Mutant of apo2 gene promoter and its application in improving yield of oryza sativa

Abstract

The present application relates to the technical field of plant breeding, and particularly relates to a mutant of APO2 gene promoter and application thereof in improving yield of rice plants. Compared with the wild-type rice APO2 gene promoter, the mutant has 21bp deletion from 787bp to 766bp upstream of the start codon and 641bp insertion of base A. The application further includes: i) improving yield of rice plants, or preparing a kit for improving yield of rice plants; ii) cultivating transgenic rice plants; iii) improving rice plant varieties related to yield; and iv) improving germplasm resources of rice plants. In the research process of the present application, a mutant of APO2 gene promoter is obtained, and the expression level of the APO2 gene can be effectively improved by mutating the wild-type APO2 gene promoter in rice plants into the mutant, so as to improve the yield of rice plants, and the present application has important value in the field of rice plant breeding.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to a mutant of the APO2 gene promoter and its application in increasing the yield of rice plants. Background Technology

[0002] In grasses, inflorescence branches develop into panicle peduncles, which in turn determine panicle size and grain number. In rice, panicle grain diameter and grain number are important agronomic traits that directly affect rice yield. The number of grains per panicle is primarily determined by panicle structure, which refers to the number and length of primary and secondary branches, as well as the number of branches on secondary and higher-order branches. Currently, several genes involved in regulating panicle size and grain number have been identified in rice, but our understanding of the genetic and molecular mechanisms controlling these traits is limited. Therefore, further exploration of superior and usable rice panicle development genes is of great significance for rice breeding improvement.

[0003] Two mechanisms are involved in regulating panicle development. One mechanism alters the differentiation time of meristems, delaying or advancing the transition from inflorescence meristems to spikelet meristems, resulting in more or fewer inflorescence branches and spikelets. Examples include LAXPANICLE1 (LAX1), LAX2, ABERRANT SPIKELET AND PANICLE1 (ASP1), and FRIZZY PANICLE (FZP). The other mechanism alters meristem activity by affecting cytokinin (CK) levels, leading to differences in panicle structure. For example, downregulation of the cytokinin oxidase gene GRAIN NUMBER1a (Gn1a) and GY3, which encodes the cytokinin peptide nucleotide phosphohydrolase, increases secondary branching, thereby increasing the number of grains per panicle. Both regulatory mechanisms have been confirmed in multiple species, and the functions of most genes involved in these regulations are considered conserved. ABERRANT PANICLEORGANIZATION 2 (APO2) is an ortholog of Arabidopsis leafy (LFY) in rice. The loss-of-function mutant apo2 exhibits spikelets with reduced branching and fewer grains per spike, and increased APO2 protein stability leads to enhanced meristem activity. Therefore, APO2 not only regulates spike size and grain number by inhibiting the transition from inflorescence meristem to floral meristem, but also actively controls meristem activity and cell proliferation after reproductive transition. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a mutant of the APO2 gene promoter and its application in increasing the yield of rice plants.

[0005] In a first aspect, the present invention provides a mutant of the APO2 gene promoter, which, compared with the wild-type rice APO2 gene promoter, has a deletion 787 bp to 766 bp upstream of the start codon and an insertion of base A at 641 bp.

[0006] Furthermore, the promoter of the wild-type rice APO2 gene includes any of the following nucleotide sequences:

[0007] i) The nucleotide sequence shown in SEQ ID NO.1;

[0008] ii) Nucleotide sequences with the same promoter function obtained by inserting, deleting, or replacing one or more nucleotides from the nucleotide sequence shown in i).

[0009] The nucleotide sequence shown in SEQ ID NO.1:

[0010]

[0011] Furthermore, the mutant comprises the following nucleotide sequence (SEQ ID NO.15):

[0012]

[0013] Secondly, the present invention provides a gRNA comprising any of the following nucleotide sequences:

[0014] i) The nucleotide sequence shown in SEQ ID NO.2;

[0015] ii) Nucleotide sequences with the same targeting function obtained by inserting, deleting, or replacing one or more nucleotides as shown in i).

