Application of OsmiR396 GRF-MAPK6 signaling pathway in regulating rice grain shape and thousand-grain weight

Abstract

The application discloses application of an OsmiR396 / GRF-MAPK6 signal path in regulating rice grain type and thousand-grain weight, and particularly relates to application of mutual interaction of a target gene OsGRF4 (GS2) coding protein of OsmiR396 and an OsMAPK6 gene coding protein in regulating rice grain type and thousand-grain weight. By crossing a transgenic line with overexpression of the OsGRF4 gene and a transgenic plant with overexpression of a phosphorylated form of the OsMAPK6 gene, the grain type of the offspring is further increased compared with that of the parents on the basis of the increased grain type of the parents, and the breeding method obtained based on the research has the effect of significantly increasing the yield of rice.

Description

Technical Field

[0001] This invention belongs to the field of plant gene breeding technology, specifically involving the application of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and thousand-grain weight. Background Technology

[0002] As one of the world's major food crops, the yield and quality of rice (Oryza sativa L.) are of paramount importance.

[0003] Rice grains are the direct carriers of rice yield and quality. Among the many factors that determine rice yield, such as plant type, panicle shape, and grain shape, grain shape has the most direct impact on yield. The components of grain shape include grain length, grain width, and grain thickness. Grain shape is mainly determined by genetic factors. After decades of effort, scientists have used forward genetics to reveal several quantitative trait loci affecting grain shape and weight and have cloned many of the genes involved.

[0004] These include genes that regulate grain length (GS3, GL3.1, An-1, GLW7, and GS2), genes that regulate grain width (GW2, GW5, GS5, GW8, and GW7), and genes that regulate grain weight (GIF1, GE, TGW6, GW6a, BG1, XIAO, and GS9), among others. Extensive research into different regulatory mechanisms across various species has gradually revealed some common molecular pathways in grain shape regulation, primarily including the ubiquitination-proteasome pathway, the G-protein signaling pathway, the mitogen-activated protein kinase (MAPK) signaling pathway, plant hormone signaling pathways, and transcriptional regulatory pathways (including miRNA regulatory pathways), further demonstrating that numerous factors participate in regulating grain shape development.

[0005] Furthermore, miRNAs have attracted more attention due to their broad functional scope and simple regulatory mechanisms. Since their discovery, miRNAs have served as concise and effective gene expression regulators, demonstrating broad functions and promising applications in crop yield regulation and stress physiology.

[0006] For example, ideal plant architecture is an important screening indicator for increasing crop yield in modern agriculture. IPA1, a target gene of OsmiR156, promotes growth and development under normal conditions, but is induced to phosphorylate and enhance the immune response upon infection with rice blast fungus. Therefore, it can increase both rice yield and rice blast resistance, providing an important theoretical basis and application value for high-yield and high-resistance breeding. However, research on the molecular pathway of the OsGRF4 (GS2) target gene of OsmiR396 is still lacking. Therefore, this paper presents an application of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain type and thousand-grain weight. Summary of the Invention

[0007] To fill the current research gap in the molecular pathway of OsGRF4 (GS2), a target gene of OsmiR396, this application provides an application of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and thousand-grain weight, which has significant economic benefits in increasing rice yield.

[0008] In one aspect, this application provides an application of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and thousand-grain weight.

[0009] Specifically, this refers to the application of the interaction between the target genes of OsmiR396, OsGRF4 (GS2) and OsMAPK6, in regulating rice grain shape and thousand-grain weight.

[0010] Furthermore, there is an interaction between the carboxyl terminus of the OsGRF4 (GS2) protein, a target gene of OsmiR396, and the full-length interaction between the OsMAPK6 gene and the protein encoded by the OsGRF4 (GS2) gene.

[0011] Furthermore, the interaction between the target gene OsGRF4 protein and the gene-encoded protein OsMAPK6 of OsmiR396 is the phosphorylation modification of amino acids at positions 288 and 308 of the carboxyl terminus of the OsGRF4 protein by OsMAPK6.

[0012] Furthermore, by screening for OsGRF4 gene overexpression and hybridizing vector plants with OsGRF4-encoded protein modified by OsMAPK6 phosphorylation, the increase in rice grain shape and thousand-grain weight was achieved.

[0013] Rice grain shape and thousand-grain weight were downregulated by screening for OsGRF4 gene repression expression and hybridizing vector plants whose OsGRF4 encoded protein was not modified by OsMAPK6 phosphorylation.

