Application of OsmiR396 GRF-MAPK6 signal channel in regulation and control of grain shape and thousand grain weight of rice

Abstract

The invention discloses an application of an OsmiR396 / GRF-MAPK6 signal channel in regulation and control of rice grain shape and thousand grain weight, and particularly relates to an application of interaction of a target gene OsGRF4 (GS2) encoding protein of OsmiR396 and an OsMAPK6 gene encoding protein in regulation and control of rice grain shape and thousand grain weight. By hybridizing an OsGRF4 gene overexpressed transgenic line and an OsMAPK6 gene phosphorylated overexpressed transgenic plant, the grain shape of a progeny is further increased compared with that of a parent on the basis that the grain shape of the parent is increased, and the breeding method obtained based on the research has the effect of remarkably increasing the rice yield.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic breeding, and particularly relates to the application of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and 1000-grain weight. Background Art

[0002] Rice (Oryza sativa L.), as one of the main food crops in the world, is of crucial importance in terms of its yield and quality.

[0003] Rice grains are the direct carriers of rice yield and quality. Among the many factors determining 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 efforts, scientists have revealed several quantitative trait loci affecting grain shape and grain weight through forward genetics methods and cloned many of these genes.

[0004] These include genes such as GS3, GL3.1, An-1, GLW7, and GS2 that regulate grain length, genes such as GW2, GW5, GS5, GW8, and GW7 that regulate grain width, genes such as GIF1, GE, TGW6, GW6a, BG1, XIAO, and GS9 that regulate grain weight, and so on. Extensive research on different regulatory mechanisms in different species has gradually revealed some common molecular pathways for grain shape regulation, mainly including the ubiquitin-proteasome pathway, G-protein signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, phytohormone signaling pathway, transcriptional regulation pathway (including the miRNA regulation pathway), etc., further indicating that many factors are involved in regulating the development of grain shape.

[0005] In addition, due to the wide range of miRNA functions and the simplicity of the regulatory mechanism, it has received more attention. Since its discovery, miRNA has been a simple and effective gene expression regulator and has shown extensive functions and good application prospects in aspects such as crop yield regulation and stress physiology.

[0006] For example, the ideal plant type is an important screening index for increasing crop yield in modern agriculture. IPA1, a target gene of OsmiR156, promotes growth and development under normal conditions, while being phosphorylated and induced to improve the immune response during Magnaporthe oryzae infection. Therefore, it can not only increase rice yield but also improve rice blast resistance, providing an important theoretical basis and application value for high-yield and high-resistance breeding. However, at present, the research on the molecular pathway of the OsmiR396 target gene OsGRF4 (GS2) is still blank. Therefore, the present invention specifically provides the application of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and 1000-grain weight. Summary of the Invention

[0007] To fill the research gap in the molecular pathway of the target gene OsGRF4 (GS2) of OsmiR396 at the current stage, the present application provides an application of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and 1000-grain weight, and this application has the economic benefit of significantly increasing rice yield.

[0008] In the first aspect, the present application provides an application of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and 1000-grain weight.

[0009] Specifically, it is the application of the interaction between the protein encoded by the target gene OsGRF4 (GS2) of OsmiR396 and the protein encoded by the OsMAPK6 gene in regulating rice grain shape and 1000-grain weight.

[0010] Furthermore, the carboxyl terminus of the protein encoded by the target gene OsGRF4 of OsmiR396 interacts with the full length of the protein encoded by the OsMAPK6 gene.

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

[0012] Furthermore, the increase in rice grain shape and 1000-grain weight is achieved by screening the hybrid plants of the overexpressed OsGRF4 gene and the vector plants with the protein encoded by OsGRF4 phosphorylated by OsMAPK6:

[0013] The decrease in rice grain shape and 1000-grain weight is achieved by screening the hybrid plants of the repressed expression of the OsGRF4 gene and the vector plants without the protein encoded by OsGRF4 phosphorylated by OsMAPK6.

