Application of rice monosaccharide transporter gene OsPLT1 and its transporters in regulating rice growth period and amplification primers

Abstract

This invention belongs to the field of biological genes and discloses the application of a rice monosaccharide transporter gene OsPLT1 and its transporter in regulating rice growth period. The rice monosaccharide transporter gene... OsPLT1 The nucleotide sequence is shown in SEQ ID NO: 1. This invention also discloses a gene for a rice monosaccharide transporter. OsPLT1 Amplification primers. This invention provides rice. OsPLT1 Novel applications of genes and their encoded proteins in regulating rice growth stage; knockout of wild-type ZH11 using CRISPR / Cas9. OsPLT1 At this time, it can accelerate the growth process of rice without affecting the yield of rice, therefore, OsPLT1 Genes can be used to regulate plant growth periods and increase annual and seasonal yields, and have high economic and application value.

Description

Technical Field

[0001] This invention belongs to the field of biological genes, and particularly relates to a rice monosaccharide transprotein gene. OsPLT1 The application of its transporters in shortening the growth period of rice. Background Technology

[0002] Rice contains three major families of sugar transporters: sucrose transporters (SUTs), sugar efflux transporters (SWEETs), and monosaccharide transporters (MSTs), encompassing a total of 92 genes. Among these, MST proteins have relatively few functional reports and can be further classified based on their transport substrates into plasma membrane monosaccharide transporters (STPs), inositol transporters (INTs), vacuole membrane monosaccharide transporters (TMTs), vacuole membrane glucose transporters (VGTs), plastid monosaccharide transporters (GMTs), early response dehydration analogs (ERDs), and polyol transporters (PLTs). Arabidopsis thaliana has six polyol transport genes (…). PLT1-6 : At2g16120 , At2g16130 , At2g18480 , At2g20780 , At3g18830 , At4g36670 Studies have shown that these genes, in addition to transporting alcohols, are also involved in the transport of monosaccharides such as glucose and xylose. AtPLT1 ( At2g16120 )and AtPLT2 ( At2g16130 Xylitol and fructose are transporters, primarily expressed in pollen and young xylem; AtPLT5 ( At4g36670 It transports sorbitol, xylitol, erythritol, and xylose, and its expression is most intense in the vascular tissues of roots and leaves, as well as in specific floral organs. Currently, Arabidopsis thaliana... PLT Related studies have shown that its transport substrates are not limited to sugar alcohols, but research mainly focuses on tissue-specific expression, subcellular localization, and transport substrates. AtPLT1 / 2 / 5 The mutant materials of these genes showed no significant difference from the wild type, so the biological function of these genes is unclear. PLT Biological functions and mechanisms of action in plants are also rarely reported, especially in rice. PLT Related research is in a completely blank area. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a rice monosaccharide transporter gene. OsPLT1 The application of its transporters in regulating rice growth period and amplification primers.

[0004] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0005] A rice monosaccharide transporter gene OsPLT1 Application in regulating rice growth period, the rice monosaccharide transporter gene OsPLT1 The nucleotide sequence is shown in SEQ ID NO: 1.

[0006] Based on a general inventive concept, the present invention also provides a method for constructing a rice monosaccharide transporter gene. OsPLT1 The application of the encoded transporter in regulating the growth period of rice, the amino acid sequence of the transporter is shown in SEQ ID NO: 2.

[0007] The preferred application described above is the knockout of wild-type rice using the CRISPR / Cas9 method. OsPLT1, Short-breeding-period rice was obtained.

[0008] The above applications, preferably, are from OsPLT1 The gDNA sequence was selected from the monosaccharide transporter gene. OsPLT1 The target sequence was edited, the Cas9-OsPLT1 vector was constructed, and then transformed into Agrobacterium tumefaciens using heat shock transformation. Finally, it was cloned into rice for expression through genetic transformation. OsPLT1 Gene-edited transgenic lines.

[0009] In the above-described applications, preferably, the monosaccharide transporter gene... OsPLT1 The edited target sequences are SEQ ID NO:3 and SEQ ID NO:4.

