A temperature-responsive rice chalky gene PGWC3, its encoded protein, and its application

Abstract

The invention discloses a temperature-responsive rice chalky gene <h2 style=";text-align:left;direction:ltr">PGWC3 The invention relates to an endosperm-specific expression gene and its encoded protein and application. The gene encodes a protein containing a PCO domain and has a nucleotide sequence shown in SEQ ID NO.1. The gene can regulate the chalkiness of rice, and the chalkiness phenotype is affected by temperature. <h2 style=";text-align:left;direction:ltr"> PGWC3 In applications of changing rice grain chalkiness and improving rice quality, the PGWC3 gene, or a vector or host cell containing the PGWC3 gene, is transformed into rice cells or tissues and cultivated, thereby changing the expression level of the PGWC3 gene in the target rice variety, thereby obtaining rice plants with different chalkiness phenotypes. <h2 style=";text-align:left;direction:ltr"> PGWC3 The gene provides a molecular mechanism for temperature-regulated chalkiness and provides a useful genetic resource for breeding rice germplasm that can regulate the chalkiness phenotype in response to temperature.

Description

Technical Field

[0001] The present invention belongs to the field of plant genetic engineering, and in particular relates to a temperature-responsive rice chalkiness gene PGWC3, its encoded protein and application. Background Art

[0002] High yield has long been the primary goal of rice breeding in my country. In recent years, with improvements in living standards, the demand for high-quality rice has gradually increased. Rice appearance is a primary indicator for consumers to judge rice quality, with chalkiness being the most critical factor. Therefore, improving the chalkiness trait in rice is a key goal in high-quality rice breeding in my country. Chalkiness is the opaque part of the endosperm and is generally caused by abnormal accumulation of starch and storage proteins in the endosperm. Starch and protein are the primary storage substances in the endosperm, and their accumulation involves a series of transcription factors, enzymes, and other factors. Mutations in these genes inevitably affect starch and protein accumulation (Zhao et al. 2022).

[0003] Extreme weather conditions, such as high temperatures, have become a significant factor influencing rice production. High temperatures during the grain-filling period can severely impact rice yield and quality, particularly the formation of chalkiness. It is generally believed that high temperatures inhibit the accumulation of starch and protein during grain filling, leading to chalkiness (Zhao et al. 2022). The physiological mechanisms by which high temperatures influence rice chalkiness have been extensively studied, including involvement in energy metabolism and stress responses. Identifying QTLs for high-temperature chalkiness tolerance is crucial for understanding the molecular mechanisms by which high temperatures influence chalkiness formation. However, limited reports exist, with only two QTLs, Apq1 and OsSFq3, identified (Takehara et al. 2018; Park et al. 2021).

[0004] Therefore, in the current context of global warming, further analyzing the molecular mechanism by which high temperature affects chalkiness is of great significance for the cultivation of high-temperature tolerant high-quality rice. Summary of the Invention

[0005] In response to the above-mentioned problems, the present invention provides a temperature-regulated rice chalkiness gene PGWC3, its encoded protein, and its application, providing genetic resources for studying the molecular mechanism of rice chalkiness response to temperature regulation and cultivating high-temperature tolerant high-quality rice.

[0006] To achieve the above-mentioned purpose, the present invention solves the technical problem by the following technical solutions:

[0007] In its first aspect, the present invention provides a temperature-responsive rice chalkiness gene, PGWC3. The nucleotide sequence of the PGWC3 gene (LOC_Os03g45210 under MSU nomenclature or Os03g0654700 under RAP-DB nomenclature) is shown in SEQ ID NO. 1. Loss-of-function mutants of pgwc3 are particularly sensitive to high temperatures, with elevated temperatures during the grain-filling stage significantly increasing chalkiness.

[0008] The amino acid sequence encoded by the PGWC3 gene is shown in SEQ ID NO.2.

[0009] In a second aspect, the present invention provides an application of a temperature-responsive rice chalkiness regulatory gene PGWC3 in changing rice grain chalkiness and improving rice quality.

