CN102628059B

CN102628059B - Paddy rice OsSPX1 protein and application of encoding gene of paddy rice OsSPX1 protein to regulation and control of plant seed maturing rate

Info

Publication number: CN102628059B
Application number: CN 201210101772
Authority: CN
Inventors: 苏震; 徐文英; 魏强; 王玲; 刘凤霞; 于静娟; 赵琳娜; 张群莲; 王春超
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2012-04-09
Filing date: 2012-04-09
Publication date: 2013-09-25
Anticipated expiration: 2032-04-09
Also published as: CN102628059A

Abstract

The invention discloses a paddy rice OsSPX1 protein and an application of an encoding gene of the paddy rice OsSPX1 protein to regulation and control of plant seed maturing rate. The amino acid sequence of the protein is shown as a sequence 3 in a sequence table, and can be used for regulating and controlling the seed maturing rate of a target plant or regulating and controlling a substance of a protein expression amount shown as a sequence 3 in the sequence table, or the method can be used for regulating and controlling the seed maturing rate of a target plant. As proved by an experiment, the relative expression amount of the paddy rice OsSPX1 protein of a pCOU OsSPX1-antisense paddy rice strain is remarkably lowered, and the seed maturing rate and effective grain number are reduced remarkably in comparison to non-transgenic nipponbare. The paddy rice OsSPX1 protein has an important application value on the aspect of research on the yield, attribute and formation of plant seeds.

Description

The application in regulation and control plant seed setting percentage of rice Os SPX1 albumen and encoding gene thereof

Technical field

The present invention relates to the application in regulation and control plant seed setting percentage of a kind of rice Os SPX1 albumen and encoding gene thereof.

Background technology

The seed-setting rate is an important factor that forms crop yield.It is many to influence rice paddy seed setting percentage factor, comprises the nature-nurture factor.Excavating the excellent gene relevant with the seed-setting rate in paddy rice, and they are applied to have important theoretical meaning and more practical value in the rice breeding production, also is an effective way of current raising rice high yield breeding.

Inventor research group finds the usefulness that this gene has simultaneously influences the rice paddy seed setting percentage when coercing relevant paddy rice SPX gene function with responding low-phosphor in that research is low temperature resistant, may exert an influence to rice yield, have the gene of strong using value in agriculture production.

Have some and studies show that the gene that contains SPX (SYG1/Pho81/XPR1) structural domain participates in signal transduction and the adjusting approach of phosphorus.SPX is about 180 amino acid longs, is present in a structural domain of 3 kinds of known proteins (SYG1, Pho81 and XPR1) N-terminal, and its title derives from the initial of these three kinds of albumen.As far back as nineteen ninety-five, thereby discovering the N-terminal of yeast SYG1, people such as Spain can suppress the signal transduction of mating pheromone (mating pheromone) with the G-protein binding; Under the phosphate starvation condition, there is report CDK to suppress the enzymic activity that sub-Pho81 can suppress PHO80-PHO85 and Pcl7-Pho85 complex body; People such as Poleg (1996) find in fungi Neurospora crassa, but with the existence of the NUC-2 albumen perception phosphorus of PHO81 structural similitude and participate in the adjusting approach of phosphorus transporter.2002, people such as Hamburger also found a class SPX gene in Arabidopis thaliana, i.e. PHO1 (having SPX structural domain and EXS structural domain) gene and PHO1 albuminoid, they can participate in phosphorus by the transportation of root to stem, and in the microtubule fasolculus cell of root specifically expressing.

Summary of the invention

An object of the present invention is to provide the new purposes of rice Os SPX1 albumen, the aminoacid sequence of this protein is shown in sequence table sequence 3, described new purposes is the seed-setting rate that albumen shown in the sequence table sequence 3 can be used for regulating and control the purpose plant, or regulates the material of expressing quantity shown in the sequence table sequence 3 or the seed-setting rate that method can be used for regulating and control the purpose plant.

Another object of the present invention provides a kind of method of cultivating low setting percentage or low seed production transgenic plant, this method is to reduce the expression of the encoding gene of albumen shown in the sequence table sequence 3 in the purpose plant, obtains the transgenic plant that at least a proterties in seed-setting rate and the real grain number is lower than described purpose plant.