[0010] The above-mentioned applications, preferably, are as follows: OsPLT1 Gene-edited transgenic lines were identified by PCR amplification. The identification primers were Cas9::OsPLT1, with the upstream primer sequence being 5'-CGTGTTAGCTCGGATCTGTA-3' (as shown in SEQ ID NO: 5) and the downstream primer sequence being 5'-GGCGAAGTTGGAGACGTA-3' (as shown in SEQ ID NO: 6).

[0011] Based on a general inventive concept, the present invention also provides a gene for rice monosaccharide transporters. OsPLT1 The amplification primers, preferably G::OsPLT1, are shown in SEQ ID NO: 7 and SEQ ID NO: 8, respectively. This amplification primer G::OsPLT1 is used to amplify rice monosaccharide transport genes. OsPLT1 The specific CDS sequence is suitable for verifying monosaccharide transporter genes. OsPLT1The expression profile of the rice monosaccharide transporter gene OsPLT1 The nucleotide sequence is shown in SEQ ID NO: 1.

[0012] Based on a general inventive concept, the present invention also provides a gene for rice monosaccharide transporters. OsPLT1 The amplification primers, OE::OsPLT1-GFP, are shown in SEQ ID NO: 9 and SEQ ID NO: 10, respectively. These primers are used to amplify the CDS sequence of the rice monosaccharide transporter OsPLT1, and are suitable for subcellular localization experiments of the monosaccharide transporter OsPLT1 in rice.

[0013] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0014] This invention provides rice OsPLT1 Novel applications of genes and their encoded proteins in regulating rice growth stage; knockout of wild-type ZH11 using CRISPR / Cas9. OsPLT1 At this time, it can accelerate the growth process of rice without affecting the yield of rice, therefore, OsPLT1 Genes can be used to regulate plant growth periods and increase annual and seasonal yields, and have high economic and application value. Attached Figure Description

[0015] Figure 1 It is obtained in Embodiment 1 of the present invention. OsPLT1 The spatiotemporal expression pattern results (where P: spikelet, YP: young spikelet, LB: leaf blade, LS: leaf sheath, S: stem, N: node, SB: stem base);

[0016] Figure 2 This refers to the OsPLT1 subcellular localization result obtained in Example 1 of this invention;

[0017] Figure 3 It is obtained in Embodiment 2 of the present invention. OsPLT1 Two different mutation types;

[0018] Figure 4 It is obtained in Embodiment 2 of the present invention. OsPLT1 The tillering phenotype of the mutant and wild type;

[0019] Figure 5 It is obtained in Embodiment 2 of the present invention. OsPLT1 The phenotypes of mutants and wild-type hydroponic seedlings;

[0020] Figure 6 It is obtained in Embodiment 2 of the present invention. OsPLT1Statistics on heading date phenotype and heading rate of mutants and wild-types;

[0021] Figure 7 It is obtained in Embodiment 2 of the present invention. OsPLT1 The mutant and wild-type ear development phenotypes;

[0022] Figure 8 The maturity period obtained in Embodiment 2 of the present invention OsPLT1 Statistics on yield-related agronomic traits of mutants and wild types. Detailed Implementation

[0023] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0024] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0025] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0026] Example 1:

[0027] A rice monosaccharide transporter gene OsPLT1 Its nucleotide sequence is shown in SEQ ID NO: 1; the amino acid sequence of the transporter encoded by it is shown in SEQ ID NO: 2.

[0028] SEQ ID NO: 1:

[0029]

[0030] SEQ ID NO: 2:

[0031] MTKDDAVPVAVAPAKRPPINKYAFGCALLASMNSVLLGYDISVMSGAQIFMKEDLKITDTQIEILAGVINIYSLFGSLAAGMTSDWLGRRYTMVLAAAIFFTGALLMGLAPNYAFLMAGRFVAGIGVGY ALMIAPVYTAEVAPTSARGFLTSFPEVFNNSGILLGYVSNFAFARLPVHLSWRAMFLVGAVPPIFLGIAVLAMPESPRWLVMRGRIEDARRVLLKTSDSPDEAEDRLLDIKKAVGIPEDASDGEDVVAIV RANKASQGEGVWKELLLNPTRPVRRMLVAGLGLMFIQQATGVDCVVMYSPRVFERAGIKSKTNSLGASMAVGVCKTFFIPIATLLLDRVGRRPLLLASGGGMAIFLFTLATSLLMMDRRPEGEAKALGA ISIAAMLSFVASFASGLGPVAWVYTSEIYPVRLRAQAAAIGTGLNRLMSGATTMSFLSLSNAITIAGSFYLYASIAAAGWVFMYFFLPETKGKSLEDTVKLFGKDTDDDDDVDTSRHERKRSTELSAQH*