[0010] The application method is as follows: the PGWC3 gene, or a vector or host cell containing the PGWC3 gene, is transformed into rice cells or tissues and cultivated, so that the expression level of the PGWC3 gene in the target rice variety is changed, thereby obtaining rice plants with different chalky phenotypes.

[0011] Furthermore, the rice plants with different chalky phenotypes are PGWC3 gene overexpression plants or PGWC3 gene knockout plants.

[0012] The present invention provides a temperature-responsive rice chalkiness gene, PGWC3, which is dominantly expressed in the endosperm. Its loss-of-function mutant, pgwc3, significantly increases grain chalkiness under high temperature conditions without affecting other traits. Transgenic lines overexpressing the PGWC3 gene can reduce grain chalkiness. This invention provides a useful genetic resource for elucidating the molecular mechanism of temperature-responsive rice chalkiness and for cultivating rice germplasm with a temperature-responsive chalkiness phenotype. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the expression pattern analysis of the PGWC3 gene in rice in Example 1 of the present invention;

[0014] Figure 2 Schematic diagram of the PGWC3 gene editing target sites and mutation types in Example 2 of the present invention;

[0015] Figure 3 The rice chalky phenotype of the pgwc3 knockout strain under the natural high temperature environment of Yangzhou in Example 3 of the present invention;

[0016] Figure 4 The rice chalky phenotype of the pgwc3 knockout strain under the natural low temperature environment of Hainan in Example 3 of the present invention;

[0017] Figure 5This is the analysis of rice chalkiness of the pgwc3 knockout strain under normal temperature treatment in the artificial climate chamber in Example 3 of the present invention; "**" indicates a very significant difference;

[0018] Figure 6 This is the analysis of rice chalkiness in the pgwc3 knockout strain under high temperature treatment in an artificial climate chamber in Example 3 of the present invention; "**" indicates a very significant difference;

[0019] Figure 7 This is the target gene expression analysis of the PGWC3 gene overexpression strain in Example 5 of the present invention; "**" indicates a very significant difference;

[0020] Figure 8 This is an analysis of rice chalkiness in the PGWC3 gene overexpression line in Example 5 of the present invention. DETAILED DESCRIPTION

[0021] The present invention will be further described below in conjunction with specific implementations. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0022] In the following examples, the experimental methods without specific conditions were all carried out according to conventional procedures, and the materials and reagents used were all commercially available conventional biochemical reagents unless otherwise specified.

[0023] Example 1: Analysis of expression pattern of PGWC3 gene in rice

[0024] 1. Obtaining the PGWC3 gene sequence

[0025] This study focuses on the impact of rice endosperm-dominantly expressed genes on rice quality. Based on a rice gene chip expression database and high-temperature transcriptome data, a series of endosperm-dominantly expressed genes in response to high temperatures were screened for functional studies. One endosperm-dominantly expressed gene, PGWC3, was identified. Its encoded protein consists of 360 amino acids (SEQ ID NO. 2), and the corresponding gene contains 5443 nucleotides (SEQ ID NO. 1). Both the nucleotide and amino acid sequences of this gene were derived from the genome of the rice variety Nipponbare.

[0026] 2. Verification of PGWC3 gene expression pattern

[0027] To verify the endosperm-dominant expression characteristics of the PGWC3 gene, a pair of exon-spanning quantitative analysis primers were designed in the exon region of the PGWC3 gene using the commonly used software Primer Premier 5. The primer sequences are as follows:

[0028]

[0029] Using wild-type japonica rice Zhonghua 11 as the material, rice tissues at different developmental stages (roots, stems, leaves, leaf sheaths, and panicles) and caryopsis at different developmental stages (0, 5, 10, 15, 20, 25, and 30 days after flowering) were collected. Total RNA was extracted from the plant using a plant rapid RNA extraction kit, and first-strand cDNA was synthesized using a reverse transcription kit. The expression level of PGWC3 in different tissues was detected using a quantitative PCR kit and the above-designed quantitative PCR primers (SEQ ID NO.3 and SEQ ID NO.4). The test results are shown in Figure 2. Figure 1 As shown, the PGWC3 gene is highly expressed only during the rice filling period, especially at 15 days after flowering, and is the dominant endosperm-expressed gene, indicating that this gene may have a very important biological function in the endosperm development process.

