CN110257422B - Application and application method of OsGPT1 gene - Google Patents

Abstract

An application and an application method of OsGPT1 gene, belonging to the field of biotechnology. The invention provides an OsGPT1 gene on one hand, and provides application and an application method of the OsGPT1 gene on the other hand. The invention has the following beneficial effects: the applicant discovers that the rice seed increase and thousand kernel weight increase can be realized by overexpression of the OsGPT1 gene in rice through research on the OsGPT1 gene, and based on the discovery, the invention realizes the improvement of rice varieties by performing overexpression on the OsGPT1 gene in rice to regulate the size and the thousand kernel weight of the rice.

Description

Application and application method of OsGPT1 gene

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application and an application method of an OsGPT1 gene.

Background

Rice is the food crop with the largest edible population on the earth. It is estimated that the rice yield must reach 8 million tons to solve the problem of global population satiety by 2025 years, whereas the rice yield per year in 2003 is 5.85 million tons (Virk PS,2004), so that the average rice yield per hectare worldwide needs to be increased by 5-8.5 tons from twenty years to 2025 years. And as the cultivated land area is reduced year by year, the rice planting area is also reduced year by year, so the yield increase per hectare is far higher than the expected yield by 2025 to solve the problem of the world population satiety.

The organelle that stores starch in the endosperm of higher plants is a highly specialized plastid, Amyloplast (Amyloplast), mainly used for the synthesis and storage of starch (Sakamoto et al, 2008). Starch assembles into transparent granules, Starch Granules (SGs), in the powder-making body. Since the inner space of the amyloplast is mostly filled with starch, the volume of the amyloplast is almost equal to the volume of the Starch Granules (SGs). Starch Granules (SGs) in rice endosperm have a diameter of about 10-20 μm (Matsushima et al, 2010), and each amyloplast contains only one Starch Granule (SGs), but each Starch Granule (SGs) consists of a plurality of water-caltrop-defined polyhedral Starch granules (Starch granules) having a diameter of about 3-8 μm.

Glucose-6-phosphate/phosphate transporter (GPT) can specifically transport Glucose-6-phosphate (Gluose-6-phosphate, Glc6P) into the plastid, while inorganic phosphate (P) is simultaneously transported_i) Or Triose Phosphates (TPs) out of the plastids. In plastids, Glc6P can be used as an important precursor for starch or fatty acid synthesis. Rice is important grain crop, starch in plastidThe synthesis and accumulation are of great significance not only for rice yield, but also for maintaining normal fertility of male gametophyte pollen. AtGPT1 is important in the function of Arabidopsis pollen development, but the functional research of homologous genes in rice is blank.

RNA-seq data analysis showed that: expressing genes related to ribosome assembly, shearing and oxidative phosphorylation at early and middle stages of rice endosperm development; the plant hormone, galactose metabolism and carbon fixation related genes are obviously up-regulated in the middle development stage of endosperm; genes associated with plant disease resistance, stress response and sugar or starch metabolism are highly expressed in late endosperm development (Gao et al, 2013). OsGPT1 in rice (Oryza sativa L.) was cloned by virtue of the amino acid sequence of maize GPT. In addition, two other genes OsGPT2-1(LOC _ Os07g33954) and OsGPT2-2(LOC _ Os07g34006) are predicted in the rice genome database (OsGPT2-1 only lacks 60aa at the N-terminal than OsGPT2-2, the remaining amino acid sequences are completely identical, and the 60aa segment deleted from OsGPT2-1 appears in the predicted 5' -UTR region, and theoretically, the two genes can be transcribed and translated into completely identical protein sequences, but the promoter regions are different in sequence due to the difference in the position on the chromosome, so that the two genes are named as OsGPT2-1 and OsGPT 2-2). Since CDS sequences of OsGPT2-1 and OsGPT2-2 are completely identical and cannot be distinguished, the results obtained by the fluorescent quantitative PCR experiment are the sum of the expression levels of OsGPT2-1 and OsGPT2-2 (marked as OsGPT2-1& 2-2). However, only OsGPT1 was highly expressed in rice seeds, while OsGPT2 was much lower in rice mature seeds than OsGPT1 and OsGPT2 was not expressed in endosperm as shown by our fluorescent quantitative PCR analysis (FIG. 1). The fluorescent quantitative PCR result shows that OsGPT1 is more important than OsGPT2-1& OsGPT2-2 in the development process of rice seed endosperm.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme of application and an application method of an OsGPT1 gene.

The invention adopts the following specific technical scheme:

the method for increasing the rice seeds and the thousand kernel weight is characterized in that the OsGPT1 gene is overexpressed in rice to obtain rice plants with the increased seeds and the thousand kernel weight.

The method for increasing the rice seed size and the thousand seed weight is characterized in that the OsGPT1 gene sequence is shown as SEQ ID No. 1.

The method for increasing the rice seeds and the thousand kernel weight is characterized by comprising the following steps of:

1) constructing an expression vector of OsGPT1 and eYFP fusion protein started by a promoter of OsGPT1, wherein the vector skeleton is pCAMBIA 3300;

2) using Agrobacterium to mix pCAMBIA3300-P_OsGPT1OsGPT1-eYFP vector, P_OsGPT1OsGPT1-eYFP sequence is shown as SEQ ID NO.4, and is transformed into rice callus, and rice plants expressed by OsGPT1-eYFP fusion protein are obtained by screening.

The OsGPT1 gene overexpression is applied to promotion of rice seed enlargement and thousand seed weight increase.

The OsGPT1 gene is applied to preparation of rice plants with improved seeds.

The application is characterized in that the OsGPT1 gene is overexpressed in rice to obtain rice plants with enlarged seeds and increased thousand seed weight.

The construction method of the overexpression vector for promoting the rice seed enlargement and thousand seed weight increase is characterized by comprising the following steps: amplification of P from the Rice genome_OsGPT1OsGPT1, eYFP is amplified in pBTYWG2 vector containing pLAT52-eYFP, the terminator of OsGPT1 is amplified from rice genome, and P is added by seamless assembly kit_OsGPT1The terminators of OsGPT1, eYFP and OsGPT1 and pCAMBIA3300 linearized by BamH I and Xba I are seamlessly assembled to obtain the over-expression vector for promoting rice seed enlargement and thousand seed weight increase.

The overexpression vector for promoting the rice seed to increase and the thousand seed weight to increase.