[0032] This rice monosaccharide transporter gene OsPLT1 The gene and its transporter were discovered in rice using CRISPR-Cas9 technology, along with plant molecular genetics and physiological and biochemical methods. Our research also revealed that mutations in this gene significantly shorten the rice's growth period without affecting yield. This provides a practical basis for enhancing the transport and distribution of photosynthetic products through varietal genetic improvement, thereby continuously increasing the annual yield of rice.

[0033] 1. Spatiotemporal expression patterns of rice monosaccharide transporter genes

[0034] To obtain the rice monosaccharide transporter gene OsPLT1RNA was extracted from samples taken from various parts of rice ZH11 at different growth stages, with three replicates for each sample. Total RNA was extracted using TRIzol, and first-strand cDNA was synthesized using the Hiscript II Q RT SuperMix kit (Vazyme). Real-time quantitative PCR analysis was performed on a StepOnePlus instrument using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) qPCR premix. The PCR amplification primers were G::OsPLT1, with forward and reverse primers of 5'-CTTCCTCTCGCTCTCCAA-3' (as shown in SEQ ID NO: 7) and 5'-TCATCGTCGTCGTCTGT-3' (as shown in SEQ ID NO: 8), respectively. Ubiquitin was used as an internal standard with primers 5'-GCTCCGTGGCGGTATCAT-3' (as shown in SEQ ID NO: 11) and 5'-CGGCAGTTGACAGCCCTAG-3' (as shown in SEQ ID NO: 12).

[0035] The specific steps are as follows:

[0036] Total RNA extraction: 1. After tissue sampling, flash freeze in liquid nitrogen and grind into powder. Add 0.1 g of sample to a 1.5 mL centrifuge tube, add 1 mL of TRIzol, mix quickly, and let stand at room temperature for 5 min; 2. Add 200 µL of chloroform to the centrifuge tube, shake vigorously for 30 s, and let stand at room temperature (25℃) for 10 min; 3. Centrifuge at 12000 rpm at 4℃ for 10 min; 4. Carefully transfer 500 µL of the supernatant to a new 1.5 mL centrifuge tube; 5. Add an equal volume of isopropanol, gently invert to mix, and let stand at room temperature (25℃) for 10 min; centrifuge at 12000 rpm at 4℃ for 10 min, and discard the supernatant; 6. Add 1 mL of 75% ethanol, gently invert to suspend the precipitate; 7. Centrifuge at 8000 rpm at 4℃ for 5 min, and discard the supernatant; 8. Repeat steps 6 and 7, and in a clean bench, blow the RNA precipitate to a semi-transparent gel state, add 40 Dissolve RNA in µL of DEPC water.

[0037] RNA quality testing and concentration determination: 1. Electrophoresis to detect RNA integrity: Rapid electrophoresis on a 1% agarose gel was used to detect RNA molecule integrity. The 18S and 28S bands were observed. The 28S band was approximately twice as bright as the 18S band, indicating good RNA integrity. 2. Nucleic acid analyzer to detect RNA purity and concentration: BLANK readings were taken using DEPC water, and RNA concentration was determined using NanoDrop. Nucleic acids have an absorption peak at 260 nm, while proteins have a maximum absorption peak at 280 nm. OD... 260 / 280 RNA samples with a purity between 1.8 and 2.0 meet the purity standard and can be used for subsequent quantitative fluorescence experiments.

[0038] First-strand cDNA synthesis: First-strand cDNA was synthesized from total RNA using the HiScript® II qRT SuperMix reverse transcription kit. The total cDNA volume for each sample was 1 µg per reverse transcription run. The cDNA was stored at -20°C for later use. The reverse transcription system was as follows: Total RNA (1 µg), 4 × gDNA wiper Mix 4 µL, 5 × HiScript® II qRT SuperMix II 4 µL, and RNA-free water to a final volume of 20 µL. Reaction conditions: Incubation at 42°C for 15 min, followed by inactivation at 85°C for 5 s. After the reaction, an equal volume of RNA-free water was added to dilute the cDNA by half, and the mixture was thoroughly mixed before storage at -20°C for later use.