[0030] Example 2: PGWC3 gene knockout vector construction and genetic transformation

[0031] 1. Gene Editing Site Design

[0032] In this study, based on existing CRISPR / Cas9-related experimental methods, a specific sequence containing NGG as a PAM recognition site was selected as the knockout target site on the first exon of PGWC3 (5'-GCCGTCAGTACCGTCCATGCTGG-3', sequence as shown in Figure 2 Primers Primer3 and Primer4 (SEQ ID NO. 5 and SEQ ID NO. 6) were designed using the online tool targetDesign software (http: / / skl.scau.edu.cn / targetdesign / ) for gene knockout vector construction. The primer sequences for CRISPR / Cas9 gene knockout vector construction are as follows:

[0033]

[0034] 2. CRISPR / Cas9 vector construction and genetic transformation methods

[0035] The CRISPR / Csa9 vector system used in this study was provided by Researcher Wang Kejian from the China National Rice Research Institute. The system includes the intermediate vector SK-gRNA and the final vector pC1300-Cas9, whose DNA backbones are derived from the pBlueScript (SK+) vector and the pCAMBLA1300 vector, respectively.

[0036] First, using the designed knockout target site, the recombinant vector pC1300-Cas9-PGWC3 was generated according to the method described in the literature (Wang et al., J Genet Genomics, 2015, 42: 703-706). The specific steps were as follows: primers 3 and 4 were annealed to generate a double-stranded sequence containing the knockout target site. The annealed primers were mixed with the linearized intermediate vector SK-gRNA, which had been cut with the AarI restriction endonuclease, and ligated using T4 DNA ligase. The ligation product was transformed into Escherichia coli DH5α competent cells, and positive clones were selected and sequenced to verify the correct target site sequence, thereby obtaining the SK-gRNA-PGWC3 recombinant vector.

[0037] The SK-gRNA-PGWC3 plasmid was digested with the restriction endonucleases Kpn I and Bgl II. A 300-bp fragment was purified and mixed with the gel-recovered product of pC1300-Cas9, which had been double-digested with Kpn I and BamH I. The fragment was then ligated with T4 DNA ligase and transformed into Escherichia coli. A single clone was selected for sequencing, and the correctly sequenced vector was named pC1300-Cas9-PGWC3. EHA105 Agrobacterium competent cells were then transformed, and positive single clones were selected for PCR analysis to obtain Agrobacterium containing the pC1300-Cas9-PGWC3 recombinant vector.

[0038] The recombinant vector was introduced into rice Zhonghua 11 callus tissue using the Agrobacterium-mediated rice transformation method (Liu Qiaoquan et al., Acta Physiologica Sinica, 1998, 24:259-271). Successfully transformed callus cells were screened for hygromycin resistance and subsequently differentiated into transgenic rice seedlings. When the seedlings reached approximately 10 cm in height, they were tested, identified, and transplanted to obtain T0 generation rice plants.

[0039] Example 3: Phenotypic Analysis of PGWC3 Gene Knockout Strains

[0040] 1. Detection of genetically modified seedlings

[0041] A total of 30 T0 generation seedlings were obtained through genetic transformation. First, positive seedlings were screened using hygromycin detection primers. Then, a pair of PCR sequencing primers, primer5 (SEQ ID NO.7) and primer6 (SEQ ID NO.8), were designed upstream and downstream of the genomic sequence of the knockout target site to detect sequence mutations near the target site. After sequence amplification and sequencing analysis based on the target site, a total of 4 gene mutation types were obtained (such as Figure 2 T0 seedlings were continuously planted and self-pollinated to obtain homozygous T2 lines. Homozygous lines were also tested using the same sequencing method. The PCR sequencing primer sequences are as follows:

[0042]

[0043] 2. Phenotypic Analysis of pgwc3 Knockout Lines

[0044] During the T2 generation, agronomic traits of different strains were investigated and it was found that the field agronomic traits of the pgwc3 knockout strains were not significantly different from those of the wild type Zhonghua 11. Under the same field planting conditions in Hainan and Yangzhou, the chalkiness and chalky grain rate of pgwc3 were significantly increased compared to the wild type under the high summer temperatures in Yangzhou ( Figure 3 ), while in Hainan, under relatively low temperature conditions, the degree of chalkiness is relatively weak ( Figure 4 ).

