Use of osmyb52 protein, or its coding gene, or biological material containing its coding gene in regulating the heading date of plants

Abstract

The present application relates to the technical field of plant molecular biology, and particularly relates to application of OsMYB52 protein, or a coding gene thereof, or a biological material containing the coding gene of the OsMYB52 protein in regulating a heading stage of a plant. The present application discloses a MYB transcription factor for negatively regulating a heading stage of rice OsMYB52 . The gene enhances transcription activation of Hd1 on Ghd7 , Ghd7.1 by interacting with Hd1, inhibits transcription activation of Hd1 on Ehd1 , Hd3a , and further negatively regulates the heading stage of rice and causes change of yield of rice. OsMYB52 The function and regulation mechanism of regulating the heading stage of rice are reported for the first time, and the function and regulation mechanism have important guiding significance for perfecting a molecular mechanism of a flowering regulation network of rice and improving yield and quality of rice.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, and in particular to the application of OsMYB52 protein, or its encoding gene, or biological materials containing its encoding gene in regulating the heading stage of plants. Background Technology

[0002] The rice heading date refers to the time from sowing to heading, reflecting the transition from the vegetative to the reproductive stage. It determines the regional and seasonal adaptability of rice varieties and is a crucial factor influencing rice yield and quality. In recent years, with the rapid development of molecular marker technology, an increasing number of rice heading date-related genes have been mapped. Currently, 618 heading date-related QTLs have been mapped in rice, with the highest number on chromosome 3, followed by chromosome 7, and the lowest on chromosome 10. While research has provided a preliminary understanding of the regulatory network of rice heading date, the molecular mechanisms of many heading date-regulating genes remain poorly elucidated. Therefore, continued exploration and research into rice heading date-related genes, and further refinement of the molecular mechanisms of the rice flowering regulatory network, are of significant guiding importance for improving rice yield and regional adaptability.

[0003] The regulatory network for rice heading stage mainly includes two pathways, one of which is... Hd1 Dependency pathway, i.e. OsGI - Hd1 - Hd3a way: OsGI Promote Hd1 The expression, under long-day conditions, Hd1 inhibition Hd3a / RFT1 The expression of this extends the heading period of rice, especially under short-day conditions. Hd1 Promote Hd3a / RFT1 The expression of this shortens the heading period of rice; another is... Ehd1 Dependency pathway, i.e. Ghd7 - Ehd1 - Hd3a / RFT1 way. Ghd7 - Ehd1 - Hd3a / RFT1 The pathway is a unique flowering pathway for rice under long-day conditions. Ehd1 Through integration Ghd7 , Ghd7.1 Multiple flowering signals are induced under blue light to generate the florigen gene. Hd3a / RFT1 The expression of this, in turn, promotes rice flowering.

[0004] MYB transcription factors are among the core transcription factors for plant growth and development, and are widely involved in regulating plant growth and development, hormone signal transduction, secondary metabolism, and biotic and abiotic stress processes. However, there is still relatively little research on the role of MYB transcription factors in regulating plant flowering time. Summary of the Invention

[0005] This invention discovers a novel MYB transcription factor that negatively regulates rice heading, and through gene editing technology, creates... OsMYB52 Gene knockout materials, in field phenotypic studies, showed that under long-day conditions, OsMBY52 Knockout materials showed earlier heading and increased yield, while excess materials showed delayed heading and decreased yield. Biochemical experiments confirmed that OsMBY52 can interact with Hd1 at the protein level and simultaneously inhibit Hd1 at the transcriptional level. Hd3a , RFT1 , Ehd1 The expression and enhancement Ghd7 and Ghd7.1 The expression of these factors regulates the heading stage of rice, ultimately affecting rice yield.

[0006] Based on this, the following technical solution is proposed.

[0007] In a first aspect, the present invention provides the application of OsMYB52 protein, or its encoding gene, or biological material containing its encoding gene in regulating the heading stage of plants; the amino acid sequence of the OsMYB52 protein is shown in SEQ ID No. 2.

