Rice chalkiness-related gene Chalk3 as well as encoding protein and application of rice chalkiness-related gene Chalk3

Abstract

The invention discloses a gene Chalk3 related to chalkiness of rice grains as well as an encoded protein and application of the gene Chalk3. The gene Chalk3 provided by the invention is 1) a DNA (Deoxyribose Nucleic Acid) molecule as shown in SEQ ID NO.1; 2) a DNA molecule as shown in SEQ ID NO. 2; 3) a DNA molecule which is hybridized with the DNA sequence limited by 1) or 2) under strict conditions and is used for coding the protein; and 4) a DNA molecule which has more than 90% of homology with the DNA sequence limited by 1) or 2) or 3) and is used for coding the rice grain chalkiness related protein. The invention further provides a protein coded by the gene, the protein affects the chalkiness degree of rice grains, and the expression quantity of the Chalk3 gene is in negative correlation with the chalkiness degree of the rice grains. Therefore, the Chalk3 gene participates in rice grain chalkiness regulation and control, and a useful gene resource is provided for rice quality improvement.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a rice chalkiness-related gene Chalk3, its encoded protein, and its applications. Background Technology

[0002] Rice is one of my country's most important food crops. Through several "green revolutions," rice yields have achieved a qualitative leap. With the continuous improvement of living standards, people's consumption demands for rice have gradually shifted from "eating enough" to "eating well and eating healthily," leading to a sustained increase in the market demand for high-quality rice. Appearance quality is the most direct indicator of rice quality, and chalkiness is a key factor restricting rice quality and market value. It not only affects appearance but also has a certain impact on taste, and its level directly determines the commodity value. To meet consumers' demand for high-quality rice, rapidly improving chalkiness traits and cultivating varieties with excellent appearance has become an urgent task in the field of rice breeding, which is of great significance for improving rice quality and yield. At the same time, with the development of specialized rice breeding, special categories such as sake brewing rice and low-gluten rice are gradually gaining attention. Unlike edible rice, high chalkiness actually has a positive effect in sake brewing rice, providing a new direction for rice quality improvement and diversified breeding.

[0003] Chalkiness refers to the opaque, loose white portion of rice endosperm, which can be divided into heart white, ventral white, and back white, with ventral white being the most common. Chalkiness degree (DEC) and chalky grain ratio (PGWC) are the main indicators for measuring chalkiness. Chalkiness is a complex quantitative trait, controlled by multiple genes and significantly influenced by genetic background and environmental conditions. In recent years, scholars both domestically and internationally have made some progress in chalkiness trait research, exploring its mechanisms from the perspectives of QTL detection and localization, gene expression regulation, related protein activity, and cultivation conditions. However, most studies still focus on the physiological regulation of chalkiness and its correlation with other traits, and the understanding of its genetic basis remains to be strengthened. The formation of the chalky phenotype involves multiple genetic regulatory pathways, mainly including starch synthesis, protein synthesis and transport, transcription factors, and organelle development, directly or indirectly affecting starch and protein accumulation. Therefore, further exploration and cloning of new chalkiness regulatory genes, elucidating their molecular mechanisms of action, and applying them to breeding practices are of great theoretical significance and practical value for promoting rice quality improvement and molecular breeding. Summary of the Invention

[0004] The purpose of this invention is to disclose a rice chalkiness-related gene Chalk3, its encoded protein, and its applications.

[0005] This invention provides a rice chalkiness regulation gene Chalk3, which is located on rice chromosome 9 and has the gene number Os03g0248600 (RAP-DB naming rule) or C (MSU naming rule).

[0006] The gene Chalk3 provided by this invention is a DNA molecule as described in 1) or 2) or 3) or 4) below:

[0007] 1) The DNA molecule shown in SEQ ID NO.1;

[0008] 2) The DNA molecule shown in SEQ ID NO.2;

[0009] 3) A DNA molecule that hybridizes to the DNA sequence defined in 1) or 2) under stringent conditions and encodes the protein;

[0010] 4) A DNA molecule that has more than 90% homology with the DNA sequence defined in 1), 2), or 3) and encodes a chalky white protein associated with rice grains.

[0011] The present invention also provides a protein encoded by the above-mentioned gene Chalk3.

[0012] Specifically, the protein provided by this invention is selected from any one shown in (a) or (b):

[0013] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.3;

[0014] (b) A protein derived from SEQ ID NO.3 with substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence of SEQ ID NO.3 and associated with chalkiness of rice grains.

[0015] This invention also provides a recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacteria containing the Chalk3 gene. Recombinant expression vectors containing any of the genes described above are also within the scope of protection of this invention.

[0016] Recombinant expression vectors containing the gene can be constructed using existing plant expression vectors.

[0017] The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microbombardment. These vectors may also contain the 3' untranslated region of a foreign gene, i.e., a polyadenylated signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylated signal can guide the addition of polyadenylated acid to the 3' end of the mRNA precursor; similar functions exist in the untranslated regions transcribed at the 3' end of Agrobacterium crown gall tumor-inducing (Ti) plasmid genes (such as the Nos gene for lipase) and plant genes (such as the soybean storage protein gene).

