Trehalose-6-phosphate synthetase gene OsTPS9 for regulating and controlling chalkiness of rice and application of trehalose-6-phosphate synthetase gene OsTPS9

Abstract

The invention belongs to the technical field of biotechnology and plant genetic engineering, and particularly relates to a trehalose-6-phosphate synthase gene OsTPS9 for regulating and controlling chalkiness of rice and application of the trehalose-6-phosphate synthase gene OsTPS9. The invention discloses a trehalose-6-phosphate synthase gene OsTPS9 for regulating and controlling chalkiness of rice, the nucleotide sequence of the trehalose-6-phosphate synthase gene OsTPS9 is as shown in SEQ ID NO.1, the CDS sequence is as shown in SEQ ID NO.2, and the amino acid sequence of encoded protein is as shown in SEQ ID NO.3. The invention further discloses a preparation method of the trehalose-6-phosphate synthase gene The gene is highly specifically expressed in rice development endosperm, and compared with a wild type, the knockout mutant tps9-6 has the advantages that the grain length of a mutant seed is remarkably increased, the grain thickness and thousand grain weight are remarkably reduced, and the chalky grain rate and chalkiness are remarkably increased. The invention can provide new important gene reserve, germplasm resources and theoretical basis for genetic improvement of rice quality and yield.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and plant genetic engineering, specifically relating to a trehalose-6-phosphate synthase gene that regulates chalkiness in rice. OsTPS9 And its applications. Background Technology

[0002] Chalkiness in rice affects its appearance, processing quality, milled rice yield, and market value. Discovering more genes regulating chalkiness and elucidating their molecular mechanisms is of significant scientific value and production guidance for the genetic improvement of rice quality.

[0003] Trehalose (Tre) is a non-reducing disaccharide widely found in many plants and animals, playing a crucial role in plant growth, development, and stress resistance. The biosynthesis of Tre in plants is catalyzed through a two-step reaction pathway involving trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate-phosphatase (TPP) located in the cytoplasm (Raza et al., 2023). The flow of glucose and sucrose produced by plant photosynthesis, serving as carbon "sources," to "sinks" such as grains determines yield. Trehalose-6-phosphate (T6P), as a signaling molecule in plant sugar metabolism, may serve as a key link between sources and sinks (Griffiths et al., 2025). Recently, Chinese scientists discovered a... OsbHLH001 - OsTPS8 The -Tre6P-α-amylase signaling cascade plays a dual role in regulating grain chalkiness and seed vigor, revealing the molecular link between the appearance quality and seed vigor of indica and japonica rice (Chen et al., 2025). However, there are few reports on other genes in the TPS family involved in regulating the chalkiness trait in rice. Therefore, it is necessary to explore genes in this domain family and analyze their biological functions to provide new and important gene reserves and germplasm resources for the genetic improvement of rice yield and quality, lay a scientific theoretical foundation for breeding better rice varieties, and provide reference for similar research on other crops. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to design and provide a trehalose-6-phosphate synthase gene that regulates chalkiness in rice. OsTPS9 and the technical solutions for their application.

[0005] The present invention is implemented using the following technical solutions: The first aspect of this invention provides a trehalose-6-phosphate synthase for regulating chalkiness in rice. OsTPS9 Genes, the ones mentioned OsTPS9The nucleotide sequence of the gene is shown as SEQ ID NO. 1, the CDS sequence is shown as SEQ ID NO. 2, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 3.

[0006] The second aspect of the present application provides the above-mentioned OsTPS9 gene or mutant tps9-6 , wherein the mutant tps9-6 The nucleotide sequence of the mutant is shown as SEQ ID NO. 4, and the CDS sequence is shown as SEQ ID NO. 5.

[0007] The third aspect of the present application provides the above-mentioned OsTPS9 gene or mutant tps9-6 for use in regulating chalkiness in rice.

[0008] The fourth aspect of the present application provides a method for regulating chalkiness in rice, the method comprising knocking out the above-mentioned OsTPS9 gene of claim 1, so that the expression level of the OsTPS9 gene in the target rice is reduced, and a rice plant with increased chalky kernel rate and chalkiness is obtained.

[0009] Further, the knockout is specifically inserting one base C to cause a frameshift mutation.

[0010] Further, the target site of the knockout is 20bp in the first exon of the OsTPS9 gene, and CGG is used as the PAM sequence.

[0011] Further, the vector used in the knockout process is VK005-01, and the vector system is CRISPR / Csa9.

[0012] The fifth aspect of the present application provides the above-mentioned method for use in increasing chalkiness in rice.