[0008] Secondly, the present invention provides the application of OsMYB52 protein, or its encoding gene, or biological materials containing its encoding gene in regulating plant seed yield; the amino acid sequence of the OsMYB52 protein is shown in SEQ ID No. 2.

[0009] In some embodiments, sequences that have undergone substitution, deletion, and / or addition of one or more amino acid residues of the sequence shown in SEQ ID NO.2 and have the function of regulating the heading period and / or regulating the seed yield of plants are also within the scope of protection of this invention.

[0010] Preferably, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 1.

[0011] In some embodiments, sequences that regulate plant heading time and / or plant seed yield by substituting one or more bases and / or inserting one or more bases and / or deleting or inserting / deleting / shifting / inverting large nucleotide sequences based on SEQ ID NO.1 are also within the scope of protection of this invention.

[0012] Preferably, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable plant cells or tissues.

[0013] In some implementations, the heading time or seed yield of plants is regulated by adjusting the content or activity of the OsMYB52 protein.

[0014] In some implementations, the content or activity of the OsMYB52 protein is regulated through transgenic or gene-editing technologies.

[0015] In some implementations, by reducing the content or activity of the OsMYB52 protein, the heading date of the plant is advanced, resulting in increased yield; or by increasing the content or activity of the OsMYB52 protein, the heading date of the plant is delayed, resulting in decreased yield.

[0016] In some embodiments, the OsMYB52 protein interacts with Hd1 while simultaneously repressing it at the transcriptional level. Hd3a , RFT1 and Ehd1 The expression and enhancement Ghd7 and Ghd7.1 The expression of this information regulates the heading period, which in turn affects the yield.

[0017] In some embodiments, the plant is a cereal crop, preferably rice.

[0018] Thirdly, the present invention provides a method for preparing a transgenic plant, comprising: introducing or knocking out the OsMYB52 protein, or its encoding gene, or biological material containing its encoding gene into a plant.

[0019] In some implementations, CRISPR / Cas9 technology was used to obtain [the desired result] in the context of ZH11. OsMYB52 The knockout material, whose transgenic positive plants showed earlier heading and increased yield under long-day conditions. OsMYB52 Excessive yield results in delayed heading and reduced yield.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention discovers a MYB transcription factor that negatively regulates the heading stage of rice. OsMYB52 This gene enhances Hd1's response to Hd1 by interacting with Hd1. Ghd7 , Ghd7.1 The transcriptional activation effect of Hd1 inhibits the effect of Hd1 on transcriptional activation. Ehd1 , Hd3a Transcriptional activation negatively regulates the heading stage of rice, leading to changes in rice yield. OsMYB52The function and regulatory mechanism of rice heading stage are reported for the first time in this invention, which has important guiding significance for improving the molecular mechanism of rice flowering regulation network and increasing rice yield and quality. Attached Figure Description

[0022] Figure 1 This is the overall technical roadmap of the present invention.

[0023] Figure 2 It is used for OsMYB52 Map and positive results of the transformed CRISPR vector pYLCRISPR / Cas9Pubi-H OsMYB52 Mutation types of knockout mutants; among which, Figure 2 Graph A in the diagram is used to construct OsMYB52 pYLCRISPR / Cas9Pubi-H vector spectrum of the knockout material; Figure 2 Figure B in the design is OsMYB52 Schematic diagram of the knockout target location; Figure 2 Figure C in the diagram is positive. OsMYB52 Mutation type of knockout mutant.

[0024] Figure 3 In this invention OsMYB52 Validation of the heading phenotype in knockout and overpopulation mutants; among which, Figure 3 Figure A in the figure is a control of wild-type ZH11 and OsMYB52 Phenotypic comparison of the entire plant at the heading stage of the knockout mutant; Figure 3 Figure B in the diagram is a comparison of wild-type ZH11 and... OsMYB52 Phenotypic comparison of the entire plant at the heading stage of the overproduce mutant. Figure 3 Figure C in the diagram is a control of wild-type ZH11 and OsMYB52 Statistical results of heading time of knockout mutants; Figure 3 Figure D in the diagram is a control of wild-type ZH11 and OsMYB52 Statistical results of heading time of excess mutants; Figure 3 Figure E in the figure is a comparison of wild-type ZH11 and OsMYB52 In the leaves of the super-mutant at 35 days old OsMYB52 mRNA levels.