[0018] When constructing recombinant plant expression vectors using the aforementioned genes, any type of enhancing or constitutive promoter can be added before the transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter or the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0019] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0020] By using any vector capable of guiding the expression of exogenous genes in plants, the gene encoding the stated protein can be introduced into plant cells to obtain transgenic cell lines and transgenic plants. The expression vector carrying the stated gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants. The plant host being transformed can be either monocotyledonous or dicotyledonous, such as tobacco, birdsfoot, Arabidopsis, rice, wheat, corn, cucumber, tomato, poplar, turfgrass, and alfalfa.

[0021] Secondly, this invention provides the application of the rice chalkiness-related gene Chalk3 in regulating rice grain chalkiness and improving rice quality.

[0022] The application method is as follows: the target gene Chalk3 is knocked out or overexpressed, so that the expression level of Chalk3 gene in the target rice material is changed, thereby obtaining rice plants with different chalky phenotypes.

[0023] The recombinant overexpression vector can be a recombinant plasmid obtained by inserting the gene (Chalk3) into the recombination site of the vector pCAMBIA1390 after double digestion with restriction endonucleases KpnI and PstI. The recombinant vector pCAMBIA1390 containing Chalk3 is named 1390-Ubi-Chalk3-GFP.

[0024] Preferably, the primer sequence is as follows:

[0025]

[0026] The recombinant vector pOs-Cas9-Chalk3 used in the gene editing process contains the Chalk3 gene. The vector system is the CRISPR / Cas9 system.

[0027] Preferably, the specific target sequence for editing the Chalk3 gene using the CRISPR / Cas system is 5'-CAGCCTGTTGAAATAACAAA-3', as follows:

[0028]

[0029] This invention reveals that disrupting the biological function of the Chalk3 gene-encoded protein can significantly increase chalkiness in rice grains, affecting the appearance quality of rice; while overexpression of Chalk3 can reduce chalkiness in rice grains to some extent. This invention provides useful genetic resources for the regulation of chalkiness traits in rice and for breeding high-chalkiness rice varieties specifically for brewing.

[0030] Beneficial Effects: This invention belongs to the field of plant genetic engineering technology, specifically relating to a rice chalkiness regulatory gene Chalk3, its encoded protein, and its applications. This gene encodes an enolase involved in regulating rice grain chalkiness and affecting the physicochemical properties of rice. By using conventional methods to edit or overexpress the Chalk3 gene, its expression level can be altered, thereby obtaining new rice germplasm with different chalkiness levels. Therefore, the Chalk3 gene regulates the degree of rice chalkiness, providing a useful genetic resource for the genetic improvement of rice quality and possessing significant breeding value. Attached Figure Description

[0031] Figure 1 This is a partial Manhattan plot of the genome-wide association analysis of the Chalk3 gene.

[0032] Figure 2 The expression level of the Chalk3 gene in different chalky germplasm materials.

[0033] Figure 3 A schematic diagram of the Chalk3 gene editing target and mutation types.

[0034] Figure 4 Chalky grain photographs and statistical analysis of Chalk3 gene knockout lines; "Indicates a highly significant difference."

[0035] Figure 5 Chalky grain photographs and statistical analysis of Chalk3 gene overexpression lines; "Indicates a highly significant difference."

[0036] Figure 6 Map of the overexpression vector 1390-Ubi-Chalk3-GFP plasmid.

[0037] Figure 7 Map of the pOs-Cas9-Chalk3 knockout vector plasmid. Detailed Implementation

[0038] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0039] Example 1: Genome-wide association analysis of chalky rice

[0040] Sixty-two rice germplasm resources were collected and stored at room temperature for three months after harvest. The chalkiness and chalky grain rate were statistically analyzed according to the national standard GB / T1354-2018 for rice. Approximately 200 grains of polished rice were randomly selected from each rice plant for chalkiness measurement. The chalky grains were scanned using the ScanWizard EZ, and the chalkiness characteristics were analyzed using the Wanshen SC-E rice appearance quality detector. The chalkiness data for the 622 rice accessions were then compiled.

[0041] Genome-wide association analysis using a mixed linear model was performed using EMMAX software, and a significant locus was located on chromosome 3. Figure 1 Named Chalk3, analysis of candidate genes at significant loci revealed a negative correlation between the expression level of the LOC_Os03g14450 gene within the interval and chalkiness in rice germplasm. Figure 2 LOC_Os03g14450 was initially identified as a candidate gene for Chalk3.

