Rice ferritin mutant with improved thermal stability, coding gene and application of rice ferritin mutant

Abstract

The invention discloses a rice ferritin mutant with improved thermal stability, a coding gene and application of the rice ferritin mutant, the mutant is rice ferritin, an extended peptide EP segment amino acid sequence of the mutant is replaced by an EP segment amino acid sequence of soybean ferritin, and the thermal stability of the rice ferritin is improved; after the gene encoding the mutant is introduced into rice, the head rice rate of transgenic rice grains is increased, the chalkiness degree is reduced, the total starch content and amylose content are increased, the total protein content and the soluble sugar content are reduced, the iron content of the transgenic rice grains is remarkably increased, the iron content of cooked rice is increased by 1.57-1.95 times compared with that of wild rice, and the rice quality is improved. The problem of serious iron loss of natural ferritin in the cooking process is partially solved. The thermal stability of the obtained rice ferritin is remarkably improved, and the rice ferritin can be directly used for producing high-quality polished rice, can also be used as a raw material for preparing functional foods such as high-iron high-amylose rice flour and low-glycemic-index rice products, and has important industrialization value and wide market application prospects.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and crop breeding technology, and in particular relates to a rice ferritin mutant with improved thermal stability, its encoding gene, and its application. Background Technology

[0002] Rice ( Oryza sativa L. Rice is the staple food for more than half of the world's population, but its grains have a low iron content, making it difficult to meet the body's nutritional needs. Therefore, it is of great significance to increase the iron content of rice through biofortification.

[0003] Ferritin is an ideal target for iron biofortification. Plant ferritin precursors contain a transport peptide (TP) and an extension peptide (EP) at their N-terminus: TP guides the precursor into the plastid and where it is cleaved, while EP is a domain specific to mature proteins. Natural rice ferritin has poor thermal stability and is easily denatured and inactivated during cooking, resulting in decreased iron binding and retention capacity, thus limiting its practical applications.

[0004] Current technologies mostly employ endosperm-specific promoters to drive the overexpression of natural ferritin, focusing solely on increasing iron content without comprehensively considering the impact on rice quality, thus failing to achieve synergistic improvement of multiple traits. Currently, there is a lack of rice ferritin mutants with improved thermal stability, making it impossible to simultaneously increase grain iron content while synergistically improving head rice yield, chalkiness, soluble sugars, total protein, and other indicators, as well as improving iron retention after cooking. Therefore, related technologies are urgently needed in this field to address these issues. Summary of the Invention

[0005] In view of this, the present invention aims to provide a rice ferritin mutant with improved thermal stability, its encoding gene, and its application, in order to solve at least one technical problem in the background art.

[0006] To achieve the above objectives, the technical solution of the present invention is implemented as follows: A rice ferritin mutant with improved thermal stability, wherein the mutant is rice ferritin in which the amino acid sequence of its extended peptide EP segment is replaced with the amino acid sequence of the EP segment of soybean ferritin; the mutant exhibits significantly improved thermal stability compared to wild-type rice ferritin. Preferably, its denaturation temperature is 81.9℃, which is 27.5℃ higher than that of the wild type.

[0007] Furthermore, the mutant also lacks the transport peptide TP segment. Compared with wild-type rice ferritin, the rice ferritin mutant lacks the transport peptide (TP segment), and its extended peptide (EP segment) amino acid sequence is replaced with the EP segment amino acid sequence of soybean ferritin. Compared with wild-type rice ferritin, the mutant has significantly improved thermal stability, with a denaturation temperature of 81.9℃, which is 27.5℃ higher than that of wild-type. It can correctly fold and be expressed in large quantities in the rice endosperm cytoplasm, avoiding the iron storage limitation problem caused by the wild-type ferritin TP segment guiding it into the plastid.

[0008] Furthermore, the amino acid sequence of the mutant is shown in SEQ ID NO:1.

[0009] SEQ ID NO:1 is: MASNAPAPLAGVIFEPFQELKKDYLAVPIAHNVSLARQKFVDECEAAINEQINVEYNASYAYHSLFAYFDRDNVALKGFAKFFKESSDEERDHAEKLIKYQNMR GGRVRLQSIVTPLTEFDHPEKGDALYAMELALALEKLVNEKLHNLHSVASRCNDPQLTDFVESEFLEEQVEAIKKISEYVAQLRRVGKGHGVWHFDQKLLEEEA; A coding gene encoding the above-mentioned rice ferritin mutant, preferably, the nucleotide sequence of the gene is shown in SEQ ID NO:2.

