Rice endosperm white gene WCG1 and application thereof

Abstract

The application provides a rice heart-white gene WCG1 and application thereof, and particularly relates to positioning, cloning, function verification and application of a WCG1 gene for controlling a rice kernel heart-white phenotype. A C / A natural variation exists at a ninth exon 986 site of the WCG1 gene, the variation site makes the rice kernel present a heart-white phenotype, that is, a kernel middle heart-white and edge transparent phenotype, and the phenotype is an excellent phenotype of "sake brewing good suitable rice" specially selected in sake brewing.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to the rice heart-white gene WCG1 cloned by map-based cloning technology and its applications, including the nucleotide sequence of the gene, the encoded protein and its functional analogues. Background Technology

[0002] Rice is one of the major food crops in China and the world. Chalk refers to the white, opaque part of the rice endosperm and is an important indicator of appearance quality. Currently, there are two main theories regarding the formation and mechanism of chalkiness. One is the cell theory, which states that insufficient grain filling in the rice endosperm results in loosely packed starch and protein bodies, creating gaps of varying sizes that form an optical characteristic, thus exhibiting chalkiness. The other is the "source-sink-flow" theory, which states that when the "source" is small and the "sink" is large, the supply of organic assimilates produced by the functional leaves is mismatched with the storage capacity of the grain endosperm, leading to chalkiness. The degree of tissue development and the ability and rate of nutrient transport related to "flow" also have a certain influence on the formation of chalkiness in rice. Based on the location of chalkiness, it can be divided into ventral white, central white, and dorsal white.

[0003] The chalky white trait in rice is regulated by multiple genes and is also easily affected by external environmental factors. It is a biological process involving multiple factors, and it is difficult to study its formation mechanism in depth using only traditional analytical methods. Therefore, most current research on chalky white related traits uses artificial mutagenesis technology to obtain corresponding mutants, construct genetic populations, and then combine map-based cloning and other related technologies to locate and clone the relevant genes.

[0004] In edible rice, chalky grain rate not only reduces the appearance quality of rice but also lowers the milling quality and cooking taste. In sake brewing, however, specialized "sake brewing rice" is selected, which has a high chalkiness rate and degree. Currently, my country's sake industry lacks dedicated fermentation strains and rice varieties, resulting in a relatively lagging development. Research on the cloning of genes related to rice chalkiness and their regulatory network molecular mechanisms is also weak. Therefore, in-depth exploration of key genes regulating rice chalkiness and identifying superior alleles from natural variations can provide important genetic resources and material reserves for sake-producing functional rice. Summary of the Invention

[0005] The purpose of this invention is to provide a gene, WCG1, that controls the white center phenotype of rice grains and its application, in response to the existing needs of sake rice breeding.

[0006] This invention provides a gene, WCG1, that controls the white-heart phenotype of rice grains. This gene can be directly used to identify and breed rice varieties for sake making, which can advance the breeding process of rice for sake making and provide new gene resources for the improvement of rice germplasm.

[0007] The present invention provides a rice grain white kernel gene WCG1, wherein the WCG1 gene has the nucleotide sequence shown in SEQ ID No: 1.

[0008] Preferably, the nucleotide sequence further includes mutants, alleles, and derivatives generated by adding, substituting, inserting, and deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID No: 1.

[0009] This invention provides a protein encoded by the rice grain white kernel gene WCG1, the amino acid sequence of which is shown in SEQ ID No: 2.

[0010] Preferably, the above-mentioned amino acid sequence also includes derivatives generated by adding, substituting, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID No: 2.

[0011] The present invention provides plasmids containing the above-mentioned genes and plant expression vectors containing the above-mentioned genes.

[0012] The present invention provides a host cell containing the above-mentioned gene sequence.

[0013] Preferably, the host cell is an Escherichia coli, Agrobacterium, or a plant cell.

[0014] This invention also provides the application of the rice heart-white gene WCG1 in controlling the heart-white phenotype of rice grains.

[0015] This invention also provides the application of the rice heart-white gene WCG1 in the identification and breeding of rice varieties for sake production.