[0016] The nucleotide sequence shown in SEQ ID NO.2:

[0017] ATACATTAATTATCCAGAA.

[0018] Thirdly, the present invention provides a biological material, including the aforementioned mutant or the aforementioned gRNA; the biological material is an expression cassette, vector, cell or recombinant viral particle.

[0019] The expression cassette of this invention includes: a promoter (corresponding to the aforementioned mutant), a coding sequence, and a termination signal for terminating the transcription process (e.g., including a terminator and a polyadenylation signal). It can also guide the cell to add a poly(A) tail to the end of the mRNA to increase mRNA stability and translation efficiency. Common examples include SV40 polyA and BGH polyA. Furthermore, various elements can be added to the expression cassette, such as enhancers, introns, Kozak sequences, Shine-Dalgarno sequences, or selectable marker genes.

[0020] The vectors described in this invention include: plasmid vectors (extrachromosomal circular DNA molecules derived from bacteria or yeast), viral vectors (modified viruses that have had their pathogenicity and self-replication capabilities removed, but retain their ability to efficiently infect cells and deliver genetic material into cells), bacteriophage vectors, or artificial chromosome vectors (e.g., bacterial artificial chromosome BAC or yeast artificial chromosome YAC).

[0021] The transgenic cells described in this invention are cells whose genetic material has undergone stable artificial alterations, such as the introduction of the mutants provided in this application. The transgenic cells described in this invention include animal cells, plant cells, or microbial cells, wherein the animal and plant cells do not have the potential to develop into a complete individual (and do not belong to any animal or plant species).

[0022] The recombinant viral particles described in this invention are virus-like particles, consisting of a protein coat (viral capsid) encapsulating genetic material. For example, the recombinant viral particles are prepared by transfecting the aforementioned viral vector and other helper plasmids into a packaging cell line (e.g., HEK293T cells), which will then complete the expression and assembly of viral proteins and recombinant genes to obtain complete recombinant viral particles.

[0023] Those skilled in the art, having the mutants disclosed in this application, are fully aware of the preparation methods of the aforementioned expression cassettes, vectors, transgenic cells, and recombinant viral particles based on existing technology. There are no technical obstacles involved, and therefore, expression cassettes, vectors, transgenic cells, and recombinant viral particles containing the aforementioned gene mutants are also within the scope of this invention.

[0024] Fourthly, the present invention provides the application of the aforementioned mutants or APO2 gene promoter editing reagents in increasing the expression level of the APO2 gene, or in preparing kits for increasing the expression level of the APO2 gene;

[0025] The editing reagent is used to edit the APO2 gene promoter, causing it to mutate into the aforementioned mutant.

[0026] Fifthly, the present invention provides the use of the aforementioned mutants or APO2 gene promoter editing reagents in any of the following:

[0027] i) Increase the number of secondary branches and spikelets per panicle in rice plants, or prepare a kit for increasing the number of secondary branches and spikelets per panicle in rice plants.

[0028] ii) Increase the yield of rice plants, or prepare a kit for increasing the yield of rice plants;

[0029] iii) Cultivating transgenic rice plants;

[0030] iv) Improvement of rice varieties related to yield;

[0031] v) Germplasm resource improvement of Oryza species;

[0032] The editing reagent is used to edit the APO2 gene promoter, causing it to mutate into the aforementioned mutant.

[0033] Furthermore, the editing reagents include one or more of the following: CRISPR-Cas9 system, TALENs system, or ZFNs system;

[0034] Preferably, the editing reagent is a CRISPR-Cas9 system.

[0035] Furthermore, the CRISPR-Cas9 system includes one or more of the following: Cas9, Cas12a, dCas9-base editor, or nCas9-lead editor systems.

[0036] Furthermore, the CRISPR-Cas9 system includes gRNA as described above.