In aforesaid method, the expression of the encoding gene of albumen shown in the sequence table sequence 3 is to import in the purpose plant by the complementary sequence with protein coding gene shown in the sequence table sequence 3 to realize in the described reduction purpose plant.

The present invention also provides another kind of method of cultivating high setting percentage or high seed production transgenic plant, this method is that the encoding gene of albumen shown in the sequence table sequence 3 is imported in the purpose plant, obtains the transgenic plant that at least a proterties in seed-setting rate and the real grain number is higher than described purpose plant.

In above-mentioned two kinds of methods, the encoding gene of albumen is following 1 shown in the described sequence table sequence 3) or 2) or 3) gene:

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

2) with 1) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have the dna molecular of albumen shown in 99% identity and the code sequence tabulation sequence 3 at least at least;

3) under stringent condition with 1) or 2) dna molecular of albumen shown in the dna sequence dna hybridization that limits and the code sequence tabulation sequence 3;

Described stringent condition can be as follows: 50 ℃, and at 7% sodium lauryl sulphate (SDS), 0.5M Na ₃PO ₄With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 2 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na ₃PO ₄With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 1 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na ₃PO ₄With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 0.5 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na ₃PO ₄With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 0.1 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na ₃PO ₄With hybridize in the mixing solutions of 1mM EDTA, at 65 ℃, 0.1 * SSC, rinsing among the 0.1%SDS; Also can be: at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of hybridization down, use 2 * SSC then, 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

In above-mentioned application and method, described purpose plant can be monocotyledons or dicotyledons.

In above-mentioned application and method, described monocotyledons specifically can be paddy rice.

Another object of the present invention provides a kind of dna molecular, the nucleotides sequence of this dna molecular is classified the complementary sequence of protein coding gene shown in the described sequence table sequence 3 as, this dna molecular can reduce the expression of the encoding gene of albumen shown in the sequence table sequence 3 in the purpose plant after importing the purpose plant, thereby reduces seed-setting rate and the real grain number of purpose plant.

The recombinant vectors, expression cassette, transgenic cell line, reorganization bacterium or the recombinant virus that contain described dna molecular also belong to protection scope of the present invention;

The described recombinant vectors that contains described dna molecular specifically can be pCOU OsSPX1_antisense, described pCOUOsSPX1_antisense be dna fragmentation that nucleotide sequence is following through inserting the pCambia1301-UbiN multiple clone site behind restriction enzyme BglII and the KpnI double digestion BglII and the KpnI restriction enzyme site between the recombinant vectors that obtains: be followed successively by nucleotide sequence complementary sequence shown in restriction enzyme BglII recognition sequence, the sequence table sequence 2 and restriction enzyme KpnI recognition sequence by 5 ' to 3 ' end.

The present invention also provides a kind of PCR primer for the identification of the expression of protein coding gene shown in the sequence table sequence 3 right, and described PCR primer is to being made up of the single stranded DNA shown in the single stranded DNA shown in the sequence table sequence 4 and the sequence table sequence 5; Or formed by the single stranded DNA shown in the single stranded DNA shown in the sequence table sequence 6 and the sequence table sequence 7.

Experiment showed, the rice strain and fine the comparing of non-transgenic Japan that change pCOU OsSPX1_antisense, the relative expression quantity of its rice Os SPX1 gene obviously reduces, and seed-setting rate and real grain number also significantly reduce simultaneously.The present invention has significant application value aspect the formation of research plant seed yield traits.

Description of drawings

Fig. 1 is the structural representation in the positive antisense recombinant expression vector T-DNA district of rice Os SPX1 gene.Wherein, figure A is just recombinant expression vector, figure B is the antisense recombinant expression vector, left arm and right arm that LB among figure A and the B and RB represent T-DNA respectively, Nos represents nopaline synthase terminator, and Hpt II represents hygromycin phosphotransferase gene, and CaMV35s represents cauliflower mosaic virus 35s promotor, Ubi represents the corn ubiquitin promoter, and OsSPX1 represents the rice Os SPX1 encoding gene shown in the sequence table sequence 2.

