A rice chalkiness regulating gene Chalk9 and its encoded protein and application

Abstract

The present invention relates to a rice chalkiness regulating gene Chalk9, its encoded protein and application. The gene is dominantly expressed in seeds, encodes an unknown protein involved in regulating rice chalkiness, and affects the physical and chemical quality of rice. Conventional methods are used to perform gene editing on Chalk9, thereby changing the expression level of the gene, and thereby obtaining a new rice germplasm with a functional deficiency of the Chalk9 gene. Compared with the parental control, the rice created by the present invention has no significant differences in plant growth and development and basic agronomic traits, but significantly affects the chalky grain rate and chalkiness of rice, and the physical and chemical quality indicators of rice are also changed. Therefore, the effect of the Chalk9 gene in regulating rice chalkiness provides a useful gene resource for the genetic improvement of rice quality and has important breeding utilization value.

Description

Technical Field

[0001] The invention relates to a rice chalkiness regulating gene Chalk9 and its encoded protein and application, belonging to the technical field of plant genetic engineering. Background Art

[0002] Rice is an important staple crop in my country. While rice production has shown a steady upward trend in recent years, the development of high-quality rice has lagged behind. As living standards improve, demand for high-quality rice is increasing. Rice appearance is a key factor in evaluating rice quality and also influences other rice properties. The degree of rice chalkiness directly determines its consumer appeal and commercial value. Chalkiness is the primary limiting factor in achieving high-quality rice varieties in my country. Therefore, improving the chalkiness trait in rice is a pressing challenge in rice (table rice) breeding in my country. In addition to the most common edible rice, specialized rice varieties, such as sake-brewing rice and low-gluten rice, have also entered the focus of rice breeders. Unlike edible rice, high chalkiness can have a beneficial effect on sake-brewing rice (Zhang et al., Molecular Plant 2023).

[0003] Chalkiness is the opaque part of the endosperm of the grain, caused by the abnormal accumulation of starch and storage proteins in the endosperm, and is closely related to the grain filling process. It generally occurs in the abdomen, back, and heart of rice, resulting in the phenotypes of belly white, back white, and heart white. The chalky phenotype of rice is synergistically influenced by multiple genetic regulatory pathways, mainly involving the starch synthesis pathway, protein synthesis and transport pathway, and other transcriptional regulators, organelle development, etc., which indirectly affect starch and protein accumulation (Zhao et al., Biotechnology Advances 2022). There are still many gaps in the discovery of chalkiness regulatory genes, and the available genes are still very limited (Wu et al., The Plant Cell 2022), which is far from meeting the needs of high-quality edible and specialized rice breeding. Therefore, it is necessary to continue to strengthen the discovery of chalkiness regulatory genes so as to use valuable genes to cultivate varieties with ideal rice quality. Summary of the Invention

[0004] The purpose of the present invention is to address the above-mentioned problems and provide a rice chalkiness regulating gene Chalk9 and its encoded protein and application, so as to provide new gene resources for improving the chalkiness trait of rice.

[0005] In order to achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is as follows: a gene Chalk9 that regulates chalkiness of rice, characterized in that the nucleotide sequence of the Chalk9 gene coding region is shown in SEQ ID NO.1.

[0006] The amino acid sequence encoded by Chalk9 is shown in SEQ ID NO.2.

[0007] The application of the gene Chalk9 regulating rice chalkiness in changing rice grain chalkiness and improving rice quality.

[0008] The application method is as follows: the rice chalky gene Chalk9 is edited to change the expression level of the Chalk9 gene in the target rice variety, thereby obtaining rice plants with different chalky phenotypes.

[0009] The recombinant vector pC1300-Chalk9-Cas9 used for the Chalk9 gene contains the Chalk9 gene; the vector system is CRISPR / Cas9, which includes the intermediate vector SK-gRNA and the final vector pC1300-Cas9.

[0010] The recombinant vector pC1300-Chalk9-Cas9 was prepared as follows: the intermediate vector SK-gRNA was digested with the restriction endonuclease Aar I, and then ligated with the denatured and annealed target gene complementary primers (SEQ ID NO. 3 and SEQ ID NO. 4) using T4 ligase to obtain the intermediate vector SK-gRNA-Chalk9; the SK-gRNA-Chalk9 intermediate vector identified by sequencing was double-digested with Kpn I and Bgl II, and ligated with the final vector pC1300-Cas9 double-digested with Kpn I and BamH I to obtain the recombinant vector pC1300-Chalk9-Cas9.