[0037] In a sixth aspect, the present invention provides a method for increasing the expression level of the APO2 gene, comprising: mutating the promoter of the APO2 gene to obtain the aforementioned mutant.

[0038] In a seventh aspect, the present invention provides a method for preparing transgenic rice plants, or increasing the number of secondary branches and spikelets per panicle in rice plants, or increasing the yield of rice plants, comprising: mutating the promoter of the APO2 gene in the rice plant to obtain the aforementioned mutant.

[0039] The present invention has the following beneficial effects:

[0040] This invention screened and obtained the promoter region of the APO2 gene, which is closely related to the development of rice panicles. The promoter region was then edited at specific sites using CRISPR / Cas9 technology to obtain a mutant that can increase the expression level of the APO2 gene. Based on this mutation, the number of panicle branches, panicle length and yield of rice can be increased.

[0041] The APO2 gene promoter regulation strategy provided by this invention can be widely applied to the breeding of high-yield rice varieties, with significant yield-increasing potential and stability. It can be used to improve rice germplasm resources and has important application value in the breeding of high-quality, high-yield, and lodging-resistant rice varieties. Attached Figure Description

[0042] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0043] Figure 1 This is the APO2 promoter structure, CRISPR / Cas9 target information, and APO2 promoter editing information provided in Embodiment 1 of the present invention; the orange area in the figure represents the promoter position where the target is located; the mutation mode is shown on the right side of each mutant.

[0044] Figure 2This is a graph showing the changes in APO2 expression levels in young spikelets from different families of the APO2 promoter editing materials (pAPO2-CR1, pAPO2-CR2, pAPO2-CR3, pAPO2-CR4, pAPO2-CR5, pAPO2-CR6) provided in Example 2 of this invention. ZH11 was used as a control, and UBQ was used as the internal reference gene. The bar chart reflects the upregulation or downregulation trend of expression levels in each family, and the error bars represent the standard deviation (SD). The graph shows the changes in APO2 expression levels in young spikelets from different families of the APO2 promoter editing materials (pAPO2-CR1, pAPO2-CR2, pAPO2-CR3, pAPO2-CR4, pAPO2-CR5, pAPO2-CR6). "" indicates a significant difference, P≤0.05, the same applies below.

[0045] Figure 3 These are plant phenotypic diagrams and ear phenotypic diagrams of different families of the APO2 promoter editing materials (pAPO2-CR1, pAPO2-CR2, pAPO2-CR3, pAPO2-CR4, pAPO2-CR5, pAPO2-CR6) provided in Embodiment 3 of the present invention; where A shows the complete plant morphology of the six edited families in the late heading stage under natural field conditions, and B shows the corresponding mature ear morphology; the scale bar of A is 20cm, and the scale bar of B is 10cm.

[0046] Figure 4 This is a statistical result of the spikelet structure data of different families of APO2 promoter editing materials (pAPO2-CR1, pAPO2-CR2, pAPO2-CR3, pAPO2-CR4, pAPO2-CR5, pAPO2-CR6) provided in Embodiment 3 of the present invention. Among them, the spikelet length, number of primary branches, number of secondary branches, and number of spikelets per spike of each material are statistically presented in the form of bar charts. A is a comparison of spikelet length between ZH11 and each editing material; B is a comparison of the number of primary branches between ZH11 and each editing material; C is a comparison of the number of secondary branches between ZH11 and each editing material; D is a comparison of the number of spikelets per spike between ZH11 and each editing material. Each indicator is based on field sampling statistics, and the data are expressed as mean ± standard deviation. Detailed Implementation

[0047] 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 embodiments of this invention, not all embodiments. 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.

[0048] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0049] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0050] Example 1: Analysis of APO2 gene promoter and identification of APO2 gene promoter editing materials

[0051] CRISPR / Cas9 editing targets were screened within the upstream promoter region (-1500 bp to the ATG start codon) of the rice APO2 gene. Three target sites located within the conserved regulatory region of the promoter were ultimately selected; the sequences of these gene editing targets are shown in Table 1.