Fig. 2 identifies electrophorogram for the PCR of transgenic paddy rice strain system.Wherein, figure A is for changeing the T of pCOU 0sSPX1_antisense ₀For the qualification result of transgenic paddy rice strain system, swimming lane M is molecular weight standard, and fragment from top to bottom is followed successively by 5kb, 3kb, and 2kb, 1kb, 750bp, 500bp, 250bp, 100bp, swimming lane 8 is that non-transgenic Japan is fine, swimming lane 1-7 is T to be identified ₀In generation, changeed pCOU OsSPX1_antisense rice strain; Figure B is for changeing the T of pCOU OsSPX1 ₀For the qualification result of transgenic paddy rice strain system, swimming lane M is molecular weight standard, and fragment from top to bottom is followed successively by 5kb, 3kb, and 2kb, 1kb, 750bp, 500bp, 250bp, 100bp, swimming lane 9 is that non-transgenic Japan is fine, swimming lane 1-8 is T to be identified ₀In generation, changeed pCOU OsSPX1 rice strain.

Fig. 3 detects relative expression's level of OsSPX1 gene in the transgenic paddy rice strain system for real-time quantitative RT-PCR.Wherein, A is the rice strain of adopted recombinant expression vector of becoming a full member, and B is the rice strain of antisense recombinant expression vector.

Fig. 4 is T ₁In generation, changes the rice plant of pCOU OsSPX1 and changes the fine performance of planting in the land for growing field crops of rice plant and non-transgenic Japan of pCOU OsSPX1_antisense.

Fig. 5 is T ₁The small ear phenotype of gathering in the crops on the rice plant of generation commentaries on classics pCOU OsSPX1 and the rice plant of commentaries on classics pCOU OsSPX1_antisense and non-transgenic Japan are fine.

Fig. 6 is T ₁Seed bearing phenotype the rice plant of generation commentaries on classics pCOU OsSPX1 and the rice plant of commentaries on classics pCOU OsSPX1_antisense and non-transgenic Japan are fine.

Fig. 7 is T ₁Generation change the rice plant of pCOU OsSPX1 and change the rice plant of pCOU OsSPX1_antisense fine with non-transgenic Japan on seed bearing total the number of institute, the bar graph of a number, setting percentage and thousand grain weigth in fact, Nipponbare wherein is that non-transgenic Japan is fine.

Embodiment

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

Method therefor is ordinary method if no special instructions among the following embodiment, and the primer synthesizes and examining order is finished by Beijing AudioCodes biotechnology limited liability company.

Japan fine (Oryza sativa L.cv.Nipponbare): China Agricultural University guarantees to provide to the public; Reference: Gene expression profiles deciphering rice phenotypic variation between Nipponbare (Japonica) and 93-11 (Indica) during oxidative stress.PLoS One.2010Jan 8,5 (1).

Agrobacterium tumefaciens EHA105 (Agrobacterium tumefaciens EHA105): China Agricultural University guarantees to provide to the public; Reference: Toki S, Hara N, Ono K, Onodera H, Tagiri A, Oka S, Tanaka be infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice.Plant.J.47 (6) H.2006.Early: 969-76.

The acquisition of embodiment 1, rice Os SPX1 gene and albumen thereof

Fine (Oryza sativa L.cv.Nipponbare) the bud phase of paddy rice Japan with 4 ℃ of subzero treatment 12h, (germination 7-10 days seedling) plant was material, utilize TRIZOL reagent to extract total RNA, and be template with total RNA, use the M-MLV ThermoScript II to carry out reverse transcription and obtain cDNA, be template with this cDNA again, under the guiding of primer CDS-PF and primer CDS-PR, cDNA sequence with conventional PCR method amplifying rice SPX gene, after reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, reclaim the also dna fragmentation of the about 900bp of purifying, this fragment is checked order.

Primer CDS-PF:5 '- ATAGATCTATGAAGTTTGGGAAGAGCCTG-3 ' (band underscore partial sequence is restriction enzyme BglII recognition site and protection base);

Primer CDS-PR:5 '- GCGGTACCTCATTTGGCGGCCTGCTCAAT-3 ' (band underscore partial sequence is restriction enzyme KpnI recognition site and protection base).

