CN114807175B - Monosaccharide transduction protein gene OsSTP15 and its transporter and its application in improving rice yield, amplification primers - Google Patents

Abstract

The present invention discloses a rice monosaccharide transporter gene OsSTP15, whose nucleotide sequence is shown in SEQ ID NO: 1; and also discloses a transporter encoded by the gene, whose amino acid sequence is shown in SEQ ID NO: 2; and the application of the gene or transporter in cultivating high-yield rice and amplification primers used for the application. The rice monosaccharide transporter gene OsSTP15 of the present invention can synthesize a rice glucose transporter; after the rice monosaccharide transporter gene OsSTP15 is mutated/knocked out, it is proved that the gene and its transporter can affect sugar metabolism, leading to high accumulation of glucose and sucrose at the base of the stem, and by increasing the number of rice tillers, thereby increasing rice yield, without affecting fertility, providing an important gene resource for enhancing rice tillering and increasing rice yield through variety genetic improvement, and having a good application prospect in cultivating high-yield rice.

Description

Monosaccharide transduction protein gene OsSTP15 and its transporter and its application in improving rice yield, amplification primers

Technical Field

The invention belongs to the field of biotechnology, and in particular relates to a rice monosaccharide transduction protein gene OsSTP15 and a transporter thereof, and an application thereof in improving rice yield, and an amplification primer.

Background technique

Rice yield is a complex agronomic trait determined by multiple factors, among which the number of tillers, number of grains per panicle, and grain weight are the decisive indicators of yield. The regulation of the number of tillers has lower heritability than the number of grains per panicle and grain weight, and is regulated by multiple genes and signal pathways. The hormones involved in regulating plant tillering are mainly auxin, cytokinin, and strigolactone. In recent years, the role of sugar in plant tillering has attracted people's attention, and the mechanism of cross-action between sugar and hormone signals to synergistically regulate plant tillering has become a hot topic. The regulation of lateral bud formation by sugar depends on cytokinin. Sugar promotes the accumulation of D53 protein, a key negative regulatory factor of strigolactone, and antagonizes the inhibitory effect of strigolactone on rice tillering. Sugar promotes the synthesis of auxin, but in rice, which is a weak apical dominant crop, the auxin concentration of lateral buds also has a great influence on tillering, and the inhibitory effect of auxin on plant branching is partly mediated by strigolactone. OsTB1 is a core negative regulatory transcription factor that controls tillering. In rice, strigolactones indirectly regulate OsTB1, while cytokinins negatively regulate the expression of OsTB1. Therefore, sugar and cytokinins positively regulate rice tillering, while auxin and strigolactones negatively regulate rice tillering.

Sugar transporters play an important role in the transport and distribution of carbohydrates, and also affect the sugar metabolism process, so they play an important regulatory role in plant growth and development. The sucrose transporter OsSUT2 located in the tonoplast has the function of sucrose transport. The loss of this gene leads to an increase in the content of soluble sugars such as sucrose, fructose, and glucose in the leaves, but has no effect on the starch content. The tiller number and yield per plant of the sut2 mutant are significantly lower than those of the wild type. The monosaccharide transporter AtSTP1 located in the cell membrane of Arabidopsis can transport glucose, xylose, mannose, and galactose. Changing the expression of this gene causes differences in carbon distribution, which leads to changes in the number of branches in Arabidopsis. Therefore, sugar transporters play an important role in the regulation of plant tillering. Discovering sugar transporters that can regulate rice tillering can provide an important theoretical reference for high-yield rice breeding.

OsSTP15 belongs to the monosaccharide transporter family gene, and its function has not been reported. The present invention uses plant molecular and physiological technical means to clarify the biological function of OsSTP15 and analyze its regulatory effect on rice tillering.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the above background technology and provide a rice monosaccharide transporter gene OsSTP15 and its transporter and application, and amplification primers.

In order to solve the above technical problems, the technical solution proposed by the present invention is:

The invention provides a rice monosaccharide transporter gene OsSTP15, the nucleotide sequence of which is shown as SEQ ID NO:1.

The amino acid sequence of the monosaccharide transporter encoded by the rice monosaccharide transporter gene OsSTP15 is shown in SEQ ID NO:2.

The present invention also provides an application of the rice monosaccharide transporter gene OsSTP15 in cultivating high-yield rice.

The above-mentioned application, preferably, comprises the following steps: selecting a specific target sequence suitable for the CRISPR-Cas9 system from the gDNA sequence of OsSTP15, constructing a Cas9-OsSTP15 vector, transferring the target vector into Agrobacterium by heat shock transformation, and finally cloning it into rice by genetic transformation for expression, thereby obtaining a transgenic strain containing the rice monosaccharide transport gene OsSTP15.

The target sequence PCR amplification method is used to identify the editing of the target gene, and the missense mutation can be used to further explore the gene function. The target sequence for editing the monosaccharide transporter gene OsSTP15 is SEQ ID NO: 3, and the expression vector is Cas9-OsSTP15.

The CRISPR/Cas9 identification amplification primers for the rice monosaccharide transporter gene OsSTP15 used in the above application are Cas9::OsSTP15, and the sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

Based on a general inventive concept, the present invention also provides the following amplification primers for the rice monosaccharide transporter gene OsSTP15:

The first pair of amplification primers is G::OsSTP15, and the sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 6 and SEQ ID NO: 7, respectively. The amplification primer G::OsSTP15 is used to amplify the specific CDS sequence of the rice monosaccharide transport gene OsSTP15, and is suitable for verifying the expression profile of the monosaccharide transport protein gene OsSTP15.

The second pair of amplification primers is GUS::OsSTP15, and the sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 8 and SEQ ID NO: 9, respectively. The amplification primers GUS::OsSTP15 are used to amplify the promoter sequence of the rice monosaccharide transport gene OsSTP15, and are suitable for use in transforming tissue expression materials GUS::OsSTP15.

The third pair of amplification primers is OE::OsSTP15-GFP, and the sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively. The amplification primer OE::OsSTP15-GFP is used to amplify the CDS sequence of the rice monosaccharide transport gene OsSTP15, and is suitable for the subcellular localization experiment of the monosaccharide transporter OsSTP15.

The fifth pair of amplification primers is Y::OsSTP15, and the sequences of the forward primer and the reverse primer are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively. The amplification primer Y::OsSTP15 is used to amplify the CDS sequence of the rice monosaccharide transporter gene OsSTP15, and is suitable for yeast absorption experiments of the monosaccharide transporter OsSTP15.