[0052] Table 1 Target sequences, detection methods, and qRT primer sequences for APO2 gene promoter editing materials

[0053]

[0054] CRISPR / Cas9 editing vectors were constructed using corresponding gRNA primers, and genetic transformation of rice variety ZH11 was performed using Agrobacterium-mediated transformation. After screening and validation, this invention obtained six homozygous mutants with different editing types of the APO2 promoter (e.g., ...). Figure 1 (As shown). Among them, the mutation type of pAPO2-CR1 is a deletion of 128bp from 769bp to 641bp before the start codon; the mutation type of pAPO2-CR2 is an insertion of a single base A 496bp before the start codon; the mutation type of pAPO2-CR3 is a deletion of 355bp; the mutation type of pAPO2-CR4 is a deletion of 25bp from 502bp to 477bp before the start codon; the mutation type of pAPO2-CR5 is a deletion of 21bp from 787bp to 766bp upstream of the start codon and an insertion of a base A 641bp; the mutation type of pAPO2-CR6 is a deletion of 8bp from 503bp to 495bp before the start codon.

[0055] Example 2: Detection of APO2 expression level in young spikelets of APO2 gene promoter-edited materials

[0056] To verify the regulatory effect of promoter editing on APO2 gene expression levels, this study analyzed the expression levels of APO2 in multiple promoter-edited families during the young panicle stage of rice. Six APO2 promoter-edited families with different editing types (pAPO2-CR1 (SEQ ID NO.11), pAPO2-CR2 (SEQ ID NO.12), pAPO2-CR3 (SEQ ID NO.13), pAPO2-CR4 (SEQ ID NO.14), pAPO2-CR5 (SEQ ID NO.15), and pAPO2-CR6 (SEQ ID NO.16) obtained in Example 1 were selected, with wild-type ZH11 as a control. Young panicle tissue was collected at the critical differentiation stage when the panicle length was approximately 0-2 mm. Total RNA was extracted using TRIzol reagent, treated with DNase, and then reverse transcribed to synthesize cDNA as a template. The relative expression level of APO2 in each material was detected using quantitative real-time PCR (qRT-PCR), with UBQ as the internal reference gene. The primer sequences for qRT-PCR are shown in Table 1.

[0057] The results show that ( Figure 2 The expression levels of the APO2 gene varied significantly among different promoter-edited materials. In the pAPO2-CR5 material, APO2 expression was significantly upregulated compared to the wild type, increasing to approximately 1.15-fold; while in other materials, expression levels were significantly downregulated, with the lowest level decreasing to approximately 0.6-fold. These results indicate that site-directed mutations in the promoter region can significantly alter APO2 expression activity, and different editing types have significant differences in expression regulation.

[0058] Example 3: Phenotypic diagrams and statistical data of APO2 gene promoter edited materials plants

[0059] To further verify the effects of APO2 gene promoter editing on panicle development and yield traits in rice, this embodiment conducted field planting trials on several promoter-edited families (pAPO2-CR1, pAPO2-CR2, pAPO2-CR3, pAPO2-CR4, pAPO2-CR5, and pAPO2-CR6) obtained earlier, and systematically observed and statistically analyzed plant phenotypes and panicle traits. This invention investigated the plant and panicle types of wild-type and six APO2 promoter-edited materials (see...). Figure 3 Subsequently, key agronomic traits of the panicle were statistically analyzed, and the measured indicators included panicle length, number of primary branches, number of secondary branches, and number of spikelets per panicle (see...). Figure 4 ).