[0016] The mechanism of this invention is as follows: Based on the donor parent YD998, whose grains exhibit a milky-white center and transparent edges, near-isogenic lines NY6 (NIP) and YN2 (YD998) were constructed through multiple generations of backcrossing and self-crossing with the recipient parent Nipponbare (NIP). The rice white-heart phenotype gene WCG1 was cloned using map-based cloning technology. This gene exhibits a natural C→A variation at position 986 of exon 9, changing from proline to glutamine, resulting in partial loss of gene function and causing the rice grains to exhibit a white-heart phenotype.

[0017] SEQ ID NO.1:

[0018]

[0019] SEQ ID NO.2:

[0020] MQFMMPLDTNACAQPMRRAGEGAGTERLMERLNIGGMTQEKALRKRCFGDGVTGTARCVFTSDADRDTPHLRTQSSRKNYADASHVSAVILGGGTGVQLFPLTSTRATPAVPVGGCYRLIDIPMSNCFNS GINKIFVMTQFNSASLNRHIHHTYLGGGINFTDGSVQVLAATQMPDEPAGWFQGTADAIRKFMWILEDHYNQNNIEHVVILCGDQLYRMNYMELVQKHVDDNADITISCAPIDGSRASDYGLVKFDDSGR VIQFLEKPEGADLESMKVDTSFLSYAIDDKQKYPYIASMGIYVLKKDVLLDILKSKYAHLQDFGSEILQRAVLEHNVKACVFTEYWEDIGTIKSFFDANLALTEQPPKFEFYDPKTPFFTSPRYLPPARL EKCKIKDAIISDGCSFSECTIEHSVIGISSRVSIGCELKDTMMMGADQYETEEETSKLLFEGKVPIGIGENTKIRNCIIDMNARIGRNVIIANTQGVQESDHPEEGYYIRSGIVVILKNATIKDGTVI*.

[0021] The beneficial effects of this invention are:

[0022] This invention provides a gene, WCG1, that controls the white-center phenotype of rice grains. The WCG1 gene exhibits a natural C / A variation at position 986 of exon 9, resulting in a white-center phenotype in rice grains, characterized by a white center and transparent edges. This phenotype is a superior phenotype specifically selected for sake brewing as "suitable rice for sake brewing." This gene can be directly used to identify and breed functional rice varieties for sake production, advancing the breeding process of rice varieties specializing in sake production. It provides new gene resources for the improvement of functional rice germplasm and helps to better understand the mechanism of action of WCG1. The cloning of WCG1 lays the foundation for further understanding the rice chalkiness regulatory network and genetic analysis. The utilization of the WCG1 allele will have significant application value in the breeding of functional rice for sake production. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show the embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Preliminary localization of the rice heart-white gene;

[0025] Figure 2 To finely map the rice heart white gene;

[0026] Figure 3 Here is a diagram of the WCG1 gene structure;

[0027] Figure 4 The spectrum of the WCG1 vector containing NIP;

[0028] Figure 5 Phenotypic diagram of T0 generation transgenic complementary line;

[0029] Figure 6 Phenotypic diagram of T0 generation seeds from single-base edited transgenic plants. Detailed Implementation

[0030] Unless otherwise specified, the methods used in the following embodiments are conventional methods.

[0031] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0032] The parental materials used in this invention are the imported varieties YD998 and Nipponbare (NIP). YD998 is an indica rice variety with long, flat grains, a light black husk at maturity, and a large, white endosperm phenotype. NIP is a japonica rice variety with plump, thick grains, a yellowish-brown husk at maturity, and a transparent endosperm. Using Nipponbare as the female parent and YD998 as the male parent, a BC4F2 population was constructed. From this population, the stably heritable white-endosperm phenotype line YN2 and the transparent endosperm phenotype line NY6 were identified.

