the guiding of primer HX1 (molecule marker), article No.: MD102-01), swimming lane 1-12 is respectively Milyang 46, and Yunnan surpasses No. 3, C418, in spend 17, osmanthus is towards No. 2, IR24, Asominori, bright extensive 63, Zhenshan 97B, special blue or green, 93-11, tri1.

Fig. 4 is the PCR qualification result of microbiotic positive plant, CK: in spend 17; The PL:pTCK303/JL1460-TRI1 plasmid; H ₂O: the empty map that does not add any template; Swimming lane 1,2 positive transfer-gen plant.

Fig. 5 is the phenotypic evaluation result of TRI1 transfer-gen plant.CK: turn the empty carrier adjoining tree; ER2: transfer-gen plant.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.The primer synthesizes and examining order is finished by Beijing AudioCodes biotechnology limited liability company.

93-11:: national germplasm resource bank.

In spend 17: national germplasm resource bank.

Carrier pTCK303/JL1460: China Agricultural University; Reference: Wang Z, Chen CG, Xu YY, JiangRX, Han Y, Xu ZH and Chong K.A Practical Vector for Efficient Knockdown of GeneExpression in Rice (Oryza sativa L.) .Plant Molecular Biology Reporter, 2004,22:409-417)

The acquisition of embodiment 1, control rice glume development gene TRI1

93-11 through EMS (methylsulphonic acid second fat) processing and after through inbreeding of more generation and character observation, is formed the pure and mild sudden change system of genotype.By a mutant that glume development is unusual has been found in the screening of these sudden change systems, the concrete manifestation of this mutant is: mutant grain husk shell is than normal wild-type grain husk shell narrowed width, namely 9311 clever shell width is 2.84 ± 0.08mm, and the clever shell width of mutant is 2.33 ± 0.11mm.But the clever shell indifference on length of the two.The most obvious feature is the variation that shows triangular shape at its tip, therefore called after tri1 (Fig. 1).

With tri1 respectively with 93-11, special blue or green, osmanthus is towards No. 2, the middle conventional variety such as 17, C418 of spending is hybridized hybridization F ₁The existing wild-type grain husk of representative shell feature, thus can infer that the gene that this mutant character of control is stealthy.Hybridization F ₁Produce F through after the selfing ₂Recessive individual among the offspring, the individual plant that namely phenotype is identical with tri1 and the individual plant ratio of wildtype phenotype were near 3: 1 (X _{2 * C}＜X _{2 * 0.05,1}=3.84).Can draw thus paddy rice triangle grain husk shell proterties by a recessive Dominant gene, this gene is named as TRI1.

At first use tri1 and osmanthus towards the little F of No. 2 hybridization ₂, gene TRI1 just slightly is positioned near the second chromosomal SSR mark RM213.Afterwards, with tri1 and the C418 F that backcrosses ₂In stealthy individual with gene TRI1 Primary Location between two mark RM8048 and near RM3850 (RM213).After this, use the discrepant SSR mark of tool between two parents (RM3774, RM14165, RM3248, RM1255) and a CAPs mark (C1) and two order-checking mark (C2 between four marks, C3) assignment of genes gene mapping is between a CAPs mark (C1) and one order-checking mark the most at last, and middle physical distance is 60kb approximately.

Primer pair HX1 (HX1F/HX1R) is designed in the zone of above-mentioned acquisition respectively, amplification tri1 and 93-11 genomic dna, the difference (Fig. 2) of the gene order aspect that searching EMS mutagenesis produces.The reaction system of pcr amplification is: oryza sativa genomic dna template 20ng, Taq Plus archaeal dna polymerase 0.5U, 2.0 μ l, 10 * PCR damping fluid (100mMTrisCl pH9.0,500mM KCl, 15mM Mg ²⁺, 1%Triton X-100), 100 μ M dNTPs, forward primer 0.2 μ M, reverse primer 0.2 μ M uses ddH ₂O postreaction system to 20 μ l.The PCR reaction conditions is: 94 ℃ of 4min of elder generation; 94 ℃ of 45sec subsequently, 58 ℃ of 45sec, 72 ℃ of 2min, totally 31 circulations; 72 ℃ of 10min again.Be that 1% agarose gel electrophoresis detects in the amplified production whether the purpose band is arranged by concentration, send again AudioCodes order-checking (sequencing primer is HX1R) after detecting bright single purpose band.Sequencing result is compared, finds to compare with 93-11, C base deletion among the tri1 (gagacgca---gagacga), thereby cause encoding sequence generation frameshit.