Compared with the prior art, the present invention has the following beneficial effects:

1. The rice monosaccharide transporter gene OsSTP15 of the present invention can synthesize rice glucose transporter; after the rice monosaccharide transporter gene OsSTP15 is mutated/knocked out, it is proved that the gene and its transporter can affect sugar metabolism, resulting in high accumulation of glucose and sucrose at the base of the stem, and by increasing the number of rice tillers, thereby increasing rice yield, without affecting fertility, providing an important gene resource for enhancing rice tillering and increasing rice yield through variety genetic improvement, and having a good application prospect in cultivating high-yield rice.

2. The present invention provides several PCR amplification primers for the rice monosaccharide transport gene OsSTP15, which can effectively and quickly amplify the gene OsSTP15 from the rice cDNA.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

FIG1 is the spatiotemporal expression pattern result of OsSTP15 obtained in Example 1 (wherein, R: root, S: stem, SB: stem base, LS: leaf sheath, LB: leaf blade, n: node, P: panicle);

FIG2 is the tissue localization result of OsSTP15 obtained in Example 1 (A and D: leaf sheath, B and E: stem internode, C and F: root);

FIG3 is the subcellular localization result of OsSTP15 obtained in Example 1;

FIG4 is the yeast absorption experimental result of OsSTP15 obtained in Example 1;

FIG5 shows two different mutation types of OsSTP15 obtained in Example 2;

FIG6 shows the tillering phenotype and the yield-related agronomic traits of the OsSTP15 mutant obtained in Example 2;

FIG7 is an analysis of starch content in different tissues of the OsSTP15 mutant and wild type obtained in Example 2 during the grain filling period;

FIG8 is a statistical diagram of the dynamic changes in tiller buds and tiller bud length of the OsSTP15 mutant and wild type hydroponic plants obtained in Example 2;

Figure 9 Sugar content at the stem base of the OsSTP15 mutant and wild type obtained in Example 2 after hydroponics for 30 days;

FIG. 10 shows the sugar-promoted tillering of OsSTP15 mutant and wild-type rice obtained in Example 2.

Detailed ways

In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and carefully below in conjunction with the accompanying drawings and preferred embodiments of the specification, but the protection scope of the present invention is not limited to the following specific embodiments.

Unless otherwise defined, all the professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

Embodiment 1:

A rice monosaccharide transporter gene OsSTP15, whose nucleotide sequence is shown in SEQ ID NO: 1; the amino acid sequence of the monosaccharide transporter encoded by it (hereinafter referred to as "monosaccharide transporter OsSTP15") is shown in SEQ ID NO: 2.

The monosaccharide transport gene OsSTP15 and its monosaccharide transporter OsSTP15 were discovered in rice using CRISPRE-CAS9 technology, as well as plant molecular genetics and physiological and biochemical methods. After research, we also found that its knockout can increase tillering and significantly increase rice yield per unit area, which provides a practical basis for enhancing rice tillering and continuously improving rice yield per unit area through genetic improvement of varieties.

1. Spatiotemporal expression pattern of the monosaccharide transporter gene OsSTP15 of the present invention

In order to obtain the expression pattern of the monosaccharide transport gene OsSTP15, RNA was extracted from various parts of rice at different growth stages, with three replicates for each sample. Total RNA was extracted using TRIzol, and the first-strand cDNA was synthesized using the Hiscript II QRT SuperMix kit (Vazyme). Real-time fluorescence quantitative PCR analysis was performed on a StepOnePlus instrument using the qPCR premix ChamQ Universal SYBRqPCR Master Mix (Vazyme). The PCR amplification primer was G::OsSTP15, and its forward primer and reverse primer were 5'-TACCGGCACTACCTGGTGAT-3' (as shown in SEQ ID NO: 6) and 5'-GGTGAAGGAAGACACGACGA-3' (as shown in SEQ ID NO: 7), respectively. Ubiquitin was used as an internal standard with primers 5'-GCTCCGTGGCGGTATCAT-3' (as shown in SEQ ID NO: 14) and 5'-CGGCAGTTGACAGCCCTAG-3' (as shown in SEQ ID NO: 15).

Specific steps are as follows:

Total RNA extraction: 1. After tissue sampling, freeze it in liquid nitrogen and grind it into powder. Take 0.1g sample and add it to a 1.5mL centrifuge tube, add 1mL TRIzol, mix quickly, and let it stand at room temperature for 5 minutes; 2. Add 200μL chloroform to the centrifuge tube, shake vigorously for 30 seconds, and let it stand at room temperature (25℃) for 10 minutes; 3. Centrifuge at 4℃12000rpm for 10 minutes; 4. Carefully aspirate 500μL of supernatant and transfer it to a new 1.5mL centrifuge tube; 5. Add an equal volume of isopropanol, gently invert and mix, let it stand at room temperature (25℃) for 10 minutes; centrifuge at 4℃12000rpm for 10 minutes, and discard the supernatant; 6. Add 1mL 75% ethanol, gently invert to suspend the precipitate; 7. Centrifuge at 4℃8000rpm for 5 minutes, and discard the supernatant; 8. Repeat steps 6 and 7, blow the RNA precipitate to a translucent gel in a clean bench, and add 30-40μL DEPC water to dissolve the RNA.

RNA quality detection and concentration determination: 1. Electrophoresis detection of RNA integrity: 1% agarose gel fast electrophoresis, detection of RNA molecular integrity, observation of 18S and 28S bands, 28S band brightness is about 2 times the brightness of 18S band, indicating good RNA integrity; 2. Nucleic acid analyzer detection of RNA purity and concentration: DEPC water is used to read BLANK, and Nano Drop is used to determine RNA concentration. The absorption peak of nucleic acid is at 260nm, and the maximum absorption peak of protein is at 280nm. RNA samples with OD260/280 between 1.8-2.0 meet the purity standards and can be used for subsequent fluorescence quantitative experiments.

Synthesis of the first strand of cDNA: The total RNA was synthesized using the reverse transcription kit Hiscript II Q RT SuperMix. The total amount of cDNA for each sample was 1 μg. The cDNA was stored at -20°C for future use. The reverse transcription system was as follows: Total RNA (1 μg), 4×gDNA wiper Mix 4 μL, II qRT Super Mix II 4μL, add RNA-free water to 20μL. Reaction conditions: incubate at 42℃ for 15min, inactivate at 85℃ for 5s. After the reaction is completed, add an equal volume of RNA-free water to dilute by half, mix well and store in a refrigerator at -20℃ for later use.