[0025] Figure 4 yes OsMYB52 Expression pattern analysis and subcellular localization in different tissues of rice database; among which... Figure 4 Figure A in the image is from the Rice Expression Profiling Database (http: / / ricexpro.dna.affrc.go.jp). OsMYB52 Expression spectrum; Figure 4 Figure B in the diagram shows the subcellular localization of the OsMYB52 protein in rice protoplasts.

[0026] Figure 5 Under long daylight conditions OsMYB52 Remove material, OsMYB52 mRNA detection results of important genes in the flowering pathway in the excess material and the control wild-type ZH11 material; among them, Figure 5 Figure A in the text is OsMYB52 In the knockout material and the control wild-type ZH11 material RFT1 , Hd3a , Ehd1 , Ghd7 , Ghd7.1 and Hd1 mRNA detection results; Figure 5 Figure B in the text is OsMYB52 Flowering period gene in excess material and control wild-type ZH11 RFT1 , Hd3a , Ehd1 , Ghd7 , Ghd7.1 and Hd1 The mRNA detection results.

[0027] Figure 6 This involves the interaction verification between OsMYB52 and Hd1; among which, Figure 6 Figure A in the figure shows the results of yeast double hybridization verification of the protein interaction between OsMYB52 and Hd1; Figure 6 Figure B in the diagram shows the results of dual-luciferase assay for the protein-protein interaction between OsMYB52 and Hd1. Figure 6 Figure C in the figure shows the results of the immunoprecipitation experiment verifying the protein-protein interaction between OsMYB52 and Hd1.

[0028] Figure 7 This refers to the results of OsMYB52 and Hd1, as well as the individual regulation of transcriptional activity of downstream gene promoters by OsMYB52; among them, Figure 7 Figure A in the diagram is a schematic diagram of the vector element for the luciferase transcription activity experiment; Figure 7 Figure B in the diagram shows OsMYB52 and Hd1, as well as OsMYB52 alone. Ghd7 , Ghd7.1 , Ehd1 , Hd3a The transcriptional activity regulation of the promoter was studied, with rLUC as an internal control and the LUC / rLUC ratio as the relative luciferase activity; significance analysis was performed using a t-test. p<0.1, , p<0.05. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0030] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and instructive reference books for those skilled in the art when performing experiments related to molecular biology. In addition, depending on the experimental purpose, those skilled in the art may conduct the corresponding experiments under the guidance of the operating manuals accompanying various commercial kits or using the conventional methods described in the references, or may entrust the experiments to specialized companies.

[0031] according to Figure 1 The technical route first obtains through genetic engineering technology. OsMYB52 Knockout and excess materials were selected, and phenotypic evaluation was conducted at the heading stage. After phenotypic confirmation, expression profile data and subcellular localization results were used to obtain... OsMYB52 The basic expression of [the information]. Subsequently, [the system] was tested. OsMYB52 The mRNA expression of key heading stage genes in knockout and over-produced materials, as well as in the Zhonghua 11 (ZH11) material, was preliminarily determined. OsMYB52 The heading stage regulatory pathway was determined. Then, through yeast library screening, the interacting protein Hd1 of OsMYB52 was obtained. The interaction between OsMYB52 and Hd1 was verified and the interacting segment was identified through yeast two-hybrid, two-luciferase complementation, and immunoprecipitation experiments. Finally, the transcriptional activity regulation of downstream gene promoters by OsMYB52 and Hd1, as well as by OsMYB52 alone, was examined to determine... OsMYB52 A regulatory network for the heading stage of rice.