[0010] SEQ ID NO:2 is: ; A recombinant expression vector containing the above-mentioned encoding gene.

[0011] Furthermore, the recombinant expression vector also includes a rice endosperm-specific promoter operably linked to the gene, preferably a rice 10kDa prolysin promoter; this can drive the mutant gene to be efficiently expressed only in the endosperm cytoplasm, avoiding any impact on other organs of the plant.

[0012] A method for synergistically improving the quality of rice grains includes the following steps: introducing the above-mentioned gene or the above-mentioned recombinant expression vector into rice cells to obtain transgenic rice plants.

[0013] Preferably, the rice is indica rice or japonica rice, and the japonica rice includes Zhonghua 11 and Nipponbare.

[0014] Furthermore, the step of introducing the recombinant expression vector into rice cells was achieved through Agrobacterium-mediated genetic transformation.

[0015] Preferably, the Agrobacterium-mediated genetic transformation method includes: infecting and co-culturing callus tissue with an engineered strain of Agrobacterium containing a recombinant expression vector, obtaining positive resistant callus tissue through resistance screening, and then obtaining transgenic rice plants after differentiation, rooting, and hardening.

[0016] Compared with wild-type rice, the genetically modified rice plants showed an increase of 11.56%–15.45% in head rice yield, a decrease of 3.4%–4.45% in chalkiness, an increase of 3.06%–4.06% in total starch content, an increase of 3.35%–6.32% in amylose content, a decrease of 1.64%–2.89% in total protein content, a decrease of 2.09%–3.76% in soluble sugar content, and an increase of 1.31–2.10 μg / g in iron content. Furthermore, the iron content in cooked rice increased by 0.66–1.09 μg / g.

[0017] The above-mentioned rice ferritin mutant, the above-mentioned coding gene, or the above-mentioned recombinant expression vector are used to simultaneously improve at least two of the following traits of rice grains: head rice rate, chalkiness, total starch content, amylose content, total protein content, soluble sugar content, and iron content; preferably, the rice is indica rice or japonica rice.

[0018] The above-mentioned rice ferritin mutant, the above-mentioned encoding gene, or the above-mentioned recombinant expression vector are used in the preparation of high-iron, high-amylose rice flour or low-glycemic index rice products.

[0019] Compared with existing technologies, the rice ferritin mutant with improved thermal stability, its encoding gene, and its application described in this invention have the following advantages: 1. Significantly improved thermal stability of ferritin: The rice ferritin mutant obtained by deleting the TP segment and replacing the EP segment in this invention exhibits significantly enhanced thermal stability compared to the wild type, with a denaturation temperature 1.51 times that of the wild type. The deletion of the TP segment allows the mutant to be located in the endosperm cytoplasm, enabling accurate folding in large quantities and avoiding the spatial limitations of plastid localization. The replacement of the EP segment further enhances thermal stability, enabling the mutant to maintain a stable conformation and iron-binding capacity during rice cooking, partially solving the problem of easy denaturation and inactivation, and severe iron loss, in natural ferritin during heat treatment.

[0020] 2. Synergistic Improvement of Multiple Quality Traits in Rice: This invention transforms rice with a ferritin mutant gene that exhibits improved thermal stability and lacks the TP segment under the control of an endosperm-specific promoter, unexpectedly achieving synergistic improvement of multiple quality traits: For processing quality, the head rice rate increases by 1.16–1.21 times, breakage resistance is enhanced, and rice processing adaptability is significantly improved; for appearance quality, chalkiness decreases by 1.64–2.05 times, grain transparency is improved, and marketability is enhanced; for physicochemical quality, total… The starch content increased by 1.04–1.05 times, the amylose content increased by 1.43–1.82 times, the total protein content decreased by 1.20–1.41 times, and the soluble sugar content decreased by 1.16–1.32 times, which is beneficial to improving the taste and digestibility of rice. Regarding nutritional quality, the iron content of the grains was significantly increased, by 1.32–1.51 times compared to the wild-type control. After cooking, the iron content in the rice was 1.57–1.95 times higher than the wild-type control, and the iron retention rate was greatly improved.