Sequencing result shows, the dna fragmentation of above-mentioned about 900bp contains the 888bp nucleotide sequence shown in the ordered list sequence 2, this 888bp nucleotides sequence is classified the encoding sequence of OsSPX1 gene as, coding has the albumen of being made up of 295 amino acid shown in the sequence table sequence 3, with this albumen called after OsSPX1, this albumen is the SPX protein structure domain from aminoterminal (N end) 1-169 amino acids residue.The genomic dna of OsSPX1 is positioned paddy rice No. six chromosomal 23,875,408bp to 23,879,965bp (its sequence is shown in sequence table sequence 1), its NCBI location number is NP_001058013, and NCBI gene number (GENE ID) is: Os06g0603600, TIGR number is LOC_Os06g40120.

The structure of embodiment 2, the positive antisense recombinant expression vector of rice Os SPX1 gene

The dna fragmentation that contains nucleotide sequence shown in the ordered list sequence 2 of about 900bp that pcr amplification in the step 1 is obtained carries out double digestion with BglII and KpnI, be inserted into plant expression vector pCambia1301-UbiN (GenBank number: AF234297) between the BglII of multiple clone site and the KpnI restriction enzyme site after reclaiming purifying, obtain the just recombinant expression vector of OsSPX1 gene, should the justice recombinant expression vector carry out the enzyme evaluation of cutting and check order, evaluation is shown the just recombinant expression vector called after pCOU OsSPX1 that contains nucleotide sequence shown in the ordered list sequence 2, and the structural representation in its T-DNA district is shown in the A among Fig. 1.

Simultaneously, make up the antisense recombinant expression vector of OsSPX1, concrete grammar is as follows: design primer 5 '- GCGGTACCATGAAGTTTGGGAAGAGCCTG-3 ' (band underscore partial sequence is restriction enzyme KpnI recognition site and protection base) and 5 '- ATAGATCTTCATTTGGCGGCCTGCTCAAT-3 ' (band underscore partial sequence is restriction enzyme BglII recognition site and protection base); carry out pcr amplification according to embodiment 1 identical method; reclaim the PCR product of the about 900bp of purifying; carry out double digestion with BglII and KpnI; insert between the BglII and KpnI restriction enzyme site of plant expression vector pCambia1301-UbiN multiple clone site after reclaiming purifying; obtain the antisense recombinant expression vector of OsSPX1 gene; this antisense recombinant expression vector is carried out the enzyme evaluation of cutting and check order; evaluation is shown the antisense recombinant expression vector called after pCOU 0sSPX1_antisense that contains with the sequence of nucleotide sequence complementation shown in the sequence table sequence 2, and the structural representation in its T-DNA district is shown in the B among Fig. 1.

The acquisition of embodiment 3, transgenic paddy rice

The callus that just recombinant expression vector pCOU OsSPX1, the antisense recombinant expression vector pCOU OsSPX1_antisense of the rice Os SPX1 that embodiment 2 is made up induces with the mature embryo of Agrobacterium-mediated Transformation method rice transformation Japan fine (Oryza sativa L.cv.Nipponbare), concrete grammar is as follows:

1, pCOU OsSPX1 and pCOU OsSPX1_antisense are transformed agrobacterium tumefaciens EHA105 with electric shocking method respectively and obtain the reorganization Agrobacterium EHA105/pCOU OsSPX1_antisense that contains the reorganization Agrobacterium EHA105/pCOU 0sSPX1 of pCOU OsSPX1 and contain pCOU OsSPX1_antisense totally two kinds of reorganization Agrobacteriums.

Above-mentioned two kinds of reorganization Agrobacteriums are inoculated in respectively in the YEB liquid medium (containing 100 μ g/ml kantlex and 75 μ g/ml Rifampins), and 28 ℃ of shaking culture are to OD ₆₀₀Be 0.6-0.8; With the centrifugal 1min of 10,000rpm room temperature, with MS salts solution (pH 5.8) resuspended thalline and the 20-50 that is diluted to the original fluid bacteria concentration doubly, obtain the bacteria suspension of two kinds of reorganization Agrobacteriums.