Real-time fluorescence quantitative PCR primer design and specificity analysis: According to the full length of gene cDNA, qPCR primers were designed by using the software primer5. Primers with good specificity and no complementary bases were selected and sent to Qingke Biotechnology Co., Ltd. for synthesis to obtain the amplification primer G::OsSTP15. The cDNA of rice at different stages was used as a template, and the primer specificity was detected by agarose gel electrophoresis. The reaction system was as follows: cDNA 1μL, G::OsSTP15 forward primer (10μM) 0.5μL, G::OsS TP15 reverse primer (10μM) 0.5μL, 1.1×T3 Super PCR Mix (Qingke Biology) 18μL. Reaction conditions: 95℃, 3 minutes; 95℃, 10 seconds; 55℃, 10 seconds; 72℃, 10 seconds; 36 cycles in total, 72℃, 5 minutes.

After the fluorescent quantitative PCR primers were detected correctly, the first strand of cDNA synthesized by reverse transcription was used as a template and the PCR product was purified by Roche Light The two-step method was used for quantification on a 96 real-time fluorescence quantitative PCR instrument. Three technical replicates were set for each gene and each sample. The reaction system was as follows: cDNA 1μL, G::OsSTP15 forward primer (10μM) 0.5μL, G::OsSTP15 reverse primer (10μM) 0.5μL, 2X ChamQ Universal SYBR qPCR Master Mix 10μL, dd H ₂ O 8μL. Reaction conditions: (1) Pre-denaturation stage: 95℃, 10 minutes; (2) Amplification stage: 95℃, 10 seconds, 60℃, 30 seconds, a total of 40 cycles; (3) Melting curve stage: 96℃ 15s, 60℃ 60s, 95℃ 15s.

The results are shown in Figure 1. OsSTP15 is expressed in all tissues at all stages, with relatively high expression in roots at the seedling stage, leaves and leaf sheaths at the tillering stage, and nodes and panicles at the heading stage.

2. Localization of the monosaccharide transport gene OsSTP15 of the present invention

In order to study the tissue specificity of the expression of the monosaccharide transport gene OsSTP15, we constructed a transformation vector carrying ProOsSTP15. The promoter of OsSTP15 (2.0 kb) was obtained by PCR amplification. The forward primer and reverse primer of the amplification primer GUS::OsSTP15 were 5'-CGACGGCCAGTGCCAAGCTTCACTAATGGCAACTTGTATACCCAG-3' (HindIII recognition site is italicized) (as shown in SEQ ID NO: 8) and 5'-GACTGACCACCCGGGGATCCTTGCGCGGGGAGCCGCGC-3' (as shown in SEQ ID NO: 9) (BamHI recognition site is italicized). The fragment was cut using HindIII and BamHI endonucleases, and the amplified fragment was cloned into the pCAMBIA1300 vector to generate the ProOsSTP15 vector. The ProOsSTP15 vector was transformed into the Agrobacterium tumefaciens EHA101 strain and then introduced into a wild-type rice (cv. ZH11) mediated by Agrobacterium tumefaciens.

The specific steps are as follows: Based on the 2.0 kb sequence before the start codon ATG of OsSTP15 obtained from the sequenced rice database (https://rapdb.dna.affrc.go.jp/index.htmL), specific primers for the OsSTP15 promoter were designed, and two restriction sites, HindⅢ and BamHI, were added. The genomic DNA of young leaves of rice seedlings cultured in nutrient solution was used as a template. Max high-fidelity polymerase (Vazyme) was used for PCR amplification. The reaction system was as follows: template DNA 1 μL, Max Master Mix 15μL, GUS::OsSTP15 forward primer 1.5μL, GUS::OsSTP15 reverse primer 1.5μL, ddH ₂ O 11μL, add the above ingredients in sequence, flick to mix, and centrifuge instantaneously. The PCR reaction conditions are 95℃ pre-denaturation for 5 minutes; 95℃ denaturation for 30 seconds, 58℃ annealing for 30 seconds, 72℃ extension for 1.5 minutes, 40 cycles; 72℃ extension for 10 minutes. The PCR amplification product of the OsSTP15 promoter sequence was recovered using a gel recovery kit after electrophoresis detection. The expression vector pC AMBIA1300 plasmid was double-digested with HindⅢ and BamHI, and then gel electrophoresis was performed to detect and recover the large fragment. Use II recombination cloning kit (Vazyme) to recombinant the recovered pCAMBIA1300 double enzyme digested large fragment and OsSTP15 promoter sequence small fragment. The reaction system is as follows: pCAMBIA1300 plasmid large fragment 200ng, proSTP15 PCR amplification product recovered fragment 80ng, 5X CE II Buffer 4μL, Exnase II 4μL, ddH ₂ O added to 20μL. Add the above components in sequence, use a pipette to gently mix, and centrifuge to collect the reaction solution to the bottom of the tube. React at 37℃ for 30 minutes, and immediately cool on ice after the reaction is completed. The recombinant plasmid was transferred into DH5ɑ Escherichia coli competent cells. The antibiotic used for the screening plate was kanamycin. The positive clones were picked for bacterial liquid PCR detection and then sent for sequencing. After sequencing identification, the bacterial liquid was expanded and the plasmid was extracted. The plasmid was named proOsSTP15-pCAMBIA1300 and transformed into Agrobacterium GV3101. The antibiotics were kanamycin and rifampicin. After the bacterial liquid was positively identified, it was used for genetic transformation of wild-type rice mature embryo callus genetic transformation. The specific steps are as follows:

(1) Callus culture of mature embryonic rice: Select healthy seeds of Zhonghua 11, remove the shells, sterilize the surface with 75% ethanol for 3 minutes, rinse twice with sterile water; sterilize with sodium hypochlorite solution for 20-30 minutes, shake gently every few minutes to ensure full disinfection, and rinse 6-8 times with sterile water; move the seeds to sterile filter paper to dry, and then inoculate them into NBM induction medium, 20-25 seeds per dish; culture at 25-26℃ in the dark for 8-10 days to induce callus. Select light yellow callus with dry surface and dense structure, remove the grains and buds, and transfer to J3 subculture medium, and culture at 25-26℃ in the dark for 5-7 days.