[0014] In the second aspect, the present application provides a method for breeding high-yield rice, including the following steps:

[0015] a) Obtain the first rice line: the transgenic plant MIM396 with down-regulated OsmiR396;

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

[0017] c) Hybridize the first line with the second line for several generations, and screen the plants with high expression of OsGRF4 and the protein encoded by OsGRF4 phosphorylated by OsMAPK6 in the offspring, which are the high-yield rice plants.

[0018] Furthermore, the sites of the OsGRF4 gene phosphorylated and modified by OsMAPK6 are S288 and T308, respectively.

[0019] In a third aspect, the present application protects a vector with overexpression or repression expression of the target gene OsGRF4 (GS2) and / or the OsMAPK6 gene of any one of the above-mentioned OsmiR396, or a host cell containing the vector.

[0020] The present application has the following beneficial effects:

[0021] The target gene OsGRF4 (GS2) of OsmiR396 in the present application is an important QTL for regulating grain type development in rice. One of the QTLs was cloned from rice large grains. Further research found that it plays a role in maintaining the balance of plant carbon-nitrogen metabolism and has great value for increasing yield;

[0022] Meanwhile, OsmiR396 may regulate the rice grain type by forming a network with its target genes to regulate other downstream target genes (including other miRNAs), which was verified in the present application;

[0023] The proteins in the MAPK signaling pathway can regulate their activities through phosphorylation / dephosphorylation states, mediate the regulation of rice grain type. OsMAPK6 has the function of regulating grain type, and they form a signaling pathway, affecting cell proliferation and brassinolide signal and homeostasis, playing an important role in regulating rice grain size; the phosphatase OsMKP1 can negatively regulate the MAPK signaling pathway;

[0024] In summary, we draw the conclusion that the protein encoded by the target gene OsGRF4 (GS2) of OsmiR396 can interact with the protein encoded by the OsMAPK6 gene. The transgenic plants with down-regulated OsmiR396 (MIM396) (patent ZL201510746467.5 that has been authorized) are crossed with CAMAPK6 (transgenic plants with overexpression of the 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; at the same time, the transgenic plants with overexpression of the GS2 gene are crossed with the transgenic plants of CAMAPK6, and the grain type of the offspring plants is also further increased on the basis of the increased grain type of the parents. Description of the Drawings

[0025] Figure 1 - Panel A is the interaction diagram of detecting the interaction between the full length, N-terminal (GRF4N) and C-terminal (GRF4C) of OsMAPK6 and GS2 by the yeast two-hybrid system;

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

[0027] Figure 2 -A is the interaction diagram of the interaction between OsMAPK6 and OsGRF4C verified by the LCA experiment;

[0028] Figure 2 -B is the interaction diagram of the interaction between OsMAPK6 and OsGRF4 verified by the CoIP experiment;

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

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

[0031] Figure 4 is the mRNA sequence diagram of the Arabidopsis IPS gene;

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

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

[0034] Figure 5 -C is the bar graph of the 1000-grain weight of wild-type ZH11, MIM396, DN-MAPK6, and the corresponding hybrid strain Cross;

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

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

[0037] Figure 5 -F is the bar graph of the 1000-grain weight of wild-type ZH11, MIM396, CA-MAPK6, and the corresponding hybrid strain Cross;

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

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

[0040] Figure 6 -C is a column chart of 1000-grain weight of wild-type ZH11, GS2OE, DN-MAPK6, and the corresponding hybrid strain Cross;

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

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

[0043] Figure 6 -F is a column chart of 1000-grain weight of wild-type ZH11, GS2OE, CA-MAPK6, and the corresponding hybrid strain Cross. Detailed implementation mode

[0044] The following further explains the present application through specific examples 1-2 and comparative examples 1-2 in combination with the attached Figures 1-6 diagrams, but does not limit the present application. The sources of each strain used are as follows:

[0045]

[0046] Application test - Interaction between GS2 and OsMAPK6

[0047] By dividing the GS2 protein into the amino (N)-terminal and carboxyl (C)-terminal, and then detecting the interactions between the full-length, N-terminal, and C-terminal of GS2 and the full-length of OsMAPK6 respectively through the yeast two-hybrid system and the BIFC system, the test results are referred to Figure 1 -A and Figure 1 -B;

[0048] Analysis Figure 1 -A and Figure 1 -B can draw the conclusion that whether in the yeast two-hybrid system ( Figure 1 -A), or in the BIFC system ( Figure 1 -B), the OsMAPK6 protein can interact with the C-terminal of GS2 at the protein level, rather than the full-length or N-terminal.

