Application of rice quality regulation gene CHALK10 and protein encoded thereby

Abstract

The application discloses an application of a rice quality regulation gene CHALK10 and a protein coded by the gene. The application clones the CHALK10 gene (Seq ID No. 1) by using MutMap, the gene codes an F-box protein (Seq ID No. 2), the gene and the protein can be used for regulating cooking taste quality (amylose content, gel consistency and / or gelatinization temperature) of rice, and can also be used for regulating appearance quality (chalky grain rate and / or chalkiness) of rice, and can be used for improving cooking taste quality and appearance quality of rice, and improving added value of a rice industry and product competitiveness.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, and in particular to the application of a rice quality regulating gene CHALK10 and its encoded protein. Background Technology

[0002] Rice quality exhibits diversity, mainly encompassing four aspects: cooking and eating quality, appearance quality, milling quality, and nutritional quality. These quality traits directly determine the commercial and nutritional value of rice, as well as consumer behavior. Improving rice quality has always been a research goal for scientists.

[0003] Cooking and eating quality is the most important quality indicator in rice quality composition. Since endosperm starch is the main edible part of rice, the composition and structure of starch are the most important factors determining the cooking and eating quality of rice. Because it is impossible to accurately identify the cooking and eating quality of rice through manual tasting, three physicochemical indicators are usually used to evaluate the cooking and eating quality of rice: amylose content, gel consistency, and gelatinization temperature.

[0004] Endosperm starch accounts for over 80% of the dry weight of rice endosperm, mainly composed of amylose and amylopectin. The amylose and amylopectin content varies significantly among different rice varieties and even among rice of the same variety at different growth stages. Amylose content is the most important factor affecting the cooking and eating quality of rice. Generally, rice with low amylose content is considered to have less expansion after cooking, a glossy appearance, and better taste, because when the amylose content is too high, the cooked rice is fluffy and the grains are harder. Gel consistency is a colloidal property of rice starch, referring to the length of the rice paste after gelatinization and cooling, indicating the tendency of starch gelatinization and retrogradation upon cooling. Gel consistency is usually negatively correlated with amylose content and is an important factor affecting the cooking quality, softness, and texture of high-quality rice. Rice with high gel consistency has a moist and smooth texture, does not retrograde upon cooling, and can increase appetite; rice with low gel consistency has a rough texture and is difficult to swallow. Generally, the higher the gel consistency of rice, the softer and better the quality of the cooked rice. Starch granules do not dissolve in cold water, forming a suspension. When heated to a certain temperature, the starch granules undergo irreversible expansion, and the internal crystalline structure is destroyed—a phenomenon known as gelatinization. Gelatinized starch is more palatable in terms of viscosity, strength, and toughness. Furthermore, because gelatinized starch is more easily hydrolyzed by amylases, it is more easily digested and absorbed by the human body. Generally, rice with lower amylose content and gelatinization temperature but higher gel consistency is relatively softer and stickier after cooking, making it more competitive in the market. Therefore, reducing the amylose content and gelatinization temperature of rice, and increasing its gel consistency, are of great significance for improving the cooked and flavorful quality of rice and increasing its commercial value.

[0005] The appearance quality of rice is evaluated using indicators such as chalky grain rate and chalkiness degree. Chalkiness in rice refers to the white, opaque portion that appears due to insufficient grain filling during the rice grain-filling stage, resulting in loose, diffused light in the endosperm. The chalky grain rate is the percentage of chalky grains out of all rice grains in a sample, and is an important quality indicator. The chalkiness degree is the percentage of chalky area out of the total area of ​​all rice grains in the sample; similar to the chalky grain rate, it is also an important quality indicator. Chalkiness not only reduces the head rice yield and the appearance quality of rice but also deteriorates its palatability.