[0014] Secondly, this application provides a method for breeding high-yield rice, comprising the following steps:

[0015] a) Obtained the first rice line: the transgenic plant MIM396 with OsmiR396 downregulated;

[0016] b) Obtain the second rice line: the CAMAPK6 line, which is obtained by phosphorylation modification of the OsGRF4 gene of the DNMAPK6 line with OsMAPK6;

[0017] c) Cross the first line with the second line for several generations, and screen the offspring plants that express OsGRF4 highly and whose OsGRF4 encoded protein is phosphorylated by OsMAPK6. These plants are high-yielding rice plants.

[0018] Furthermore, the OsGRF4 gene is phosphorylated by OsMAPK6 at sites S288 and T308, respectively.

[0019] Thirdly, this application protects vectors containing the target genes OsGRF4(GS2) and / or OsMAPK6 of any of the above-described OsmiR396, or host cells containing said vectors.

[0020] This application has the following beneficial effects:

[0021] The target gene OsGRF4(GS2) of OsmiR396 in this application is an important QTL in rice that regulates grain development. One of the QTLs was cloned from giant rice grains. Further research has found that it plays a role in maintaining the carbon-nitrogen metabolic balance in plants and has great yield-increasing value.

[0022] Meanwhile, OsmiR396 may regulate rice grain shape by regulating other downstream target genes (including other miRNAs) through its target genes to form a network. This application has verified this.

[0023] Proteins in the MAPK signaling pathway can regulate their activity through phosphorylation / dephosphorylation, mediating the regulation of rice grain shape. OsMAPK6 has the function of regulating grain shape, and they constitute a signaling pathway, affecting cell proliferation and brassinolide signaling and homeostasis, playing an important role in regulating rice grain size; while the phosphatase OsMKP1 can negatively regulate the MAPK signaling pathway.

[0024] In summary, we conclude that the protein encoded by the target gene OsGRF4 (GS2) of OsmiR396 can interact with the protein encoded by the OsMAPK6 gene. When transgenic plants with downregulated OsmiR396 (MIM396) (patent already granted ZL201510746467.5) are crossed with CAMAPK6 (a transgenic plant overexpressing the phosphorylated form of the OsMAPK6 gene), the offspring have a larger grain size than both parents, on top of the increased grain size of the parents. At the same time, when transgenic plants with overexpressed GS2 gene are crossed with transgenic plants with CAMAPK6, the offspring also have a larger grain size on top of the increased grain size of the parents. Attached Figure Description

[0025] Figure 1 -A is a diagram showing the full-length, N-terminal (GRF4N), and C-terminal (GRF4C) interactions of OsMAPK6 and GS2 in a yeast two-hybrid system.

[0026] Figure 1-B is a diagram showing the full-length, N-terminal (GRF4N), and C-terminal (GRF4C) interactions of OsMAPK6 and GS2 detected by the BIFC system;

[0027] Figure 2 -A is a graph showing the interaction between OsMAPK6 and OsGRF4C as verified by LCA experiments;

[0028] Figure 2 -B is a diagram showing the interaction between OsMAPK6 and OsGRF4 as verified by CoIP experiments;

[0029] Figure 3 -A is a schematic diagram of the sites where OsGRF4 is phosphorylated and modified by OsMAPK6 in vitro.

[0030] Figure 3 -B represents the in vitro phosphorylation reaction analysis of OsMAPK6 on the phosphorylation modification of OsGRF4 and OsGRF4A;

[0031] Figure 4 This is a diagram of the mRNA sequence of the Arabidopsis thaliana iPS gene.

[0032] Figure 5 -A shows the grain shape of wild-type ZH11, MIM396, DN-MAPK6, and the corresponding hybrid Cross;

[0033] Figure 5 -B is a bar chart of grain length for wild-type ZH11, MIM396, DN-MAPK6, and the corresponding hybrid Cross;

[0034] Figure 5 -C is a bar chart of the thousand-grain weight of wild-type ZH11, MIM396, DN-MAPK6, and the corresponding hybrid Cross;

[0035] Figure 5 -D represents the grain shape diagram of wild-type ZH11, MIM396, CA-MAPK6, and the corresponding hybrid Cross;

[0036] Figure 5 -E is a bar chart of grain length for wild-type ZH11, MIM396, CA-MAPK6, and the corresponding hybrid Cross;