The prescription of above-mentioned MS salts solution: NH ₄NO ₃1.65g, KNO ₃1.9g, KH ₂PO ₄0.17g, MgSO ₄7H ₂O 0.7g, CaCl ₂2H ₂O 0.44g, MnSO ₄4H ₂O 22.3mg, ZnSO ₄7H ₂O8.6mg, H ₃BO ₃6.2mg, KI 0.83mg, Na ₂MoO ₄2H ₂O 0.25mg, CoCl ₂6H ₂O 0.025mg, CuSO ₄5H ₂O 0.025mg, FeSO ₄7H ₂O 27.8mg, Na ₂-EDTA2H ₂O 37.3mg, inositol 100mg, nicotinic acid (B ₅) 0.5mg, pyridoxine hydrochloride (vitamins B ₆) 0.1mg, vitamin (vitamins B ₁) 0.5mg, glycine 2.0mg, water is settled to 1L.

2, paddy rice Japan fine (WT, the Oryza sativa L.cv.Nipponbare) seed after will sterilizing is inoculated on the N6D substratum (pH5.8), cultivates 1-5 days for 32 ℃, removes behind bud and the residual endosperm succeeding transfer culture 10-20 days again, obtains mature embryo callus.

The prescription of above-mentioned N6D substratum: KNO ₃2.83g/L, (NH ₄) ₂SO ₄0.463g/L, MgSO ₄7H ₂O 0.185g/L, CaCl ₂0.166g/L, KH ₂PO ₄0.4g/L; MnSO ₄4H ₂O 4.4mg/L, ZnSO ₄7H ₂O 1.5mg/L, KI 0.8mg/L, H ₃BO ₃1.6mg/L, FeSO ₄7H ₂O 27.8mg/L, Na ₂EDTA2H ₂O 37.3mg/L; 2,4-D 2mg/L, CH:0.3g/L, L-proline(Pro) 2.878g/L, sucrose 30g/L, solidifying agent 4g/L.

3, the mature embryo callus that step 2 is obtained is divided into 2 groups, every group is dipped in respectively in two kinds of Agrobacterium bacteria suspensions that step 1 obtains, rock about 1.5min gently, be inoculated on the N6D substratum (DH=5.2) that contains the 10-20mg/L Syringylethanone, be lined with one deck on this substratum in advance and be soaked with 0.5 milliliter of AAM substratum (Hiei, Y., S.0hta, TKomari andT.Kumashiro.1994.Efficient transformation of rice (Oryza sativa L.) mediated by agrobacterium and sequence analysis of theboundaries of the T-DNA.Plant is J.6:271-282.) aseptic filter paper, cultivated altogether 3 days under 25 ℃ of dark conditions.

4, will be inoculated in the middle 1-2 of cultivation of the RE division culture medium (pH 5.8) that contains 50mg/L Totomycin (hygromycin B) and 250mg/L Pyocianil (carbenicillin) through the callus that step 3 is cultivated altogether after week, choose that eugonic resistant calli breaks up, strong sprout, obtain to change respectively the T of pCOU OsSPX1 and pCOUOsSPX1_antisense ₀For the transgenic paddy rice strain be.

The prescription of above-mentioned RE division culture medium: NH ₄NO ₃1.65g/L, KNO ₃1.9g/L, KH ₂PO ₄0.17g/L, MgSO ₄7H ₂O 0.7g/L, CaCl ₂2H ₂O 0.44g/L, MnSO ₄4H ₂O 22.3mg/L, ZnSO ₄7H ₂O 8.6mg/L, H ₃BO ₃6.2mg/L, KI 0.83mg/L, Na ₂MoO ₄2H ₂O 0.25mg/L, CoCl ₂6H ₂O 0.025mg/L, CuSO ₄5H ₂O 0.025mg/L, FeSO ₄7H ₂O 27.8mg/L, Na ₂-EDTA2H ₂O 37.3mg/L, inositol 100mg/L, nicotinic acid (B ₅) 0.5mg/L, pyridoxine hydrochloride (vitamins B ₆) 0.5mg/L, vitamin (vitamins B ₁) 0.1mg/L, sucrose 30g/L, sorbyl alcohol 30g/L, acid hydrolyzed casein 2g/L, a-naphthylacetic acid 0.02mg/L, kinetin 2mg/L, furfuryladenine 2mg/L, plant gel 4g/L.