[0013] The present application has the following beneficial effects: Compared with the wild type, the mutant seed has a significantly increased kernel length, a significantly reduced kernel thickness and thousand kernel weight, and a significantly increased chalky kernel rate and chalkiness, and the present application can provide new important gene reserves, germplasm resources and theoretical basis for genetic improvement of rice quality and yield. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Rice trehalose-6-phosphate synthase gene OsTPS9 Gene encoding protein domain prediction map and phylogenetic tree construction map; Figure 2 Rice trehalose-6-phosphate synthase gene OsTPS9 Gene expression level map in different tissues; Figure 3Gene knockout of trehalose-6-phosphate synthase gene OsTPS9 And identification diagrams of mutant phenotypes; Figure 4 Scanning electron microscope image of starch granules in the endosperm of mature seeds of wild type and mutant. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1: Prediction of the protein domain and construction of a phylogenetic tree for rice trehalose-6-phosphate synthase OsTPS9 This invention first identifies rice trehalose-6-phosphate synthase, which is specifically highly expressed in rice seeds, through bioinformatics analysis. OsTPS9 Genes were then input into the rice gene using the SMART website (http: / / smart.embl-heidelberg.de / ). OsTPS9 Domain prediction analysis was performed on the protein sequences corresponding to the genes. (Downloaded from the National Rice Data Center) OsTPS9 The protein sequences corresponding to the genes were entered into BLASTP on the NCBI website for homology search. Protein sequences from different species were downloaded and saved in Fasta format. Multiple alignment of amino acids was performed using the Clustal (http: / / www.clustal.org) program. Then, a phylogenetic tree was constructed using the bootstrap method with MEGA (version 5.0) software.

[0017] like Figure 1 As shown, the rice of the present invention OsTPS9 Predicted domain diagrams and phylogenetic tree constructions of gene-encoded proteins, where A represents... OsTPS9 A) shows the domain diagram of the protein sequence corresponding to the gene; B) shows the phylogenetic tree constructed using the bootstrap method with MEGA (version 5.0) software. Figure 1 As indicated by A, this protein contains a Glyco_transf_20 domain between amino acids 47-538 and a Trehalose_PPase domain between amino acids 595-847. Figure 1 As can be seen from B, this protein is relatively conserved in different rice genera (Indica, Japonica, glutinous rice, wild rice, etc.) and other plants.

[0018] OsTPS9The nucleotide sequence of the gene is shown in SEQ ID NO. 1. OsTPS9 The CDS sequence is shown in SEQ ID NO.2.

[0019] Example 2: Rice Trehalose-6-phosphate synthase gene OsTPS9 Organizational expression patterns like Figure 2 As shown, the rice of the present invention OsTPS9 A graph showing gene expression levels in different tissues of rice. (Source: [Insert Source Here]) Figure 2 It is evident that this gene is highly expressed during the early grain-filling stage, and its expression level gradually decreases as the seed matures, but it is almost not expressed in the roots, stems, leaves, and leaf sheaths. Therefore, this indicates... OsTPS9 The gene is highly specifically expressed in the rice grain-filling endosperm.

[0020] Example 3: Rice Trehalose-6-phosphate synthase gene OsTPS9 Gene knockout, genotype and mutant phenotype identification 1. Construction and transformation of gene knockout vectors A CRISPR / Cas9 gene knockout plasmid construction kit was purchased from a biotechnology company. The vector used was VK005-01, and the prokaryotic resistance was kanamycin. Hygromycin resistance was used to detect positive plants. Rice starch branching enzymes were selected using the CRISPRdirect website through the National Rice Data Center website. OsTPS9 The first exon of the gene contains a 20bp target site, and CGG is the PAM sequence. Primer sequences are shown in the attached table.

[0021] Table 1 lists the primers involved in this invention. .

[0022] After receiving the kit, centrifuge the test tubes before use to avoid leaving solution residue on the tube walls, and then conduct the experiment. The specific experimental procedures are as follows: (1) Preparation of dimers After mixing the reagents in Table 2, heat at 95℃ for 3 minutes, cool to room temperature, and let stand at 16℃ for 5 minutes.

[0023] Table 2 Reaction system for dimer preparation .

[0024] (2) The dimer is attached to the carrier. Connect the reagents in Table 3 by incubating at 16°C overnight.

[0025] Table 3 Reaction system of dimer-linked carrier .

[0026] (3) Transformation The product from (2) was added to DH5α competent cells and mixed well. The cells were then incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, and placed on ice for 2 min. 500 μL of antibiotic-free LB was then added, and the cells were placed in a 37℃ constant temperature shaker at 170 rpm for 1 h of recovery. After recovery, the kanamycin resistance (K) was measured. + LB plates were incubated overnight at 37°C inverted.