HX1F：5’-gtactggcaaagcaagatgg-3’；

HX1R：5’-atcgggaagcagattcatcc-3’。

For verifying that whether above-mentioned deletion mutantion is control rice glume development gene TRI1, take HX1 as Marker to the F by HM13 (HM13 is the title of the stable strain of tri1 mutant strain) and C418 structure ₂In 15 recessive exchange individual plants (14 at C1 place, 1 at C2 place) carried out genotype detection, sequencing result finds that the C base deletion identical with mutant tri1 all occured in the mutational site all exchange individual plants.

For further verifying above-mentioned deletion mutantion and affiliated gene thereof and the dependency of controlling rice glume development, according to the method described above 10 normal rice varieties of typical glume development and local kind, 93-11 and tri1 are extracted respectively its genomic dna, under the guiding of primer HX1, carry out pcr amplification, order-checking, analyze its sequence in 10 rice varieties.The electrophorogram of pcr amplification product is seen Fig. 3.The result shows, all 10 rice varieties (glume development is normal) are all consistent with wild-type 93-11 sequence at the nucleotide variation place.

With protein called after TRI1 shown in the sequence 1 of sequence table, with its encoding gene called after TRI1, its encoding sequence is shown in the sequence 2 of sequence table, and genome sequence is shown in the sequence 3 of sequence table.

The acquisition of embodiment 2, TRI1 transgenic paddy rice and glume development phenotypic evaluation thereof

One, the structure of TRI1 plant interference vector

1, the acquisition of dna fragmentation A and dna fragmentation B

(1) design primer

5 ' end length according to the ORF of the LOC_Os02g56610 of 93-11 is the non-conservative primers of 355bp, and introduces respectively restriction enzyme BamHI, KpnI and Spe I, SacI recognition site at the primer two ends, and primer sequence is as follows:

7BHF:5 '- GGATCCAtggatcgtcaccatcacc-3 '; Band underscore base is restriction enzyme BamHI recognition site;

7KNR:5 '- GGTACCActtgaggaactccagcacg-3 '; Band underscore base is restriction enzyme KpnI recognition site;

7SEF:5 '- ACTAGTActtgaggaactccagcacg-3 '; Band underscore base is restriction enzyme SpeI recognition site;

7SCR:5 '- GAGCTCAtggatcgtcaccatcacc-3 '; Band underscore base is restriction enzyme SacI recognition site.

(2) acquisition of dna fragmentation A and dna fragmentation B

Take the genomic dna (this gene does not have intron in the ORF zone) of 93-11 as template, the primer pair that forms with 7BHF and 7KNR carries out pcr amplification, obtains dna fragmentation A; Take the genomic dna of 93-11 as template, the primer pair that forms with 7SEF and 7SCR carries out pcr amplification, obtains dna fragmentation B; PCR parameter: 94 ℃ of 4min of elder generation; 94 ℃ of 30sec subsequently, 58 ℃ of 30sec, 72 ℃ of 1min, totally 31 circulations; 72 ℃ of 10min again.

(3) sequence verification

Dna fragmentation A and dna fragmentation B are carried out respectively 1% agarose gel electrophoresis, be connected respectively to PMD18 carrier (TAKARA) after the recovery, send AudioCodes company to check order.Sequencing result shows: dna fragmentation A, and two ends have respectively BamHI recognition site and KpnI recognition site, hold the DNA shown in the 1st to 355 Nucleotide for the sequence 2 of sequence table from 5 ' between two sites; Dna fragmentation B, two ends have respectively SpeI recognition site and SacI recognition site, hold the DNA shown in the 1st to 355 Nucleotide for the sequence 2 of sequence table from 5 ' between two sites.