[0039] Real-time quantitative PCR primer design and specificity analysis: qPCR primers were designed using Primer 5 software based on the full-length cDNA of the gene. Primers with good specificity and no base complementarity were selected and synthesized by Qingke Biotechnology Co., Ltd., resulting in the amplification primer G::OsPLT1. Using cDNA from different rice growth stages as templates, primer specificity was detected by agarose gel electrophoresis. The reaction system was as follows: cDNA 1 µL, G::OsPLT1 forward primer (10 µM) 0.5 µL, G::OsPLT1 reverse primer (10 µM) 0.5 µL, 1.1×T3 Super PCR Mix (Qingke Biotechnology) 18 µL. Reaction conditions: 95℃, 3 min; 95℃, 10 s; 55℃, 10 s; 72℃, 10 s; 36 cycles total, 72℃, 5 min.

[0040] After the primers for quantitative real-time PCR were correctly detected, the first strand of cDNA synthesized by reverse transcription was used as a template for quantification using a two-step method on a Roche LightCycler® 96 real-time quantitative PCR instrument. Three technical replicates were set up for each gene and each sample. The reaction system was as follows: 1 µL cDNA, 0.5 µL G::OsPLT1 forward primer (10 µM), 0.5 µL G::OsPLT1 reverse primer (10 µM), 10 µL 2 × ChamQ Universal SYBR qPCR Master Mix, and 8 µL ddH2O. Reaction conditions: (1) Pre-denaturation stage: 95℃, 10 min; (2) Amplification stage: 95℃, 10 s, 60℃, 30 s, for a total of 40 cycles; (3) Melting curve stage: 96℃, 15 s, 60℃, 60 s, 95℃, 15 s.

[0041] The results are as follows Figure 1 As shown, OsPLT1 It is expressed in all stages and tissues, with relatively high expression in leaves and leaf sheaths during the tillering stage, leaves, leaf sheaths and stems during the jointing stage, stems, nodes and panicles during the booting and heading stages, and panicles during the grain-filling stage.

[0042] 2. Rice monosaccharide transporter gene OsPLT1 Subcellular localization

[0043] In order to study OsPLT1 To achieve cell-specific expression, we constructed a transformation vector carrying OsPLT1-GFP. The resulting vector was amplified by PCR. OsPLT1 The CDS sequence (excluding the terminator), and the forward and reverse primers for the amplification primer OE::OsPLT1-GFP are 5'-Gagctcggtacccgg. ggatcc ATGACCAAGGACGACGCCG-3' (as shown in SEQ ID NO: 9) BamH I (identification site shown in italics) and 5'- CTTCTCCTTTGCCCAT GTCGAC GTGCTGAGCGCTCAGCTC -3' (as shown in SEQ ID NO: 10) Sal I. Identification sites are shown in italics. (Use...) BamH I and Sal The fragment was cut by an I restriction enzyme, and the amplified fragment was cloned into the pCAMBIA1300-GFP vector to generate the OsPLT1-GFP vector, which was then transformed into rice protoplasts via PEG-mediated transformation.

[0044] The specific steps are as follows: shake the OsPLT1-GFP bacterial culture vigorously, extract the OsPLT1-GFP plasmid using the Solarbio plasmid extraction kit, and concentrate the plasmid. The concentration steps are as follows: (1) mix all plasmids with the same gene name into one tube; (2) place the centrifuge tube containing the plasmid in -80℃ freeze; (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 ddH2O to adjust the concentration to ≥1 µg, and store at -20℃ for later use.