T ₀Transgenosis plant in the present age, T are shown in representative ₁T is shown in representative ₀The seed that produces for selfing reaches the plant that is grown up to by it, T ₂T is shown in representative ₁The seed that produces for selfing reaches the plant that is grown up to by it, T ₃T is shown in representative ₂The seed that produces for selfing reaches the plant that is grown up to by it.

5, the PCR of transgenic paddy rice strain system identifies

All plant leafs in two kinds of paddy rice transgenic lines of step 4 acquisition are extracted genomic dna respectively, carry out pcr amplification, primer is HPT-F:5 '-TACTTCTACACAGCCATC-3 ' and HPT-R:5 '-CGTCTGTCGAGAAGTTTC-3 ', target sequence is the partial sequence of hygromycin phosphotransferase gene, prediction purpose product sheet segment length 947bp, the result conforms to expection, and the agarose gel electrophoresis result of part amplified production as shown in Figure 2.Obtain the T of the positive pCOU of commentaries on classics OsSPX1 altogether ₀Be 8 for the transgenic paddy rice strain, the positive T that changes pCOUOsSPX1_antisense ₀It is 9 for the transgenic paddy rice strain.

The real-time quantitative RT-PCR of embodiment 4, transgenic paddy rice detects

Get respectively non-transgenic Japan fine, identify the T that is positive according to the PCR of step 5 among the embodiment 3 ₁In generation, changes 2 of the rice strains of pCOUOsSPX1 and identifies that according to the PCR of step 5 among the embodiment 32 of the rice strains of the commentaries on classics pCOUOsSPX1_antisense that is positive are at the about 120 days plant boot leaf of field planting, utilize TRIZOL reagent to extract total RNA, be template with this RNA, use the M-MLV ThermoScript II to carry out reverse transcription and obtain cDNA, be template with this cDNA, utilize the primer special shown in the table 1, carry out real-time quantitative RT-PCR and detect.

The real-time quantitative RT-PCR amplimer of table 1, each transgenic line of paddy rice

Reaction system: 4 μ l cDNA templates (2ng/ μ l), 4.5 μ l SYBR Green Master Mix, 1 μ l special primer, 0.5 μ l ddH ₂O.

Amplification program (detection system of utilizing MJ Research company to produce): 95 ℃ of 5min, 1 circulation, 95 ℃ of 30s, 60 ℃ of 30s read plate (read plate), and 85 ℃ of 1s read plate (read plate), totally 40 circulations, 72 ℃ of 2min make solubility curve every 0.2 ℃ for 65 ℃-95 ℃.3 repetitions are established in above-mentioned experiment.

Primer

1 and 2 is to detect OsAPX1 expression of gene amount in the rice strain that changes pCOU OsSPX1, and target sequence is the characteristic fragment of OsSPX1 gene coding region;

Primer

3 and 4 is the degree that are suppressed that detect its endogenous OsSPX1 genetic expression of rice strain of changeing OsSPX1_antisense, disturbs for getting rid of, and target sequence is the characteristic fragment on the OsSPX1 gene 3 ' URT.

The result is shown in A and the B among Fig. 3 among Fig. 3: change that OsSPX1 expression of gene amount is significantly improved than non-transgenic is Japanese fine in the rice strain (Ox-line1 and Ox-line2) of pCOU OsSPX1, and that the middle OsSPX1 expression of gene amount of rice strain (Anti-line1 and Anti-line2) of changeing pCOU OsSPX1_antisense is starkly lower than non-transgenic Japan is fine.

The seed-setting rate statistics of embodiment 5, transgenic paddy rice strain system

PCR according to step 5 among the embodiment 3 identifies the T that is positive ₁In generation, changeed rice plant and the T of pCOU OsSPX1 ₁In generation, changeed fine normal the plantation in big Tanaka of rice plant and non-transgenic Japan of pCOU OsSPX1_antisense, observes the situation of its upgrowth situation and seed-setting.With fine the comparing of non-transgenic Japan, rice strain's heading evening, grouting of changeing pCOU OsSPX1_antisense late, slowly ripe, and it is better or close to change the growth situation of rice strain of pCOU OsSPX1, as shown in Figure 3.