[0049] Subsequently, we further carried out LCA experiments ( Figure 2 -A) and Co-IP experiments ( Figure 2 -B), and the corresponding analysis results Figure 2-A and 2-B, it can be concluded that through different in vivo and in vitro biochemical experiments, it is proved that OsMAPK6 can interact with the C-terminal of OsGRF4 at the protein level, that is, OsMAPK6 can interact with OsGRF4 at the protein level. 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] Through preliminary in vitro phosphorylation experiments, it was proved that OsMAPK6 can phosphorylate and modify OsGRF4. Further, through liquid chromatography-mass spectrometry analysis, the sites of OsGRF4 phosphorylated and modified by OsMAPK6 were determined as Figure 3 shown in -A (in vitro phosphorylation modification site), which are S288 and T308 respectively;

[0052] Subsequently, after mutating the amino acids of OsGRF4 phosphorylated and modified by OsMAPK6 to alanine (OsGRF4A), the Western blotting results after in vitro phosphorylation reaction are shown as Figure 3 shown in -B;

[0053] Among them, the top is the detection result of Anti-Thiophosphate ester antibody, and the middle and bottom bands are the detection results of the protein content in the reaction system with MBP and OsGRF4 antibodies respectively.

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

[0055] Example 1

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

[0057] a) Obtain the first rice line: a transgenic line with overexpression of the OsGRF4 gene, and the transgenic line with overexpression of the OsGRF4 gene is the MIM396 plant;

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

[0059] The CAMAPK6 line is obtained by phosphorylating and modifying the 288th and 308th sites at the carboxyl terminus of the OsGRF4 gene of the DN-MAPK6 line with OsMAPK6;

[0060] c) Hybridize the first strain with the second strain, that is, during the flowering period of rice, pollinate the CAMAPK6 plants with the pollen of MIM396 plants to obtain the hybrid F1 generation;

[0061] Then detect the presence of the backbone IPS gene of the MIM396 plasmid, and the backbone IPS gene refers to Figure 4 , prove the success of hybridization, and obtain positive hybrid plants, that is, high-yield rice plants.

[0062] Comparative Example 1

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

[0064] a) Obtain the first rice strain: a transgenic strain with overexpression of the OsGRF4 gene, and the transgenic strain with overexpression of the OsGRF4 gene is MIM396 plants;

[0065] b) Obtain the second rice strain: the DNMAPK6 strain;

[0066] c) Hybridize the first strain with the second strain, that is, during the flowering period of rice, pollinate the DNMAPK6 plants with the pollen of MIM396 plants to obtain the hybrid F1 generation;

[0067] Then detect the presence of the backbone IPS gene of the MIM396 plasmid, and the backbone IPS gene refers to Figure 4 , prove the success of hybridization, and obtain positive hybrid plants, that is, high-yield rice plants.

[0068] Then detect the plants obtained in Example 1 and Comparative Example 1, and the test results are as follows: Statistical table of the traits of the grain shape diagrams of the wild type ZH11, MIM396, CA-MAPK6, DN-MAPK6 corresponding to the hybrid strain Cross

[0069]

[0070] Applying the t-test shows significant differences (*P<0.05, **P<0.01).

[0071] Combined with the above table and Figure 5 (A-F) shows that the grain length of the hybrid plant CAMAPK6-MIM396 is 10.98±0.331, which is increased by 48.18%, 21.33%, and 23.37% compared with the wild type ZH11, MIM396, and CA-MAPK6 respectively; the 1000-grain weight of the hybrid plant CAMAPK6-MIM396 is 43.73±0.618, which is increased by 48.39%, 21.81%, and 22.84% compared with the wild type ZH11, MIM396, and CA-MAPK6 respectively;

[0072] Specific analysis of the above data shows that: for the grain shape of the hybrid plants of MIM396 plants and CAMAPK6 - CAMAPK6 - MIM396, on the basis that MIM396 and CAMAPK6 are respectively larger than the wild type, a further increase was obtained ( Figure 6 -D), and the grain length and 1000 - grain weight also further increased ( Figure 6 -E, 6 - F). This hybrid line CAMAPK6 - MIM396 has extremely high economic value.

