Rice SS3a allele and application thereof in increasing amylose content and grain weight of rice

Abstract

The invention belongs to the field of molecular genetics, and relates to a rice SS3a allele and application thereof in increasing amylose content and grain weight of rice. The 5th basic group G of the 7th intron of the rice SS3a gene of the gene is replaced by A. The invention also relates to application of the new SS3a allele in rice breeding. The allele significantly improves the amylose content and grain weight of rice, and also changes the amylopectin structure, starch gelatinization characteristics and rice viscosity characteristics. The allele is introduced through a crossbreeding method, or the protein coded by the allele is introduced through a genetic engineering method, or the original SS3a gene is subjected to site-directed mutagenesis to form the allele, so that the amylose content of rice is changed, and the rice quality is improved. The allele can also be used in other cereals with similar metabolic pathways, is used for increasing the amylose content of the seeds and synchronously improving the quality of the seeds, and provides a molecular design modification target for genetic improvement breeding of crops.

Description

Rice SS3a allele and its application in increasing rice amylose content and grain weight Technical Field

[0001] This invention belongs to the field of molecular genetics and relates to the rice SS3a allele and its application in increasing the amylose content and grain weight of rice. Background Technology

[0002] Rice is the staple food of more than half the world's population, and its quality and yield are of paramount importance. Amylose content is a core indicator determining the cooking quality, texture, and processing adaptability of rice. Rice with high amylose content (typically >25%) has characteristics such as high hardness and low stickiness, making it particularly suitable for the development of processed foods such as rice noodles and rice vermicelli, as well as certain health foods.

[0003] Grain weight is one of the three key factors constituting rice yield and is directly related to the crop's economic output. However, in traditional breeding and existing genetic resources, high amylose content and low grain weight often show a negative correlation. For example, allelic variations or mutants of many known starch synthesis-related genes (such as the SBEIIb gene) often increase amylose content but are accompanied by a decrease in grain weight and yield, which severely limits the breeding of rice varieties that combine high quality and high yield.

[0004] Soluble starch synthase 3a (SS3a) in rice is one of the key enzymes in amylopectin synthesis, responsible for the elongation of long branched side chains in amylopectin. Existing research mainly focuses on the effects of SS3a loss of function on starch composition and amylopectin structure, but its comprehensive regulatory effect on amylose content and grain weight remains unclear, and no SS3a allele has been found that can simultaneously and significantly increase amylose content and grain weight. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides the rice SS3a allele and its application in increasing rice amylose content and grain weight. It can simultaneously improve rice quality and yield, and has strong application prospects in rice breeding.

[0006] The technical solution provided by this invention is as follows:

[0007] The rice SS3a allele, wherein the 5th base G on the 7th intron of the rice SS3a allele is replaced by A; the nucleotide sequence of the rice SS3a allele is shown in SEQ ID No. 1.

[0008] The present invention also provides a protein encoded by the above-mentioned rice SS3a allele, wherein amino acids 1378-1390 of the SS3a protein are deleted; the amino acid sequence of the protein is shown in SEQ ID No. 2.

[0009] The present invention also provides the application of the above-mentioned rice SS3a allele or the protein encoded by the above-mentioned rice SS3a allele in the improvement of rice quality.

[0010] Furthermore, the rice quality improvement includes increasing the amylose content and grain weight of the rice.

[0011] The present invention also provides the application of the above-mentioned rice SS3a allele or the protein encoded by the above-mentioned rice SS3a allele in reducing the long branched side chains of rice amylopectin.

[0012] The present invention also provides the application of the above-mentioned rice SS3a allele or the protein encoded by the above-mentioned rice SS3a allele in improving the short branched side chains of rice amylopectin.

[0013] The present invention also provides the application of the above-mentioned rice SS3a allele or the protein encoded by the above-mentioned rice SS3a allele in reducing the average chain length of rice amylopectin.