Simultaneously, observe T ₁Fringe grain situation result such as Fig. 5 and Fig. 6 that the rice plant of generation commentaries on classics pCOU OsSPX1 and the rice plant of commentaries on classics pCOU OsSPX1_antisense and non-transgenic Japan are fine, T ₁The fringe of rice strain (Anti-line1 and Anti-line2) that generation is changeed pCOU OsSPX1_antisense is little, the fringe grain less, flat grain is many, and T ₁Then fringe is big, the fringe grain is many, plumpness is high in the fine rice strain of rice strain (Ox-line1 and Ox-line2) and background material non-transgenic Japan of generation commentaries on classics pCOU OsSPX1.

Add up T respectively ₁In generation, changeed rice strain and the T of pCOU OsSPX1 ₁Generation the rice strain that changes pCOU OsSPX1_antisense with the fine not homophyletic system (getting 10-15 individual plant at random in each strain system) of non-transgenic Japan on average total the number of institute's seed of tie, a real number, setting percentage (setting percentage=reality number/always a number) and thousand seed weight, result such as table 2 and shown in Figure 7.With fine the comparing of non-transgenic Japan, the real grain of the seed number, the setting percentage that change the rice strain of pCOU OsSPX1_antisense obviously reduce, and show that the OsSPX1 gene has vital role aspect the setting percentage of adjusting and controlling rice seed.

Table 2T ₁Seed production proterties investigation result (mean+SD) for transgenic paddy rice strain system

Strain system	Total grain number	Real grain number	Setting percentage	Thousand seed weight (gram)
					Non-transgenic Japan is fine	987±116	733±85	75.2％±1.6％	20.0±0.40
Anti-line1	773±75	244±59 ^**	30.8％±6.0％ ^**	18.0±0.61
					Anti-line2	1169±139	438±51 ^**	37.6％±1.3％ ^**	20.4±0.47
Ox-line1	1340±164	1044±125 ^*	78.2％±2.0％	20.2±0.44
					Ox-line2	1206±155	848±114	70.7％±2.4％	18.1±0.40

Annotate: it is remarkable that * * represents P＜0.01 level difference; * it is remarkable to represent P＜0.05 level difference; Fine the comparing of the result of transgenic line and non-transgenic Japan.

Claims

1. the application of albumen shown in the sequence table sequence 3 in regulation and control purpose plant seed setting percentage; Described purpose plant is paddy rice.

2. application according to claim 1 is characterized in that: the nucleotide sequence of the encoding gene of albumen is the dna molecular shown in the sequence table sequence 2 shown in the described sequence table sequence 3.

3. cultivate the method for hanging down setting percentage or low seed production transgenic plant for one kind, be to reduce the expression of the encoding gene of albumen shown in the sequence table sequence 3 in the purpose plant, obtain the transgenic plant that at least a proterties in seed-setting rate and the real grain number is lower than described purpose plant;

Described purpose plant is paddy rice.

4. method according to claim 3 is characterized in that: the expression of the encoding gene of albumen shown in the sequence table sequence 3 is to import in the purpose plant by the complementary sequence with protein coding gene shown in the sequence table sequence 3 to realize in the described reduction purpose plant.

5. according to claim 3 or 4 described methods, it is characterized in that: the nucleotide sequence of the encoding gene of albumen is the dna molecular shown in the sequence table sequence 2 shown in the described sequence table sequence 3.

6. a method of cultivating high setting percentage or high seed production transgenic plant is in the encoding gene importing purpose plant with albumen shown in the sequence table sequence 3, obtains at least a transgenic plant that are higher than described purpose plant in seed-setting rate and the real grain number;

Described purpose plant is paddy rice.

7. method according to claim 6, it is characterized in that: the nucleotide sequence of the encoding gene of albumen is the dna molecular shown in the sequence table sequence 2 shown in the described sequence table sequence 3.