[0073] However, the grain length of the hybrid plants DNMAPK6 - MIM396 is only 8.03 ± 0.165, showing changes of ±8.4%, ±11%, and ±33% respectively compared with the wild type ZH11, MIM396, and DN - MAPK6; the 1000 - grain weight of the hybrid plants DNMAPK6 - MIM396 is 32.33 ± 0.34, showing changes of ±9.7%, ±9.94%, and ±32.12% respectively compared with the wild type ZH11, MIM396, and DN - MAPK6;

[0074] Therefore, further derivation in combination with the data reveals that: for the grain shape of the hybrid plants of MIM396 plants and DNMAPK6 - DNMAPK6 - MIM396, it is similar to that of the wild type ZH11, and the grain length and 1000 - grain weight also return to a level similar to that of the wild type ZH11, indicating that the large - grain phenotype of MIM396 depends on the phosphorylation of OsMAPK6, that is, the performance of Comparative Example 1 without phosphorylation significantly decreases.

[0075] Example 2

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

[0077] a) Obtaining a first rice line: a transgenic line with over - expression of the OsGRF4 gene, and the transgenic line with over - expression of the OsGRF4 gene is the GS2OE plant;

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

[0079] The CAMAPK6 line is obtained by phosphorylating the 288th and 308th sites at the carboxyl - terminal of the OsGRF4 gene of the DN - MAPK6 line by OsMAPK6;

[0080] c) Crossing the first line with the second line, that is, during the flowering period of rice, pollinating the pollen of the GS2OE plant to the CAMAPK6 plant to obtain the hybrid F1 generation;

[0081] Obtaining the gene sequence at the GS2 gene position by PCR amplification, by comparing with GS2AA Compare and confirm whether the hybridization is successful, and subsequently obtain homozygous plants in the F2 generation by the same detection method.

[0082] Comparative Example 2

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

[0084] a) Obtain the first rice line: a transgenic line with overexpression of the OsGRF4 gene, and the transgenic line with overexpression of the OsGRF4 gene is the GS2OE plant;

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

[0086] c) Hybridize the first line with the second line, that is, during the flowering period of rice, pollinate the pollen of the GS2OE plant to the DNMAPK6 plant to obtain the hybrid F1 generation;

[0087] Obtain the gene sequence at the GS2 gene locus by PCR amplification, compare and confirm whether the hybridization is successful by comparing with GS2AA, and subsequently obtain homozygous plants in the F2 generation by the same detection method.

[0088] Then, the plants obtained in Example 2 and Comparative Example 2 were detected, and the test results are as follows in the table:

[0089] Statistical table of the traits of the grain shape diagrams of wild-type ZH11, GS2OE, CA-MAPK6, DN-MAPK6 and the corresponding hybrid strains Cross

[0090]

[0091] The application of t-test shows significant differences (*P<0.05, **P<0.01).

[0092] Combined with the above table and Figure 6 (A-F) It can be seen that the grain length of the hybrid plant CAMAPK6-GS2OE is 11±0.161, which is increased by 32.4%, 20%, and 23.6% compared with the wild-type ZH11, GS2OE, and CA-MAPK6, respectively; the 1000-grain weight of the hybrid plant CAMAPK6-GS2OE is 44.13±0.899, which is increased by 49.75%, 11.4%, and 24% compared with the wild-type ZH11, GS2OE, and CA-MAPK6, respectively;

[0093] Specifically analyzing the above data, it can be known that for the grain shape of the hybrid plants of GS2OE plants and CAMAPK6 - CAMAPK6-GS2OE, on the basis that GS2OE and CAMAPK6 are larger than the wild type respectively, compared with the hybrid strain DNMAPK6-MIM396, its grain shape has also increased (Figure 6 -D), and its grain length and 1000-grain weight also further increased. Figure 6 -E, 6-F);