[0014] The present invention also provides the application of the above-mentioned rice SS3a allele or the protein encoded by the above-mentioned rice SS3a allele in reducing the gelatinization temperature and gelatinization enthalpy of rice.

[0015] The present invention also provides the application of the above-mentioned rice SS3a allele or the protein encoded by the above-mentioned rice SS3a allele in reducing the peak viscosity, hot paste viscosity, disintegration viscosity and final viscosity of rice.

[0016] The present invention also provides a method for increasing the amylose content and grain weight of rice by introducing the above-mentioned rice SS3a allele through hybridization breeding, or by introducing the protein encoded by the above-mentioned rice SS3a allele through genetic engineering, or by site-directed mutation of the original SS3a gene into the above-mentioned rice SS3a allele.

[0017] Beneficial effects

[0018] There are currently no reports of this allelic variation in the SS3a gene. The new SS3a allele in rice provided by this invention, which increases amylose content and grain weight, is a novel allelic mutation. This new SS3a allele mutant, screened from an EMS-mutated library of non-floury endosperm mutants of Nipponbare japonica rice, has been confirmed through years of field trials in multiple locations to exhibit stable superior traits controlled by this allele under different growth environments. Specifically, the apparent amylose content increased from 16.5±1.3% in the wild type to 25.6±1.3%, an increase of 56.0±10.4%, and the 100-grain weight increased from 2.09±0.05 g to 2.14±0.05 g, an increase of 2.6±0.8%. The novel allele encoding starch synthase 3a provided by this invention plays a key role in regulating starch composition and amylopectin structure. It increases amylose content from 13.90% to 22.82%, an increase of 64.2%, while simultaneously increasing the contents of amylopectin DP6-12 and DP13-24 from 28.26% and 46.87% to 32.31% and 52.61%, respectively, an increase of 14.3% and 12.2%. Meanwhile, the contents of DP≥37 and the average side chain length decrease from 13.93% and 21.11 DP to 5.41% and 18.01 DP, respectively, a decrease of 61.2% and 14.7%. In this study, the SS3a allele mutant exhibited reduced starch gelatinization initiation, peak, and termination temperatures from 62.6, 69.7, and 77.4 °C to 56.4, 65.7, and 73.6 °C, respectively, and a reduction in gelatinization enthalpy from 11.0 J / g to 9.6 J / g. Furthermore, the peak viscosity, hot paste viscosity, disintegration viscosity, and final viscosity of the rice decreased from 4942, 2516, 2426, and 4164 mPas to 2629, 1898, 732, and 3529 mPas, representing reductions of 46.8%, 24.6%, 69.8%, and 15.2%, respectively. In conclusion, this allele can not only be directly used to breed new rice varieties with high amylose content and high yield, but its unique starch characteristics and rice quality also offer special applications in the food processing industry. Attached Figure Description

[0019] Figure 1 shows the screening and propagation of the rice NM8 mutant. A shows the identification of the brown rice half-grain urea gelatinization-iodine solution colorimetric method of the NM8 single-plant progeny M3; B shows the identification of the brown rice half-grain urea gelatinization-iodine solution colorimetric method of the NM8 single-plant progeny M4.

[0020] Figure 2 shows the iodine absorption spectrum of M4 brown rice starch from NM8.

[0021] Figure 3 shows the molecular weight distribution of M4 brown rice starch in NM8.

[0022] Figure 4 shows the amylopectin chain length distribution of NM8 in M4 brown rice; where A is the molar percentage and B is the difference in molar percentage between the mutant and WT.

[0023] Figure 5 shows the differential scanning calorimetry (DSC) spectrum of NM8 M4 brown rice starch.

[0024] Figure 6 shows the mutation analysis of the SS3a gene in NM8. A is a schematic diagram of the primer distribution for SS3a gene sequencing; B is the sequencing peak diagram of SS3a gene mutation sites; C is the WB detection of SS3a protein in developing endosperm; D is the agarose gel electrophoresis detection of SS3a mRNA reverse transcription cDNA in developing endosperm; E is the sequencing peak diagram of SS3a mRNA reverse transcription cDNA in developing endosperm; F is the mutation sequence analysis of SS3a protein in NM8.