[0021] 3. Multiple effects from a single factor, resulting in significant comprehensive benefits: This invention, through endosperm-specific expression of a single ferritin mutant (with TP segment deletion and EP segment replacement), simultaneously regulates key pathways of carbon and nitrogen metabolism and starch synthesis in grains, achieving a unified approach to nutritional fortification and quality improvement. Compared to traditional breeding or single-trait gene engineering methods, this invention avoids the cumbersome operation of multi-gene aggregation, and is characterized by its simplicity, significant effects, and excellent comprehensive traits.

[0022] 4. The genetically modified rice grains obtained by this invention have high iron content, excellent processing quality and appearance quality. They can be used directly for the production of high-quality refined rice, or as raw materials to prepare functional foods such as high-iron, high-amylose rice flour and low-glycemic index rice products. They have important industrialization value and broad market application prospects. Attached Figure Description

[0023] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings…

[0024] Figure 1 The images show the SDS-PAGE analysis results of the wild-type OsFer1(△TP) and mutant OsFer1M protein after treatment at different temperatures (A is the SDS-PAGE analysis result of wild-type OsFer1(△TP), and B is the SDS-PAGE analysis result of mutant OsFer1M protein). Figure 2The images show the Native-PAGE analysis results of wild-type OsFer1(△TP) and mutant OsFer1M protein after treatment at different temperatures (A is the Native-PAGE analysis result of wild-type OsFer1(△TP), and B is the Native-PAGE analysis result of mutant OsFer1M protein). Figure 3 Differential scanning calorimetry (DSC) thermograms and denaturation temperatures of wild-type OsFer1 (ΔTP) and mutant OsFer1M proteins described in this invention. T m Comparison results graph; Figure 4 This is a comparison chart of the iron content of wild-type Zhonghua 11 and transgenic rice lines (OsFer1M-OE3, OsFer1M-OE9, OsFer1M-OE11) before and after cooking. Detailed Implementation

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0026] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0027] Example 1: Obtaining and Determining the Thermal Stability of Rice Ferritin Mutants 1.1 Construction of mutants With rice ( Rice Wild-type ferritin gene OsFer1 Using the CDS sequence of (GenBank accession number: AF519570.1) as a template, the nucleotide sequence encoding the N-terminal EP segment (amino acids 49-80) of the mature protein was synthesized by the company and replaced with the EP segment coding sequence of soybean ferritin (GenBank accession number: AB062754.1) (its nucleotide sequence is shown in SEQ ID NO:3, and the encoded amino acid sequence is shown in SEQ ID NO:4; the EP segment coding sequence was synthesized by the company).

[0028] SEQ ID NO:3 is: ATGGCTTCAAATGCACCCGCACCACTCGCTGGGGTCATATTTGAACCATTTCAAGAGCTCAAGAAGGATTATCTTGCTGTTCCGATTGCACACAATGTT; SEQ ID NO:4 is: MASNAPAPLAGVIFEPFQELKKDYLAVPIAHNV; Simultaneously, to facilitate in vitro expression and thermostability assays, the sequence encoding the transport peptide (TP, amino acids 1-48) was removed, retaining only the coding region of the mature protein. After sequencing verification, the mutant gene was obtained. OsFer1M Its nucleotide sequence is shown in SEQ ID NO:2, and the amino acid sequence of the mature protein it encodes is shown in SEQ ID NO:1.

[0029] SEQ ID NO:1 is: MASNAPAPLAGVIFEPFQELKKDYLAVPIAHNVSLARQKFVDECEAAINEQINVEYNASYAYHSLFAYFDRDNVALKGFAKFFKESSDEERDHAEKLIKYQNMR GGRVRLQSIVTPLTEFDHPEKGDALYAMELALALEKLVNEKLHNLHSVASRCNDPQLTDFVESEFLEEQVEAIKKISEYVAQLRRVGKGHGVWHFDQKLLEEEA; SEQ ID NO:2 is: ; wild type OsFer1(△TP) The nucleotide sequence of the gene (with only the TP region removed and the EP segment not replaced) is shown in SEQ ID NO:5, and the amino acid sequence of the encoded mature protein is shown in SEQ ID NO:6.