[0094] However, the grain length of the hybrid plant DNMAPK6-GS2OE was only 7.07±0.203, showing changes of ±4.59%, ±22.9%, and ±16.7% compared with the wild type ZH11, GS2OE, and DN-MAPK6, respectively; the 1000-grain weight of the hybrid plant DNMAPK6-GS2OE was 24.07±0.525, showing changes of ±18.3%, ±39.2%, and ±1.63% compared with the wild type ZH11, GS2OE, and DN-MAPK6, respectively.

[0095] Further combining the above data and deducing, it can be known that: the grain shape of the hybrid plant -DNMAPK6-GS2OE of GS2OE plant and DNMAPK6 is similar to that of the wild type ZH11, and the grain length and 1000-grain weight also return to a level similar to that of the wild type ZH11, indicating that the large-grain phenotype of GS2OE also depends on the phosphorylation of OsMAPK6. Therefore, the performance of Comparative Example 2 without phosphorylation decreased significantly.

[0096] In summary, combining Examples 1-2, Comparative Examples 1-2, Figure 6 and their corresponding data, it can be known that:

[0097] This application effectively fills the research gap in the interaction between the protein encoded by the target gene OsGRF4 (GS2) of OsmiR396 and the protein encoded by the OsMAPK6 gene. Moreover, this interaction has extremely high application value in regulating rice grain shape and 1000-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 only an explanation of this application, and it is not a limitation of this application. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, but as long as it is within the scope of the claims of this application, it is protected by the patent law.

Claims

1. Application of OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and 1000-grain weight.

2. The use of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and thousand-grain weight according to claim 1, characterized in that: Application of the interaction between the protein encoded by the target gene OsGRF4 of OsmiR396 and the protein encoded by the gene OsMAPK6 in regulating rice grain shape and 1000-grain weight.

3. The use of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and thousand-grain weight according to claim 2, characterized in that: The carboxyl terminus of the protein encoded by OsGRF4, a target gene of OsmiR396, interacts with the full-length protein encoded by OsMAPK6 gene.

4. The use of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and thousand-grain weight according to claim 3, characterized in that: The interaction between the protein encoded by the target gene OsGRF4 of OsmiR396 and the protein encoded by the gene OsMAPK6 is the amino acid phosphorylation modification of the 288th and 308th positions of the carboxyl terminal of the protein encoded by OsGRF4 by OsMAPK6.

5. The use of the OsmiR396 / GRF-MAPK6 signaling pathway in regulating rice grain shape and 1000-grain weight according to any one of claims 1 to 4, characterized in that: The increase of rice grain size and 1000-grain weight was achieved by screening the overexpression of OsGRF4 gene and the hybridization of the vector plants after the OsGRF4 encoding protein was modified by OsMAPK6 phosphorylation: The downregulation of rice grain shape and 1000-grain weight was achieved by screening the vector plants in which the OsGRF4 gene expression was suppressed and the OsGRF4 encoding protein was not phosphorylated by OsMAPK6.

6. A method for breeding high-yield rice, characterized in that: The following steps are involved: a) obtaining the first rice line: OsmiR396 down-regulated transgenic plant MIM396; b) obtaining a second rice strain: a CAMAPK6 strain, wherein the CAMAPK6 strain is obtained by phosphorylating and modifying the OsGRF4 gene of the DNMAPK6 strain by OsMAPK6; c) hybridizing the first strain with the second strain for several generations, and selecting the plants in which OsGRF4 is highly expressed and the protein encoded by OsGRF4 is phosphorylated by OsMAPK6 in the offspring, so as to obtain high-yield rice plants.

7. The method for breeding high-yield rice according to claim 6, characterized in that: The sites where the OsGRF4 gene is phosphorylated and modified by OsMAPK6 are S288 and T308 respectively.

8. A vector for overexpression or repression of the target gene OsGRF4 and / or OsMAPK6 of OsmiR396 according to any one of claims 1 to 5, or a host cell containing the vector.

Images