[0006] As can be seen from the above, discovering new rice quality regulatory genes (such as cooking and appearance quality), cloning and functional analysis of related genes, clarifying the molecular mechanisms of action and regulatory mechanisms of these genes, and thus carrying out targeted research on the improvement of rice quality (such as cooking and appearance quality) is indeed one of the important research and development topics at present. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide a gene and its protein that can regulate rice quality, wherein the rice quality refers to the cooking and eating quality of rice and / or the appearance quality of rice.

[0008] This invention mainly utilizes MutMap to obtain the genes and proteins, and the specific technical steps are as follows:

[0009] 1. Isolation and genetic analysis of the mutant chalk10:

[0010] The rice starch synthesis defect mutant chalk10 of this invention is derived from the EMS (Ethyl Methyl Sulfonate) mutagenesis of the japonica rice variety Zhonghua 11 (Oryza sativa L.cv ZH11). Reciprocal crosses with the wild type demonstrated that this mutant is controlled by a recessive single gene.

[0011] 2. Comparison of cooking taste and appearance quality between mutant chalk10 and wild-type polished rice:

[0012] Compared to the wild type, the chalk10 mutant exhibits a significantly higher chalky grain rate and chalkiness in its polished rice. Furthermore, the cooked rice from the chalk10 mutant demonstrates superior taste and quality compared to the wild type, specifically with a significantly reduced amylose content, a significantly increased gel consistency, and a significantly lower gelatinization temperature.

[0013] 3. Cloning of the CHALK10 gene:

[0014] (1) Positioning of CHALK10:

[0015] To isolate the CHALK10 gene, this invention first constructed a segregating population. The F1 generation was obtained by crossing the mutant CHALK10 with the japonica rice variety Zhonghua 11. The F1 offspring were then self-crossed to obtain the F2 segregating population. From the F2 segregating population, a certain number of individuals exhibiting extreme phenotypes were selected to construct both a mutant population DNA pool and a wild-type population DNA pool. Whole-genome sequencing was performed on both pools to identify SNP sites that differed between them. MutMap analysis revealed a non-synonymous mutation site with a high SNP index on chromosome 10, which was associated with the phenotype. Genome sequencing analysis showed that this SNP site was located in the coding region of a candidate gene; therefore, this gene was identified as a candidate gene for CHALK10.

[0016] (3) Identification and functional analysis of the CHALK10 gene:

[0017] Through transgenic technology, the results show that the present invention has obtained transgenic rice that restores the cooking and appearance quality of CHALK10 mutant rice to the wild type phenotype, proving that the present invention has correctly cloned the CHALK10 gene.

[0018] Based on the above research results, the present invention has developed its corresponding applications.

[0019] On one hand, the present invention provides an application of the rice quality regulating gene CHALK10, which is used to regulate rice quality, and the sequence of the gene is shown in (a) or (b):

[0020] (a) The nucleotide sequence encoded by Seq ID No. 1;

[0021] (b) A mutant gene, allele, or derivative that encodes a protein that regulates rice quality by adding and / or substituting and / or deleting one or more nucleotides in the nucleotide sequence shown in (a).

[0022] The rice quality refers to the cooked and flavor quality of the rice and / or the appearance quality of the rice.

[0023] The steaming and cooking quality of the rice includes amylose content, gel consistency and / or gelatinization temperature;

[0024] The appearance quality of the rice includes chalky grain rate and / or chalkiness.

[0025] The application involves mutating the gene, transforming rice cells with the mutated gene, and then cultivating the transformed rice cells into plants to improve the cooking and eating quality of rice. The improvement in the cooking and eating quality of rice includes reducing the amylose content, increasing the gel consistency, and / or lowering the gelatinization temperature.

[0026] Alternatively, the application may involve using rice plants containing the mutant gene to improve the cooking and eating quality of rice through traditional breeding; the improvement in the cooking and eating quality of rice includes a reduction in amylose content, an increase in gel consistency, and / or a decrease in gelatinization temperature.

[0027] The application involves using the gene to transform rice cells, and then cultivating the transformed rice cells into plants to reduce the chalky grain rate and chalkiness of rice.