(2) Co-cultivation of Agrobacterium and callus tissue: Pick light yellow callus tissue with dry surface and dense structure, place it on sterile filter paper and air dry until the surface turns white; transfer the callus tissue into the bacterial solution with adjusted OD value, soak for 30 minutes, and shake it gently every 5 minutes; pour out the bacterial solution, wash it with sterile water for 3 to 5 times, and place the callus tissue on the filter paper to dry until the surface turns white; take out the NBM (containing As) solid culture medium, spread a piece of sterile filter paper on the surface of the culture medium, transfer the callus tissue, and then place it at 25 to 26°C and culture it in the dark for 2 to 3 days.

(3) Screening culture: After 2-3 days of co-culture, the callus tissue was removed and rinsed with sterile water for 6-8 times until the rinsed liquid was not turbid; soaked in 50 mL of sterile water containing antibiotics (500 mg/L cephalosporin and 400 mg/L carbenicillin) for 30 min, and gently shaken every 5 min; the callus tissue was transferred to sterile filter paper and dried until the surface turned white, and then transferred to the screening medium (J3 + 500 mg/L cephalosporin + 400 mg/L carbenicillin + 50 mg/L hygromycin) and cultured at 25-26°C in the dark. After 12-17 days of screening culture, active callus tissue was picked and transferred to a new screening medium for a second screening.

(4) Predifferentiation culture: Carefully transfer the callus tissue that has grown resistant callus to the predifferentiation medium (Y + 500 mg/L cephalosporin + 400 mg/L carbenicillin) and place it in a light incubator at 25-26°C and 14 h light for 3-7 days.

(5) Differentiation culture: Carefully transfer the green callus tissue from pre-culture to differentiation medium (DL + 500 mg/L cephalosporin + 400 mg/L carbenicillin) and culture at 25-26°C with 14 h light intensity. Replace the medium every 15-20 days.

(6) Rooting culture: The differentiated green seedlings (about 4-6 cm in height) were transferred to the rooting medium (R) and cultured at 25-26°C with 14 h light intensity to allow them to take root. The medium was replaced every 15-20 days.

(7) Hardening and transplanting: After the roots have grown for 10 to 20 days, open the culture bottle cap, add sterile water to slightly cover the culture medium, and harden the seedlings at room temperature for 7 to 12 days. Rinse the culture medium attached to the roots of the seedlings with tap water, transplant them into a small pot filled with soil, and transplant them into the experimental field for cultivation after the seedlings survive.

(8) Identification of transgenic plants: Extract the DNA of transgenic plants, design hygromycin identification specific primers based on the sequence of the selection marker hygromycin gene carried on the vector, and finally obtain transgenic positive seedlings.

The strains obtained by genetic transformation were cultured throughout the growth period, and the positive plants were added to the prepared GUS dye solution to ensure that all tissues of the sample were completely immersed in the GUS dye solution. They were placed in a 37°C incubator and stained with tin foil for about 10 hours in the dark. When the sample showed obvious blue color, the dye solution was poured out and recovered. The materials were decolorized with 30%, 50%, and 70% alcohol in turn. When the chlorophyll was completely removed, the materials were placed under a microscope for observation and photography. The results of the GUS protein expression site driven by the OsSTP15 promoter are shown in Figure 2.

As shown in Figure 2, during the tillering stage, the GUS protein driven by the STP15 promoter is mainly expressed in the vascular bundle sheath and xylem of the leaf sheaths, stems and roots.

3. Subcellular localization of the monosaccharide transporter OsSTP15 of the present invention

In order to study the cell specificity of OsSTP15 expression, we constructed a transformation vector carrying OsSTP15-GFP. The CDS sequence of OsSTP15 (excluding the terminator) was obtained by PCR amplification. The forward primer and reverse primer of the amplification primer OE::OsSTP15-GFP were 5'-GAGCTCGGTACCCGGGGATCCATGGCGGCAGGGACAG AG-3' (as shown in SEQ ID NO: 10, BamHI recognition site is italicized) and 5'-CTTCTCCTTTGCCCATGTCGACGAGGCAGTTCACTTGGGCG-3' (as shown in SEQ ID NO: 11, SalI recognition site is italicized). The fragment was cut using HindIII and SalI endonucleases, and the amplified fragment was cloned into the pCAMBIA1300-GF P vector to generate the OsSTP15-GFP vector. The generated vector was transformed into rice protoplasts by PEG-mediated method.

The specific steps are as follows: shake the OsSTP15-GFP bacterial suspension, extract the OsSTP15-GFP plasmid using the Solebow plasmid extraction kit and concentrate the plasmid. The concentration steps are as follows: (1) mix all plasmids of the same gene into one tube; (2) freeze the centrifuge tube containing the plasmid at -80℃; (3) seal the centrifuge tube with sealing film and poke two holes in the tube cap with a needle; (4) freeze dry the plasmid using a freeze dryer; (5) add ddH ₂ O to adjust the concentration to ≥1μg and store at -20℃ for later use.

Preparation of rice protoplasts: (1) 1/2MS cultured rice medium flower 11, grew in the dark for 11-14 days, and then selected 15 yellow flower seedlings with good growth status, and cut them into thin strips with a primary blade, the thinner the better; (2) placed in a culture dish containing 10 mL of enzymatic solution, vacuumed for 5 minutes, and enzymolyzed in a shaker in the dark for 4-5 hours (28°C, 45 rpm); (3) Filtered the enzymatic solution into a 10 ml round-bottom centrifuge tube using a 150-mesh sieve; (4) 200×g, 1 min, 4°C, discarded the supernatant, gently added 1 mL of W5 along the wall, and gently resuspended the protoplasts until there was no precipitation at the bottom; (5) Incubated on ice for 40 minutes; (6) Removed the supernatant, added 2 mL W5 was gently inverted to mix; (7) The tip of the yellow pipette was cut off, 12 μL of the mixed sample was taken and gently injected into a hemocytometer and placed under a microscope for counting; (8) The remaining protoplasts were incubated on ice for 40 min and the supernatant was discarded.