[0030] SEQ ID NO:5 is: ATGGCCGGGAAAGGGAAGGAGGTGCTCAGCGGCGTGGTCTTCCAGCCATTCGAGGAGCTCAAGGGGGAGCTCTCCCTCGTCCCCCAGGCCAAGGACCAGTCTCTCGCTAGGCAAAAGTTCGTCGACGAGTGCGAGGCCGCCATCAACGAGCAGATCAATGTGGAGTACAATGCATCGTACGCGTACCACTCCCTTTTCGCCTACTTTGATCGTGACAACGTTGCTCTCAAGGGATTCGCCAAATTCTTCAAAGAATCCAGCGATGAGGAGAGGGATCACGCAGAGAAACTCATCAAGTACCAGAACATGCGTGGAGGCAGGGTGCGGCTCCAGTCCATCGTCACACCTTTGACAGAGTTCGACCATCCTGAGAAAGGGGATGCCTTGTATGCTATGGAGTTGGCCTTGGCTCTCGAAAAGCTTGTAAATGAGAAGTTGCACAACCTGCACAGTGTGGCATCAAGGTGCAATGATCCACAGCTGACCGACTTCGTTGAGAGCGAATTCCTTGAGGAGCAGGTTGAAGCCATCAAGAAGATCTCTGAGTATGTCGCCCAGCTGAGAAGAGTGGGAAAGGGGCATGGGGTGTGGCACTTTGATCAGAAGCTGCTTGAGGAAGAAGCTTGA; SEQ ID NO:6 is as follows: MAGKGKEVLSGVVFQPFEELKGELSLVPQAKDQSLARQKFVDECEAAINEQINVEYNASYAYHSLFAYFDRDNVALKGFAKFFKESSDEERDHAEKLIKYQNMRGGRVRLQSIVTPLTEFDHPEKGDALYAMELALALEKLVNEKLHNLHSVASRCNDPQLTDFVESEFLEEQVEAIKKISEYVAQLRRVGKGHGVWHFDQKLLEEEA; 1.2 Prokaryotic expression and purification Primers were designed based on SEQ ID NO:2. Using the synthesized gene as a template, PCR amplification was performed using a commercially available high-fidelity DNA polymerase with forward primers (taagaaggagatataccatgCACCACCACGCTTCAAATGCACCCGCACC) and reverse primers (tggtggtggtggtgctcgagTCAAGCTTCTTCCTCAAGCAG) to obtain genes containing homologous arms. OsFer1M The gene sequence has a three-histidine tag added to its N-terminus. Using the same method, PCR amplification was performed with the forward primer (taagaaggagatataccatgCACCACCACGCCGGGAAAGGGAAGGAGGTG) and the reverse primer (tggtggtggtggtgctcgagTCAAGCTTCTTCCTCAAGCAG); wild-type genes containing homologous arms were obtained. OsFer1(△TP) The gene sequence also has three histidine tags added to its N-terminus. Using the empty pET28a plasmid vector as a template, forward primers (CTCGAGCACCACCACCACC) and reverse primers (CATGGTATATCTCCTTCTTAAAGTTAAACA) were used for PCR amplification with commercially available high-fidelity DNA polymerase to obtain the full-length linear fragment of the pET28a high-efficiency expression vector containing homologous arm sequences and the T7 promoter.

[0031] Using a commercially available seamless cloning kit, the gene fragment containing the above-mentioned homologous arms ( OsFer1M or OsFer1(△ TP) Seamless cloning and ligation were performed with the pET28a linear vector, which also has homologous arms, to construct the recombinant expression vector pET28a- OsFer1M and pET28a- OsFer1(△TP) After sequencing verification confirmed its correctness, the recombinant plasmid was transformed into... Escherichia coli BL21(DE3) competent cells. Positive single clones were picked and inoculated into LB medium containing 50 μg / mL kanamycin, and cultured at 37°C with shaking until... Fire 600 =0.6, IPTG was added to a final concentration of 1 mM, and expression was induced at 20℃ for 16 h. The bacterial cells were collected by centrifugation, and the supernatant was collected after sonication. The recombinant protein was purified using a Ni-NTA affinity chromatography column. SDS-PAGE analysis showed that the purity of the recombinant protein was greater than 90%.