[0033] Example 1: Genetic analysis of white core in rice endosperm

[0034] Using YN2 and NY6 as parents, an F2 population of 425 plants was constructed. The seed phenotypes of the F2 generation were analyzed. 104 plants had entirely white seeds, 211 plants had heterozygous seeds, and 110 plants had completely transparent seeds. The segregation ratio was approximately 1:2:1, consistent with Mendelian classical segregation. Self-pollination of the white-sperm seeds resulted in all offspring exhibiting the white-sperm phenotype. Self-pollination of the transparent-sperm seeds resulted in offspring containing both transparent and white-sperm varieties. Simultaneously, three genetic populations were constructed using YN2 as the female parent and NIP, Wuyunjing 7, and Chunjiang 19B as the male parents. In all three hybrid combinations, the segregation ratio of transparent-sperm to white-sperm seeds in the F2 generation was approximately 3:1, consistent with Mendelian classical segregation. Therefore, it can be deduced that the white heart trait in rice endosperm is controlled by a pair of single recessive nuclear genes.

[0035] Example 2: Preliminary localization of the rice heart-white gene WCG1

[0036] Resequencing of NIP, YD998, YN2, and NY6 revealed that, regarding NIL... YD998 and NIL NIP In terms of the two strains, there are 6 substitution regions and 3 non-substitution regions. Comparative analysis with the genome sequences of NIP and YD998 shows that at position 1 of substitution region (… Figure 1 The genome sequence of the gene (-A) matches the phenotype, and the rice heart-white gene is preliminarily located in this region.

[0037] Individuals with the central white phenotype were selected as the localization population. A total of 171 pairs of SSR markers evenly distributed throughout the genome were used for parental polymorphism screening. The results showed that parents exhibited polymorphism at markers M1-11 and M1-12. These two markers were also used to detect central white individuals in the F2 population. Figure 1 -B), and found that the cardiac white phenotype is linked to these two markers.

[0038] The two markers selected by map-based cloning overlapped with the resequencing results, thus allowing the rice heart-white gene to be preliminarily located at the position of substitution region 1.

[0039] Example 3: Fine mapping of the rice heart-white gene WCG1

[0040] Using the F2 population as the localization population, polymorphic markers were designed based on resequencing Indel data for fine localization.

[0041] Genotyping was performed on 691 individuals from the F2 population. Phenotypic analysis revealed a low exchange rate in this region. Therefore, the population was expanded using: 1) germination of seeds with the white-centered phenotype from heterozygous individuals for fine mapping; and 2) seeds with the transparent phenotype from heterozygous individuals for further population expansion. The percentage of white-centered seeds in heterozygous individuals ranged from 25.22% to 36.04%. Figure 2 -A, Figure 2 -B). A total of 19,880 individual plants were used to map the rice heart-white gene to the marker Whc6 (24,950,804 bp) and the marker Whc44 (25,751,426 bp). Figure 2 -C), and this interval was used to locate the candidate region for the rice grain white heart gene for further analysis.

[0042] Example 4: Gene Prediction and Functional Analysis

[0043] Gene annotation was performed using the Nipponbare Genome Annotation website (http: / / rice.plantbiology.msu.edu / ), and tissue expression analysis was conducted on candidate genes in candidate regions. The results showed that the WCG1 gene, located on chromosome 1 (25354796 bp - 25361949 bp), was specifically highly expressed in seed endosperm within this region. Genome sequence analysis revealed single-base substitutions in exons 9 and 10 of this gene in the YN2 material. Specifically, the 986th base C in the CDs sequence corresponding to exon 9 was replaced with A, resulting in a change in the encoded amino acid sequence. The 329th amino acid P (proline, Pro) was replaced with Q (glutamine, Gln). Similarly, the 1071st base T in the CDs sequence corresponding to exon 10 was replaced with C, but the encoded amino acid sequence remained unchanged. Figure 3 Therefore, it is speculated that the WCG1 gene is very likely the target gene controlling the white phenotype of rice grains.