[0060] The results showed significant differentiation in panicle development traits among materials with different promoter editing. The pAPO2-CR5 material significantly outperformed the control in both the number of secondary branches and the number of spikelets per panicle, demonstrating a yield advantage. Conversely, the pAPO2-CR1 and pAPO2-CR6 materials showed significant reductions in panicle length, the number of secondary branches, and the number of spikelets per panicle. The changes in yield traits in the promoter-edited materials were consistent with changes in APO2 expression levels. This result indicates that promoter editing can achieve targeted regulation of phenotypic traits without altering the gene coding sequence, providing an effective means for molecular design breeding of rice.

[0061] Example 4: Yield statistics of single plants of APO2 gene promoter editing material

[0062] To further evaluate the impact of APO2 promoter editing on rice yield traits, this embodiment statistically analyzes the yield per plant of different promoter-edited families under plot conditions to verify their breeding application potential.

[0063] Six promoter-edited families (pAPO2-CR1, pAPO2-CR2, pAPO2-CR3, pAPO2-CR4, pAPO2-CR5, and pAPO2-CR6) obtained in the aforementioned examples were selected and subjected to a small-plot experiment along with the wild-type control ZH11. The experiment was conducted under natural field conditions using a randomized block design, with three replicates for each material and 36 plants per replicate plot. Management practices were consistent with conventional field practices. After harvest, each plant was threshed individually, and yield per plant was measured. Statistical results showed significant differences in yield per plant among the different promoter-edited materials under plot cultivation conditions. The pAPO2-CR5 material showed an 8.15% increase in yield per plant compared to the wild type, demonstrating significant yield potential. In contrast, the yields of other families decreased. This example further verifies the feasibility of applying the APO2 promoter editing strategy provided by this invention under actual breeding conditions. It can be applied to improve rice yield and provides an effective technical path for improving crop yield traits through precise regulation of gene expression levels.

[0064] Table 2. Yield statistics of individual plants from different APO2 promoter editing families under plot planting conditions.

[0065]

[0066] This means P < 0.05. This means P < 0.01.

[0067] 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. A mutant of the APO2 gene promoter, characterized in that, The nucleotide sequence of the mutant is shown in SEQ ID NO.

15.

2. The mutant according to claim 1, characterized in that, The nucleotide sequence of the promoter of the wild-type rice APO2 gene corresponding to the mutant is shown in SEQ ID NO.

1.

3. A gRNA, characterized in that, The nucleotide sequence of the gRNA is shown in SEQ ID NO.

2.

4. A biomaterial, characterized in that, Includes the mutant as described in claim 1 or 2, or the gRNA as described in claim 3; the biological material is an expression cassette, vector, cell, or recombinant viral particle.

5. The use of the mutant or APO2 gene promoter editing reagent according to claim 1 or 2 in increasing the expression level of the APO2 gene or in preparing a kit for increasing the expression level of the APO2 gene; The editing reagent is used to edit the APO2 gene promoter to mutate it into the mutant described in claim 1 or 2.

6. The use of the editing reagent for the mutant or APO2 gene promoter as described in claim 1 or 2 in any of the following: i) Increase the number of secondary branches and spikelets per panicle in rice plants, or prepare a kit for increasing the number of secondary branches and spikelets per panicle in rice plants. ii) Increase the yield of rice plants, or prepare a kit for increasing the yield of rice plants; iii) Cultivating transgenic rice plants; iv) Improvement of rice varieties related to yield; v) Germplasm resource improvement of Oryza species; The editing reagent is used to edit the APO2 gene promoter to mutate it into the mutant described in claim 1 or 2.

7. The application according to claim 5 or 6, characterized in that, The editing reagents include one or more of the following: CRISPR-Cas9 system, TALENs system, or ZFNs system.

8. A method for increasing the expression level of the APO2 gene, characterized in that, include: The promoter of the APO2 gene is mutated to obtain the mutant described in claim 1 or 2.

9. A method for preparing transgenic rice plants or increasing the number of secondary branches and spikelets per panicle in rice plants or increasing the yield of rice plants, characterized in that, include: The promoter of the APO2 gene in the rice plant was mutated to obtain the mutant described in claim 1 or 2.

Images