[0032] 1.3 Thermal stability determination Purified wild-type rice ferritin OsFer1(ΔTP) and mutant OsFer1M protein were treated at 0, 60, 70, 80, 90, and 100℃ for 10 min, respectively. After treatment, the corresponding loading buffers were added, and the results were identified by SDS-PAGE and Native-PAGE. The results are as follows: Figure 1 and Figure 2 As shown, the wild-type OsFer1(△TP) protein was significantly degraded after treatment at 60℃, while the mutant OsFer1M protein was rapidly degraded after treatment at 80℃.

[0033] The denaturation temperature of the purified recombinant protein was determined by differential scanning calorimetry (DSC). T m Protein samples were dialyzed three times with deionized water, deep-frozen at -80°C, and then freeze-dried. 3–5 mg of freeze-dried protein was weighed and sealed in an aluminum sample pan, and analyzed at a heating rate of 10°C / min within the range of 30°C to 150°C. Using an empty aluminum pan as a reference, the heat flow difference between the sample and the reference was recorded. The peak temperature of the melting process was obtained from the resulting thermogram. T m (Unit: °C). Each sample was measured three times. Wild-type OsFer1 (ΔTP) was used as a control. Results are as follows: Figure 3 As shown, the mutant OsFer1M protein exhibits significantly improved thermal stability compared to wild-type rice ferritin OsFer1 (ΔTP), with a denaturation temperature of 81.9℃, compared to 54.4℃ for the wild-type, representing an increase of 27.5℃.

[0034] The above results demonstrate the superior thermal stability of the OsFer1M mutant constructed in this invention.

[0035] Example 2: Construction of rice recombinant expression vector and obtaining transgenic plants 2.1 Construction of recombinant expression vectors Specifically, the rice endosperm-specific 10kDa prolysin promoter was used. P10KDa (GenBank accession number: AY427572) was used as the control element, as obtained in Example 1. OsFer1M Using the gene sequence (a rice ferritin mutant gene with deleted TP and replaced EP segments, as shown in SEQ ID NO:2) as a template, the gene was amplified by PCR technology. OsFer1M The complete coding sequence of the gene. Using overlap extension PCR technology, the complete coding sequence of the gene was obtained. P10KDa promoter sequence, OsFer1M The encoded sequence and the Tnos termination sequence are seamlessly fused to obtain P10KDa : OsFer1M:Tnos Fusion gene fragments (as shown in SEQ ID NO:7).

[0036] SEQ ID NO:7 is: The fusion product was directionally inserted into the plant expression vector pCAMBIA1300 using a seamless cloning method. Hin dⅢ and Bam At position HⅠ, the recombinant expression vector was constructed and named p1300- P10KDa : OsFer1M .

[0037] 2.2 Agrobacterium-mediated genetic transformation of rice The recombinant expression vector p1300- P10KDa:OsFer1M Agrobacterium strain EHA105 was introduced via electroporation, and after resistance screening and molecular biological identification, an engineered Agrobacterium strain containing the recombinant expression vector was obtained. Mature seeds of rice variety Zhonghua 11 were selected, and after aseptic treatment, mature embryos were peeled off and inoculated into callus induction medium to induce embryogenic callus. The callus was then infected and co-cultured with the engineered Agrobacterium strain for 2 days to achieve Agrobacterium-mediated genetic transformation.

[0038] After co-culture, the callus tissue was transferred to a selection medium containing 50 mg / L hygromycin for two rounds of resistance screening, with each round lasting two weeks, to obtain positive resistant callus tissue. The resistant callus tissue was then transferred to a differentiation medium containing 30 mg / L hygromycin and induced to differentiate into regenerated plants under suitable light conditions. After the regenerated seedlings had fully developed their root systems, they were hardened off, transplanted to a greenhouse for cultivation, and managed with conventional water and fertilizer until the plants matured, at which point the transgenic rice seeds were harvested.