[0037] Figure 5 -F is a bar chart of the thousand-grain weight of wild-type ZH11, MIM396, CA-MAPK6, and the corresponding hybrid Cross;

[0038] Figure 6 -A shows the grain shape of wild-type ZH11, GS2OE, DN-MAPK6, and the corresponding hybrid Cross;

[0039] Figure 6 -B is a bar chart of grain length for wild-type ZH11, GS2OE, DN-MAPK6, and the corresponding hybrid Cross;

[0040] Figure 6 -C is a bar chart of the thousand-grain weight of wild-type ZH11, GS2OE, DN-MAPK6, and the corresponding hybrid Cross;

[0041] Figure 6 -D represents the grain shape diagram of wild-type ZH11, GS2OE, CA-MAPK6, and the corresponding hybrid Cross;

[0042] Figure 6 -E is a bar chart of grain length for wild-type ZH11, GS2OE, CA-MAPK6, and the corresponding hybrid Cross;

[0043] Figure 6 -F is a bar chart of the thousand-grain weight of wild-type ZH11, GS2OE, CA-MAPK6, and the corresponding hybrid Cross. Detailed Implementation

[0044] The following examples, 1-2, and comparative examples, along with the appendix, illustrate this further. Figure 1-6 This application will be further elaborated upon, but not limited thereto. The sources of the various strains used are shown below:

[0045]

[0046] Application Testing - Interaction between GS2 and OsMAPK6

[0047] By dividing the GS2 protein into its N-terminal and C-terminal segments, and then using a yeast two-hybrid system and a BIFC system, the interactions between the full-length GS2 protein, its N-terminus, and C-terminus, and the full-length OsMAPK6 protein were detected. The test results were referenced from [reference needed]. Figure 1 -A and Figure 1 -B;

[0048] analyze Figure 1 -A and Figure 1 -B leads to the conclusion that: regardless of the yeast two-hybrid system ( Figure 1 -A), or in the BIFC system ( Figure 1 -B) OsMAPK6 protein can interact at the C-terminus of GS2, rather than the full-length or N-terminus.

[0049] Subsequently, we further conducted LCA experiments ( Figure 2 -A) and Co-IP experiments ( Figure 2 -B), and corresponding analysis results Figure 2Based on -A and 2-B, we can conclude that, through different in vivo and in vitro biochemical experiments, it has been confirmed that OsMAPK6 can interact with the C-terminus of OsGRF4 at the protein level. That is, OsMAPK6 and OsGRF4 can interact at the protein level, and this test result provides a molecular basis for the phosphorylation of OsGRF4 by OsMAPK6.

[0050] In vitro phosphorylation assay - verification of phosphorylation modification of OsGRF4 by OsMAPK6

[0051] Preliminary in vitro phosphorylation experiments demonstrated that OsMAPK6 can phosphorylate OsGRF4. Furthermore, liquid chromatography-mass spectrometry analysis identified the phosphorylation sites of OsGRF4 by OsMAPK6, such as... Figure 3 -A (in vitro phosphorylation modification sites) are shown, namely S288 and T308;

[0052] Subsequently, the amino acid modified by OsGRF4 phosphorylation by OsMAPK6 was mutated to alanine (OsGRF4A), and the Western spectral analysis results after the in vitro phosphorylation reaction showed the following... Figure 3 -B is shown;

[0053] The top band shows the results of the Anti-Thiophosphate ester antibody test, while the middle and bottom bands show the results of the protein content detection in the reaction system using MBP and OsGRF4 antibodies, respectively.

[0054] In summary, it can be seen that, under the same protein content, the phosphorylation modification signal of the mutant OsGRF4A protein by OsMAPK6 is significantly weakened, indicating that the S288 and T308 sites are effective sites for phosphorylation modification of OsGRF4 by OsMAPK6. Therefore, this provides a theoretical basis for hybridization of vector plants overexpressing the OsGRF4 and OsMAPK6 genes.

[0055] Example 1

[0056] A method for breeding high-yield rice includes the following steps:

[0057] a) Obtain the first rice line: a transgenic line overexpressing the OsGRF4 gene, wherein the transgenic line overexpressing the OsGRF4 gene is the MIM396 plant;

[0058] b) Obtained a second rice line: the CAMAPK6 line;

[0059] The CAMAPK6 strain was obtained by phosphorylating the OsGRF4 gene of the DN-MAPK6 strain at sites 288 and 308 of its carboxyl terminus with OsMAPK6.