[0042] Table 1. Different rice varieties and chalky grain rate

[0043]

[0044]

[0045]

[0046]

[0047]

[0048]

[0049]

[0050]

[0051] Example 2: Transgenic Validation of Chalk3 Candidate Gene LOC_Os03g14450

[0052] The knockout target site for the Chalk3 gene was designed using the website (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR), with the target site selected based on exon location. Knockout primers Chalk3-cas9-F and Chalk3-cas9-R were designed, and the pOs-Cas9-Chalk3 vector was constructed using PCR primer preparation. Figure 7 After obtaining the correct recombinant vector through sequencing, it was used to transform wild-type rice Kitaake. After obtaining positive seedlings through tissue culture, DNA was extracted from the transgenic rice seedlings, and PCR amplification was performed using fragments around the specific target site. The target product was then subjected to Sanger sequencing. Two independent knockout mutants, Cr-5 and Cr-7, were obtained, with a 1 bp insertion and a 12 bp deletion in the exon, respectively. Figure 3 This leads to varying expression levels of the LOC_Os03g14450 gene. Primers are as follows:

[0053]

[0054] Seeds were harvested from the wild-type Kitaake germplasm and two independent knockout lines (Cr-5 and Cr-7) under field conditions. Compared with the wild type, the knockout lines had large white centers in the kernels, and statistical analysis revealed that the chalky kernel rate in the knockout lines was significantly higher than that in the wild type. Figure 4 This indicates that the gene controls chalkiness in rice.

[0055] Example 3: Construction and Phenotypic Analysis of Chalk Gene Overexpression Lines

[0056] The plant overexpression vector used in this study was pCAMBIA1390-Ubi-GFP, which contains a constitutively high expression of the Ubi promoter. The recombinant vector 1390-Ubi-Chalk3-GFP was constructed using amplification primers. Figure 6 ).

[0057] The primers for amplifying the coding sequence of the Chalk3 gene are as follows:

[0058]

[0059] For transgenic rice carrying Chalk3-GFP, two lines (OE-1 and OE-2) with significantly upregulated expression levels were obtained in the T2 generation for candidate phenotypic analysis. Compared with the wild type, the chalkiness of the overexpressing lines was significantly reduced. Figure 5 This indicates that overexpression of Chalk3 can improve the appearance quality of rice, providing genetic resources for the genetic improvement of rice quality traits.

[0060] The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention. Those skilled in the art can make possible changes and modifications to the present invention based on the above-disclosed technical content without departing from the scope of the technical solution of the present invention, or modify it into equivalent embodiments with equivalent changes. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. A rice chalkiness gene Chalk3, characterized by: The gene is a DNA molecule as shown in 1) or 2) or 3) or 4) below: 1) The DNA sequence shown in SEQ ID NO.1; 2) The DNA sequence shown in SEQ ID NO.2; 3) A DNA sequence that hybridizes under stringent conditions to the DNA sequence defined in 1) or 2) and encodes the protein described in SEQ ID NO. 3; 4) A DNA sequence that has more than 90% homology with the DNA sequence defined in 1), 2), or 3) and encodes a protein associated with the chalky trait of rice grains.

2. A protein encoded by the rice chalkiness-related gene as described in claim 1.

3. The protein as described in claim 2, characterized in that, Choose from either (a) or (b): (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.3; (b) A protein derived from SEQ ID NO.3 by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence of SEQ ID NO.3 and associated with the chalky trait of rice grains.

4. A recombinant expression vector, expression cassette, transgenic cell line, or recombinant bacteria containing the gene described in claim 1.

5. The recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector is a recombinant plasmid obtained by inserting the gene of claim 1 between the multiple cloning sites of the pCAMBIA1390 vector.

6. Primers for amplifying the full length of the gene or any fragment thereof as described in claim 1.

7. The gene as described in claim 1, the protein encoded therein, and the application of recombinant expression vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the gene in the preparation of transgenic rice with different chalky phenotypes, or in the improvement of rice quality; specifically, editing, knocking out or overexpressing the rice chalky gene Chalk3 in target rice materials to change the expression level of the Chalk3 gene, thereby obtaining rice plants with different chalky phenotypes, thereby improving the quality of rice.

8. The application according to claim 7, characterized in that, The application involves editing or knocking out the chalky gene Chalk3 in rice, thereby altering the expression level of the Chalk3 gene in the target rice variety and obtaining rice plants with different chalky phenotypes. The gene knockout was performed using the CRISPR / Cas9 system.

9. The application according to claim 7, characterized in that, The starting rice is a plant with abnormal endosperm development caused by a defect in the gene shown in SEQ ID NO.2, i.e., an abnormal increase in rice chalkiness. The application involves introducing the gene as described in claim 1 into starting rice with abnormal endosperm development caused by a gene defect in SEQ ID NO. 2 to obtain transgenic rice with normal endosperm development; or obtaining transgenic rice with improved rice quality.

10. A method for cultivating transgenic plants with normal endosperm development, characterized in that, The method involves introducing the gene described in claim 1 into a plant with abnormal endosperm development caused by a gene defect in SEQ ID NO. 2 to obtain a transgenic plant with normal endosperm development. Specifically, the gene is introduced into the plant with abnormal endosperm development through the recombinant expression vector described in claim 4 or 5.

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