The OsGPT1 gene provided by the invention is a glucose-6-phosphate/phosphate transporter gene on the No. 8 chromosome of rice, and has the gene number of Os08g0187800(NCBI number) and LOC _ Os08g08840(MSU number). The genomic DNA of OsGPT1 has a total length of 2632bp, contains 5 exons in total, and has the sequence shown in SEQ ID NO.1 (wherein capital letters represent exons (exon) and lower case letters represent introns (intron)), and the structure diagram is shown in FIG. 2. The CDS full field of the coding region of the OsGPT1 gene is 1164bp, and the sequence is shown as SEQ ID NO. 2; the OsGPT1 gene codes 387 amino acids, and the specific sequence is shown as SEQ ID NO. 3. The OsGPT1 protein contains 6 putative transmembrane α -helices (I-VI), the arrow indicates the transporter cleavage site, and an alignment of OsGPT1 with GPT proteins in other species is shown in FIG. 3.

The full-length sequence of the genomic DNA of OsGPT1 is as follows: OsGPT1 genome sequence (Genomic DNA), wherein capital letters represent exons (exon) and lower case letters represent introns (intron).

ATGCTACCTGCTGTGAAGCTCTCTCCTGGCCCTGTGGCCTTCGCTGGCACCAATCTACGGTCCAGATCAGCTTCGGTTTCATCTGTCTCAAGTCTCAAACCATCCAAATTTGTGGTCTCTTCACTCAGACCACTCTACCTTGCACCACTAGATGGCCCAAGAGCTGCTGGGCAAAAGGCTCAGAGGCAGCCACTTGAGTTCAGGTGTGCTGCTTCCGCAGCCGATGACAAGGAGTCTAAGACCGAGGTGGTGCCCGTCCGCTCGGAAGCTGCCCAGAAGCTGAAGATCTCCATCTATTTCGCGACATGGTGGGCGCTTAATGTGATCTTTAACATCTACAACAAGAAGGTTCTCAATGCTTTCCCATACCCCTGGCTCACTTCTACGCTCTCCCTTGCCTGCGGCTCTGCGATGATGCTTGTCTCATGGGCCACTCGCCTTGTTGAGGCCCCCAAGACTGACCTAGATTTCTGGAAAGTTCTTTTCCCGGTgagtggaatctttttcttggcaattatttgtatatttgctgtgcctgcttgggcaagcatgattgattttggctattttccttttgaaggtTGCTGTGGCTCATACAATTGGGCATGTTGCTGCGACAGTGAGTATGTCAAAAGTAGCAGTGTCATTCACACACATTATCAAAAGTGCAGAGCCTGCATTCAGTGTTTTGGTATCAAGGTTCCTTCTTGGGGAGACGTTTCCGGTTCCTGTATATCTTTCTCTTCTTCCAATCATTGGTGGATGTGCTCTTGCTGCTGTCACAGAGCTGAACTTTAACATGGTTGGtaagtatatatttcgaaatcgatgcatattctgctatgagcttatgaaccattatagccgcaatgctaatttgtataaacgatgagtagccgtgagccaactgaagtggttgctttagtaatcaatactttaatactatgttttaacacaaaatggcttattagatgattattttgccccatctggatgtctgtttttcaggaattgcaatgttcttgagattagaatttacaggaaataataaacagtaacagatcatagatacaacctattaaccttatctaatttaattgttatgaaaacaggtgcaattatattctcctttataccagaagatacatgcttaagtctttttttctcatttcttcctgaagttccatgcgtagatttatgtttaagaacaaaacgaaataggagtcataggctctgtaattgtttccagtggtctggtcttgctcaggttagctcaggcaggtcttctcctgttctctttctcttgatgttaacagttgttgtttctcctcataccaggATTCATGGGTGCCATGATATCAAACCTTGCATTTGTTTTCCGCAACATCTTCTCAAAGAGGGGCATGAAGGGGAAATCTGTCAGTGGCATGAATTACTATGCCTGCCTGTCGATAATGTCCCTTGTCATACTCACACCATTCGCTATCGCTATGGAGGGCCCTCAAATGTGGGCTGCTGGTTGGCAAAAGGCTCTTGCGGAAGTTGGTCCCAATGTTGTCTGGTaagcaataaaataaaccaagcatcctttaatttttcttcagcaattgctattttgtagcccgtagttttgtacttgtgttacataggacaactcagaatgaacattattttcaccttaggagatatcattcatatgcttacatatttaacacacttttaagagaccattggtttcactcttgtgctggacttagtttgggattttggccagtctttgctggcatgtattgatatattgtgttttatgtactggacatattttggaatttacccagtctttgctggcatggattgatatattgtatttgatgatttacaacatactacagcgtatgcagcaacacttgagtattttattgatattacttaaattttggggatgttgaatattgtaggtGGGTTGCTGCACAGAGCGTTTTCTACCACTTATACAACCAGGTGTCTTACATGTCTCTGGATGAGATTTCTCCATTGACATTTAGCATTGGCAATACGATGAAGCGTATATCTGTGATTGTTTCGTCAATCATTATCTTCCACACACCTGTCCGCCCTGTCAATGCACTAGGAGCTGCCATTGCCATCCTTGGCACATTCCTGTATTCTCAGGtaaattcttgctttgatattttactttagccgtgcttgccctgtgtatacttcaaacaaactttcttttccctgcacgagaaggatgacattttgtatatagtattgtccacgtgtggtttgacattgatgtggaaaaacaggcagctactttgtgaatatgcttgattttaaatcctttccgctcaattataagcaggatggattggtaaatgatatgttactatcaactgtaattagactgcagagtattttagttcattcttttgctataggaggtaatatcacttgaaagaggaaatctgaaaacatgagggagtaaatacgactgcataagcttctgctgcacctgacttattcctccaatgccatatttatcctaaacatttttttttggttgtagctgacgttttcttttctttacttacttttttaatttctgtttcaggCAAAGCAGTGA。

The invention has the following beneficial effects: the applicant discovers that the rice seed increase and thousand kernel weight increase can be realized by overexpression of the OsGPT1 gene in rice through research on the OsGPT1 gene, and based on the discovery, the invention realizes the improvement of rice varieties by performing overexpression on the OsGPT1 gene in rice to regulate the size and the thousand kernel weight of the rice.

Drawings

FIG. 1 expression profiles of OsGPT1 and OsGPT2 in rice seeds; OsGPT1 is highly expressed in rice seeds, while OsGPT2 is much lower in rice mature seeds than OsGPT1, and OsGPT2 is not expressed in endosperm. Actin1 was used as an internal reference gene. Statistical analysis the method of the Student's t test was used (3 independent biological replicates, 3 technical replicates per biological replicate, P < 0.001).

FIG. 2 structural diagram of OsGPT1 gene.

