Application of gene line DgCsp2-DrWhy in regulation and control of rice tillering phenotype
By introducing the DgCsp2 and Drwhy genes into rice and regulating protein abundance, the number of effective tillers and plant height were increased, solving the problem of rice tiller regulation and improving rice yield and population expansion capacity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
- Filing Date
- 2026-01-23
- Publication Date
- 2026-05-19
AI Technical Summary
Existing technologies are insufficient to effectively regulate the number of rice tillers and plant height, thus affecting rice yield and population expansion.
By introducing the DgCsp2 and Drwhy genes, the abundance of DgCsp2 and Drwhy proteins in rice was regulated, increasing the number of effective tillers and plant height. Genetic transformation was then carried out using recombinant plasmids and Agrobacterium to obtain transgenic rice plants.
It significantly increased the number of effective tillers and plant height in transgenic rice, thereby improving rice yield and population expansion capacity.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to the application of the gene circuit DgCsp2-DrWhy in regulating rice tillering phenotype. Background Technology
[0002] Tillering is a branching phenomenon unique to gramineous crops (such as rice and wheat). In rice, tillering refers to the new branches that grow from axillary buds (tillering buds) on the basal nodes (tillering nodes) of the main stem. Under suitable conditions, these branches can further develop into more advanced tillers (secondary tillers, tertiary tillers). The location of rice tillering: It occurs at the base of the stem underwater, near the nodes close to the soil. The essence of rice tillering: It is a way for rice to clone itself and expand its population.
[0003] The number of effective panicles per unit area is directly determined by the number of panicles produced by tillers. If the main stem is damaged (e.g., by pests, diseases, or frost), tillers can compensate for the damage and ensure a certain yield. When there are insufficient basic seedlings, this can be made up for by promoting tillering.
[0004] Early tillering stage: Tillering begins after transplanting and the plant turns green again (about 10-15 days), and the growth rate is relatively slow.
[0005] Peak tillering stage: Tillering speed increases dramatically, and the number of tillers increases exponentially (approximately 30-40 days after transplanting). The maximum number of tillers is reached at this time.
[0006] Late tillering stage (ineffective tillering stage): As the plant population closes and nutrient competition intensifies, the young tillers begin to stop growing and die, and the number of stem tillers decreases. This process is called tiller extinction.
[0007] Heading stage: Tillering stops, and the surviving tillers begin to differentiate into young spikelets, and then head and bear grain, becoming effective tillers; those that fail to head are ineffective tillers. Summary of the Invention
[0008] The purpose of this invention is to provide the application of the gene circuit DgCsp2-DrWhy in regulating rice tillering phenotype.
[0009] This invention provides a method for preparing transgenic plants with increased effective tiller number and / or increased plant height, comprising the following steps: introducing the DgCsp2 gene and the Drwhy gene into a recipient plant to obtain transgenic plants with increased effective tiller number and / or increased plant height.
[0010] This invention also protects the use of the DgCsp2 gene and the Drwhy gene in the preparation of transgenic plants with increased effective tillering number and / or increased plant height.
[0011] This invention also protects the use of DNA molecules containing the DgCsp2 gene and the Drwhy gene in the preparation of transgenic plants with increased effective tillering number and / or increased plant height.
[0012] This invention also protects the use of plasmids containing the DgCsp2 gene and the Drwhy gene in the preparation of transgenic plants with increased effective tillering number and / or increased plant height. Exemplarily, the plasmid containing the DgCsp2 gene and the Drwhy gene is the plasmid shown in SEQ ID NO: 5.
[0013] This invention also protects the use of recombinant microorganisms possessing the DgCsp2 and Drwhy genes in the preparation of transgenic plants with increased effective tillering number and / or increased plant height. Exemplarily, the recombinant microorganism possessing the DgCsp2 and Drwhy genes is a recombinant Agrobacterium obtained by introducing the plasmid shown in SEQ ID NO: 5 into Agrobacterium EHA105.
[0014] This invention also protects the application of DgCsp2 and Drwhy proteins in regulating the number of effective tillers and / or plant height in plants. The regulation is positive regulation. Increased abundance of both DgCsp2 and Drwhy proteins increases the number of effective tillers and / or plant height.
[0015] This invention also protects the application of the DgCsp2 and Drwhy genes in regulating the number of effective tillers and / or plant height in plants. The regulation is positive regulation. Increased abundance of both the DgCsp2 and Drwhy genes increases the number of effective tillers and / or plant height.
[0016] Specifically, any of the plants mentioned above belong to the Poaceae family.
[0017] Specifically, any of the plants mentioned above belong to the genus *Rice*.
[0018] Specifically, any of the plants mentioned above is indica rice.
[0019] For example, any of the above-mentioned plants is rice 9311.
[0020] For example, the plant height is the plant height during the grain-filling stage.
[0021] For example, the effective number of tillers is the effective number of tillers during the grouting period.
[0022] This invention also protects the DgCsp2 protein.
[0023] This invention also protects the Drwhy protein.
[0024] This invention also protects the DgCsp2 gene.
[0025] This invention also protects the Drwhy gene.