[0028] Alternatively, rice plants containing the aforementioned regulatory genes can be used to reduce the chalky grain rate and chalkiness of rice through traditional breeding methods.

[0029] On the other hand, the present invention also provides an application of a protein encoded by the rice quality regulating gene CHALK10, said protein being used to regulate rice quality, said protein having the sequence shown in (A) or (B):

[0030] (A) The amino acid sequence shown in Seq ID No. 2;

[0031] (B) A protein derived from (A) with the addition and / or substitution and / or deletion of one or more amino acids in the amino acid sequence defined in (A) and having the same function.

[0032] The rice quality includes the cooking and eating quality of the rice and / or the appearance quality of the rice.

[0033] The steaming and cooking quality of the rice includes amylose content, gel consistency and / or gelatinization temperature;

[0034] The appearance quality of the rice includes chalky grain rate and / or chalkiness;

[0035] The protein is used to positively regulate the amylose content and gelatinization temperature of rice, and to negatively regulate the gel consistency, chalky grain rate, and chalkiness of rice.

[0036] Furthermore, the present invention also provides a mutant gene of the rice quality regulating gene CHALK10, wherein the mutant gene sequence is: based on the coding nucleotide sequence shown in Seq ID No.1, the base at position 442 is replaced by t instead of g.

[0037] On the other hand, the present invention also provides a mutant protein of the rice quality regulation gene CHALK10, wherein the mutant protein sequence is: based on the coding amino acid sequence shown in Seq ID No.2, the amino acid at position 148 is replaced by glutamic acid with a stop codon 0.

[0038] In another aspect, the present invention also provides an application of the above-mentioned rice quality regulating gene CHALK10 mutant gene or mutant protein, for the application in regulating the cooking and eating quality of rice, wherein the cooking and eating quality of rice includes amylose content, gel consistency and / or gelatinization temperature.

[0039] On the other hand, the present invention also provides an application of an SNP site on the nucleotide sequence of the rice quality regulation gene CHALK10, wherein there is one SNP site, and the base at position 442 in the coding nucleotide sequence shown in Seq ID No.1 is replaced by t; the application is to assist in the selection of the cooking and / or appearance quality of rice; the cooking and / or appearance quality includes amylose content, gel consistency and / or gelatinization temperature, and the appearance quality includes chalky grain rate and / or chalkiness.

[0040] This invention utilizes rice quality mutants and, for the first time, cloned the CHALK10 gene in rice using MutMap. This gene encodes an F-box protein that regulates changes in amylose content, gel consistency, and gelatinization temperature, as well as the formation of chalkiness in rice, thereby affecting the cooked and cooked taste and appearance quality of rice. Functional analysis of the CHALK10 gene further clarifies the genetic mechanisms underlying changes in amylose content, gel consistency, gelatinization temperature, and chalkiness formation in plants, particularly rice (a member of the Poaceae family). This lays the foundation for improving the cooked and cooked taste and appearance quality of crops, enhancing the added value and competitiveness of the rice industry. Attached Figure Description

[0041] The above is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0042] Figure 1 The comparison of the rice phenotype (A) and cooking and appearance quality (BF) of the rice of the rice starch synthesis defect mutant chalk10 and the wild type material is as follows:

[0043] Figure 2 This is a clone diagram of the CHALK10 gene;

[0044] Figure 3 This is the pCAMBIA2300-CHALK10 vector spectrum;

[0045] Figure 4 This study compares the phenotypes (A) of transgenic overexpression complementary T1 generation rice and the mutant chalk10 rice, as well as the cooking and appearance qualities (BF) of the rice. Detailed Implementation

[0046] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are all within the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the biochemical reagents, carriers, consumables, etc. used in the embodiments are commercially available products.

[0047] Example 1. Cloning of CHALK10, a gene regulating rice quality (cooking and eating quality, appearance quality).