FIG. 3 highly conserved GPTs in rice, Arabidopsis, maize, sorghum, potato and poplar, 6 putative transmembrane α -helices (I-VI) in the figure, and the arrow indicates the transporter cleavage site.

FIG. 4 pCAMBIA3300-P_OsGPT1OsGPT1-eYFP vector map.

FIG. 5P_OsGPT1Electrophoretograms of PCR products of terminators of OsGPT1, eYFP and OsGPT 1.

FIG. 6 overexpression of OsGPT1 seeds were enlarged, in which (A) P_OsGPT1OsGPT1-eYFP Nip #5 seed enlargement; (B) p_OsGPT1The thousand grain weight of OsGPT1-eYFP Nip #5 is increased by 12.7 percent; (C) p_OsGPT1OsGPT1-eYFP Nip #5 is used as a female parent, a wild type is used as a male parent, and hybrid seeds are enlarged; (D) p_OsGPT1OsGPT1 expression level in OsGPT1-eYFP Nip #5 material is about 19 times of that of wild type, and Bar is 2 mm.

FIG. 7P_OsGPT1OsGPT1-eYFP is expressed on the plastid membrane of rice protoplast, and P is shown in the figure_OsGPT1OsGPT1-eYFP fusion gene and fluorescent probe plasmid marker (pt-rk CD3-999) are co-transformed into rice protoplast, and observed by Confocal, wherein Bar is 10 mu m.

FIG. 8P_OsGPT1OsGPT1-eYFP is expressed on Nipponbare seed amyloplast membrane, Bar 10 μm.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

The following examples were conducted in accordance with conventional procedures, and materials and reagents used therein were commercially available.

Example 1: localization of OsGPT1

Although GPTs are isolated, purified or cloned from rice, Arabidopsis, maize, sorghum, potato and poplar species, the subcellular localization of GPTs in living cells is rarely reported. We have found thatP was transformed by PEG-mediated transformation of rice protoplasts (Zhang et al, 2011)_OsGPT1OsGPT1-eYFP and fluorescent probe plastid marker (pt-rk CD3-999) (Nelson et al, 2007) were co-transformed into rice protoplasts, and the photograph of the subcellular localization is shown in FIG. 7. The specific experimental method is as follows:

(1) preparation and transformation of rice protoplasts.

Preparation and transformation of rice protoplasts Rice protoplasts were isolated from stem and leaf sheaths of etiolated rice seedlings by enzymatic methods and transformed by PEG-mediated transformation (Zhang et al, 2011). The specific method comprises the following steps:

1. the rice seeds were sterilized and sown in sterile solid media made of Phytagel and MES, approximately 2-3 seeds were sown per glass tube.

2. The rice seeds are cultured in the dark for 4-5 days after being cultured for one week in the light.

3. The etiolated rice seedlings were removed, the leaves and roots were cut off, and the remaining tissue was cut into pieces of about 0.1mm, which were not repeatedly cut at the incision, to prevent the destruction of the cells at the incision.

4. Adding a proper volume of the enzymolysis liquid into the minced tissue, vacuumizing for 1h, then shaking and incubating for 5h at 40rpm in the dark, adding an equal volume of W5 solution, shaking gently by hand for 2min, mixing uniformly and releasing protoplasts.

5. The mixture was filtered through a 4-layer 400 mesh nylon net and centrifuged at 1500rpm for 4min at room temperature. Carefully pour off the supernatant, add W5 to resuspend, repeat centrifugation, add W5 to resuspend, ice for 30min, repeat centrifugation.

6. Discarding the supernatant, adding a certain volume of Suspension medium to resuspend the protoplast, calculating with a hemocytometer and adjusting the protoplast concentration to 2.5-5X 10⁶/mL。

7. To 200. mu.L of protoplast of the adjusted concentration, 6 to 8. mu.g of plasmid and 220. mu.L of 40% PEG were added, incubated at room temperature for 2min in the dark, and 1mL of W5 was added to terminate the reaction.

Cultured overnight at 8.28 ℃ in the dark. Fluorescence signals in protoplasts were detected using Nikon C2 confocal. P_OsGPT1OsGPT1-eYFP was observed using the FITC channel at 500-550nm, and plasmid marker was observed using the PI channel at 575-615 nm.

The experimental results are as follows: p_OsGPT1OsGPT1-eYFP fusion gene and fluorescent probe plasmid marker are co-transformed into rice protoplast, and P can be clearly seen through the result of Confocal observation (figure 7)_OsGPT1OsGPT1-eYFP is expressed on the protoplast membrane of rice, which is consistent with the expression positions of GPTs separated and purified from other species and GPTs predicted. P_OsGPT1The OsGPT1-eYFP sequence is shown in SEQ ID NO. 4.

(2)pCAMBIA3300-P_OsGPT1Construction of OsGPT1-eYFP vector (see FIG. 4 for vector). The specific method comprises the following steps:

amplification of P from the Rice genome_OsGPT1OsGPT1, eYFP amplified from the existing pBTYGG 2 vector containing pLAT52-eYFP, and the terminator of OsGPT1 amplified from the rice genome. P is prepared by using seamless assembly kit_OsGPT1The terminators of OsGPT1, eYFP and OsGPT1 were seamlessly assembled with pCAMBIA3300 linearized with BamH I and Xba I. The electrophoretogram of each cloned fragment is shown in FIG. 5. The primer sequence is shown in SEQ ID NO. 5-10.

Genomic DNA and Terminator were amplified with high fidelity DNA polymerase Phusion using genomic DNA from wild type Nip as template.

The PCR reaction program is:

the 30 reactions are carried out in the 2 nd to 4 th steps

72℃ 10min

eYFP was amplified using high fidelity DNA polymerase Phusion using the pBTYGG 2 vector containing pLAT52-eYFP, available in the laboratory as a template.

The PCR reaction program is:

the 30 reactions are carried out in the 2 nd to 4 th steps

72℃ 10min

The PCR product was subjected to 1% agarose electrophoresis, and the target band was excised and purified by using a crude gel recovery kit.

Seamless tissue system 10 μ L:

wherein n (fragment of interest): n (carrier) ═ 2: 1, the ratio of target fragments is 1: 1.

the experimental steps are as follows:

1. mixing the target fragment and the carrier, standing in a water bath at 45 deg.C for 30min, and transferring to ice.

2. mu.L of the reaction solution was added to the competent large intestine dissolved in 50. mu.L of ice, and left on ice for another 20 min.

Standing in water bath at 3.42 deg.C for 1min, and standing on ice for 2 min.

4. 1mL of liquid LB medium was added, and the mixture was thawed at 37 ℃ and 200rpm for 1 hour.