[0025] Figure 7 is a statistical analysis chart of the brown rice grain weights of M5 and M6 of NM8 in multiple planting areas.

[0026] Figure 8 shows the iodine colorimetric analysis of brown rice starch from M5 and M6 of NM8; A, B, C, and D are four different planting sites: 2024JD, 2025YZ(1), 2025YZ(2), and 2025GY, respectively.

[0027] Figure 9 shows the viscosity characteristics (RVA) of NM8 M5 rice. Detailed Implementation

[0028] The implementation scheme of the present invention will be described in detail below with reference to specific implementation steps.

[0029] 1. Experimental materials

[0030] The wild-type japonica rice variety Nipponbare and its mutant NM8 were used as experimental materials.

[0031] NM8 was obtained from an EMS-mutated library of Nipponbare non-floury endosperm mutants. The screening method was as follows: Mature brown rice was transversely cut in half with a sharp blade, and the half-grain without the embryo was taken as a gelatinized sample. This half-grain was placed horizontally in a flat-bottomed 96-well plate, and 200 μL of 2.5 M and 3.0 M urea solutions were added (wild-type grains at 2.5 M concentration did not gelatinize, and the iodine staining solution was yellow; wild-type grains at 3.0 M concentration gelatinized, and the iodine staining solution was blue). The plate was then incubated at 24℃ for 16 h for gelatinization. After gelatinization, 20 μL of iodine solution (0.2% iodine, 1% potassium iodide, 0.5% acetic acid) was added to the 96-well plate for staining, and the mixture was stirred thoroughly. After color development, the color of the staining solution was observed, and mutants with altered starch composition whose staining solution color did not match the WT were screened. The corresponding half-grain with the embryo was then used for seed propagation. Figure 1 shows that NM8-5 (M3 brown rice) is fully homozygous. We will use NM8-5 for breeding and related measurements in the future.

[0032] 2. Analysis of M4 brown rice starch components in NM8

[0033] The starch composition of Nipponbare and NM8 was analyzed using starch-iodine absorption spectroscopy and starch molecular weight distribution. The OD620 of the starch-iodine complex reflects the absorbance of the long side chains of amylose and amylopectin bound to iodine molecules and is commonly used to assess apparent amylose content. The iodine colorimetric determination results (Figure 2 and Table 1) showed that the apparent amylose content of NM8 increased from 18.3% to 26.1%, an increase of 42.6%. The molecular weight distribution of debranched starch was analyzed by size exclusion chromatography (Figure 3 and Table 2). The results showed that the amylose content of NM8 increased from 13.90% to 22.82%, an increase of 64.2%, while the contents of long and short side chains of amylopectin decreased from 19.11% and 66.99% to 13.42% and 63.76%, respectively, a decrease of 29.8% and 4.8%. The ratio of short to long side chain content, which reflects the degree of branching of amylopectin, increased from 3.50 to 4.75, an increase of 35.7%, indicating that the degree of branching of amylopectin in NM8 was significantly higher than that in Nipponbare.

[0034] Table 1. Starch iodine uptake parameters and apparent amylose content

[0035]

[0036] The data in the table are mean ± standard deviation (n=3). * indicates the difference between mutant and WT data by t-test (**, p<0.01; ***, p<0.001).

[0037] Table 2. Starch molecular weight distribution parameters

[0038]

[0039] The data in the table are mean ± standard deviation (n=2). * indicates the difference between mutant and WT data by t-test (*, p<0.05; **, p<0.01).