[0032] The following embodiments further illustrate the present invention and describe it. OsMYB52 Gene function and regulatory pathways. SEQ ID NO.1 is the gene cloned in this invention. OsMYB52 The coding region nucleotide sequence; SEQ ID NO.2 is the gene cloned in this invention. OsMYB52 The encoded protein sequence.

[0033] Example 1: Construction of OsMYB52 transgenic material

[0034] Using the CRISPR / Cas9 gene editing system ( Figure 2 )exist OsMYB52 The coding region is designed to cut the target for knockout, while simultaneously using CaMV35S Startup driver OsMYB52 An overexpression vector was constructed, and the genetic transformation method employed was Agrobacterium-mediated transformation, with the wild-type ZH11 as the transgenic recipient. The transgenic materials were planted in a transgenic experimental field and managed according to standard field practices. A survey of the heading phenotype of the transgenic materials revealed (…). Figure 3 Under long-day conditions, compared to the control wild-type ZH11, OsMYB52 The heading period of the knockout material was significantly advanced. OsMYB52 The significant delay caused by excess material indicates OsMYB52 It is a negative regulatory factor for the heading stage of rice.

[0035] Example 2: Subcellular localization of OsMYB52

[0036] Will OsMYB52 The coding sequence of 35S:OsMYB52:YFP was fused with the coding sequence of yellow fluorescent protein (YFP) to construct the vector 35S:OsMYB52:YFP. This vector, along with the nuclear localization marker vector 35S:NLS:CFP, was then transformed into rice protoplasts via PEG·CaCl2 induction. After overnight incubation, the results were observed using an FV1200 laser confocal microscope. Figure 4 As shown, 35S:OsMYB52:YFP and 35S:NLS:CFP were found to co-localize, indicating that OsMYB52 is a nuclear localized protein.

[0037] Example 3: OsMYB52 Detection of expression levels of key flowering period genes in transgenic materials

[0038] Hd3a / RFT1 It is the rice flowering gene. Ehd1 , Ghd7 , Ghd7.1 , Hd1 It is a key gene in the rice flowering pathway. Using qRT-PCR, it was detected under long-day conditions. OsMYB52 mRNA levels of key genes in the flowering pathway in knockout and wild-type ZH11 materials. mRNA was collected from flag leaf samples taken at 35 days of age under long-day conditions (14h light / 10h dark). Figure 5 As shown, compared with the wild type (WT), under long-day conditions OsMYB52 The florigen gene in knockout strains Hd3a ,as well as Ehd1 , RFTThe expression level of 1 was significantly upregulated, while Ghd7 , Ghd7.1 and Hd1 The expression level was downregulated. Additionally, this embodiment also tested the expression level under long-day conditions. OsMYB52 Expression levels of the above genes in overexpression lines and wild-type ZH11 materials. Compared with wild type, Hd3a , Ehd1 , RFT1 The expression level of was significantly downregulated, while Ghd7 , Ghd7.1 and Hd1 The expression level of OsMYB52 was upregulated. qRT-PCR results showed that OsMYB52 promoted... Ghd7 , Ghd7.1 The expression of [something] is suppressed, thereby inhibiting [something]. Hd3a , Ehd1 , RFT1 The expression of this ultimately inhibits rice flowering.

[0039] Example 4: Verification of protein interaction between OsMYB52 and Hd1

[0040] Will OsMYB52 The protein was fully fused into the yeast pGBKT7 vector, and the interacting protein LOC_Os06g16370, or Hd1, was identified through yeast library screening. Subsequently, the interaction between these two proteins was verified using a yeast two-hybrid assay, and a yeast truncation assay demonstrated that OsMYB52 interacts with the zinc finger domain of Hd1 through its MYB domain. Further verification of the interaction between OsMYB52 and Hd1 was achieved through dual luciferase complementation and co-immunoprecipitation assays. Figure 6 ).