5.4850 rpm, 2min, were smeared onto Kan-resistant plates and incubated overnight at 37 ℃ in an inverted position.

(3)P_OsGPT1Obtaining OsGPT1-eYFP Nip transgenic material.

Using a solution containing pCAMBIA3300-P_OsGPT1The rice callus is infected by EHA105 of OsGPT1-eYFP carrier, co-cultured in a culture room at 22 ℃ for 3 days, washed away by liquid culture medium to remove agrobacterium, cultured on a selective culture medium containing appropriate antibiotics, and subjected to resistance screening twice. After resistance screening, resistance callus can be obtained, the resistance callus is differentiated into plantlets, and the plantlets are planted in a rice greenhouse, and the method specifically comprises the following steps:

1. rice seed disinfection:

taking mature rice seeds, manually or mechanically shelling, selecting full, smooth and plaque-free seeds, and disinfecting according to the following steps:

1) placing the seeds into a 100mL sterile beaker, and pouring 70% alcohol (15mL) for disinfection for 2 min;

2) pouring off alcohol, adding 100mL of 30% sodium hypochlorite (NaClO) solution, and soaking for 30 min;

3) the sodium hypochlorite solution was poured off, the seeds were washed 4-5 times with sterile distilled water and soaked for the last 30 min.

2. Induction and subculture: (the following procedure requires aseptic manipulation)

1) The seeds are put on sterile filter paper for suction drying and are put into an adult embryo induction culture medium, and 20 to 30 seeds are placed in each dish;

2) sealing film (Micropore) after completion of operation^TMSurgical Tape) sealed and incubated at 28 ℃ in an incubator. And (5) induction culture of japonica rice for 3-4 weeks.

3) The culture dish was opened on an ultraclean bench, and the naturally-divided embryogenic callus (pale yellow, dense and spherical) was picked up with forceps and placed in a subculture medium, and the incubator was irradiated with light at 28 ℃ for subculture for 1 week.

3. Agrobacterium culture

Picking electric transfer pCAMBIA3300-P_OsGPT1Agrobacterium monoclonality of OsGPT1-eYFP vector or the deposited transformed pCAMBIA3300-P_OsGPT150 mu L of OsGPT1-eYFP vector agrobacterium liquid is put into 5mL of LB liquid culture solution (containing 50mg/L kanamycin and 50mg/L streptomycin), and the mixture is subjected to shaking culture at 28 ℃ and 250rpm for 12-16h until the bacterial liquid OD is reached₆₀₀Is 0.8-1.0.

4. Co-culturing the infectious microbes: taking the cultured transformed pCAMBIA3300-P _OsGPT1500 mu L of OsGPT1-eYFP carrier bacterium liquid is put in a 1.5mL centrifuge tube, centrifuged for 2min at the room temperature and 4000rpm, and the supernatant is removed. Preparing suspension with 30mL of AAM infection-inducing liquid containing 200 μmol/L acetosyringone, and obtaining final concentration OD of the liquid₆₀₀Is 0.01; picking out the rice callus growing to a certain size, putting the rice callus into the agrobacterium tumefaciens suspension, and shaking for 5min on a horizontal shaking table at 80 rpm; taking out the callus, placing on sterile filter paper, and draining for 30-40 min; the calli were placed on co-culture medium with a sterile filter paper. Dark culture was carried out at 25 ℃ for 3 days.

5. Selecting and culturing: taking out the callus, and washing with sterile water for 5-6 times without continuous oscillation. And then washed with sterile water containing 300mg/L carbenicillin sodium for 2 times, each time shaking on a horizontal shaker for 30 min. Finally placing the mixture on sterile filter paper and draining for 2 hours; the air-dried callus was transferred to a selection medium containing 300mg/L carbenicillin sodium and corresponding selection pressure for the first round of selection, and cultured at 28 ℃ under light for 14 days. The initial calli with resistant calli were transferred to medium containing 300mg/L carbenicillin sodium and corresponding selection pressure for a second round of selection at 28 ℃ with light until granular resistant calli grew out (about 14 days).

6. Induced differentiation and rooting of resistant callus: 3-5 resistant calli with bright yellow color from different calli are picked, transferred into a plastic wide-mouth bottle filled with a differentiation medium, sealed by a sealing film, placed into a constant-temperature (25 ℃) culture chamber (photoperiod: 16h illumination) and wait for differentiation into seedlings (about 40 days). After the seedling grows to about 3cm, old roots and callus are cut off from the base of the seedling by scissors and placed in a rooting medium to strengthen the seedling (about 1 week).

OsGPT1 is a phosphate transporter positioned on a plastid membrane, and in order to prove the expression condition of OsGPT1 on rice endosperm amyloplasts, P is taken out_OsGPT1The endosperm of seeds 4 days after the OsGPT1-eYFP Nip material blooms was observed under Confocal. The observation result shows that P_OsGPT1OsGPT1-eYFP is expressed on the membrane of the amyloplast in the endosperm, but not on the membrane of Starch granules (Starch granules) assembled into the amyloplast (FIG. 8).

Example 2: obtaining OsGPT1 over-expression material, enlarging seeds and increasing thousand seed weight

In planting P_OsGPT1OsGPT1-eYFP Nip #5 material was found to have an increased seed size with a thousand kernel weight increase of about 12.7%. Detect P_OsGPT1Expression of OsGPT1 in OsGPT1-eYFP Nip #5 is up-regulated, which is about 19 times of the expression of OsGPT1 in Nip (FIG. 6). Furthermore, we will P_OsGPT1OsGPT1-eYFP Nip #5 material is used as a female parent and pollinated by the pollen of Nip, and the seed is found to be greatly enlarged compared with the hybrid seed which uses Nip as the female parent and Nip as the male parent, and the size of the original glume is broken through (figure 6).

The specific experimental method is as follows:

(1) method for extracting RNA from rice seeds rich in starch

TransGen TransZol Plant (Cat. No.: ET101-01) reagent was used. Rice seeds were ground to a powder sufficiently with a precooled mortar, and then RNA extraction was performed according to the protocol of the TransGen TransZol Plant.

(2) Digestion of DNA in RNA

The extracted RNA is contaminated with genomic DNA, and the DNA is digested by DNase treatment. The DNase treatment system is as follows:

total 25. mu.L system, where RNA can be treated as required at 5-10. mu.g, the remaining volume with DEPC-H₂And (4) complementing O.

The experimental procedure was as follows:

mu.L of the DNase-treated system was mixed in a PCR tube and left at 37 ℃ for 1 hour.