[0040] 3. Fine structural analysis of M4 brown rice amylopectin from NM8

[0041] The chain length distribution of amylopectin was determined by fluorescence-assisted capillary electrophoresis. The difference in chain length distribution between NM8 and Nipponbare amylopectin samples showed an increase in the content of short-branched side chains and a decrease in the content of long-branched side chains (Figure 4B). Statistical analysis of the chain length distribution parameters revealed that the contents of DP6-12 and DP13-24 increased from 28.26% and 46.87% to 32.31% and 52.61%, respectively, representing increases of 14.3% and 12.2%. Conversely, the contents of DP≥37 and the average side chain length decreased from 13.93% and 21.11 DP to 5.41% and 18.01 DP, respectively, representing decreases of 61.2% and 14.7% (Table 3).

[0042] Table 3. Distribution of amylopectin chain length

[0043]

[0044] The data in the table are mean ± standard deviation (n=2), * indicates the difference between mutant and WT data by t test (**, p<0.01).

[0045] 4. Analysis of the gelatinization characteristics of NM8 M4 brown rice

[0046] The gelatinization characteristics of M4 brown rice were analyzed using differential scanning calorimetry. The results showed that the gelatinization initiation temperature, peak temperature and termination temperature decreased from 62.6, 69.7 and 77.4℃ to 56.4, 65.7 and 73.6℃, ​​respectively, and the gelatinization enthalpy decreased from 11.0 J / g to 9.6 J / g (Figure 5).

[0047] 5. SS3a mutation analysis of NM8

[0048] SS3a is believed to be primarily responsible for the elongation of long branched side chains in amylopectin. Inhibition of SS3a activity typically leads to a significant decrease in the proportion of long branched side chains in endosperm starch, while simultaneously increasing the content of amylose. Furthermore, mutations in the rice SS3a gene generally result in a decrease in starch gelatinization temperature. Therefore, this invention first sequenced the full-length exons and intron splicing sites of the SS3a gene in NM8. A total of 12 primer pairs were designed, and their distribution in the SS3a gene is shown in Figure 6A. Primer information is shown in Table 4. The genomic DNA of leaves from Nipponbare and NM8 plants was extracted using the CTAB method. PCR amplification was performed using leaf DNA as a template. The amplification system and procedure are shown in Tables 5 and 6. After identification by agarose gel electrophoresis, the amplified products were sequenced by Nanjing Qingke Biotechnology Co., Ltd. The sequencing results showed that the 5th base G in the 7th intron of SS3a in NM8 was replaced by A (Figure 6B). The gene sequence of NM8 is shown in SEQ ID No. 1. Total protein was extracted from the developing endosperm. Western blotting analysis showed that the SS3a protein of NM8 had a smaller molecular weight (Figure 6C). Since the mutation was located on an intron, RNA was extracted and reverse-engineered into cDNA for transcript analysis. The cDNA gel image showed that NM8 had one transcript with a smaller molecular weight (Figure 6D). Sequencing analysis showed that 39 bases (-1 to -39) after exon 7 of the SS3a gene in NM8 were excised as an intron (Figure 6E), resulting in a 13-amino acid deletion at positions 1378-1390 of the SS3a protein (Figure 6F). The amino acid sequence encoded by the SS3a allele of NM8 is shown in SEQ ID No. 2.

[0049] Table 4 Primer information for SS3a sequencing detection

[0050]

[0051] Table 5 PCR amplification system

[0052]

[0053] Table 6 PCR Amplification Procedure

[0054]

[0055] 6. Analysis of M5 and M6 brown rice grain weight, starch iodine uptake parameters, and apparent amylose content of NM8.