[0048] (1). Rice material:

[0049] The rice starch synthesis defect mutant chalk10 has a wild type that is the japonica rice variety Zhonghua 11 (Oryza sativa L.cv ZH11). Figure 1 As shown, compared with the wild type, the chalk10 mutant exhibits a highly significant increase in the chalky grain rate and chalkiness of polished rice (e.g., ...). Figure 1 (As shown in B, 1C). Compared with the wild type, the cooked rice of mutant chalk10 has a superior taste quality, specifically manifested in a highly significant reduction in amylose content, a highly significant increase in gel consistency, and a highly significant decrease in gelatinization temperature (e.g., ...). Figure 1 (As shown in D, 1E, 1F).

[0050] (2). Genetic analysis and mapping populations:

[0051] The homozygous mutant chalk10 was crossed with the wild-type japonica rice variety Zhonghua11. The F1 generation was self-crossed to obtain the F2 population. From the F2 population, 50 individuals with extreme phenotypes were selected to construct the mutant population DNA pool and the wild-type population DNA pool, respectively.

[0052] (3). Location of the CHALK10 gene:

[0053] Whole-genome sequencing was performed on the two constructed pools to identify SNP sites that differed between them. MutMap analysis revealed a non-synonymous mutation site with a high SNP index on chromosome 10, which was associated with the phenotype. Candidate genes were obtained by comparing the published rice whole-genome sequence with the SNP sites. Figure 2 ).

[0054] (4). Gene sequencing analysis:

[0055] Based on the localization results, sequencing primers for the candidate gene were designed. PCR was used to amplify the candidate gene from the genomes of the mutant chalk10 and the wild-type variety Zhonghua 11, respectively, for sequencing analysis. It was found that in one DNA fragment of the candidate gene, the product amplified from the mutant chalk10 had a base substitution compared to the wild-type variety Zhonghua 11. In the nucleotide sequence encoded by Seq ID No. 1, the base at position 442 was replaced by t instead of g. In the amino acid sequence encoded by Seq ID No. 2, the amino acid at position 148 was replaced by a stop codon instead of glutamic acid. The sequencing process was repeated three times, yielding the same results each time. Therefore, this candidate gene was named CHALK10. Based on gene annotation information in the MSU database, it was predicted that this candidate gene encodes an F-box protein.

[0056] Example 2. Construction of pCAMBIA2300-CHALK10 plant expression vector

[0057] Based on the complete genome sequence of the rice variety Nipponbare (Oryza sativa L cv. Nipponbare) provided in NCBI, specific primers for amplifying the full-length sequence of the CHALK10 candidate gene were designed. Specific restriction enzyme sites were added to both ends of the specific primers according to the characteristics of the selected pCAMBIA2300 expression vector and the CHALK10 candidate gene sequence. Figure 3 The specific primers designed are as follows: the forward primer (CHALK10-F) has an XbaI restriction site (TCTAGA) added to the 5' end, and the reverse primer (CHALK10-R) has a PstI restriction site (CTGCAG) added to the 5' end. The primer sequences are as follows:

[0058] CHALK10-F forward primer: 5'-CCGGGGATCCTCTAGAATGAGGTGGACGATGCCTC-3'

[0059] CHALK10-R reverse primer: 5'-GAGCCCTGGCATGCCTGCAGTTATGTGATAAGGCTGCTTT-3'

[0060] Then, genomic cDNA was extracted from the rice variety Nipponbare, and using the Nipponbare genomic cDNA as a template, the CHALK10 candidate gene CDS sequence, totaling 2859 bp, was amplified using the primers (CHALK10-F and CHALK10-R) designed above. The following amplification program was used: 94℃ pre-denaturation for 3 minutes; 98℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 68℃ extension for 3 minutes, for 35 cycles; and a final extension at 68℃ for 10 minutes. The target fragment amplified by PCR was recovered; simultaneously, the pCAMBIA2300 empty vector was linearized by double digestion with XbaI and PstI, and the pCAMBIA2300 backbone was recovered. Then, an infusion enzyme, the recovered CHALK10 candidate gene fragment, and the digested pCAMBIA2300 empty vector were used to prepare the reaction mixture. The specific reaction system consisted of: 1 μL of 5× In-fusion enzyme premix; 3 μL of linearized vector; 6 μL of the target fragment; incubation at 50°C for 20 minutes; transformation into *E. coli* DH5α competent cells; screening for positive clones by colony PCR; and sequencing of positive clones by Hangzhou Youkang Company. The final result was the CHALK10 complementary overexpression vector pCAMBIA2300-CHALK10. Figure 3 The pCAMBIA2300-CHALK10 expression vector was transformed into Agrobacterium tumefaciens EHA105 using the liquid nitrogen method.