[0027] Positive clones were selected and sent to the company for Sq-primer sequencing (sequences are shown in Table 1, and bacterial PCR results are shown in Tables 4 and 5). The sequencing results were analyzed using DNAMAN or SNAP software to determine whether the target gene site Target was constructed into the VK005-01 vector.

[0028] Table 4. Bacterial PCR Reaction System .

[0029] Table 5. PCR reaction procedure for bacterial culture .

[0030] Agrobacterium-mediated transformation steps: Select plasmids with correct sequencing and send them to a biotechnology company for Agrobacterium transformation and infection experiments, using Zhonghua 11 as the recipient and EHA105 Agrobacterium. （ Agrobacterium tumefaciens was screened using hygromycin resistance selective medium.

[0031] 2. Extraction of DNA from leaves (SDS method) (1) Reagent preparation 1M Tris-HCl: 121.1g Tris base and 800mL ddH2O, adjust the pH to 8.0 with concentrated HCl, and bring the volume to 1L.

[0032] 0.5M EDTA: 186.1g EDTA and 600mL ddH2O, adjust the pH to 8.0 with NaOH, and add water to a final volume of 1L.

[0033] SDS extraction solution: 100 mL 1M Tris-HCl (pH 8.0), 50 mL 0.5M EDTA (pH 8.0), 29.25 g NaCl and 12 g SDS, dissolved in 800 mL ddH2O at 65℃, and brought to a final volume of 1 L.

[0034] KAC: 29.5 mL glacial acetic acid, adjust pH to 4.8 with KOH, and bring the volume to 100 mL.

[0035] Solvent ratio 24:1:500mL chloroform and 21mL isoamyl alcohol.

[0036] (2) DNA extraction Cut the collected leaf samples into small pieces and place them in a 2mL centrifuge tube. Add a steel ball, freeze in liquid nitrogen, and grind into powder using a sample grinder. Add 600μL of SDS extraction buffer preheated to 65℃, mix well, and then place in a 65℃ water bath and cook for half an hour, shaking twice during the process. Add 150μL of KAC, shake well, and then place in a... - Let stand at 20℃ for half an hour; add 750 μL of 24:1 solution and shake on a shaker for half an hour; centrifuge at 12,000 rpm for 10 min; transfer 400 μL of supernatant to a new 1.5 mL centrifuge tube; add 800 μL of pre-chilled ethanol, shake well, and place in a centrifuge tube. - Let stand at 20℃ for half an hour; centrifuge at 12,000 rpm for 10 min, slowly discard the supernatant and wait for the liquid to evaporate; add 100 μL ddH2O and wait for the precipitate to dissolve before use.

[0037] 3. PCR amplification PCR amplification and sequencing were performed using the KOD-FX method. The amplification system and procedure are shown in Tables 6 and 7.

[0038] Table 6. KOD-FX method amplification system .

[0039] Table 7. PCR reaction procedure for bacterial culture .

[0040] Note: The Tm value of 55℃ in step 3 can be adjusted; the extension time of 68℃ in step 4 can be adjusted according to the size of the amplified fragment; the amplification rate of KOD-FX is 1kb / min.

[0041] 4. Identification of transgenic positive plants and their genotypes Positive seedlings were detected using hygromycin Hyg-F / R (amplification system and procedure are shown in Tables 8 and 9, sequence is shown in Table 1). Sequencing primers Seq-F / R (see Table 1) were designed near the knockout target site for PCR amplification of a 244bp fragment including the target sequence, which was then sent to the company for sequencing. The genotype and mutation type of the transgenic single plant were determined by comparing it with the wild-type target sequence and by observing the peak diagram of the mutation site.

[0042] Table 8 PCR system for transgenic positive plants .

[0043] Table 9 PCR Procedure for Transgenic Positive Plants .

[0044] like Figure 3 As shown in Figure A, the rice trehalose-6-phosphate synthase gene was first analyzed.OsTPS9 Basic information: The gene is 3238 bp in length, with a CDS of 2661 bp, encoding 886 amino acids. Its sequence is shown in SEQ ID NO.3. A 20 bp sequence with good specificity was selected from the first exon of this gene as the knockout target site, and CGG was used as the PAM sequence. Experimental procedures were performed according to the instructions for vector construction using kit VK005-01. The successfully sequenced vector (plasmid or bacterial culture) was sent to a biotechnology company for Agrobacterium-mediated transformation. The japonica rice variety Zhonghua 11 (ZH11) was used as the transgenic recipient, and it also served as a wild-type control for subsequent result analysis. T0 generation transgenic single-plant test-tube seedlings were collected, and hygromycin gene detection was performed to determine if the seedlings were positive. After the test-tube seedlings stabilized, leaf DNA was extracted. Sequencing primers were designed near the knockout target site for PCR amplification, and the DNA was sent to the company for sequencing. The genotype and mutation type of the transgenic single plant were determined by comparing it with the wild-type target sequence and observing the peak diagram of the mutation site. Homozygous mutants with different mutation patterns were detected in T0 generation single plants. Then, in May of the following year, stable homozygous mutant types were planted, and all relevant experiments were conducted. Compared with the wild type, such as... Figure 3 Mutant shown in B tps9-6 Insertion of a single carbon base resulted in a frameshift mutation. This mutation was then selected for phenotypic identification and all subsequent related experiments.