[0011] In the preparation method of the recombinant vector pC1300-Chalk9-Cas9, the specific target site sequence for editing the Chalk9 gene by the CRISPR / Cas9 system is 5'-GCTCGAGCAAGTACAGACGC-3', and the primer sequences are as follows:

[0012] sequence name sequence Sequence number Chalk9-cas9-F 5'-GGCAGCTCGAGCAAGTACAGACGC-3' SEQ ID NO.3 Chalk9-cas9-R 5'-AAACGCGTCTGTACTTGCTCGAGC-3' SEQ ID NO.4 .

[0013] The present invention is an advanced and scientific method. In its first aspect, the present invention provides a rice chalkiness regulating gene, Chalk9. The Chalk9 gene is located on rice chromosome 9 and has the gene number Os09g0368900 (RAP-DB nomenclature) or LOC_Os09g20340 (MSU nomenclature). The Chalk9 gene coding region has a 2061-bp nucleotide sequence, as shown in SEQ ID NO. 1. The Chalk9 gene encodes a 686-amino acid amino acid sequence, as shown in SEQ ID NO. 2.

[0014] In a second aspect, the present invention provides an application of a rice chalkiness regulating gene Chalk9 in changing the chalkiness of rice grains and improving rice quality.

[0015] The application method is as follows: editing the rice chalky gene Chalk9 to change the expression level of the Chalk9 gene in the target rice variety, thereby obtaining rice plants with different chalky phenotypes.

[0016] The recombinant vector pC1300-Chalk9-Cas9 used in the gene editing process contains the Chalk9 gene. The vector system is a CRISPR / Cas9 system, which includes the intermediate vector SK-gRNA and the final vector pC1300-Cas9.

[0017] The preparation method of the recombinant vector pC1300-Chalk9-Cas9 is as follows: the intermediate vector SK-gRNA is digested with the restriction endonuclease Aar I, and then connected with the target gene complementary primer after denaturation and annealing using T4 ligase to obtain the intermediate vector SK-gRNA-Chalk9; the intermediate vector SK-gRNA-Chalk9 identified as correct by sequencing is double-digested with Kpn I and Bgl II, the target fragment (fragment size is 300 bp) is recovered and connected with the final vector pC1300-Cas9 double-digested with Kpn I and BamH I.

[0018] Preferably, the specific target site sequence for editing the Chalk9 gene using the CRISPR / Cas9 system is 5'-GCTCGAGCAAGTACAGACGC-3', and the primer sequence is as follows:

[0019] sequence name sequence Sequence number Chalk9-cas9-F 5'-GGCAGCTCGAGCAAGTACAGACGC-3' SEQ ID NO.3 Chalk9-cas9-R 5'-AAACGCGTCTGTACTTGCTCGAGC-3' SEQ ID NO.4

[0020] Through the present invention, the present invention discovered that disrupting the biological function of the protein encoded by the Chalk9 gene can significantly increase grain chalkiness and affect the appearance quality of rice. The present invention provides a useful gene resource for regulating the chalkiness trait of rice and cultivating high-chalkiness rice specifically for winemaking.

[0021] In summary, the present invention belongs to the technical field of plant genetic engineering, and specifically relates to a rice chalkiness regulating gene Chalk9, its encoded protein and application. This gene is dominantly expressed in seeds, encodes an unknown protein involved in regulating rice chalkiness, and affects the physical and chemical quality of rice. Conventional methods are used to perform gene editing on Chalk9, thereby changing the expression level of the gene, and thereby obtaining a new rice germplasm with a functional deficiency of the Chalk9 gene. Compared with the parental control, the rice created by the present invention has no significant differences in plant growth and development and basic agronomic traits, but significantly affects the chalkiness rate and chalkiness of rice, and the physical and chemical quality indicators of rice are also changed. Therefore, the effect of the Chalk9 gene in regulating rice chalkiness provides a useful gene resource for the genetic improvement of rice quality and has important breeding utilization value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the spatiotemporal expression pattern of the Chalk9 gene in rice in Example 1 of the present invention.