PEG-mediated protoplast transformation: The number of protoplasts was estimated based on the concentration of protoplasts, and MMG was added to make the final concentration of protoplasts about 2×10 ⁵ /mL; the following reaction solution was prepared in a 1.5mL round-bottom centrifuge tube: 150μL protoplasts, 15μg GFP plasmid (1.0μg/μL), and mixed evenly; then 165μL of PEG transformation solution was added, and the mixture was gently inverted several times to promote the fusion of PEG and protoplasts, and the mixture was allowed to stand at room temperature for 5min, and 1mL of W5 was added to terminate the reaction; centrifuged at 200g for 5min, the supernatant was removed, and the protoplasts were resuspended with 200μL of W5, and cultured overnight at 22℃ in the dark; the GFP signal of the protoplasts was observed using a laser confocal microscope. The results are shown in Figure 3, and the rice monosaccharide transporter OsSTP15 is located on the cell membrane of rice protoplasts.

4. Functional analysis of OsSTP15 of the present invention in yeast absorption experiments

In order to study the specific substrate of OsSTP15, the gene OsSTP15 was amplified from rice cDNA by PCR amplification using primer Y::OsSTP15, the forward primer sequence was 5'-TCCCCCGGGCTGCAGGAATTCATGGCGGCAGGGACAGAG-3' (as shown in SEQ ID NO: 12, the EcoRI recognition site is italicized), and the reverse primer sequence was 5'-GGTACCGGGCCCCCCCTCGAGTTAGAGGCAGTTCACTTGGGCG-3' (as shown in SEQ ID NO: 13, the XhoI recognition site is italicized), and then cloned into the yeast expression vector PDR196 by homologous recombination to obtain the target vector PDR96-STP15 containing the gene OsSTP15, and an empty vector control group PDR196 (without OsSTP15) was set up. The empty vector and the target vector containing the gene OsSTP15 were transformed into the yeast mutant strain EBY.VW4000 for heterologous overexpression using the PEG/lithium acetate method, as follows: 1) The yeast strain to be transformed was streaked and activated on a complete nutrient medium (YPD medium: 20 g/L tryptic peptone, 10 g/L yeast extract, 20 g/L D-glucose, solid medium supplemented with 20 g/L agar, autoclaved for use); 2) A single clone was picked and inoculated into 5 mL of complete nutrient medium, and cultured at 28°C with shaking at 200 rpm until OD ₆₀₀ =1.0; 3) Take 0.5mL bacterial solution, 12000g, centrifuge for 30 seconds, and pour off the supernatant (about 100μL culture medium remains at the bottom); 4) Add 1μL salmon sperm DNA, mix well; add 1μg target plasmid DNA, mix well; then add 500μL yeast transformation buffer, mix well, and stand at room temperature for 8-10 hours; 5) Take 20-50μL bacterial suspension at the bottom of the tube, apply it on the selection plate SD (-Ura) + 2% maltose, 28°C, and stand and culture to obtain yeast containing the monosaccharide transporter OsSTP15 of the present invention. Then, the transport function of OsSTP15 was identified on SD (-Ura) plates with different sugars as the sole carbon source using a yeast dot experiment. Yeast transformation buffer: 90 mL 45% PEG4000, 10 mL 1 M lithium acetate, 1 mL 1 M Tris-HCl (pH 7.5), and 0.2 mL 0.5 M EDTA were mixed.

To verify the subcellular localization of OsSTP15 protein in yeast, the CDS of ST15 was cloned using primer Y::OsSTP15 and cloned into the yeast subcellular localization vector PDR196-GFP by homologous recombination to obtain the PDR96-GFP-STP15 target vector, and a control group PDR196-GFP was set up at the same time. The control vector PDR196-GFP and the target vector PDR96-GFP-STP15 containing the gene OsSTP15 were transformed into the yeast mutant strain EBY.VW4000 using the PEG/lithium acetate method. Single clones were picked and cultured to the logarithmic growth phase, and the subcellular localization of GFP fluorescence was observed using a confocal microscope.

The results are shown in Figure 4. In the strain containing PDR196-GFP, the GFP protein is expressed in the cytoplasm, and in the strain containing PDR96-GFP-STP15, the GFP protein is mainly expressed in the cell membrane and vacuole membrane, and is also expressed in the cytoplasm (Figure 4A). On the medium with maltose as the only carbon source, both the empty vector and the target gene vector can grow normally. However, on the medium with glucose as the only carbon source, the strain expressing STP15 and the positive control strain expressing MST6 can grow, and the empty vector cannot grow at all, indicating that STP15 has glucose transport activity. The strains expressing STP15 on 1% maltose + 0.2% deoxyglucose and 1% glucose + 0.2% deoxyglucose can grow (Figure 4B), indicating that the localization of its cell membrane and vacuole membrane has glucose transport function.

Embodiment 2:

The invention discloses an application of the rice monosaccharide transport gene OsSTP15 in cultivating high-yield rice, which specifically includes the construction of OsSTP15 mutant plants, and the analysis of maturity phenotype and yield factors.

1. Obtaining the OsSTP15 gene knockout material of the present invention

In order to verify the role of the monosaccharide transporter OsSTP15 of the present invention in rice, we edited OsSTP15 using CRISPR-Cas9 gene editing technology. The Cas9/g RNA vector was constructed with the target sequence 5'-GAGGCGGCGCGGGACTACGG-3' (as shown in SEQ ID NO: 3), and Agrobacterium tumefaciens GV3101 was transformed. The positive strain was used for genetic transformation of wild-type rice Zhonghua 11 to obtain the OsSTP15 gene-edited transgenic strain. The transgenic strain was identified by PCR amplification, and the identification primers were Cas9::OsSTP15, the upstream primer sequence: 5'-CTAAGCGTGGAGGAGAGGC-3' (as shown in SEQ ID NO: 4), and the downstream primer sequence: 5'-AGACGCGCAAGAGGTTGATT-3' (as shown in SEQ ID NO: 5). After identification, two knockout strains stp15-1 and stp15-2 with different mutation types were obtained. As shown in Figure 5, the TA base of stp15-1 mutated to a C base near the target site, resulting in the premature termination of the 14 amino acids encoded by the OsSTP15 gene; the single base A insertion mutation of stp15-2 near the target site resulted in the premature termination of the 10 amino acids encoded by the OsSTP15 gene.