2. Add 2.5. mu.L of DNase inactivation reagent, mix well and then let stand at room temperature for 5 min. The aim is to inactivate the DNase.

3. Centrifuging: 22 ℃, 10000rpm, 5 min. Carefully pipette 20. mu.L of the supernatant into a new PCR tube and measure the RNA concentration.

4. The RNA after DNase treatment is stored in a refrigerator at-80 ℃.

(3) Reverse transcription of RNA

RNA is subjected to reverse transcription reaction to synthesize cDNA. The method comprises the following steps:

RNA and OligdT23 primer were incubated at 1.65 ℃ for 5 min. The system is as follows:

DEPC-H₂O *

OligdT23 1μL

RNA *

RNA can be reversely transcribed by 500-1000ng according to the experimental requirements, the total system is 5 mu L, and the rest volume is DEPC-H₂And (4) complementing O.

2. RT mix was added to the above system.

The 15 μ L RT mix system was as follows:

the reaction conditions were as follows:

(4) RT-PCR analysis

The cDNA obtained by reverse transcription is diluted by 40 times according to the experimental requirements to be used as a template, and the rice Actin1 gene is used as an internal reference gene. The RT-PCR primer sequence is shown in SEQ ID NO. 11-14.

The RT-PCR reaction system is as follows:

diluted cDNA 5. mu.L

1.2μM primers 5μL

2X RT-PCR mix 10μL

(5) Investigation of rice plants over-expressing OsGPT1 on seed size and thousand kernel weight

The OsGPT1 gene provided by the invention has high application value in the aspects of rice seed size and thousand seed weight. The expression level of OsGPT1 is detected by RT-PCR after the RNA extraction and reverse transcription of the obtained seed of the transgenic material. The RT-PCR detection finds P_OsGPT1Expression of OsGPT1 in OsGPT1-eYFP Nip #5 is up-regulated, which is about 19 times of the expression of OsGPT1 in Nip (FIG. 6). P_OsGPT1OsGPT1-eYFP Nip #5 has both increased seed size and thousand seed weight compared to the control, as shown in FIG. 6.

(6)P_OsGPT1Hybridization of OsGPT1-eYFP Nip #5 material with Nipponbare

P_OsGPT1OsGPT1-eYFP Nip #5 material is used as a female parent and pollinated by the pollen of Nip, and the seed is found to be greatly enlarged compared with the hybrid seed which uses Nip as the female parent and Nip as the male parent, and the size of the original glume is broken through (figure 6).