[0044] To further verify whether the WCG1 gene is the target gene for the white heart trait in rice, complementary transgenic materials were constructed. Primers were designed based on the sequence of the WCG1 gene in NIP, and the pCAMBIA1300 vector was used to construct the following transgenic material: Figure 4 The expression vector pCAMBIA1300-WCG1 is shown. The WCG1 DNA sequence in this expression vector is 6130 bp, including the WCG1 promoter sequence (2102 bp) and the genomic sequence (4028 bp). The primers used to amplify the NIP WCG1 gene sequence are (underlined parts are restriction sites and protective bases):

[0045] WCG1-KpnI-F:acgaattcgagctcggtaccTGAACTGAGACTGGAGAGCA

[0046] WCG1-R1:AATGGCATCATGAATTGCATGGGTACTCAAAGTCCCAGACA

[0047] WCG1-F1: TGTCTGGGACTTTGAGTACCCATGCAATTCATGATGCCATT

[0048] WCG1-BamHI-R: caggtcgactctagaggatccTTATATGACGGTCCCGTCC

[0049] The specific process of vector construction is as follows: Four primers for amplifying the WCG1 gene were designed: WCG1-KpnI-F, WCG1-R1, WCG1-F1, and WCG1-BamHI-R. Using DNA extracted from NIP leaves as a template, PCR amplification was performed using primers WCG1-KpnI-F and WCG1-R1 with a high-fidelity enzyme to obtain fragment 1. PCR amplification was then performed using primers WCG1-F1 and WCG1-BamHI-R to obtain fragment 2. Both amplification products were gel-cleaved and recovered. Simultaneously, the pCAMBIA1300 vector was digested with BamHI and KpnI. The recovered and purified fragments 1 and 2, which were of the correct size, were then ligated into the digested vector via homologous recombination for E. coli transformation. Positive single clones were selected and sequenced to obtain the correct transformation vector pCAMBIA1300-WCG1.

[0050] The expression vector was transferred into the Agrobacterium tumefaciens strain EHA105 via electroporation to transform rice. We used callus induced from mature YN2 seeds, and after culturing on induction medium for 7-10 days, selected vigorous callus as recipients for transformation. Rice callus was infected with the EHA105 strain containing the expression vector and co-cultured in the dark at 25°C for 3 days, followed by 2 weeks of culture on selection medium containing 400 mg / L carbenicillin and 50 mg / L hygromycin. The selected resistant callus was induced to differentiate on differentiation medium containing 250 mg / L carbenicillin and 50 mg / L hygromycin. The differentiated seedlings were transferred to rooting medium to induce rooting. Resistant transgenic plants were obtained in approximately two months. Plant identification and analysis of the mature seed phenotype revealed that the seeds of the transgenic T0 generation plants exhibited segregation (…). Figure 5 The results obtained using the above transgenic technology showed that the WCG1 fragment in NIP could restore the white grain phenotype in YN2, and WCG1 is the target gene controlling white grains in rice.

[0051] Compared to NIP, single-base substitutions occurred at positions 986 (C→A) and 1071 (T→C) of the CDs sequence in YN2. To further determine which site mutation ultimately led to the white-heart phenotype, single-base site-directed editing was performed on these two sites in the WCG1 gene of NIP. Corresponding expression vectors were constructed and transformed into Agrobacterium EHA105 via electroporation. Using NIP as the recipient, transgenic plants were constructed. The results showed that after the C-to-A mutation at position 986 of the WCG1 gene CDs sequence in NIP, the rice grains exhibited a white-heart phenotype, while the T-to-C mutation at position 1071 of the CDs sequence did not alter the rice grain phenotype. Figure 6 Therefore, it can be determined that the control of the white-heart phenotype in rice grains is caused by a C→A mutation at position 986 of the WCG1 gene.

[0052] The above description is merely a preferred embodiment of this application, but the scope of protection of this application is not limited thereto. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art within the scope of the technology disclosed in this application are all covered within the scope of protection of this invention.

Claims

1. A method for constructing transgenic rice plants with white-hearted grains, characterized in that, After mutating the base C at position 986 of the CDS sequence of the WCG1 gene in rice to A, the rice grains exhibit a white-heart phenotype. The nucleotide sequence of the WCG1 gene CDS sequence after mutating the base C at position 986 to A is shown in SEQ ID No: 1.

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