2. Effects of OsSTP15 gene knockout on rice yield-related agronomic traits

The wild type (ZH11) and mutants (stp15-1, stp1515-2) were cultivated in the field. There was no significant difference between the stp15 mutant and the wild type at the seedling stage. The number of tillers of the mutant increased compared with the wild type at the end of tillering (Figure 6A). After maturity, the yield-related agronomic traits of the stp15 mutant were statistically analyzed. The number of tillers increased by 21.8%-39.2% and the yield per plant increased by 8.93%-12.25% compared with the wild type, but the number of grains per ear decreased by 18.47%-20.87%. There was no significant difference in other yield-related agronomic traits such as plant height, fruit setting rate, and 1000-grain weight between the wild type and the mutant. The values are the mean ± standard deviation of 15 repeated experiments, and the P value is the statistical test for significant difference Student’s t-test analysis *P<0.05, **P<0.01.

In order to verify the effect of monosaccharide transporter OsSTP15 on carbohydrate source pool allocation, we took leaves, leaf sheaths, nodes, internodes, and ears of OsSTP15 mutants and wild types during the grain filling period (30 days after flowering) and measured the starch content. The specific method is as follows: (1) Weigh 0.5% dried and 100-mesh sieve sample and place it in a 10mL centrifuge tube; (2) Add 7mL 80% ethanol, extract in 80℃ water bath for 30min, cool to room temperature, centrifuge at 3000rpm for 5min, and remove the supernatant; (3) Repeat twice and add 6mL 80% ethanol, water bath for 10 minutes, centrifuge again, and remove the supernatant; (4) Add 3 mL of distilled water to the precipitate, vortex to mix thoroughly, and gelatinize in a 100°C water bath for 15 minutes; (5) After cooling, add 2 mL of perchloric acid, shake thoroughly, and extract for 15 minutes; (6) Add distilled water to 9 mL, shake thoroughly, centrifuge for 10 minutes, and collect the supernatant in a 50 mL centrifuge tube; (7) Add 2 mL of cold 1/2 perchloric acid to the precipitate, shake thoroughly, and extract for 15 minutes Then add water to 9 mL, mix well and centrifuge for 10 minutes, collect the supernatant in a centrifuge tube; (8) Add distilled water to wash the precipitate 1-2 times, 7 mL each time, centrifuge for 10 minutes, and combine the supernatant in a 50 mL centrifuge tube; (9) Make the volume to 50 mL, take 1 mL in a 10 ml centrifuge tube, add 5 mL of anthrone reagent, and mix thoroughly; (10) Take 100 μL of the marking solution, add 1 mL of anthrone reagent respectively, and mix thoroughly; (11) Boil in water bath for 10 minutes, take out and cool. After cooling, measure the absorbance at 625 nm in an enzyme reader and adjust to zero with the 0 concentration of the standard curve. Draw a standard curve, from which the concentration of sugar in the extract is obtained, and thus the sugar content in the sample is calculated.

The results are shown in Figure 7. There was no significant difference in starch content in various tissues between stp15 and the wild type, indicating that OsSTP15 had no effect on the source-sink allocation of carbohydrates during the grain filling period.

3. Effects of OsSTP15 gene knockout on sugar content at the base of rice stems and tillering

To verify the effect of OsSTP15 on rice tillering, we hydroponic mutants and wild types and counted the length of tiller buds. The hydroponic experiment was carried out in a light culture room with a temperature set at 26°C, a photoperiod of 14h (light)/10h (darkness), a light intensity of 300-320μmol/m2/s, and a humidity of 60-75%. The nutrient solution used for hydroponics adopted the conventional nutrient solution formula of the International Rice Research Institute. The experiment was set up to culture wild-type and mutant materials with normal nutrient solution, replace the nutrient solution every 3d, and ventilate every day. The length of tiller buds was investigated at 22d, 25d, 28d, and 31d of hydroponics, and photographed with a macro zoom microscope, and imageJ was used to measure and count the length of tiller buds. The results are shown in Figure 8. Starting from 25 days, the lengths of the first and second tiller buds of the stp15 mutant were significantly greater than those of the wild type, indicating that OsSTP15 knockout promotes the formation and elongation of rice tiller buds.

After 30 days of hydroponic culture, the contents of glucose, fructose, sucrose and starch at the stem base of stp15 and the wild type were measured.

Glucose content was determined using a glucose content kit micro-method (Solarbio), the specific method is as follows:

(1) Sample processing: Take fresh leaf samples at the tillering stage and grind them into a homogenate under liquid nitrogen freezing. Quickly weigh about 0.1 g and add 1 mL of distilled water. Boil in a water bath at 100°C for 10 min, cool to room temperature, centrifuge at 25°C and 8000g for 10 min, and take the supernatant for testing.

(2) Determination steps: Add reagents to a 1.5 mL centrifuge tube according to the table below and keep warm at 25°C for 15 min. Turn on the microplate reader in advance, preheat for more than 30 min, adjust to zero with distilled water, and read the absorbance at a wavelength of 505 nm. The blank tube is marked as A1, the standard tube is marked as A2, and the determination tube is marked as A3.

Table 1: Configuration of different reagents and samples

(3) Calculate the content: glucose content (μmol·g-1 fresh weight) = (A3-A1)/(A2-A1)/W.

The fructose and sucrose contents were determined using the resorcinol colorimetric method. The specific method is as follows: (1) Preparation of the sample solution: weigh 0.5 g of the dried sample and place it in a 10 mL centrifuge tube. Add 7 mL of 80% ethanol, extract in a water bath at 80°C for 30 min, cool to room temperature and centrifuge at 3000 rpm for 5 min, and collect the supernatant. Repeat twice, add 6 mL of 80% ethanol each time, water bath for 10 min, and centrifuge again. Combine the three supernatants in a 50 mL centrifuge tube and make up to the same scale. Add an appropriate amount of activated carbon, decolorize in an oven at 85°C for 30 min, and filter the filtrate for determination of fructose and sucrose contents. (2) Preparation of the standard curve. The sucrose and fructose lines were prepared with 80% ethanol. The standard sample concentrations were 0, 10, 20, 30, 40, 60, 80, 100, 150, and 200 μg·mL-1. (3) Colorimetric determination: Determination of sucrose content: Take 0.4 mL of the marking solution and the sucrose test solution, add 200 μL of 2 mol/L NaOH, boil in a 100°C water bath for 5 min, and cool to room temperature; add 2.8 mL of concentrated HCl and 0.8 mL of 0.1% resorcinol, shake well, and place in a 80°C water bath for 10 min. After cooling, measure the absorbance at a wavelength of 480 nm in an ELISA reader, and adjust the zero value to the 0 concentration of the standard curve. Draw a standard curve, calculate the concentration of the colorimetric solution based on the marking line, and thus calculate the sucrose content. Determination of fructose content: Take 1 mL of the marking solution and the fructose test solution, add 2 mL of 0.1% resorcinol and 1 mL of concentrated HCl, shake well. React in a 80°C water bath for 10 min, and then cool to room temperature; after cooling, measure the absorbance at a wavelength of 480 nm in an ELISA reader, and adjust the zero value to the 0 concentration of the standard curve. A standard curve was drawn and the fructose concentration of the color developing solution was obtained from the standard curve to calculate the fructose content.