[0056] NM8 M4 and M5 plants were planted together with Nipponbare rice in 2024 at the Jiudian Experimental Field of Yangzhou University (2024JD) and in 2025 at Experimental Field 1 (2025YZ(1)) and Experimental Field 2 (2025YZ(2)) of Yangzhou University Wenhui Road Campus, Yangzhou City, and Experimental Field 2 (2025GY) of Yangzhou University, respectively, according to conventional cultivation and management methods. Ten individual plants were randomly harvested from each experimental field, and the 100-grain weight of each individual plant was measured. The results showed that the grain weight of NM8 in all four planting sites significantly exceeded that of its Nipponbare WT, with the 100-grain weight increasing from 2.09±0.05 g to 2.14±0.05 g, an increase of 2.6±0.8% (Figure 7). Starch was further separated from the mixed rice harvested from the four planting sites, and the iodine absorption spectrum was measured (Figure 8). The iodine absorption parameters and apparent amylose content were analyzed (Table 7). The maximum absorption wavelength, OD620, OD680, and OD620 / OD550 of the NM8 starch-iodine complex from the four planting sites increased from 569.5–572.8 nm, 0.169–0.206, 0.121–0.153, and 0.909–0.930 to 592.0–599.0 nm, 0.248–0.285, 0.202–0.240, and 1.105–1.146, respectively. The apparent amylose content increased from 14.3%–17.3% to 24.0%–27.6%, with an average increase of 59.3% (Table 7).

[0057] Table 7. Starch Iodine Absorption Parameters and Apparent Amylose Content

[0058]

[0059] Rice was cultivated and managed according to conventional methods. In 2024, it was planted in the Jiudian Experimental Field of Yangzhou University (2024JD), and in 2025, it was planted in Experimental Field 1 (2025YZ(1)) and Experimental Field 2 (2025YZ(2)) of Yangzhou University Wenhui Road Campus in Yangzhou City, and Experimental Field 2 (2025GY) of Yangzhou University Gaoyou Campus. The data in the table are mean ± standard deviation (n = 3). * indicates the difference between the mutant and WT data by t test (*, p < 0.05; ***, p < 0.001).

[0060] 7. Analysis of the viscosity characteristics of M5 rice from NM8

[0061] The viscosity properties of rice were determined using a rapid viscosity analyzer (RVA) (Figure 9). The peak viscosity, hot paste viscosity, disintegration viscosity, and final viscosity of NM8 rice decreased from 4942, 2516, 2426, and 4164 mPas to 2629, 1898, 732, and 3529 mPas, respectively, representing reductions of 46.8%, 24.6%, 69.8%, and 15.2% (Table 8).

[0062] Table 8. Rice viscosity characteristics parameters

[0063]

[0064] The data in the table are mean ± standard deviation (n=4). * indicates the difference between mutant and WT data by t-test (**, p<0.01; ***, p<0.001).

[0065] 7. Breeding applications of the NM8 mutant and its carried SS3a allele

[0066] The above experimental results demonstrate that the novel SS3a allele provided by this invention can simultaneously increase rice grain weight and amylose content. Data from multi-year, multi-field trials have proven its universality in increasing yield and improving quality traits. Furthermore, the unique starch physicochemical properties induced by this gene open new avenues for its application in high-end food processing. This allele can be introduced into target rice varieties using traditional hybridization breeding methods, or its encoded protein can be introduced through genetic engineering, or the existing SS3a gene can be site-directedly mutated to form this allele, thereby altering the amylose content and improving rice quality. This allele can also be used in other grains with similar metabolic pathways to increase seed amylose content and simultaneously improve seed quality, providing a molecular design target for crop genetic improvement breeding.

[0067] SEQ ID No.1

[0068] NM8 SS3a mutant gene sequence

[0069]

[0070] SEQ ID No.2

[0071] SS3a mutant amino acid sequence of NM8:

[0072]

[0073] SEQ ID No. 3 (primer 1F): GAGCGCTGAAGGTCGTC.

[0074] SEQ ID No. 4 (primer 1R): CGTATGAAGGGAAATCGTCC.

[0075] SEQ ID No. 5 (primer 2F): CAAGTTCAGGTTTGTGTAGGATAGC.

[0076] SEQ ID No. 6 (primer 2R): GCTTCATCCACCACATCCAC.

[0077] SEQ ID No. 7 (primer 3F): CTTCAAGCACTGTAATGTATGGG.