[0026] The present invention also protects a protein assembly; said protein assembly consists of DgCsp2 protein and Drwhy protein.
[0027] This invention also protects a gene combination; said gene combination consists of the DgCsp2 gene and the Drwhy gene.
[0028] This invention also protects DNA molecules containing the DgCsp2 gene and the Drwhy gene.
[0029] The present invention also protects plasmids containing the DgCsp2 gene and the Drwhy gene. Exemplarily, the plasmid containing the DgCsp2 gene and the Drwhy gene is the plasmid shown in SEQ ID NO: 5.
[0030] The present invention also protects recombinant microorganisms having the DgCsp2 gene and the Drwhy gene. Exemplarily, the recombinant microorganism having the DgCsp2 gene and the Drwhy gene is a recombinant Agrobacterium obtained by introducing the plasmid shown in SEQ ID NO: 5 into Agrobacterium EHA105.
[0031] The DgCsp2 protein described above is shown in SEQ ID NO: 1.
[0032] The Drwhy protein described above is shown in SEQ ID NO: 3.
[0033] The DgCsp2 gene mentioned above is a gene encoding the protein shown in SEQ ID NO: 1.
[0034] The Drwhy gene mentioned above is a gene that encodes the protein shown in SEQ ID NO: 3.
[0035] Specifically, the coding region of the DgCsp2 gene is shown in SEQ ID NO: 2.
[0036] Specifically, the coding region of the Drwhy gene is shown in SEQ ID NO: 4.
[0037] This invention has application and promotion value for cultivating new plant germplasm, especially for new rice cultivation methods that increase the number of tillers and plant height. Attached Figure Description
[0038] Figure 1 This represents the relative expression level of the DgCsp2 gene.
[0039] Figure 2 This represents the relative expression level of the Drwhy gene.
[0040] Figure 3 This is a photo of the plant during the grain-filling stage.
[0041] Figure 4 This refers to the number of effective tillers per plant during the grain-filling stage.
[0042] Figure 5 This refers to the plant height during the grain-filling stage. Detailed Implementation
[0043] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0044] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available. Unless otherwise specified, the quantitative experiments in the following embodiments are all performed in triplicate, and the results are averaged.
[0045] Rice 9311 (Indica rice), also known as Rice 93-11, is described in the following literature: N 6 -Methyladenine DNAMethylation in Japonica and Indica Rice Genomes and Its Association with
[0046] Gene Expression, Plant Development, and Stress Responses; Molecular Plant 11, 1492–1508, December 20.
[0047] Example I. Construction of recombinant plasmids The recombinant plasmid BGV003-CW is a circular plasmid formed from double-stranded DNA molecules, with a full length of 10720 bp.
[0048] The full sequence of the recombinant plasmid BGV003-CW is shown in SEQ ID NO: 5. In SEQ ID NO: 5, positions 620-883 represent the DgCsp2 gene, and positions 1845-2147 represent the Drwhy gene.
[0049] II. Performing genetic transformation and obtaining regenerated plants The recombinant plasmid BGV003-CW was introduced into Agrobacterium EHA105 to obtain recombinant Agrobacterium. Using the Agrobacterium infection method, the recombinant Agrobacterium was used to genetically transform embryogenic callus of rice 9311. Resistant callus was then screened (resistance screening used 50 mg / L kanamycin), followed by differentiation and regeneration culture, and then rooting culture to obtain T0 generation regenerated plants.
[0050] III. Screening for transgenic plants 1. Select transgenic plants from the T0 generation regenerated plants obtained in step 2, which are the T0 generation transgenic plants.
[0051] Method for screening transgenic plants: Take leaves, extract genomic DNA, and perform PCR amplification using primer pairs composed of F1 and R1. If a target band of approximately 1521 bp is displayed, the plant is a transgenic plant.
[0052] F1: 5'-GGCCGCCCCGGTGAGCAGCTACAACG-3'; R1: 5'-CTATCGATCAATCAGGATCCCTGC-3'.
[0053] 2. The T0 generation transgenic plants obtained in step 1 are self-pollinated, and the seeds are harvested. The seeds are then cultivated into plants, which are the T1 generation plants.
[0054] 3. Select transgenic plants from the T1 generation plants obtained in step 2 (using the same method as step 1), which are the T1 generation transgenic plants.
[0055] 4. The T1 generation transgenic plants obtained in step 3 are self-pollinated, and the seeds are harvested and cultivated into plants, which are the T2 generation plants. Leaves are randomly sampled from the T2 generation plants, and genomic DNA is extracted. PCR amplification is performed using primers composed of F1 and R1. If a target band of approximately 1521 bp is displayed, the plant is a transgenic plant.
[0056] For a given T1 generation transgenic plant, if the sampled T2 generation plants obtained through self-pollination are all transgenic plants, then the T1 generation plant is a homozygous transgenic plant, and its self-pollinated offspring are homozygous transgenic lines.
[0057] Two homozygous transgenic lines were obtained: CW#83 and CW#84.