The results are shown in Figure 9 . The fructose, sucrose, and glucose contents at the stem base of the stp15 mutant were higher than those of the wild type ( Figure 9 ).

In view of the fact that existing studies have shown that sugar can promote rice tillering, we germinated and cultured the stp15 mutant and ZH11 in 1/2MS medium supplemented with glucose and sucrose to verify the effect of sugar on rice tillering. The specific method is as follows: select healthy seeds of Zhonghua11 and stp15 mutant, remove the shells, sterilize the surface with 75% ethanol for 3 minutes, and rinse twice with sterile water; disinfect with sodium hypochlorite stock solution for 20-30 minutes, shake gently every few minutes to ensure full disinfection, and rinse with sterile water 6-8 times; move the seeds to sterile filter paper to dry, and inoculate them into 1/2MS, 1/2MS+3% glucose, and 1/2MS+4% sucrose solid medium for culture. Culture conditions: temperature is 26℃, light cycle is 14h (light)/10h (dark), light intensity is 300-320μmol/m ² /s, and humidity is 60-75%. Take pictures after 30 days of culture.

The results are shown in Figure 10. Adding 3% glucose or 4% sucrose can effectively promote rice tillering. Therefore, we speculate that the reason why OsSTP15 knockout promotes tillering may be the increase in sugar content at the base of the stem.

In summary, the rice monosaccharide transport gene OsSTP15 of the present invention can synthesize rice glucose transporter, localize in the cell membrane of rice protoplasts, and knock out its expression to increase the soluble sugar content at the stem base, increase the number of tillers, and increase the yield per plant by about 10%. The rice monosaccharide transport gene OsSTP15 is applied to rice to improve the yield per unit area of rice and has a significant effect.

Sequence Listing

<110> Hunan Agricultural University

<120> Monosaccharide transduction protein gene OsSTP15 and its transporter and its application in improving rice yield, amplification primers

<160> 15

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1524

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 1

atggcggcag ggacagaggc ggcgcgggac tacggcggcg gcgtgacggc cagcgtcgtg 60

gtgacctgcc tcatcgccgc ctcctgcggc ctcatcttcg gttacgacat tggcgtctca 120

ggtggtgtga cgcagatgca atcgttcctg accaagttct tcccggaggt ggtgaagggg 180

atgcgtggcg cgaagcggga cgcctactgc aggtacgaca accaggtcct gacggcgttc 240

acctcgtcgc tgtacatcgc cggcgcggtg gcgtcgctgg tggcgagccg ggtgaccagg 300

atggtgggcc gccaggccat catgctgacc ggcggcgccc tcttcctcgc cggctccgcc 360

ttcaacgctg gcgccgtcaa catcgccatg ctcatcatcg gccgcattct gctcggcgtc 420

ggcgtcggct tcaccacgca ggcggctccg ctgtatctcg ccgagacagc gccggcgagg 480

tggcgcgggg cgttcaccgc cgcgtaccat atcttcctcg tcatcggcac ggtggccgcg 540

acggccgcca actacttcac ggaccgaatc ccgggctggg ggtggcgcgt ctcgctgggc 600

ctcgcggcgg tgccggccac ggtcatcgtg gtgggcgcgc tcttcgtccc ggacacgccc 660

gctagcctcg tcctgcgcgg gcacacggag aaggcccgcg cgtcgctcca gcgcgtccgc 720

ggcgcggacg ccgacgtcga cgccgagttc aaggacatca tccgcgccgt cgaggaggcg 780

cggcggaacg acgagggcgc gttccggagg ctgcgtggcc gtgggtaccg gcactacctg 840

gtgatggtgg tggccatccc gacattcttc gacctcaccg gcatggtcgt catcgccgtc 900

ttctcgccgg tgctgttccg gacgctcggg ttcaacagcc agagggccat ccttgcctcc 960

atcgtactta ccctcgtcaa cttgtgcgcc gtcgtcgtgt cttccttcac cgtcgaccgc 1020

gtcggccgca ggttcttgtt cctcgccggc ggcaccgcca tgctcctctg ccaggtggcg 1080

gtggcgtgga tactggcgga gcatctcggg aggagccacg cggcggcgac gatggcgaag 1140

agctacgcgg cgggggtggt ggcgctgatg tgcgtgtaca cggcgagcct cggcctgtca 1200

tggggaccgc tcaagtgggt ggtgccgagc gagatctacc cggtggaggt ccggtcggcg 1260

gggcaggcgc tgggcctgtc cgtctcgctc acgctctcct tcgcgcagac gcaggtgttc 1320

atgtccatgc tctgcgccat gaagtacgcc atcttcctct tctacgccgg ctgggtcctc 1380

gccatgacgg ccttcatcgc gctgttcctc ccggagacca agggcgtccc actggaggcc 1440

atgcgcgcgg tgtgggcgaa gcactggtac tggaagcggt tcgccatgga cgccaagctg 1500

gacgcccaag tgaactgcct ctaa 1524

<210> 2

<211> 507

<212> PRT

<213> Rice (Oryza sativa L.)