[0023] Figure 2 Schematic diagram of Chalk9 gene editing target sites and mutation types in Example 2 of the present invention.

[0024] Figure 3 This is an analysis of the agronomic traits of the Chalk9 gene knockout strain in Example 3 of the present invention.

[0025] Figure 4 This is the chalkiness analysis of the Chalk9 gene knockout strain in Example 3 of the present invention.

[0026] Figure 5 This is the grain filling dynamics of the Chalk9 gene knockout strain in Example 3 of the present invention.

[0027] Figure 6 This is the physical and chemical quality analysis of rice of the Chalk9 gene knockout strain in Example 3 of the present invention. DETAILED DESCRIPTION

[0028] To facilitate understanding of the present invention, the following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention.

[0029] 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.

[0030] Example 1: Analysis of spatiotemporal expression patterns of the Chalk9 gene in rice;

[0031] This research team focused on regulating rice quality through dominantly expressed genes in rice seeds and screened a number of dominantly expressed genes for their effects on rice quality regulation. One of these dominantly expressed genes, Chalk9, encodes a protein containing 686 amino acids (SEQ ID NO. 2), and its corresponding gene contains 2061 nucleotides (SEQ ID NO. 1). Both the nucleotide and amino acid sequences of these genes were derived from the reference genome of the rice variety Nipponbare (rice.plantbiology.msu.edu).

[0032] To verify the seed-dominant expression characteristics of the Chalk9 gene, a pair of exon-spanning primers were designed in the exon region of the Chalk9 gene using Primer Premier 5 software for quantitative PCR analysis. The primer sequences are as follows:

[0033]

[0034] Using the japonica rice Zhonghua 11 parent as the material, roots, stems, leaves, leaf sheaths, panicles, and seeds at different stages after flowering were collected. The samples were placed in liquid nitrogen, ground to disrupt the cells, and total RNA was extracted. Then, a reverse transcription kit was used for first-strand cDNA synthesis. The expression level of the Chalk9 gene in different tissues was detected using a quantitative PCR kit and the above-designed quantitative PCR primers (SEQ ID NO.5 and SEQ ID NO.6). The test results are shown in the attached figure. Figure 1 As shown, the Chalk9 gene is highly abundantly expressed only during the grain filling period of rice and is a seed-dominantly expressed gene, indicating that this gene may play an important role in seed development.

[0035] Example 2: Construction of Chalk9 gene knockout vector and genetic transformation in rice;

[0036] Based on existing CRISPR / Cas9 experimental methods, this study selected the 5'-CATTGCTCTAAGGCAGTTTG-3' sequence containing NGG as the PAM site in the exon of the Chalk9 gene as the knockout target site. The online tool targetDesign software was used to design primers (SEQ ID NO. 3 and SEQ ID NO. 4) for gene editing vector construction. The primer sequences for CRISPR / Cas9 gene editing vector construction are as follows:

[0037] sequence name sequence Sequence number Chalk9-cas9-F 5'-GGCAGCTCGAGCAAGTACAGACGC-3' SEQ ID NO.3 Chalk9-cas9-R 5'-AAACGCGTCTGTACTTGCTCGAGC-3' SEQ ID NO.4

[0038] The CRISPR / Cas9 vector system used in this study was provided by Researcher Wang Kejian from the Chinese Academy of Rice Research. The system includes the intermediate vector SK-gRNA and the final expression vector pC1300-Cas9, whose DNA backbones are derived from the pBlueScript (SK+) vector and the pCAMBLA1300 vector, respectively.

[0039] The specific process is as follows: After the intermediate vector SK-gRNA is digested with the restriction endonuclease Aar I, it is connected with the target gene complementary primer (Chalk9-cas9-F / R) after denaturation and annealing using T4 ligase to obtain the intermediate vector SK-gRNA-Chalk9; the SK-gRNA-Chalk9 intermediate vector identified by sequencing is double-digested with Kpn I and Bgl II, and connected with the final expression vector pC1300-Cas9 double-digested with Kpn I and BamH I to obtain the recombinant vector pC1300-Chalk9-Cas9. Subsequently, the pC1300-Chalk9-Cas9 vector is transformed into the Agrobacterium strain EHA105 by heat shock method, and positive Agrobacterium containing the pC1300-Chalk9-Cas9 vector is obtained by kanamycin selection;

[0040] The above-mentioned positive Agrobacterium strain containing pC1300-Chalk9-Cas9 was used to transform the callus tissue cells of rice Zhonghua 11 using an Agrobacterium-mediated method. The rice callus tissue was screened for hygromycin resistance to obtain resistant calli. The resistant calli were transferred to differentiation medium, and positive transgenic seedlings were obtained after differentiation culture. Finally, after testing and identification, they were transplanted to the field to obtain T0 generation rice plants.