Two, the structure of TRI1 plant interference vector

1, BamHI and KpnI enzyme are cut dna fragmentation A;

2, BamHI and KpnI enzyme are cut carrier pTCK303/JL1460, reclaim carrier framework;

3, the enzyme of step 1 is cut the carrier framework connection that product is connected with step, obtained the recombinant vectors first;

4, SpeI and SacI enzyme are cut dna fragmentation B;

5, SpeI and SacI enzyme are cut the recombinant vectors first, reclaim carrier framework;

6, the enzyme of step 4 is cut the carrier framework connection that product is connected with step, the recombinant vectors that obtains is the plant interference vector pTCK303/JL1460-TRI1 of TRI1.

Sequence verification result shows: the skeleton carrier of pTCK303/JL1460-TRI1 is pTCK303/JL1460, has inserted the reverse complemental DNA of sequence 2 from the DNA shown in the 1st to 355 Nucleotide of 5 ' end having inserted sequence 2 between BamHI and the KpnI restriction enzyme site between DNA, the SpeI shown in the 1st to 355 Nucleotide of 5 ' end and SacI restriction enzyme site.

Two, rice transformation

PTCK303/JL1460-TRI1 with the mature embryo callus of spending 17 in the Bombardment-Mediated Transformation, is carried out the two-wheeled screening with the NB substratum that contains the 50mg/L Totomycin, whenever take turns screening 20-30 days, obtain positive plant through breaking up in advance, breaking up.Positive plant is carried out PCR to be identified.PCR primer (according to the design of the hygromycin gene on the carrier): 5 '-AAAAGT TCG ACA GCG TCT CCG ACC-3 '; 5 '-TCT ACA CAG CCA TCG GTC CAG ACG-3 '; PCR reaction conditions: 94 ℃ of 5min of elder generation; Then 94 ℃ of 30sec, 58 ℃ of 30sec, 72 ℃ of 1min, totally 31 circulations; Last 72 ℃ of 10min.After reaction finishes, amplified production is carried out 1% agarose gel electrophoresis detects, detected result as shown in Figure 4, TRI1 transgenic positive plant can amplify the approximately band of 1Kb.2 positive T that phenotypic variation is arranged have finally been obtained through the PCR evaluation in conjunction with phenotypic evaluation ₀For transfer-gen plant.

PTCK303/JL1460 with the mature embryo callus of spending 17 in the Bombardment-Mediated Transformation, is carried out 2 with the NB substratum that contains the 50mg/L Totomycin and takes turns screening, whenever take turns screening 20-30 days, obtain T through breaking up in advance, breaking up ₀In generation, turn the empty carrier adjoining tree.

Three, the PCR of transgenic paddy rice identifies and phenotypic evaluation

With T ₀Carry out selfing for plant, obtain T ₀Seed (T for plant ₁Generation).

With T ₀Cultivate under the same conditions (every kind of 30 strains) for the seed (transfer-gen plant, empty carrier adjoining tree) of plant and middle 17 the seed spent, carry out phenotypic evaluation boot stage.Found that: T ₀For transfer-gen plant the interior coetonium shell deformity similar with mutant occuring, and form triangle grain husk shell phenotype (Fig. 5), shows that TRI1 of the present invention can be used for studying the molecule mechanism of glume development; T ₀It is consistent with middle 17 the phenotype spent that generation turns the empty carrier adjoining tree.

In boot stage, middle 17 the clever shell width spent is 3.44 ± 0.14mm, and the clever shell width that turns the empty carrier adjoining tree is 3.49 ± 0.15mm, and the clever shell width of transfer-gen plant is 3.05 ± 0.18mm.

Sequence table

＜110〉China Agricultural University

＜120〉rice glume development related protein TRI 1 and encoding gene thereof and application

<130>CGGNARY102147

<160>3

<210>1

<211>248

<212>PRT

＜213〉Oryza common cultivated rice (O.sativa L.)