<400> 2

Met Ala Ala Gly Thr Glu Ala Ala Arg Asp Tyr Gly Gly Gly Val Thr

1 5 10 15

Ala Ser Val Val Val Thr Cys Leu Ile Ala Ala Ser Cys Gly Leu Ile

20 25 30

Phe Gly Tyr Asp Ile Gly Val Ser Gly Gly Val Thr Gln Met Gln Ser

35 40 45

Phe Leu Thr Lys Phe Phe Pro Glu Val Val Lys Gly Met Arg Gly Ala

50 55 60

Lys Arg Asp Ala Tyr Cys Arg Tyr Asp Asn Gln Val Leu Thr Ala Phe

65 70 75 80

Thr Ser Ser Leu Tyr Ile Ala Gly Ala Val Ala Ser Leu Val Ala Ser

85 90 95

Arg Val Thr Arg Met Val Gly Arg Gln Ala Ile Met Leu Thr Gly Gly

100 105 110

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

115 120 125

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

130 135 140

Thr Thr Gln Ala Ala Pro Leu Tyr Leu Ala Glu Thr Ala Pro Ala Arg

145 150 155 160

Trp Arg Gly Ala Phe Thr Ala Ala Tyr His Ile Phe Leu Val Ile Gly

165 170 175

Thr Val Ala Ala Thr Ala Ala Asn Tyr Phe Thr Asp Arg Ile Pro Gly

180 185 190

Trp Gly Trp Arg Val Ser Leu Gly Leu Ala Ala Val Pro Ala Thr Val

195 200 205

Ile Val Val Gly Ala Leu Phe Val Pro Asp Thr Pro Ala Ser Leu Val

210 215 220

Leu Arg Gly His Thr Glu Lys Ala Arg Ala Ser Leu Gln Arg Val Arg

225 230 235 240

Gly Ala Asp Ala Asp Val Asp Ala Glu Phe Lys Asp Ile Ile Arg Ala

245 250 255

Val Glu Glu Ala Arg Arg Asn Asp Glu Gly Ala Phe Arg Arg Leu Arg

260 265 270

Gly Arg Gly Tyr Arg His Tyr Leu Val Met Val Val Ala Ile Pro Thr

275 280 285

Phe Phe Asp Leu Thr Gly Met Val Val Ile Ala Val Phe Ser Pro Val

290 295 300

Leu Phe Arg Thr Leu Gly Phe Asn Ser Gln Arg Ala Ile Leu Ala Ser

305 310 315 320

Ile Val Leu Thr Leu Val Asn Leu Cys Ala Val Val Val Ser Ser Phe

325 330 335

Thr Val Asp Arg Val Gly Arg Arg Phe Leu Phe Leu Ala Gly Gly Thr

340 345 350

Ala Met Leu Leu Cys Gln Val Ala Val Ala Trp Ile Leu Ala Glu His

355 360 365

Leu Gly Arg Ser His Ala Ala Ala Thr Met Ala Lys Ser Tyr Ala Ala

370 375 380

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

385 390 395 400

Trp Gly Pro Leu Lys Trp Val Val Pro Ser Glu Ile Tyr Pro Val Glu

405 410 415

Val Arg Ser Ala Gly Gln Ala Leu Gly Leu Ser Val Ser Leu Thr Leu

420 425 430

Ser Phe Ala Gln Thr Gln Val Phe Met Ser Met Leu Cys Ala Met Lys

435 440 445

Tyr Ala Ile Phe Leu Phe Tyr Ala Gly Trp Val Leu Ala Met Thr Ala

450 455 460

Phe Ile Ala Leu Phe Leu Pro Glu Thr Lys Gly Val Pro Leu Glu Ala

465 470 475 480

Met Arg Ala Val Trp Ala Lys His Trp Tyr Trp Lys Arg Phe Ala Met

485 490 495

Asp Ala Lys Leu Asp Ala Gln Val Asn Cys Leu

500 505

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 3

gaggcggcgc gggactacgg 20

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 4

ctaagcgtgg aggagaggc 19

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 5

agacgcgcaa gaggttgatt 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 6

taccggcact acctggtgat 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 7

ggtgaaggaa gacacgacga 20

<210> 8

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 8

cgacggccag tgccaagctt cactaatggc aacttgtata cccag 45

<210> 9

<211> 38

<212> DNA

<213> Artificial Sequence

<400> 9

gactgaccac ccggggatcc ttgcgcgggg agccgcgc 38

<210> 10

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 10

gagctcggta cccggggatc catggcggca gggacagag 39

<210> 11

<211> 41

<212> DNA

<213> Artificial Sequence

<400> 11

cttctccttt gcccatgtcg acgaggcagt tcacttgggc g 41

<210> 12

<211> 39

<212> DNA

<213> Artificial Sequence

<400> 12

tcccccgggc tgcaggaatt catggcggca gggacagag 39

<210> 13

<211> 43

<212> DNA

<213> Artificial Sequence

<400> 13

ggtaccgggccccccctcga gttagaggca gttcacttgg gcg 43

<210> 14

<211> 18

<212> DNA

<213> Artificial Sequence

<400> 14

gctccgtggc ggtatcat 18

<210> 15

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 15

cggcagttga cagccctag 19

Claims (6)

1. An application of rice monosaccharide transporter gene OsSTP or a transporter encoded by the gene in the cultivation of high-yield rice, which is characterized in that the application method comprises the following steps: constructing a CRISPR/Cas9-OsSTP vector by selecting a specific gDNA target site of a monosaccharide transport protein gene OsSTP, transferring the target vector into agrobacterium by a thermal shock transformation method, and finally cloning the target vector into rice by genetic transformation to express the target vector to obtain a transgenic strain containing the rice monosaccharide transport gene OsSTP;

The nucleotide sequence of the rice monosaccharide transporter gene OsSTP is shown in SEQ ID NO:1, wherein the target sequence edited by the monosaccharide transporter gene OsSTP is SEQ ID NO:3, the expression vector is Cas 9-OsSTP.

2. The use according to claim 1, wherein the rice monosaccharide transporter gene OsSTP has a CRISPR/Cas9 identification amplification primer of Cas9: osSTP15, and the forward primer and the reverse primer have the sequences shown in SEQ ID NO:4 and SEQ ID NO: shown at 5.

3. The application of claim 1, wherein the amplification primer of the rice monosaccharide transporter gene OsSTP is G: osSTP15, and the forward primer and the reverse primer have the sequences shown in SEQ ID NO:6 and SEQ ID NO: shown at 7.

4. The application of claim 1, wherein the rice monosaccharide transporter gene OsSTP promoter has the amplification primer GUS: osSTP15, and the forward primer and the reverse primer have the sequences shown in SEQ ID NO:8 and SEQ ID NO: shown at 9.

5. The use according to claim 1, wherein the amplification primer of the CDS of the rice monosaccharide transporter gene OsSTP is OE: osSTP15-GFP, and the forward primer and the reverse primer have the sequences shown in SEQ ID NO:10 and SEQ ID NO: 11.

6. The application of claim 1, wherein the amplification primer of the rice monosaccharide transporter gene OsSTP is Y: osSTP15, and the forward primer and the reverse primer have the sequences shown in SEQ ID NO:12 and SEQ ID NO: shown at 13.