[0041] Example 3: Phenotypic analysis of Chalk9 knockout strains;

[0042] 1. Detection of genetically modified plants;

[0043] A total of 30 seedlings were obtained using Agrobacterium-mediated genetic transformation. First, universal PCR detection primers for the hygromycin resistance gene were used to obtain positive seedlings with hygromycin resistance through PCR detection. Then, a pair of PCR sequencing primers (SEQ ID NO.7 and SEQ ID NO.8) were designed upstream and downstream of the knockout target site sequence to detect sequence variations near the target site. After PCR amplification and sequencing analysis of the sequences on both sides of the target site, three types of Chalk9 gene mutations were obtained (see Appendix). Figure 2 ). The sequencing primer sequences are as follows

[0044]

[0045]

[0046] 2. Analysis of rice chalkiness in Chalk9 gene knockout lines;

[0047] In order to understand the biological function of the Chalk9 gene, a comprehensive agronomic character of rice was investigated. In the T0 and T1 generation, agronomic traits of different lines were investigated and it was found that the Chalk9 gene knockout lines had no difference in plant height, tiller number, main panicle length, flag leaf length, and flag leaf width compared with the wild-type parent Zhonghua 11 (see Appendix). Figure 3 There were no significant differences in 1000-grain weight, grain length, grain width, and grain thickness in the mature grains of the Chalk9 gene knockout strain (Appendix Figure 3 ); however, in terms of grain appearance quality, chalkiness and chalky kernel rate increased significantly (see Appendix Figure 4 Further tracking of the grain filling dynamics revealed that around 17 days after flowering, the grains of the Chalk9 knockout strain began to show an appearance phenotype that was inconsistent with that of the wild-type Zhonghua 11, specifically manifested by the grains becoming significantly opaque (see Appendix). Figure 5 ). This indicates that the Chalk9 gene can specifically regulate grain chalkiness.

[0048] 3. Analysis of the physicochemical quality of rice from Chalk9 gene knockout lines;

[0049] In order to evaluate the breeding value of the Chalk9 gene, the effects of chalkiness on the physicochemical quality of rice were further analyzed. Compared with the wild-type parent, Zhonghua 11, the Chalk9 gene knockout strain had increased amylose content, decreased total protein content, and decreased soluble sugar content. The results showed that after the Chalk9 gene was knocked out, in addition to chalkiness, it also led to changes in the physicochemical quality of rice (see Appendix). Figure 6 ).

[0050] 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 knocking out or inhibiting the rice chalkiness gene Chalk9 in increasing rice grain chalkiness, wherein the nucleotide sequence of the Chalk9 gene coding region is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The amino acid sequence of the Chalk9 gene coding region is shown in SEQ ID NO.

2.

3. The use according to claim 1, characterized in that The application steps are as follows: knocking out the rice chalky gene Chalk9, thereby changing the expression level of the Chalk9 gene in the target rice variety, thereby obtaining rice plants with a chalky phenotype; The recombinant vector pC1300-Chalk9-Cas9 used for the Chalk9 gene contains the Chalk9 gene; the vector system is CRISPR / Cas9, which includes the intermediate vector SK-gRNA and the final vector pC1300-Cas9; The recombinant vector pC1300-Chalk9-Cas9 was prepared as follows: the intermediate vector SK-gRNA was digested with the restriction endonuclease Aar I, and then ligated with the denatured and annealed target gene complementary primers using T4 ligase to obtain the vector SK-gRNA-Chalk9; the SK-gRNA-Chalk9 vector identified as correct by sequencing was double-digested with Kpn I and Bgl II, and ligated with the final vector pC1300-Cas9 double-digested with Kpn I and BamH I to obtain the recombinant vector pC1300-Chalk9-Cas9.

Images