Sequence listing

<110> Zhejiang university

Application and application method of <120> OsGPT1 gene

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2632

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 1

atgctacctg ctgtgaagct ctctcctggc cctgtggcct tcgctggcac caatctacgg 60

tccagatcag cttcggtttc atctgtctca agtctcaaac catccaaatt tgtggtctct 120

tcactcagac cactctacct tgcaccacta gatggcccaa gagctgctgg gcaaaaggct 180

cagaggcagc cacttgagtt caggtgtgct gcttccgcag ccgatgacaa ggagtctaag 240

accgaggtgg tgcccgtccg ctcggaagct gcccagaagc tgaagatctc catctatttc 300

gcgacatggt gggcgcttaa tgtgatcttt aacatctaca acaagaaggt tctcaatgct 360

ttcccatacc cctggctcac ttctacgctc tcccttgcct gcggctctgc gatgatgctt 420

gtctcatggg ccactcgcct tgttgaggcc cccaagactg acctagattt ctggaaagtt 480

cttttcccgg tgagtggaat ctttttcttg gcaattattt gtatatttgc tgtgcctgct 540

tgggcaagca tgattgattt tggctatttt ccttttgaag gttgctgtgg ctcatacaat 600

tgggcatgtt gctgcgacag tgagtatgtc aaaagtagca gtgtcattca cacacattat 660

caaaagtgca gagcctgcat tcagtgtttt ggtatcaagg ttccttcttg gggagacgtt 720

tccggttcct gtatatcttt ctcttcttcc aatcattggt ggatgtgctc ttgctgctgt 780

cacagagctg aactttaaca tggttggtaa gtatatattt cgaaatcgat gcatattctg 840

ctatgagctt atgaaccatt atagccgcaa tgctaatttg tataaacgat gagtagccgt 900

gagccaactg aagtggttgc tttagtaatc aatactttaa tactatgttt taacacaaaa 960

tggcttatta gatgattatt ttgccccatc tggatgtctg tttttcagga attgcaatgt 1020

tcttgagatt agaatttaca ggaaataata aacagtaaca gatcatagat acaacctatt 1080

aaccttatct aatttaattg ttatgaaaac aggtgcaatt atattctcct ttataccaga 1140

agatacatgc ttaagtcttt ttttctcatt tcttcctgaa gttccatgcg tagatttatg 1200

tttaagaaca aaacgaaata ggagtcatag gctctgtaat tgtttccagt ggtctggtct 1260

tgctcaggtt agctcaggca ggtcttctcc tgttctcttt ctcttgatgt taacagttgt 1320

tgtttctcct cataccagga ttcatgggtg ccatgatatc aaaccttgca tttgttttcc 1380

gcaacatctt ctcaaagagg ggcatgaagg ggaaatctgt cagtggcatg aattactatg 1440

cctgcctgtc gataatgtcc cttgtcatac tcacaccatt cgctatcgct atggagggcc 1500

ctcaaatgtg ggctgctggt tggcaaaagg ctcttgcgga agttggtccc aatgttgtct 1560

ggtaagcaat aaaataaacc aagcatcctt taatttttct tcagcaattg ctattttgta 1620

gcccgtagtt ttgtacttgt gttacatagg acaactcaga atgaacatta ttttcacctt 1680

aggagatatc attcatatgc ttacatattt aacacacttt taagagacca ttggtttcac 1740

tcttgtgctg gacttagttt gggattttgg ccagtctttg ctggcatgta ttgatatatt 1800

gtgttttatg tactggacat attttggaat ttacccagtc tttgctggca tggattgata 1860

tattgtattt gatgatttac aacatactac agcgtatgca gcaacacttg agtattttat 1920

tgatattact taaattttgg ggatgttgaa tattgtaggt gggttgctgc acagagcgtt 1980

ttctaccact tatacaacca ggtgtcttac atgtctctgg atgagatttc tccattgaca 2040

tttagcattg gcaatacgat gaagcgtata tctgtgattg tttcgtcaat cattatcttc 2100

cacacacctg tccgccctgt caatgcacta ggagctgcca ttgccatcct tggcacattc 2160

ctgtattctc aggtaaattc ttgctttgat attttacttt agccgtgctt gccctgtgta 2220

tacttcaaac aaactttctt ttccctgcac gagaaggatg acattttgta tatagtattg 2280

tccacgtgtg gtttgacatt gatgtggaaa aacaggcagc tactttgtga atatgcttga 2340

ttttaaatcc tttccgctca attataagca ggatggattg gtaaatgata tgttactatc 2400

aactgtaatt agactgcaga gtattttagt tcattctttt gctataggag gtaatatcac 2460

ttgaaagagg aaatctgaaa acatgaggga gtaaatacga ctgcataagc ttctgctgca 2520

cctgacttat tcctccaatg ccatatttat cctaaacatt tttttttggt tgtagctgac 2580

gttttctttt ctttacttac ttttttaatt tctgtttcag gcaaagcagt ga 2632

<210> 2

<211> 1164

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 2

atgctacctg ctgtgaagct ctctcctggc cctgtggcct tcgctggcac caatctacgg 60

tccagatcag cttcggtttc atctgtctca agtctcaaac catccaaatt tgtggtctct 120

tcactcagac cactctacct tgcaccacta gatggcccaa gagctgctgg gcaaaaggct 180

cagaggcagc cacttgagtt caggtgtgct gcttccgcag ccgatgacaa ggagtctaag 240

accgaggtgg tgcccgtccg ctcggaagct gcccagaagc tgaagatctc catctatttc 300

gcgacatggt gggcgcttaa tgtgatcttt aacatctaca acaagaaggt tctcaatgct 360

ttcccatacc cctggctcac ttctacgctc tcccttgcct gcggctctgc gatgatgctt 420

gtctcatggg ccactcgcct tgttgaggcc cccaagactg acctagattt ctggaaagtt 480

cttttcccgg ttgctgtggc tcatacaatt gggcatgttg ctgcgacagt gagtatgtca 540

aaagtagcag tgtcattcac acacattatc aaaagtgcag agcctgcatt cagtgttttg 600

gtatcaaggt tccttcttgg ggagacgttt ccggttcctg tatatctttc tcttcttcca 660

atcattggtg gatgtgctct tgctgctgtc acagagctga actttaacat ggttggattc 720

atgggtgcca tgatatcaaa ccttgcattt gttttccgca acatcttctc aaagaggggc 780

atgaagggga aatctgtcag tggcatgaat tactatgcct gcctgtcgat aatgtccctt 840

gtcatactca caccattcgc tatcgctatg gagggccctc aaatgtgggc tgctggttgg 900

caaaaggctc ttgcggaagt tggtcccaat gttgtctggt gggttgctgc acagagcgtt 960

ttctaccact tatacaacca ggtgtcttac atgtctctgg atgagatttc tccattgaca 1020

tttagcattg gcaatacgat gaagcgtata tctgtgattg tttcgtcaat cattatcttc 1080

cacacacctg tccgccctgt caatgcacta ggagctgcca ttgccatcct tggcacattc 1140

ctgtattctc aggcaaagca gtga 1164

<210> 3

<211> 387

<212> PRT

<213> Rice (Oryza sativa L.)