[0060] c) The first line was crossed with the second line, that is, during the flowering period of rice, pollen from MIM396 plants was used to pollinate CAMAPK6 plants to obtain the hybrid F1 generation.

[0061] Then, the presence of the IPS backbone gene in the MIM396 plasmid was detected, with the IPS backbone gene referenced... Figure 4 This proves that the hybridization was successful and that positive hybrid plants, i.e., high-yielding rice plants, were obtained.

[0062] Comparative Example 1

[0063] A method for breeding rice includes the following steps:

[0064] a) Obtain the first rice line: a transgenic line overexpressing the OsGRF4 gene, wherein the transgenic line overexpressing the OsGRF4 gene is the MIM396 plant;

[0065] b) Obtained a second rice line: the DNMAPK6 line;

[0066] c) The first line is crossed with the second line, that is, during the flowering period of rice, pollen from MIM396 plants is used to pollinate DNMAPK6 plants to obtain the hybrid F1 generation.

[0067] Then, the presence of the IPS backbone gene in the MIM396 plasmid was detected, with the IPS backbone gene referenced... Figure 4 This proves that the hybridization was successful and that positive hybrid plants, i.e., high-yielding rice plants, were obtained.

[0068] Then, the plants obtained in Example 1 and Comparative Example 1 were tested, and the test results are shown in the table below: Statistical table of grain shape of the crosses corresponding to wild-type ZH11, MIM396, CA-MAPK6, and DN-MAPK6.

[0069]

[0070] The t-test showed a significant difference (*P<0.05, **P<0.01).

[0071] Combining the above table with Figure 5 (AF) shows that the grain length of the hybrid plant CAMAPK6-MIM396 is 10.98±0.331 mm, which is 48.18%, 21.33%, and 23.37% higher than that of wild-type ZH11, MIM396, and CA-MAPK6, respectively; the thousand-grain weight of the hybrid plant CAMAPK6-MIM396 is 43.73±0.618 mm, which is 48.39%, 21.81%, and 22.84% higher than that of wild-type ZH11, MIM396, and CA-MAPK6, respectively.

[0072] A detailed analysis of the above data reveals that the grain size of the hybrid plant CAMAPK6-MIM396, which is larger than that of the wild type in both MIM396 and CAMAPK6, has further increased in size. Figure 6 -D), and its grain length and thousand-grain weight also further increased and expanded. Figure 6 The hybrid strain CAMAPK6-MIM396 (-E, 6-F) has extremely high economic value.

[0073] The grain length of the hybrid plant DNMAPK6-MIM396 was only 8.03±0.165 mm, which was ±8.4%, ±11%, and ±33% different from the wild types ZH11, MIM396, and DN-MAPK6, respectively. The thousand-grain weight of the hybrid plant DNMAPK6-MIM396 was 32.33±0.34 g, which was ±9.7%, ±9.94%, and ±32.12% different from the wild types ZH11, MIM396, and DN-MAPK6, respectively.

[0074] Therefore, further analysis of the data revealed that the grain shape of the hybrid plant DNMAPK6-MIM396, which is a hybrid of MIM396 and DNMAPK6, is similar to that of wild-type ZH11. The grain length and thousand-grain weight also returned to a level similar to that of wild-type ZH11. This indicates that the large grain phenotype of MIM396 depends on the phosphorylation of OsMAPK6, i.e., the unphosphorylated comparative example 1 showed a significant decrease in various properties.

[0075] Example 2

[0076] A method for breeding high-yield rice includes the following steps:

[0077] a) Obtain the first rice line: a transgenic line overexpressing the OsGRF4 gene, wherein the transgenic line overexpressing the OsGRF4 gene is the GS2OE plant.

[0078] b) Obtained a second rice line: the CAMAPK6 line;

[0079] The CAMAPK6 strain was obtained by phosphorylating the OsGRF4 gene of the DN-MAPK6 strain at sites 288 and 308 of its carboxyl terminus with OsMAPK6.

[0080] c) The first line was crossed with the second line, that is, during the rice flowering period, the pollen of the GS2OE plant was pollinated with the CAMAPK6 plant to obtain the hybrid F1 generation.

[0081] The gene sequence at the GS2 gene location was obtained by PCR amplification, and then compared with GS2.AA After comparing and confirming whether the hybridization was successful, homozygous plants were obtained in the F2 generation using the same testing method.