[0045] Preparation of rice protoplasts: (1) Rice ZH11 was cultured in 1 / 2 MS and grown in the dark for 11-14 days. 15 yellow-flowered seedlings with good growth were selected and cut into thin strips with a disposable sterile blade. The thinner the better. (2) The strips were placed in a culture dish containing 10 mL of enzymatic hydrolysate, vacuumed for 5 min, and enzymatically hydrolyzed in a shaker in the dark for 4-5 h (28℃, 45 rpm). (3) The enzymatic hydrolysate was filtered into a 10 mL round-bottom centrifuge tube using a 150-mesh sieve. (4) 200 g, 1 min, 4℃, the supernatant was discarded, and 1 mL of W5 (154 mM NaCl; 125 mM CaCl2; 5 mM KCl; 2 mM MES) was gently added along the wall. The protoplasts were gently resuspended until there was no precipitate at the bottom. (5) The protoplasts were incubated on ice for 40 min. (6) The supernatant was discarded, and 2 mL of W5 was added and gently inverted to mix. (7) The tip of the yellow pipette was cut off, and 12 mL of the protoplast was aspirated. (8) Gently inject µL of the mixed sample into a hemocytometer and place it under a microscope for counting; (9) Incubate the remaining protoplasts on ice for 40 min and then discard the supernatant.

[0046] PEG-mediated protoplast transformation: The number of protoplasts was estimated based on the protoplast concentration, and MMG (0.6 M Mannitol; 15 mM MgCl2; 4 mM MES) was added to bring the final protoplast concentration to approximately 2 × 10⁻⁶. 5 Protoplasts / mL; Prepare the following reaction solution in a 1.5 mL round-bottom centrifuge tube: 150 µL protoplasts, 15 µg GFP plasmid (1.0 µg / µL), mix thoroughly; then add 165 µL PEG transformation solution, gently invert several times to promote PEG and protoplast fusion, let stand at room temperature for 5 min, add 1 mL W5 to terminate the reaction; centrifuge at 200 g for 5 min, remove the supernatant, resuspend the protoplasts with 200 µL W5, and incubate overnight at 22°C in the dark; observe the GFP signal of the protoplasts using a laser confocal microscope.

[0047] The results are as follows Figure 2 As shown, rice monosaccharide transporter gene OsPLT1 It is located on the cell membrane of rice protoplasts.

[0048] Example 2:

[0049] The rice monosaccharide transporter gene of the present invention OsPLT1 Its application in the cultivation of short-growing-period rice specifically includes OsPLT1 Construction of mutant materials, phenotypic analysis and yield factor analysis at each growth and development stage.

[0050] 1. OsPLT1 Obtaining gene knockout materials

[0051] To verify the rice monosaccharide transporter of the present invention OsPLT1 In rice, we used CRISPR-Cas9 gene editing technology to... OsPLT1 Editing was performed. A Cas9 / g RNA vector was constructed using target sequence 1: 5'-CCCATCAACAAGTACGCCTTCGG-3' (as shown in SEQ ID NO: 3) and target sequence 2: 5'-GGGCATGACGTCCGACTGGCTGG-3' (as shown in SEQ ID NO: 4). This vector was transformed into Agrobacterium tumefaciens GV3101. Positive strains were used for genetic transformation of wild-type rice ZH11 to obtain... OsPLT1 Gene-edited transgenic lines were identified. The transgenic lines were identified using PCR amplification with Cas9::OsPLT1 primers. The upstream primer sequence was 5'-CGTGTTAGCTCGGATCTGTA-3' (as shown in SEQ ID NO: 5), and the downstream primer sequence was 5'-GGCGAAGTTGGAGACGTA-3' (as shown in SEQ ID NO: 6). Two knockout lines with different mutation types were obtained. plt1-1 , plt1-2 .

[0052] The results are as follows Figure 3 As shown, plt1-1 The deletion of a single base C near target site 1 and target site 2 respectively leads to OsPLT1 The gene encoding 31 amino acids terminated prematurely. plt1-2 Inserting a single T base near target site 1 results in OsPLT1 The gene, which encodes 39 amino acids, terminated prematurely.

[0053] 2.Os PLT1 Effects of gene knockout on rice growth

[0054] In order to evaluate Os PLT1 The effects of gene knockout on rice germination and seedling growth were compared between field-grown wild-type ZH11 and... plt1Phenotypic effects of mutant seed germination and seedling growth. Seeds were soaked in a 5000-fold diluted solution of imazalil for 16 h, rinsed with running water, and then soaked overnight in sterile distilled water at 37°C. Seeds were sown on completely moistened filter paper and cultured in an incubator at 28°C with 12 h of light and 12 h of darkness. Photographs were taken from day 1 to day 4, and germination rates were statistically analyzed. After the seeds showed signs of sprouting, they were transferred to hydroponics and cultured for 14 days. The effects of ZH11 and seedling growth were then observed. plt1 The growth of the mutants was recorded, and their plant height and dry weight were statistically analyzed. Values ​​are the mean ± standard deviation of 10 replicate experiments. The p-value is used for statistical testing of significance. t -test analysis* P <0.05,** P <0.01.