[0039] 2.3 Identification of transgenic positive lines Positive identification of harvested transgenic rice plants: Genomic DNA and total RNA were extracted from transgenic rice seeds and detected using PCR. OsFer1M The integration of genes into the genome was detected using real-time quantitative PCR (qRT-PCR). OsFer1M The transcriptional expression level of genes is used to screen for... OsFer1M Positive transgenic lines with stable gene integration and efficient expression are used for subsequent quality trait detection and related research.

[0040] Example 3: Analysis of quality traits of mature grains in transgenic rice 3.1 Rice Materials Test materials: Positive transgenic rice lines (OsFer1M-OE3, OsFer1M-OE9, OsFer1M-OE11) obtained in Example 2, with the non-transgenic wild-type rice variety "Zhonghua 11" as a blank control; all test materials were mature grains, which were dehulled and milled before use.

[0041] 3.2 Determination Method All quality traits were tested in accordance with the corresponding national standards and routine testing methods, as detailed below: (1) Determination of head rice rate and chalkiness: Refer to GB / T21719-2025 "Grain and Oil Inspection: Inspection of Head Rice Rate of Rice" and GB / T17891-2017 "High-Quality Rice" standards to test the head rice rate and chalkiness of each test material. Three biological replicates were set up for each line and the average value was calculated.

[0042] (2) Determination of total starch content: Using a commercially available kit, accurately weigh approximately 100 mg of sample pulverized through a 0.5 mm sieve and place it in a 16 × 120 mm test tube. Add 10 mL of 100 mM sodium acetate buffer (pH 5.0) containing 5 mM calcium chloride and vortex to mix. Add 0.1 mL of heat-resistant α-amylase to the sample tube and 0.1 mL of the same buffer to the blank sample tube. Vortex and incubate in a boiling water bath for 15 min, vortexing once every 5 min. Remove and transfer to a 50 °C water bath for equilibration for 5 min. Add 0.1 mL of amylase to the sample tube and 0.1 mL of buffer to the blank tube, and incubate at 50 °C for 30 min. After cooling to room temperature, take 2.0 mL of the solution and centrifuge (13,000 rpm, 5 min). Take 1.0 mL of the supernatant and add it to 4.0 mL of sodium acetate buffer (pH 5.0) and mix well. Take 0.1 mL of this solution, add 3.0 mL of LGOPOD reagent, incubate at 50 °C for 20 min, and measure the absorbance at 510 nm. Simultaneously, use 0.1 mL of LD-glucose standard solution (1.0 mg / mL) as a control and 0.1 mL of buffer as a reagent blank. The total starch content (%, dry weight) is calculated using the following formula: Starch% = ΔA × F × EV × D × 0.90 / W (where: ΔA is the sample absorbance minus the reagent blank absorbance; F is the absorbance-to-glucose conversion factor; EV is the sample extraction volume; D is the dilution factor; W is the sample weight (mg); and 0.90 is the glucose anhydride conversion factor). Set up three biological replicates for each strain and calculate the average value.

[0043] (3) Determination of amylose content: Using a commercially available kit, accurately weigh 20-25 mg of starch or cereal powder sample and place it in a 10 mL sample tube with a screw cap. Add 1 mL of dimethyl sulfoxide (DMSO), heat in a boiling water bath for 1 minute until the sample is dispersed, vortex at high speed and continue heating for 15 min. After standing at room temperature for 5 min, add 2 mL and 4 mL of 95% ethanol sequentially, invert and mix to form a precipitate, stand upright for 15 min, centrifuge at 2,000 g for 5 min, discard the supernatant, invert for 10 minutes to remove residual ethanol; add 2 mL of DMSO to the precipitate, heat in a boiling water bath for 15 min, remove and immediately add 4 mL of ConA working solution, mix well and transfer to a 25 mL volumetric flask to make up to volume, obtaining solution A; take 1.0 mL of solution A and add 0.50 mL of ConA solution, stand at room temperature for 1 minute. Centrifuge at 14,000g for 10 min; take 1 mL of supernatant and add 3 mL of 100 mM sodium acetate buffer (pH 4.5), heat in a boiling water bath for 5 min to denature ConA, transfer to a 40°C water bath for equilibration for 5 min, add 0.1 mL of amylase / α-amylase mixed solution, incubate at 40°C for 30 min, centrifuge at 2,000g for 5 min, take 1.0 mL of supernatant and add 4 mM LGOPOD reagent, incubate at 40°C for 20 min, and measure the absorbance at 510 nm; separately take 0.5 mL of solution A and add 4 mL of sodium acetate buffer (pH 4.5) and 0.1 mL of enzyme mixed solution, incubate at 40°C for 10 min, take 1.0 mL and add 4 mM LGOPOD reagent, incubate at 40°C for 20 min, and measure the absorbance at 510 nm. The amylose content (as a percentage of total starch) was calculated using the following formula: Amylose % = (Absorbance value of ConA supernatant × 6.15) / (Absorbance value of total starch per unit volume × 9.2) × 100, where 6.15 and 9.2 are the dilution factors for ConA and total starch extracts, respectively. Three biological replicates were set up for each strain, and the average value was calculated.