<400>

Met Asp Arg His His His His His His His His His His His Met Met

1 5 10 15

Ser Gly Gly Gly Gln Asp Pro Ala Ala Gly Asp Gly Gly Ala Gly Gly

20 25 30

Ala Thr Gln Asp Ser Phe Phe Leu Gly Pro Ala Ala Ala Ala Met Phe

35 40 45

Ser Gly Ala Gly Ser Ser Ser Ser Gly Ala Gly Thr Ser Ala Gly Gly

50 55 60

Gly Gly Gly Gly Pro Ser Pro Ser Ser Ser Ser Pro Ser Leu Ser Arg

65 70 75 80

Tyr Glu Ser Gln Lys Arg Arg Asp Trp Asn Thr Phe Gly Gln Tyr Leu

85 90 95

Arg Asn His Arg Pro Pro Leu Ser Leu Ser Arg Cys Ser Gly Ala His

100 105 110

Val Leu Glu Phe Leu Lys Tyr Met Asp Gln Phe Gly Lys Thr Lys Val

115 120 125

His Thr Pro Val Cys Pro Phe Tyr Gly His Pro Asn Pro Pro Ala Pro

130 135 140

Cys Pro Cys Pro Leu Arg Gln Ala Trp Gly Ser Leu Asp Ala Leu Ile

145 150 155 160

Gly Arg Leu Arg Ala Ala Tyr Glu Glu Asn Gly Gly Thr Pro Glu Met

165 170 175

Asn Pro Phe Gly Ala Arg Ala Val Arg Leu Tyr Leu Arg Glu Val Arg

180 185 190

Glu Thr Gln Ala Arg Ala Arg Gly Ile Ser Tyr Glu Lys Lys Lys Arg

195 200 205

Lys Lys Pro Ser Ser Ala Gly Ala Gly Ala Gly Pro Ser Ser Glu Gly

210 215 220

Ser Pro Pro Pro Pro Gly Gly Ser Ala Ser Gly Gly Gly Asp Thr Ser

225 230 235 240

Ala Ser Pro Gln Phe Ile Ile Pro

245

<210>2

<211>747

<212>DNA

＜213〉Oryza common cultivated rice (O.sativa L.)

<400>2

atggatcgtc accatcacca ccaccaccac catcaccatc acatgatgtc gggcggcggg 60

caagacccgg cggcggggga cggcggcgcc ggcggcgcca cgcaggacag cttcttcctc 120

ggcccggccg cggccgccat gttctccggc gccgggtcgt cgtcgtcggg cgcggggacg 180

tcggcgggag gcggcggcgg cgggccatcg ccgtccagct cgtcgccgtc gctgagccgg 240

tacgagtcgc agaagcggcg ggactggaac acgttcgggc agtacctgcg gaaccaccgg 300

ccgccgctgt ccctgtcgcg gtgcagcggc gcgcacgtgc tggagttcct caagtacatg 360

gaccagttcg ggaagacgaa ggtgcacacg ccggtgtgcc ccttctacgg ccaccccaac 420

ccgccggcgc catgcccgtg cccgctccgc caagcctggg gctccctcga cgcgctcatc 480

ggccgcctcc gcgccgccta cgaggagaac ggcggcacgc cggagatgaa ccccttcggc 540

gcccgcgccg tccgcctcta cctgcgcgag gtgcgcgaga cgcaggccag ggcgaggggg 600

atcagctacg agaagaagaa gcgcaagaag ccctcgtccg ccggcgccgg agccgggccc 660

tcgtccgagg ggagcccgcc gccacccggc ggctcggcca gcggcggcgg cgacacgtcg 720

gcgtcgccgc agttcatcat cccgtga 747

<210>3

<211>2091

<212>DNA

＜213〉Oryza common cultivated rice (O.sativa L.)