[0082] Comparative Example 2

[0083] A method for breeding rice includes the following steps:

[0084] a) Obtain the first rice line: a transgenic line overexpressing the OsGRF4 gene, wherein the transgenic line overexpressing the OsGRF4 gene is the GS2OE plant.

[0085] b) Obtained a second rice line: the DNMAPK6 line;

[0086] c) The first line was crossed with the second line, that is, during the flowering period of rice, the pollen of GS2OE plants was pollinated with DNMAPK6 plants to obtain the hybrid F1 generation.

[0087] The gene sequence at the GS2 gene location was obtained by PCR amplification. The hybridization was confirmed by comparison with GS2AA. Subsequently, homozygous plants were obtained in the F2 generation using the same detection method.

[0088] The plants obtained in Example 2 and Comparative Example 2 were then tested, and the test results are shown in the table below:

[0089] A statistical table of phenotypic traits in the grain shape diagrams of the crosses corresponding to wild-type ZH11, GS2OE, CA-MAPK6, and DN-MAPK6.

[0090]

[0091] The t-test showed a significant difference (*P<0.05, **P<0.01).

[0092] Combining the above table with Figure 6 (AF) shows that the grain length of the hybrid plant CAMAPK6-GS2OE is 11±0.161 mm, which is 32.4%, 20%, and 23.6% higher than that of wild-type ZH11, GS2OE, and CA-MAPK6, respectively; the thousand-grain weight of the hybrid plant CAMAPK6-GS2OE is 44.13±0.899 g, which is 49.75%, 11.4%, and 24% higher than that of wild-type ZH11, GS2OE, and CA-MAPK6, respectively.

[0093] A detailed analysis of the above data reveals that, based on the fact that the grain size of the hybrid CAMAPK6-GS2OE (GS2OE and CAMAPK6) is larger than that of the wild type, the grain size of the same hybrid line DNMAPK6-MIM396 also shows an increase. Figure 6 -D), and its grain length and thousand-grain weight also further increased and expanded. Figure 6 -E, 6-F);

[0094] The grain length of the hybrid plant DNMAPK6-GS2OE was only 7.07±0.203 mm, which was ±4.59%, ±22.9%, and ±16.7% different from the wild types ZH11, GS2OE, and DN-MAPK6, respectively. The thousand-grain weight of the hybrid plant DNMAPK6-GS2OE was 24.07±0.525 g, which was ±18.3%, ±39.2%, and ±1.63% different from the wild types ZH11, GS2OE, and DN-MAPK6, respectively.

[0095] Further combining the above data and deduction, it can be seen that the grain shape of the hybrid plant - DNMAPK6-GS2OE - of GS2OE is similar to that of wild type ZH11, and the grain length and thousand-grain weight have also returned to a level similar to that of wild type ZH11. This indicates that the large grain phenotype of GS2OE also depends on the phosphorylation of OsMAPK6. Therefore, the performance of the unphosphorylated comparative example 2 is significantly reduced.

[0096] In summary, combining Examples 1-2 and Comparative Examples 1-2, Figure 6 From the corresponding data, we can see that:

[0097] This application effectively fills the research gap on the interaction between the target gene OsGRF4(GS2) protein and the OsMAPK6 gene protein of OsmiR396. Moreover, this interaction has extremely high application value in regulating rice grain shape and thousand-grain weight. The method for breeding high-yield rice obtained based on this discovery can significantly increase rice yield, thereby alleviating the current food crisis.

[0098] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A method for regulating rice grain shape and thousand-grain weight using the OsmiR396 / GRF-MAPK6 signaling pathway, characterized in that, Includes the following steps: a) Obtain the first rice line: transgenic plants with OsmiR396 downregulated; b) Obtain the second rice line: the CAMAPK6 line, which is obtained by phosphorylation modification of the OsGRF4 gene of the DNMAPK6 line with OsMAPK6; c) Cross the first rice line with the second rice line for several generations, and screen the offspring plants in which OsGRF4 is highly expressed and the OsGRF4 encoded protein is phosphorylated by OsMAPK6 to obtain high-yielding rice plants.

2. The method for regulating rice grain shape and thousand-grain weight using the OsmiR396 / GRF-MAPK6 signaling pathway according to claim 1, characterized in that, The OsGRF4 gene is phosphorylated by OsMAPK6 at sites S288 and T308, respectively.

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