[0044] (3) Determination of total protein and soluble sugar content: Total protein content was determined according to GB5009.5-2025 "Determination of Protein in Food" (Kjeldahl method); soluble sugar content was determined according to GB / T37493-2019 "Grain and Oil Inspection - Determination of Soluble Sugar in Cereals and Legumes - Copper Reduction-Iodometric Method". Three biological replicates were set up for each strain, and the average value was calculated.

[0045] 3.3 Measurement Results The experimental results showed that, compared with the wild-type Zhonghua 11, the transgenic rice lines (OsFer1M-OE3, OsFer1M-OE9, OsFer1M-OE11) exhibited significant improvements in all quality traits of mature grains, as shown in Table 1. Table 1. Analysis of Grain Quality Traits Processing and appearance quality: The head rice rate of the transgenic lines is increased by at least 11.56% to 15.45% compared with the wild type, and the resistance to breakage is significantly enhanced; the chalkiness is reduced by at least 3.4% to 4.45% compared with the wild type, the rice grain transparency is significantly improved, and the marketability is improved.

[0046] (2) Starch and protein related qualities: After drying, the total starch content in the grains increased by at least 3.06% to 4.06% compared with the wild type, the amylose content increased by at least 3.35% to 6.32% compared with the wild type; the total protein content decreased by at least 1.64% to 2.89% compared with the wild type, and the soluble sugar content decreased by at least 2.09% to 3.76% compared with the wild type, effectively optimizing the eating quality and digestibility of rice.

[0047] The above results confirm that the transgenic rice lines constructed in this invention can achieve synergistic improvement in rice processing quality, appearance quality, and starch quality, which aligns with the core objective of this invention.

[0048] Example 4: Determination of iron retention rate before and after cooking 4.1 Rice Materials Test materials: The positive transgenic rice lines (OsFer1M-OE3, OsFer1M-OE9, OsFer1M-OE11) obtained by the above screening were used as blank control with the non-transgenic wild-type rice variety "Zhonghua 11"; the test rice was obtained by dehulling, milling and screening of the grains of each test material, removing impurities and broken rice, and was used for later use.

[0049] 4.2 Method for determining iron content The determination of iron content in rice grains and cooked rice was performed according to the flame atomic absorption spectrometry method in the national standard GB5009.90—2016 "Determination of Iron in Food". The specific operation is as follows: Iron content determination before cooking: Take rice samples of each test material, crush them and pass them through a 40-mesh sieve. Accurately weigh an appropriate amount of sample, and after microwave digestion, determine the iron content using a flame atomic absorption spectrometer. Set up 3 biological replicates for each strain and calculate the average value.

[0050] (2) Determination of iron content after cooking: Take the rice samples of each test material, mix rice and distilled water in a ratio of 1:1 (mass:volume), place them in a sterile glass container, cook in water for 10 minutes, cool to room temperature, grind the rice evenly, and after microwave digestion, determine the iron content using a flame atomic absorption spectrometer. Three biological replicates are set up for each strain, and the average value is calculated.

[0051] 4.3 Results of iron content determination Iron content determination results are as follows Figure 4 As shown, the details are as follows: (1) Iron content before cooking: The average iron content of wild-type Zhonghua 11 rice was 4.12 μg / g; the average iron content of transgenic lines OsFer1M-OE3, OsFer1M-OE9 and OsFer1M-OE11 rice was 5.43 μg / g, 5.44 μg / g and 6.22 μg / g, respectively. Compared with wild type, the iron content of each transgenic line increased by 1.31~2.10 μg / g, which is 1.32~1.51 times.