[0041] Example 5: Transcriptional Regulation of Downstream Genes by OsMYB52

[0042] To determine the transcriptional activity of the nuclear protein OsMYB52, OsMYB52 was fused with the yeast GAL4 binding domain to form an effector (OsMYB52-GAL4BD). This effector, along with a recombinant reporter consisting of a reporter gene and five copies of GAL4, was then co-transfected into rice protoplasts. After overnight culture, luciferase activity was measured. Figure 7 As shown, the results revealed that compared to the control group (GAL4-DB), the luciferase activity of OsMYB52-GAL4BD was significantly increased by 3-fold, indicating that OsMYB52 is a transcriptional activator in rice. Subsequently, Hd1 and OsMYB52 were fused with the effector None to form an effector... CaMV35S Promoter-driven, then with reporter genes and Ghd7 , Ghd7.1 , Ehd1 , Hd3aA 2.5 kbp sequence upstream of the start codon was fused into a reporter and co-transfected into rice protoplasts. After overnight culture, luciferase activity was measured. Results showed that, compared to the control group (none+none), the experimental group (OsMYB52+none) significantly promoted luciferase activity. pGhd7.1 -190 LUC of activity. This indicates that OsMYB52 can promote activity on its own. Ghd7.1 The expression of [a specific substance / factor] was significantly enhanced compared to the control group (none + Hd1). The experimental group (OsMYB52 + Hd1) significantly promoted the expression of [a specific substance / factor]. Ghd7.1 -190LUC and pGhd7 The transcriptional activity of -190LUC was significantly inhibited. pHd3a and pEhd1 The transcriptional activity indicates that OsMYB52 promotes transcription through interaction with Hd1. Ghd7 and Ghd7.1 The expression, inhibition Ehd1 , Hd3a The expression.

[0043] In summary, the MYB transcription factor OsMYB52 is a novel negative regulator of rice flowering. Yeast two-hybrid, tobacco LCI, and protoplast CO-IP experiments confirmed that OsMYB52 interacts with Hd1 through its MYB domain. Furthermore, qRT-PCR and luciferase transcriptional activity assays demonstrated that the interaction between OsMYB52 and Hd1 can further influence Hd1's effect on downstream genes. Ghd7 , Ghd7.1 , Hd3a and Ehd1 The expression regulation of [something]. Research has found that... OsMYB52 Possibly related to important flowering genes Hd1 Similarly, it connects the conserved flowering regulation pathways and specificities of rice. Ehd1 Flowering regulation pathways play a crucial role in regulating rice flowering. The discovery and functional analysis of OsMYB52 will not only further refine the rice flowering regulation network, but also help scientists breed high-yielding and high-quality rice varieties adapted to different regions.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of OsMYB52 protein, or its encoding gene, or biological material containing its encoding gene in regulating the heading stage of plants; the amino acid sequence of the OsMYB52 protein is shown in SEQ ID No. 2; the nucleotide sequence of the encoding gene is shown in SEQ ID No. 1; the plant is rice.

2. The application according to claim 1, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable rice cells or tissues.

3. The application according to claim 1, characterized in that, The heading period of rice can be regulated by adjusting the content or activity of OsMYB52 protein.

4. The application according to claim 3, characterized in that, The content or activity of OsMYB52 protein can be regulated through transgenic technology or gene editing technology.

5. The application according to claim 1, characterized in that, The heading date of rice can be advanced by reducing the content or activity of OsMYB52 protein; or the heading date of rice can be delayed by increasing the content or activity of OsMYB52 protein.

6. The application according to claim 1, characterized in that, The OsMYB52 protein interacts with Hd1 and simultaneously represses [something] at the transcriptional level. Hd3a , RFT1 and Ehd1 The expression and enhancement Ghd7 and Ghd7.1 The expression of this information, in turn, regulates the heading period of rice.

7. A method for preparing a transgenic plant, characterized in that, include: The OsMYB52 protein, or its encoding gene, or biological material containing its encoding gene is introduced into or knocked out in a plant; the plant is rice; the amino acid sequence of the OsMYB52 protein is shown in SEQ ID No. 2; and the nucleotide sequence of the encoding gene is shown in SEQ ID No. 1.

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