[0078] SEQ ID No. 8 (primer 3R): GACTGGCTTTTCCTATGGACAC.

[0079] SEQ ID No. 9 (primer 4F): CCAGCGTTTATCAACAAGAAGG.

[0080] SEQ ID No. 10 (primer 4R): GACTAAACGACCATTGAAGAACAC.

[0081] SEQ ID No. 11 (primer 5F): TGTCCACATCAAAGGAGCATTC.

[0082] SEQ ID No. 12 (primer 5R): AGCCAACAAAATACATCAGAACC.

[0083] SEQ ID No. 13 (primer 6F): ATCAGAGAACGGGAGGAAGC.

[0084] SEQ ID No. 14 (primer 6R): CTCCAGTTACAGAATTGTCGTAGC.

[0085] SEQ ID No. 15 (primer 7F): CAAGCACTGTTGAAGTATATGTCTAGC.

[0086] SEQ ID No. 16 (Primer 7R): CTAACTGAGAAAGGAAGGACAACAC.

[0087] SEQ ID No. 17 (primer 8F): TATCGCAGTCTAACCCA.

[0088] SEQ ID No. 18 (primer 8R): GCAGTGGAAGGAGGGAT.

[0089] SEQ ID No. 19 (primer 9F): CTTTCTTTGTTTCCATGTTTTGC.

[0090] SEQ ID No. 20 (primer 9R): AAGAGGAGTAAAGTGAATGTAAGGTG.

[0091] SEQ ID No. 21 (primer 10F): TTGCCCTTTCTCAGCCTAT.

[0092] SEQ ID No. 22 (primer 10R): GGCAACCCAAGCAAACT.

[0093] SEQ ID No. 23 (primer 11F): AGTTTGCTTGGGTTGCC.

[0094] SEQ ID No. 24 (primer 11R): TGCCAATGCTTGCTTTCT.

[0095] SEQ ID No. 25 (primer 12F): GCTTGGTGGTCTATTGTTTATGTG.

[0096] SEQ ID No. 26 (primer 12R): GGCTTTAGGAATCGTGATGG.

Claims

1. The rice SS3a allele, characterized in that, The 5th base G on the 7th intron of the rice SS3a allele is replaced by A; the nucleotide sequence of the rice SS3a allele is shown in SEQ ID No.

1.

2. The protein encoded by the rice SS3a allele according to claim 1, characterized in that, The amino acid sequence of the protein is shown in SEQ ID No.

2.

3. The application of the protein encoded by the rice SS3a allele as described in claim 1 or the rice SS3a allele as described in claim 2 in the improvement of rice quality.

4. The application according to claim 3, characterized in that, The rice quality improvement includes increasing the amylose content and grain weight of the rice.

5. The application of the protein encoded by the rice SS3a allele of claim 1 or the rice SS3a allele of claim 2 in reducing the long branched side chains of rice amylopectin.

6. The application of the protein encoded by the rice SS3a allele of claim 1 or the rice SS3a allele of claim 2 in improving the short branched side chains of rice amylopectin.

7. The application of the protein encoded by the rice SS3a allele of claim 1 or the rice SS3a allele of claim 2 in reducing the average chain length of amylopectin in rice.

8. The application of the protein encoded by the rice SS3a allele of claim 1 or the rice SS3a allele of claim 2 in reducing the gelatinization temperature and enthalpy of rice.

9. The application of the protein encoded by the rice SS3a allele of claim 1 or the rice SS3a allele of claim 2 in reducing the peak viscosity, hot slurry viscosity, disintegration viscosity and final viscosity of rice.

10. A method for increasing the amylose content and grain weight of rice, characterized in that, The rice SS3a allele of claim 1 can be introduced through hybridization breeding, or the protein encoded by the rice SS3a allele of claim 1 can be introduced through genetic engineering, or the original SS3a gene can be mutated at a specific site into the rice SS3a allele of claim 1.

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