[0052] (2) Iron content after steaming: The average iron content of rice cooked by wild-type Zhonghua 11 was 1.15 μg / g; the average iron content of rice cooked by transgenic lines OsFer1M-OE3, OsFer1M-OE9 and OsFer1M-OE11 was 1.84 μg / g, 1.81 μg / g and 2.24 μg / g, respectively. Compared with the wild-type control, the iron content of rice cooked by each transgenic line increased by 0.66~1.09 μg / g, which is 1.57~1.95 times.

[0053] The above results show that the transgenic rice lines constructed in this invention not only significantly increase the iron content of the grains, but also greatly improve the iron retention rate during the cooking process, effectively solving the technical problems of easy denaturation and severe iron loss of natural ferritin during cooking.

[0054] Example 5: Statistical Analysis All data in the above examples are expressed as mean ± standard deviation (mean ± SD). Tukey's test was used to analyze the significance of the differences in the data. Different lowercase letters represent statistically significant differences (P < 0.05) between different lines of transgenic plants and between the wild type.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A rice ferritin mutant with improved thermal stability, characterized in that: The mutant is rice ferritin, whose extended peptide EP segment amino acid sequence is replaced with the EP segment amino acid sequence of soybean ferritin. Preferably, the mutant has a thermal stability that is at least 27.5°C higher than that of wild-type rice ferritin.

2. The rice ferritin mutant with improved thermal stability according to claim 1, characterized in that: The mutant also lacks the transport peptide TP segment.

3. The rice ferritin mutant with improved thermal stability according to claim 1, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO:

1.

4. A gene encoding a gene, characterized in that: The gene encoding the rice ferritin mutant according to any one of claims 1-3, preferably, the nucleotide sequence of the gene is shown in SEQ ID NO:

2.

5. A recombinant expression vector, characterized in that: A recombinant expression vector containing the encoding gene of claim 4.

6. A recombinant expression vector according to claim 5, characterized in that: The recombinant expression vector further includes a rice endosperm-specific promoter operably linked to the gene, preferably, the rice endosperm-specific promoter is a rice 10kDa prolysin promoter.

7. A method for synergistically improving the quality of rice grains, characterized in that, The process includes the following steps: introducing the gene described in claim 4, or the recombinant expression vector described in claim 5 or 6, into rice cells to obtain transgenic rice plants; Preferably, the rice is indica rice or japonica rice.

8. The method for synergistically improving rice grain quality according to claim 7, characterized in that: The step of introducing the recombinant expression vector into rice cells is achieved through Agrobacterium-mediated genetic transformation. Preferably, the Agrobacterium-mediated genetic transformation method includes: infecting and co-culturing callus tissue with an Agrobacterium engineered strain containing a recombinant expression vector, obtaining positive resistant callus tissue through resistance screening, and then obtaining transgenic rice plants after differentiation, rooting, and hardening. Preferably, compared with wild-type rice, the mature grains of the transgenic rice plants have a 11.56%~15.45% higher head rice rate, a 3.4%~4.45% lower chalkiness, a 3.06%~4.06% higher total starch content, a 3.35%~6.32% higher amylose content, a 1.64%~2.89% lower total protein content, a 2.09%~3.76% lower soluble sugar content, and a 1.31~2.10 μg / g higher iron content. Furthermore, the iron content in the cooked rice is increased by 0.66~1.09 μg / g.

9. The application of the rice ferritin mutant according to any one of claims 1-3, the encoding gene according to claim 4, or the recombinant expression vector according to any one of claims 5 or 6 in simultaneously improving at least two traits of rice grain head rice rate, chalkiness, total starch content, amylose content, total protein content, soluble sugar content, and iron content; preferably, the rice is indica rice or japonica rice.

10. The use of the rice ferritin mutant according to any one of claims 1-3, the encoding gene according to claim 4, or the recombinant expression vector according to any one of claims 5 or 6 in the preparation of high-iron, high-amylose rice flour or low-glycemic index rice products.

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