[0058] IV. Identification of Target Gene Expression Levels Test plants: T2 generation plants of CW#83 and T2 generation plants of CW#84.
[0059] Leaves were collected from test plants 21 days after germination, and total RNA was extracted and reverse transcribed to obtain cDNA. Using cDNA as a template and Actin gene as an internal reference gene, the relative expression levels of the target genes were detected by quantitative real-time PCR (2-ΔΔCT method). The target genes were DgCsp2 and Drwhy genes.
[0060] The primers used to detect the DgCsp2 gene are as follows: F2: 5'-atggccaacggcaaggtgaa-3'; R2: 5'-ATGTTCTTGGCCTGCGGGCC-3'.
[0061] The primers used to detect the Drwhy gene are as follows: F3: 5'-ATGTTGACTTCCTTTTCACTTCC-3'; R4: 5'-TGTCCTGGCAAGCGTATGTCAG-3'; The primers used to detect the Actin gene are as follows: F5: 5'-GTTGGTGCATGTTGTAGCCTAG-3'; R5: 5'-GCCCTAGCAGTACAGGGACC-3'; The relative expression levels of the DgCsp2 gene are shown in the figure. Figure 1 The relative expression levels of the Drwhy gene are shown in [the table]. Figure 2 .
[0062] V. Identification of Characteristics The tested seeds were: self-pollinated seeds of the T2 generation of CW#83 line, self-pollinated seeds of the T2 generation of CW#84 line, and seeds of rice 9311.
[0063] Test location: Langfang City, Hebei Province.
[0064] On May 4, the test seeds were sown and cultivated under normal conditions.
[0065] See the photo of the plant taken on August 25 (when it was in the grain-filling stage). Figure 3 .
[0066] On September 15th, the number of effective tillers per plant was counted (at this time, the plants were in the grain-filling stage). The results are shown below. Figure 4 (The number of plants per plant is 10-20).
[0067] On September 15th, the plant height was measured (at this time, the plants were in the grain-filling stage). The results are shown below. Figure 5 (The number of plants per plant is 10-20).
[0068] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.
Claims
1. A method for preparing transgenic plants with increased effective tiller number and / or increased plant height, comprising the following steps: introducing the DgCsp2 gene and the Drwhy gene into a recipient plant to obtain transgenic plants with increased effective tiller number and / or increased plant height; wherein the DgCsp2 gene encodes the protein shown in SEQ ID NO: 1; and the Drwhy gene encodes the protein shown in SEQ ID NO:
3.
2. The method as described in claim 1, characterized in that: The coding region of the DgCsp2 gene is shown in SEQ ID NO: 2; the coding region of the Drwhy gene is shown in SEQ ID NO:
4.
3. The application of the DgCsp2 gene and the Drwhy gene in the preparation of transgenic plants with increased effective tillering number and / or increased plant height; wherein the DgCsp2 gene is as described in claim 1 or 2; and the Drwhy gene is as described in claim 1 or 2.
4. The use of DNA molecules containing the DgCsp2 gene and the Drwhy gene in the preparation of transgenic plants with increased effective tillering number and / or increased plant height; wherein the DgCsp2 gene is as described in claim 1 or 2; and the Drwhy gene is as described in claim 1 or 2.
5. The use of plasmids containing the DgCsp2 gene and the Drwhy gene in the preparation of transgenic plants with increased effective tillering number and / or increased plant height; wherein the DgCsp2 gene is as described in claim 1 or 2; and the Drwhy gene is as described in claim 1 or 2.
6. The application of recombinant microorganisms possessing the DgCsp2 gene and the Drwhy gene in the preparation of transgenic plants with increased effective tillering number and / or increased plant height; wherein the DgCsp2 gene is as described in claim 1 or 2; and the Drwhy gene is as described in claim 1 or 2.
7. Application of DgCsp2 and Drwhy proteins in regulating the effective number of tillers and / or plant height in plants; the DgCsp2 protein is shown in SEQ ID NO: 1; the Drwhy protein is shown in SEQ ID NO:
3.
8. The application of the DgCsp2 gene and the Drwhy gene in regulating the effective tillering number and / or plant height in plants; wherein the DgCsp2 gene is as described in claim 1 or 2; and the Drwhy gene is as described in claim 1 or 2.
9. The method or application as described in any one of claims 1 to 8, characterized in that: The plant in question is a member of the Poaceae family.
10. DgCsp2 protein, Drwhy protein, DgCsp2 gene, Drwhy gene, protein combination, gene combination, DNA molecule, plasmid or recombinant microorganism; The protein assembly consists of DgCsp2 protein and Drwhy protein; The gene combination consists of the DgCsp2 gene and the Drwhy gene; The DNA molecule contains the DgCsp2 gene and the Drwhy gene; The plasmid contains the DgCsp2 gene and the Drwhy gene; The recombinant microorganism contains the DgCsp2 gene and the Drwhy gene; The DgCsp2 protein is shown in SEQ ID NO: 1; the Drwhy protein is shown in SEQ ID NO: 3; The DgCsp2 gene is as described in claim 1 or 2; the Drwhy gene is as described in claim 1 or 2.