<400> 3

Met Leu Pro Ala Val Lys Leu Ser Pro Gly Pro Val Ala Phe Ala Gly

1 5 10 15

Thr Asn Leu Arg Ser Arg Ser Ala Ser Val Ser Ser Val Ser Ser Leu

20 25 30

Lys Pro Ser Lys Phe Val Val Ser Ser Leu Arg Pro Leu Tyr Leu Ala

35 40 45

Pro Leu Asp Gly Pro Arg Ala Ala Gly Gln Lys Ala Gln Arg Gln Pro

50 55 60

Leu Glu Phe Arg Cys Ala Ala Ser Ala Ala Asp Asp Lys Glu Ser Lys

65 70 75 80

Thr Glu Val Val Pro Val Arg Ser Glu Ala Ala Gln Lys Leu Lys Ile

85 90 95

Ser Ile Tyr Phe Ala Thr Trp Trp Ala Leu Asn Val Ile Phe Asn Ile

100 105 110

Tyr Asn Lys Lys Val Leu Asn Ala Phe Pro Tyr Pro Trp Leu Thr Ser

115 120 125

Thr Leu Ser Leu Ala Cys Gly Ser Ala Met Met Leu Val Ser Trp Ala

130 135 140

Thr Arg Leu Val Glu Ala Pro Lys Thr Asp Leu Asp Phe Trp Lys Val

145 150 155 160

Leu Phe Pro Val Ala Val Ala His Thr Ile Gly His Val Ala Ala Thr

165 170 175

Val Ser Met Ser Lys Val Ala Val Ser Phe Thr His Ile Ile Lys Ser

180 185 190

Ala Glu Pro Ala Phe Ser Val Leu Val Ser Arg Phe Leu Leu Gly Glu

195 200 205

Thr Phe Pro Val Pro Val Tyr Leu Ser Leu Leu Pro Ile Ile Gly Gly

210 215 220

Cys Ala Leu Ala Ala Val Thr Glu Leu Asn Phe Asn Met Val Gly Phe

225 230 235 240

Met Gly Ala Met Ile Ser Asn Leu Ala Phe Val Phe Arg Asn Ile Phe

245 250 255

Ser Lys Arg Gly Met Lys Gly Lys Ser Val Ser Gly Met Asn Tyr Tyr

260 265 270

Ala Cys Leu Ser Ile Met Ser Leu Val Ile Leu Thr Pro Phe Ala Ile

275 280 285

Ala Met Glu Gly Pro Gln Met Trp Ala Ala Gly Trp Gln Lys Ala Leu

290 295 300

Ala Glu Val Gly Pro Asn Val Val Trp Trp Val Ala Ala Gln Ser Val

305 310 315 320

Phe Tyr His Leu Tyr Asn Gln Val Ser Tyr Met Ser Leu Asp Glu Ile

325 330 335

Ser Pro Leu Thr Phe Ser Ile Gly Asn Thr Met Lys Arg Ile Ser Val

340 345 350

Ile Val Ser Ser Ile Ile Ile Phe His Thr Pro Val Arg Pro Val Asn

355 360 365

Ala Leu Gly Ala Ala Ile Ala Ile Leu Gly Thr Phe Leu Tyr Ser Gln

370 375 380

Ala Lys Gln

385

<210> 4

<211> 6837

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 4

ggagaagtat tataatcttt gcattaaatg catgtcttgt ctcttgactt gggaagccaa 60

gcacacgtcg tcttgatgat ggggcctgtc aaatcttgcc gcctgacacg gggggtgccc 120

ctgtcagtca ccatttaatg ggtgaagact tctgggctcc tattacaccg ggaaacagga 180

gccttgcttt gaccagagcg ggaggaccga ctccggctgg gataagagga tgagaacatc 240

tcaccatcac tatttgatgg gacatgtcag gctgcacgcc gcctggcaca cgagcagcgc 300

acaggtgcac tagccagtcc catcggtcat tcgaccggtc acagaccggt tgagtgcact 360

gtacgccgca ttaaatgcga cgtggcgcga ccgctcggga cgccataaat gcgggtaggt 420

gggcacctgg aggcggacac ctaatccacc gtacgtggtg acctagagag ggggtcgggg 480

gagcccgacc cctagtcacg ggagggggcg gccgccgccc gacctcgggc ccgatccccc 540

ctcggggggg gagccacgtg gcgcctgggg cggccttctc cctctagtct cggccagaga 600

gtggccgccg ccctacctcg ggcccgaccc ccctcggggg agggtcacgt ggcattggag 660

ggcaacctcc tccaactagt ctttgggtaa ggagtggccg ccaccccacc tcgggtctga 720

ctcccctcgg gagagggcca cgtggcatcg gggggcagcc tcctctctct aaacctgata 780

ccccgggccc atatcaccga caagtgtaat tacactataa atatatgtat tttataatgt 840

aataaaaaat atcgctcttt ttatttataa tgtaataaaa aatatcgctc ttcttgaaaa 900

atatgatgtt ataactataa ctaatcacct tttttaaaag caacacttat atagaatttt 960

tttaaaaaag cacgacaaga tgcacatgaa aaaaaaaaga gcaaatagaa actaggaaaa 1020

tgcagtatag aacgcaacgt aagttatgct cgcctccaaa tcgcttttat aacctttttc 1080

ttgtttacct tgccggtgct aatttctttt gctttacatt ttcaacaaac tttgccttct 1140

acattatctc atcatctaga caacagggaa accaaaatca aactttattt tttttttggt 1200

ttttgctaca cacaacaatt tcctcccaaa ccaaaaaccc ggaaaaatca gaaatccatg 1260

tgctcgatga cacaaatact agggatggtc tccttaaaaa gaacaacatt tgcaataaaa 1320

ccctcacact tgtccaacct taccaacaag ccaccaagaa aagatacgca aaaaacaaaa 1380

aaaacgcatt ttttttcacc taccagccca gccgccacct tctcctctcc ccctttatac 1440

tcgccggagc ggagactccc ctctccttcc cttgtgatct ccgaattccc atatcagatt 1500

ccgagggctt ggggctctcc tacgatcccc cgcctcctcc tcctcctcca tacggtatat 1560

ctctcggcgg catctcgaaa tcctcatccg tttctgttgt tgtttccgcg gcgagtttga 1620

ggggtgtgtg ttcttggaga ggaagaaccg ttcgccggtt tgattcaggg tacaggaact 1680

tgctacagag tgattggtgg gtggataagg tgagatcttt tcttctcttg atcctacaaa 1740

gttgaggttg tttttttttt taagtttatt tttaaatttc tcttttcaat ctgcggcgat 1800

gccgccgccg ccgccacgtt tgttcctccc ctcgccgcga gggcgcgagg cttgagactg 1860

gcgtcttcat tcatgcatcc attttgtttt tgtgttgttg ttgttgggta ttttgatcca 1920

agaaagaagt cttcattcaa tctgggtggg ggtggtggta aagatgggtg atttgcatgc 1980

gaaatctgcc gggctagatc acgggagttg gagaagcttg gtatctttac gtgtagatca 2040

ctcggcgaaa acgatgtgat aatttgtccc catctatgat tttgtaggct tgtagtttct 2100

gtgatttggg gatttatttg gtgacggttc tgccgtgcag gtattgtgta ctactagtac 2160

tttccgctga gtaatttggg aggaaaacta aaaaagatgg ttgttgggtc atacatacat 2220

ggtcgattca ttcattggtt tgatcagttc aagagaaata tttttgggag ccatctcaaa 2280

agaatgatgt gtaaaattta cttaaccagc tcattgagga aagttgttcc atgtcacttt 2340

cttttctggt gctgatagat caattgctct actgaggtcc tcaaagaaag tttttgttga 2400

aaggaaatgg cctgttttgt tttatttgat gacagatgat gctttaaatt gtttcctacc 2460

tgttgtcatg tacaattttg ttctttgatt tgcagaagcc atgctacctg ctgtgaagct 2520

ctctcctggc cctgtggcct tcgctggcac caatctacgg tccagatcag cttcggtttc 2580

atctgtctca agtctcaaac catccaaatt tgtggtctct tcactcagac cactctacct 2640

tgcaccacta gatggcccaa gagctgctgg gcaaaaggct cagaggcagc cacttgagtt 2700

caggtgtgct gcttccgcag ccgatgacaa ggagtctaag accgaggtgg tgcccgtccg 2760

ctcggaagct gcccagaagc tgaagatctc catctatttc gcgacatggt gggcgcttaa 2820

tgtgatcttt aacatctaca acaagaaggt tctcaatgct ttcccatacc cctggctcac 2880

ttctacgctc tcccttgcct gcggctctgc gatgatgctt gtctcatggg ccactcgcct 2940

tgttgaggcc cccaagactg acctagattt ctggaaagtt cttttcccgg tgagtggaat 3000

ctttttcttg gcaattattt gtatatttgc tgtgcctgct tgggcaagca tgattgattt 3060

tggctatttt ccttttgaag gttgctgtgg ctcatacaat tgggcatgtt gctgcgacag 3120

tgagtatgtc aaaagtagca gtgtcattca cacacattat caaaagtgca gagcctgcat 3180

tcagtgtttt ggtatcaagg ttccttcttg gggagacgtt tccggttcct gtatatcttt 3240

ctcttcttcc aatcattggt ggatgtgctc ttgctgctgt cacagagctg aactttaaca 3300