[0061] Example 3. Transforming rice with the pCAMBIA2300-CHALK10 plant expression vector.

[0062] The recombinant expression vector pCAMBIA2300-CHALK10 was transformed into mature rice embryos using an Agrobacterium-mediated callus infection transformation method. The operation steps are as follows:

[0063] (1) Pre-culture of mature rice embryo callus: The outer shell of mature mutant chalk10 and wild-type seeds was removed, and then the surface was disinfected with 75% alcohol for 1 minute, then soaked in 50% NaClO solution for 30 minutes, and repeated once. The seeds were then washed with sterile water 3-5 times. The seeds were then placed in callus induction medium and cultured in the dark at 26℃ for 30 days, and then transferred to pre-medium for callus pre-culture.

[0064] (2) Co-culture of rice callus with Agrobacterium: The EHA105 strain containing the pCAMBIA2300-CHALK10 expression vector identified in Example 2 was activated, enriched, and resuspended, with OD600 adjusted to 0.6-0.8. Callus tissue was collected in 100 mL Erlenmeyer flasks, and the resuspended Agrobacterium working culture solution was poured in. The flasks were soaked for 30 minutes, shaking several times during this period to infect the callus. The culture solution was discarded, and the surface of the callus was blotted dry with sterile absorbent paper. The callus was then inoculated into co-culture medium and cultured in the dark for 3 days.

[0065] (3) Screening of resistant callus: Co-cultured callus was transferred to a 100ml sterile conical flask, rinsed quickly with sterile water, the washing solution was discarded, and the callus was thoroughly dried on sterile filter paper. It was then inoculated into selection medium containing 50mg / ml hygromycin. It was incubated in the dark at 26℃ and subcultured every two weeks for a total of three subcultures.

[0066] (4) Differentiation of resistant callus: The newly grown resistant callus tissue on the selection medium was inoculated into the predifferentiation medium and cultured at 26°C in the dark for one week. The predifferentiated resistant callus was transferred into the differentiation medium without antibiotics and placed under long-day conditions of 16 hours of light / 8 hours of darkness and an ambient temperature of 28°C for differentiation culture until seedlings were differentiated.

[0067] (5) Rooting of differentiated seedlings: When the differentiated seedlings grow to more than 2cm, they are transferred to a rooting medium for rooting culture. Seedlings that have developed roots are then transplanted to a greenhouse or transgenic nursery for further growth.

[0068] (6) The plants were identified and continuously observed, and their cooking and appearance quality were assessed. Compared with the mutant chalk10 of the same period, the appearance quality of the polished rice harvested from the transgenic overexpression complementary T1 generation plants was not significantly different from that of the wild type. Figure 4 A) The content of amylose, gel consistency, gelatinization temperature, chalky grain rate, and chalkiness of polished rice all showed significant recovery. Figure 4 BF).

[0069] Based on the above research results, the rice quality regulating gene CHALK10 can be used to regulate rice quality, such as regulating the cooking and eating quality of rice; or regulating the appearance quality of rice; or simultaneously regulating the cooking and eating quality and appearance quality of rice. The cooking and eating quality of rice includes amylose content, gel consistency, and / or gelatinization temperature; the appearance quality of rice includes chalky grain percentage and / or chalkiness.