[0045] Among them, mutant tps9-6 The nucleotide sequence of the gene is shown in SEQ ID NO.4, and its CDS sequence is shown in SEQ ID NO.5.

[0046] 5. Phenotypic identification of mature seeds from transgenic plants Harvested wild-type ZH11 and homozygous mutants tps9-6 Mature seeds were dried in a 45°C oven until constant weight. Brown rice was obtained by hulling using a brown rice machine, and then the phenotypic characteristics of the seeds were compared and recorded by scanning. Figure 3 C).

[0047] like Figure 3 As shown in D, several representative wild-type ZH11 and mutant strains were randomly selected. tps9-6 Phenotypic comparison and photography of mature seeds from the T1 generation revealed that the mature seeds of the normal wild-type japonica rice variety Zhonghua 11 (WT or ZH11) were colorless and transparent, while the three different mutant types... tps9-6 The seeds exhibited a distinct chalky phenotype, with a highly significant increase in the chalky grain rate and chalkiness. Figure 3 I, J).

[0048] 6. Determination of rice yield traits Harvested wild-type ZH11 and mutant tps9-6Mature seeds were dried in a 45℃ oven until constant weight. Two hundred plump seeds were randomly selected, and their length and width were measured using a Wanshen automatic seed quality analyzer (SC-A1 type, Hangzhou Wanshen Testing Technology Co., Ltd.), and the weight of 1000 seeds was calculated. Seed thickness was measured using vernier calipers, with at least 10 seeds per sample. Results are expressed as mean ± SD (standard deviation).

[0049] like Figure 3 As shown in F, wild-type ZH11 and mutant tps9-6 There was no significant difference in particle width; however, the mutants tps9-6 Grain length was significantly higher than that of wild type, showing a significant increase. Figure 3 E); mutant tps9-6 Both grain thickness and thousand-grain weight were significantly lower than those of the wild type. Figure 3 G, H), indicating the rice trehalose-6-phosphate synthase gene. OsTPS9 Genes also regulate yield traits.

[0050] Example 4: Comparative analysis of scanning electron microscopy structure of starch complex in the endosperm of mature seeds of wild type and mutant. For wild type and mutant tps9-6 Scanning electron microscopy (SEM) analysis of the endosperm complex starch structure in cross-sections of mature rice seeds revealed that wild-type starch granules exhibited dense, irregular polygonal morphology, while mutant granules... tps9-6 It exhibits numerous spherical or ellipsoidal starch granules with gaps, thus producing a distinct chalky phenotype. Figure 4 Figure 4 ).

Claims

1. A trehalose-6-phosphate synthetase gene for regulating chalkiness in rice, characterized by, OsTPS9 The OsTPS9 The nucleotide sequence of the gene is shown as SEQ ID NO. 1, the CDS sequence is shown as SEQ ID NO. 2, and the amino acid sequence of the encoded protein is shown as SEQ ID NO.

3. ​ 2. The method of claim 1 OsTPS9 mutants of genes tps9-6 characterized in that, The mutant tps9-6 The nucleotide sequence of the mutant is shown as SEQ ID NO. 4, and the CDS sequence is shown as SEQ ID NO.

5.

3. As described in claim 1 OsTPS9 Gene or mutant as described in claim 2 tps9-6 Application in regulating chalkiness in rice.

4. A method of modulating chalkiness in rice, characterized by, The method includes knocking out the method described in claim 1. OsTPS9 Genes that enable the target rice to OsTPS9 Lower gene expression levels result in rice plants with increased chalky grain rate and chalkiness.

5. The method of claim 4, wherein, The knockout is specifically an insertion of 1 base C resulting in a frame shift mutation.

6. The method of claim 4, wherein, The knockout target site is OsTPS9 20 bp in the 1st exon of the gene, CGG as PAM sequence.

7. The method of claim 4, wherein, The vector used in the knockout process is VK005-01; the vector system is CRISPR / Csa9.

8. The use of the method of any one of claims 4-7 in improving rice chalkiness.

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