<400>3

atttctacct cacacaccta gctagcaagt aggctctctc actccacacc cacactactc 60

ctcccaggct gcctgcctaa aaggctagac ctatccatct cttcccccca ctcctccagc 120

atcgccatca tccactgttc atccactcca tctctctctc tctctctctc tctccatcga 180

tccctgcagg ttcttgctgc tgctgctgct gccttgcttg ctgtcaagca tggcttgacc 240

acctgagagc gaggagagca tagcgtagta cttggctagc tgctgttcaa ttcctcatgg 300

atgattggag gatcgctagc taggtcgccc ggatccacgg acacctctcc tcgtctcgtg 360

ctcgtgcatg ccaagatcga tcgatcccag ctgctgctgc gagtggagca gtggaggagg 420

agatcggctg ctacctgacc tagatcggga agcagattca tccggtacat gttatatata 480

gatatagatc gttgcttagg ttcttctctt cttgtttgat ttcgtcggag caagaaatga 540

tgcttgcttc ttgtgattca ggttgcatta cggcggcgag gcgagggcga ggatggatcg 600

tcaccatcac caccaccacc accatcacca tcacatgatg tcgggcggcg ggcaagaccc 660

ggcggcgggg gacggcggcg ccggcggcgc cacgcaggac agcttcttcc tcggcccggc 720

cgcggccgcc atgttctccg gcgccgggtc gtcgtcgtcg ggcgcgggga cgtcggcggg 780

aggcggcggc ggcgggccat cgccgtccag ctcgtcgccg tcgctgagcc ggtacgagtc 840

gcagaagcgg cgggactgga acacgttcgg gcagtacctg cggaaccacc ggccgccgct 900

gtccctgtcg cggtgcagcg gcgcgcacgt gctggagttc ctcaagtaca tggaccagtt 960

cgggaagacg aaggtgcaca cgccggtgtg ccccttctac ggccacccca acccgccggc 1020

gccatgcccg tgcccgctcc gccaagcctg gggctccctc gacgcgctca tcggccgcct 1080

ccgcgccgcc tacgaggaga acggcggcac gccggagatg aaccccttcg gcgcccgcgc 1140

cgtccgcctc tacctgcgcg aggtgcgcga gacgcaggcc agggcgaggg ggatcagcta 1200

cgagaagaag aagcgcaaga agccctcgtc cgccggcgcc ggagccgggc cctcgtccga 1260

ggggagcccg ccgccacccg gcggctcggc cagcggcggc ggcgacacgt cggcgtcgcc 1320

gcagttcatc atcccgtgag ttcttctcga tccccatcgc catctccgca tctccattga 1380

tcgatcgagc ctcgcagcaa gaaattccaa tctccttggc agaaatgcca tcttgctttg 1440

ccagtacaag gcatcatatc tcttctttcc tcctgaattg gtttcgatct ctcccatttt 1500

gcaaaaaaga tttctcctct ttttaatttc atctctgtct cgctttctaa gctaggacga 1560

aggtcatgca tgagtgagta cttagtttaa ttcttatttt tttccttagc tttgggacgt 1620

caattaattg gtagctgctg tcttcgatcg atctcgtccc gtttggttta attattttga 1680

tgcaagaact ggagtaaatt tgatgagaat ctcaagctag ctagcctaat taggagttgt 1740

aatatttgtt ttagtctggg ggatttgcaa gaacaaaagt agcagaagct gtaggcatag 1800

caagctagct agtgttagta tctgtagaat ttgttcttgt aatagcttgt cgccaagcta 1860

gccaagtgca gtggcgtcag caatgcttcc ttctatctat catgtactag tgatgatgtc 1920

ttggtcttcc tttcttggtt gagcaaaagg gcaaaaaggg agagatcgac tagctgatga 1980

gatgatgagt gatttcaatt ggtagcttaa ttaagacaga aacaacatgt gttatgtgta 2040

cttgtcctgg gaacagattc catatatatg ccaaatgctt taatttgggc t 2091

Claims (4)

1. recombinant expression vector is characterized in that: described recombinant expression vector is to insert respectively the recombinant plasmid that dna fragmentation A and dna fragmentation B obtain between the multiple clone site of pTCK303/JL1460-TRI1; The sequence 2 that described dna fragmentation A is sequence table is from the DNA shown in the 1st to 355 Nucleotide of 5 ' end; The sequence 2 that described dna fragmentation B is sequence table is from the reverse complemental DNA of the DNA shown in the 1st to 355 Nucleotide of 5 ' end.

2. recombinant expression vector as claimed in claim 1, it is characterized in that: described dna fragmentation A inserts between the BamHI and KpnI restriction enzyme site of pTCK303/JL1460-TRI1, and described dna fragmentation B inserts between the SpeI and SacI restriction enzyme site of pTCK303/JL1460-TRI1.

3. a method of cultivating the transgenic paddy rice that clever shell width diminishes is that claim 1 or 2 described recombinant expression vectors are imported in the purpose paddy rice, obtains clever shell width less than the transgenic paddy rice of described purpose paddy rice.

4. method as claimed in claim 3, it is characterized in that: described purpose paddy rice is to spend 17 in the rice varieties.

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