[0070] In practical applications, the above-mentioned genes can be mutated, and the mutated genes can be used to transform rice cells. The transformed rice cells can then be cultivated into plants to improve the cooking and eating quality of rice. Improving the cooking and eating quality of rice includes reducing the content of amylose, increasing the gel consistency, and / or lowering the gelatinization temperature. For example, the aforementioned mutated genes of this invention, or other similar mutated genes, can be used.

[0071] Alternatively, in practical applications, rice plants containing the mutant gene can be used to improve the cooking and eating quality of rice through traditional breeding. Improving the cooking and eating quality of rice includes reducing amylose content, increasing gel consistency, and / or lowering gelatinization temperature. The mutant gene can be the one described above in this invention, or other similar mutant genes.

[0072] Alternatively, in specific applications, the gene can be used to transform rice cells, and then the transformed rice cells can be cultivated into plants to reduce the chalky grain rate and chalkiness of rice.

[0073] Alternatively, in practical applications, rice plants containing the aforementioned regulatory genes can be used to reduce the chalky grain rate and chalkiness of rice through traditional breeding.

[0074] Alternatively, in specific applications, the aforementioned protein can be used to regulate rice quality, which includes the cooked and flavor quality and / or appearance quality of rice; the cooked and flavor quality includes amylose content, gel consistency, and / or gelatinization temperature; the appearance quality includes chalky grain percentage and / or chalkiness; the protein is used to positively regulate the amylose content and gelatinization temperature of rice and negatively regulate the gel consistency, chalky grain percentage, and chalkiness. Regulation can be tailored to specific needs.

[0075] Alternatively, the aforementioned specially mutated genes or mutated proteins can be used to regulate the cooking and eating quality of rice, which includes amylose content, gel consistency, and / or gelatinization temperature.

[0076] Alternatively, the SNP site on the nucleotide sequence of the rice quality regulating gene CHALK10 can be applied, wherein there is one SNP site, and the base at position 442 in the coding nucleotide sequence shown in Seq ID No.1 is replaced by t; the application is to assist in the selection of the cooking and / or appearance quality of rice; the cooking and / or appearance quality includes amylose content, gel consistency and / or gelatinization temperature, and the appearance quality includes chalky grain rate and / or chalkiness.

[0077] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes or alterations made by those skilled in the art using the disclosed technical content shall fall within the protection scope of the present invention.

Claims

1. An application of the rice quality regulating gene CHALK10, characterized in that, The gene is used to regulate rice quality, and the nucleotide sequence of the gene is shown in Seq ID No. 1; The rice quality refers to the steamed and cooked taste quality of the rice. The steaming and cooking quality of the rice includes amylose content, gel consistency and / or gelatinization temperature; The application involves replacing the base at position 442 of the nucleotide sequence shown in Seq ID No. 1 with t to obtain mutant rice plants to improve the cooking quality of rice. The improvement in the cooking quality of rice includes a reduction in amylose content, an increase in gel consistency, and / or a decrease in gelatinization temperature.

2. The application of a protein encoded by the rice quality regulating gene CHALK10, characterized in that, The protein is used to regulate rice quality, and the amino acid sequence of the protein is shown in Seq ID No. 2; The rice quality includes the steaming and cooking quality of the rice. The steaming and cooking quality of the rice includes amylose content, gel consistency and / or gelatinization temperature; By replacing amino acid position 148 of the amino acid sequence shown in Seq ID No.2 with a stop codon to render it nonfunctional, the amylose content of rice was reduced, the gelatinization temperature was lowered, and the gel consistency of rice was increased.

3. The application of a SNP site on the nucleotide sequence of the rice quality regulating gene CHALK10, characterized in that, The SNP site is one, and the base at position 442 in the coding nucleotide sequence shown in Seq ID No.1 is replaced by t; the application is to assist in the selection of the cooking and / or appearance quality of rice; the cooking and / or appearance quality includes amylose content, gel consistency and / or gelatinization temperature, and the appearance quality includes chalky grain rate and / or chalkiness.

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