[0045] To further clarify the effect of temperature on the chalkiness of the pgwc3 mutant, different temperature treatment conditions were simulated in an artificial climate chamber (high temperature: dynamic changes from 33°C during the day to 28°C at night; normal temperature: dynamic changes from 28°C during the day to 22°C at night). The wild type and mutant pgwc3 were subjected to temperature treatment (5 days after flowering to the end of grain filling). The results showed that under normal temperature conditions, the chalkiness of the pgwc3 knockout lines was less different ( Figure 5 ), while the chalkiness and chalky grain rate of the pgwc3 knockout strain increased significantly under high temperature conditions ( Figure 6 ).

[0046] Example 4: Construction of PGWC3 gene overexpression vector and genetic transformation

[0047] The plant expression vector used in this study is pC1300-Gt1, whose DNA backbone is derived from the pCAMBLA1300 vector. The promoter Gt1, a dominantly expressed rice endosperm gene GluA2, drives overexpression of downstream target genes. The primer sequences for amplifying the coding region of the PGWC3 gene are as follows:

[0048]

[0049] The specific steps are as follows: Using Nipponbare cDNA as a template, a PCR reaction system consisting of a high-fidelity DNA polymerase and primers Primer7 and Primer8 was used to amplify the coding region of PGWC3. The PCR product was examined by 1% agarose gel electrophoresis, and the gel containing the target gene fragment was excised and recovered using a gel extraction kit. The recovered product was then mixed with the recovered product of pC1300-Gt1, which had been digested with the restriction endonucleases BamH1 and Sma1, and ligated for homologous recombination. The ligation product was transformed into competent Escherichia coli DH5α cells using the heat shock method. The transformed cells were plated and cultured on LB solid medium supplemented with 100 mg / L kanamycin. Colonies were selected for sequencing, and the plasmid that correctly sequenced was designated pC1300-Gt1-PGWC3. The recombinant vector was transformed into high chalky rice variety Wuyunjing 30 using Agrobacterium-mediated rice genetic transformation method (Liu Qiaoquan et al., Acta Physiologica Sinica, 1998, 24: 259-271), and T0 generation transgenic rice seedlings were obtained.

[0050] Example 5: Phenotypic analysis of PGWC3 gene overexpressing transgenic lines

[0051] For the T0 generation transgenic rice seedlings carrying the pC1300-Gt1-PGWC3 recombinant vector, hygromycin detection primers were first used to screen positive seedlings; T0 generation seedlings were continuously planted and self-pollinated to obtain T2 generation homozygous genotype lines. At the same time, the PGWC3 gene expression detection primers Primer1 and Primer2 described in Example 1 were used to analyze the expression levels of 10 seeds developed from the PGWC3 gene overexpressing transgenic plants, and two lines with significantly increased expression levels were obtained for subsequent plant phenotypic analysis ( Figure 7 ).

[0052] Agronomic traits of the PGWC3-overexpressing transgenic lines obtained above were investigated. It was found that there were no significant differences in field agronomic traits such as plant height, tiller number, growth period, ear length, and grain shape between the PGWC3-overexpressing transgenic lines and the control parent, Wuyunjing 30. The chalky grain rate and chalkiness of the PGWC3-overexpressing transgenic lines were significantly reduced ( Figure 8 ), the results showed that overexpression of the PGWC3 gene can improve the appearance quality of rice, which has important breeding value for the genetic improvement of rice quality traits.

[0053] The above description is only a preferred embodiment of the present invention, but is not intended to limit the present invention. Those skilled in the art can use the technical content disclosed above to make possible changes and modifications to the present invention without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Without departing from the content of the technical solution of the present invention, any simple modification, equivalent change and modification made to the above embodiment based on the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. Application of a temperature-responsive rice chalkiness gene PGWC3 in changing rice grain chalkiness, PGWC3 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that The application steps are as follows: Overexpression of PGWC3 can reduce rice grain chalkiness and improve rice appearance quality.

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