tggttggtaa gtatatattt cgaaatcgat gcatattctg ctatgagctt atgaaccatt 3360

atagccgcaa tgctaatttg tataaacgat gagtagccgt gagccaactg aagtggttgc 3420

tttagtaatc aatactttaa tactatgttt taacacaaaa tggcttatta gatgattatt 3480

ttgccccatc tggatgtctg tttttcagga attgcaatgt tcttgagatt agaatttaca 3540

ggaaataata aacagtaaca gatcatagat acaacctatt aaccttatct aatttaattg 3600

ttatgaaaac aggtgcaatt atattctcct ttataccaga agatacatgc ttaagtcttt 3660

ttttctcatt tcttcctgaa gttccatgcg tagatttatg tttaagaaca aaacgaaata 3720

ggagtcatag gctctgtaat tgtttccagt ggtctggtct tgctcaggtt agctcaggca 3780

ggtcttctcc tgttctcttt ctcttgatgt taacagttgt tgtttctcct cataccagga 3840

ttcatgggtg ccatgatatc aaaccttgca tttgttttcc gcaacatctt ctcaaagagg 3900

ggcatgaagg ggaaatctgt cagtggcatg aattactatg cctgcctgtc gataatgtcc 3960

cttgtcatac tcacaccatt cgctatcgct atggagggcc ctcaaatgtg ggctgctggt 4020

tggcaaaagg ctcttgcgga agttggtccc aatgttgtct ggtaagcaat aaaataaacc 4080

aagcatcctt taatttttct tcagcaattg ctattttgta gcccgtagtt ttgtacttgt 4140

gttacatagg acaactcaga atgaacatta ttttcacctt aggagatatc attcatatgc 4200

ttacatattt aacacacttt taagagacca ttggtttcac tcttgtgctg gacttagttt 4260

gggattttgg ccagtctttg ctggcatgta ttgatatatt gtgttttatg tactggacat 4320

attttggaat ttacccagtc tttgctggca tggattgata tattgtattt gatgatttac 4380

aacatactac agcgtatgca gcaacacttg agtattttat tgatattact taaattttgg 4440

ggatgttgaa tattgtaggt gggttgctgc acagagcgtt ttctaccact tatacaacca 4500

ggtgtcttac atgtctctgg atgagatttc tccattgaca tttagcattg gcaatacgat 4560

gaagcgtata tctgtgattg tttcgtcaat cattatcttc cacacacctg tccgccctgt 4620

caatgcacta ggagctgcca ttgccatcct tggcacattc ctgtattctc aggtaaattc 4680

ttgctttgat attttacttt agccgtgctt gccctgtgta tacttcaaac aaactttctt 4740

ttccctgcac gagaaggatg acattttgta tatagtattg tccacgtgtg gtttgacatt 4800

gatgtggaaa aacaggcagc tactttgtga atatgcttga ttttaaatcc tttccgctca 4860

attataagca ggatggattg gtaaatgata tgttactatc aactgtaatt agactgcaga 4920

gtattttagt tcattctttt gctataggag gtaatatcac ttgaaagagg aaatctgaaa 4980

acatgaggga gtaaatacga ctgcataagc ttctgctgca cctgacttat tcctccaatg 5040

ccatatttat cctaaacatt tttttttggt tgtagctgac gttttctttt ctttacttac 5100

ttttttaatt tctgtttcag gcaaagcaga tggtgagcaa gggcgaggag ctgttcaccg 5160

gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag ttcagcgtgt 5220

ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca 5280

ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cttcggctac ggcctgcagt 5340

gcttcgcccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc gccatgcccg 5400

aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac aagacccgcg 5460

ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag ggcatcgact 5520

tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac agccacaacg 5580

tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag atccgccaca 5640

acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc cccatcggcg 5700

acggccccgt gctgctgccc gacaaccact acctgagcta ccagtccgcc ctgagcaaag 5760

accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc gccgggatca 5820

ctctcggcat ggacgagctg tacaagtaag ttcaaaaaga tggtaatgtt cagggccatg 5880

gatgaagaag ggatccagat gaagaatgtc tctgccaaac cagtagcaag cactgaggcc 5940

aattttgctt gtgcaactta tcttgtagat ataggggggt taggttttga tatgaggtca 6000

taataataat ggtctatgtt gctcgtttct tgttccttgt tagtgcaaac tgaactccag 6060

aggctatgaa ctgtggtcga gtgtaatctt gaaataaaaa cttgaatggg aatttgtcga 6120

tgtttcattc tgggtgtggg aaggtaacta ccatccaagt cccaggtttc aatggttgtt 6180

actacctttc ttactggcaa atgaacggag gtcacaaata tgttgcaatt tttcttgagc 6240

aacaaagata ttgcaagttt tcttgatccc ttgatattat acagatcaaa atttctgaat 6300

gctttcaacc acataattct attgggatat aacacttagc ggccccatcg ttaataattt 6360

gtgaaaaaaa acttttacat ttgtattctt aacaatctaa aagtaaaggc tggaaaataa 6420

actgcgatga aaaaacccta aaaccaactc cacatcgcca aaatcagtta aaaattcaaa 6480

tttatggttt ataagtataa acataaacgg aaagagacga ggttgtcgat ttcttcatgg 6540

cttaatttgc taagtattga atgattctgc taaagaagtt cagttgaagg acatgatcac 6600

atgtcttaca ttatagccaa aaaaaaaaaa gccttgaagg atatgtttga ggacaatata 6660

agaacataca tgcttgtttt tatctggaat atattattag tatttaggta gcactgttaa 6720

atatttttct ttagaattca taagctctaa aatgaggtgc acgcatgagt ccaaagcccg 6780

tgggcctctg tggctgggaa tgaacatgca gctggaggat ctaattgatg cctccgt 6837

<210> 5

<211> 40

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 5

agctcggtac ccggggatcc ggagaagtat tataatcttt 40

<210> 6

<211> 40

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 6

tcctcgccct tgctcaccat ctgctttgcc tgaaacagaa 40

<210> 7

<211> 40

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 7

ttctgtttca ggcaaagcag atggtgagca agggcgagga 40

<210> 8

<211> 40

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 8

acattaccat ctttttgaac ttacttgtac agctcgtcca 40

<210> 9

<211> 40

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 9

tggacgagct gtacaagtaa gttcaaaaag atggtaatgt 40

<210> 10

<211> 40

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 10

gcctgcaggt cgactctaga acggaggcat caattagatc 40

<210> 11

<211> 20

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 11

caacacccct gctatgtacg 20

<210> 12

<211> 21

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 12

catcaccaga gtccaacaca a 21

<210> 13

<211> 22

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 13

atggttggat tcatgggtgc ca 22

<210> 14

<211> 23

<212> DNA

<213> Artificial Synthesis (synthetic sequence)

<400> 14

tttgagggcc ctccatagcg ata 23

Claims (2)

1.OsGPT1Application of gene in promotion of rice seed enlargement and thousand kernel weight increaseOsGPT1The gene sequence is shown in SEQ ID No. 1.

2.OsGPT1Use of genes for the preparation of seed-modified rice plants, said plantsOsGPT1The gene sequence is shown in SEQ ID No.1, and the rice plant with improved seeds is a rice plant with enlarged seeds and increased thousand seed weight.

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