[0055] The results are as follows Figure 4 As shown, compared with the field-grown wild type ZH11, plt1 The mutant had no effect on seed germination and seedling growth.

[0056] In order to evaluate Os PLT1 Effects of gene knockout on tillering stage in rice, field-grown wild type (ZH11) plt1 mutant ( plt1-1 , plt1-2 ), Analysis of Os PLT1 The tillering phenotype of the genetic material was analyzed, and the number of tillers and plant height were statistically analyzed. Values ​​are the mean ± standard deviation of 15 replicates. The p-value is the statistical test for significance. t -test analysis* P <0.05,** P <0.01.

[0057] The results are as follows Figure 5 As shown, compared to ZH11, plt1 The number of tillers and plant height of the mutant were significantly increased.

[0058] In order to evaluate Os PLT1 The effect of gene knockout on the reproductive growth stage of rice was investigated starting 57 days after sowing for the wild-type (ZH11). plt1 mutant ( plt1-1 , plt1-2 The heading status of the materials in the plot was recorded every three days until the wild type (ZH11) was reached. plt1 mutant ( plt1-1 , plt1-2 All materials reached the heading stage, and [the following was done]: OsPLT1 Phenotypic records of genetic materials at the heading stage were taken by photograph; starting 30 days after sowing, wild-type (ZH11) samples were taken every 3 days. plt1 mutant ( plt1-1 , plt1-2 The material is a whole rice plant. Carefully peel off the leaves of the plant and take a picture of the main stem tip tissue.

[0059] The results are as follows Figure 6 and Figure 7 As shown, compared to wild-type ZH11, plt1 The mutant phenotype showed a significant earlier heading time, nearly 8 days earlier, and plt1 The development of the ear in the mutant was significantly accelerated.

[0060] 3.Os PLT1 Effects of gene knockout on agronomic traits related to rice yield

[0061] Wild-type ZH11 grown in the field plt1 mutant ( plt1-1 , plt1-2 The materials were analyzed, and yield-related agronomic traits were statistically analyzed after maturity. Compared with the wild type ZH11, plt1 The mutants showed no differences in main spike length, number of grains per spike, number of effective tillers, thousand-grain weight, seed setting rate, and yield per plant. Figure 8 The values ​​are the mean ± standard deviation of 15 repeated experiments. The p-value is the statistical test for statistical significance. Student's t-test analysis: *P<0.05, **P<0.01.

[0062] In summary, the rice monosaccharide transporter gene of this invention... OsPLT1 Located in the cell membrane of rice protoplasts, its knockout mutation significantly advances the rice growth period by approximately 8 days. This advancement is unaffected by germination rate, and... OsPLT1 Gene knockout mutations do not affect rice yield.

Claims

1. A gene for knocking out rice monosaccharide transporters OsPLT1 Its application in shortening the rice growth period is characterized by, The rice monosaccharide transporter gene OsPLT1 The nucleotide sequence is shown in SEQ ID NO:

1.

2. The application as described in claim 1, characterized in that, Knockout in wild-type rice using CRISPR / Cas9 method OsPLT1, Short-breeding-period rice was obtained.

3. The application as described in claim 1, characterized in that, from OsPLT1 The gDNA sequence was selected from the monosaccharide transporter gene. OsPLT1 The target sequence was edited, the Cas9-OsPLT1 vector was constructed, and then transformed into Agrobacterium tumefaciens using heat shock transformation. Finally, it was cloned into rice for expression through genetic transformation. OsPLT1 Gene-edited transgenic lines.

4. The application as described in claim 3, characterized in that, The monosaccharide transporter gene OsPLT1 The edited target sequences are SEQ ID NO:3 and SEQ ID NO:

4.

5. The application as described in claim 3, characterized in that, The OsPLT1 Gene-edited transgenic lines were identified by PCR amplification. The identification primers were Cas9::OsPLT1. The upstream primer sequence is shown in SEQ ID NO: 5, and the downstream primer sequence is shown in SEQ ID NO: 6.

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