Application of BPM3 gene in regulation and control of growth period and yield of plants
By knocking out or overexpressing the BPM3 gene, CRISPR/Cas9 technology is used to regulate the millet heading period, which solves the shortcomings in the regulation of plant heading period and yield in the existing technology, and achieves optimization of growth cycle and yield improvement under different light conditions.
Patent Information
- Application Number
- CN202510222639.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-27
- Publication Date
- 2025-07-11
AI Technical Summary
The prior art has failed to effectively regulate the heading period and yield of plants, especially under different light conditions, and the growth cycle and yield of crops have significant changes, and there is a lack of effective gene regulation methods.
By knocking out or overexpressing the BPM3 gene, the millet heading period is regulated by using CRISPR/Cas9 technology, extending or premature heading period, increasing plant height and ear length, and optimizing growth cycle and yield.
It realizes precise regulation of the heading stage under different light conditions, improves the growth stability and yield of crops, provides new genetic resources and breeding methods, and adapts to different environmental conditions.
Smart Images
Figure CN120290583A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of plant molecular biology, and particularly relates to the application of a BPM3 gene in regulating the growth period and yield of plants. Background Art
[0002] The heading date of plants is one of the key traits in their growth and development process, directly affecting the growth cycle and yield of crops. The regulation of the heading date involves the interaction of multiple genetic and environmental factors, among which light, temperature, and the plant's own gene regulatory network play a core role in this process. Especially under different light conditions, there will be significant changes in the growth cycle and heading time of crops. Therefore, regulating the heading date of plants to adapt to environmental changes or increase yield is an important goal of modern agricultural breeding.
[0003] The BPM3 gene belongs to the BTB-MATH (Broad Complex, Tramtrack, Bric-à-Brac-MATH) protein family, which is widely present in plants and animals and participates in the regulation of various biological processes, such as plant development, stress response, and metabolic regulation. The typical feature of BTB-MATH family members is that they have a BTB domain and a MATH domain, which can interact with multiple proteins to form an E3 ubiquitin ligase complex and regulate the stability and function of target proteins. However, it has not been reported in the prior art that the BPM3 gene can regulate the growth period and yield of plants. Summary of the Invention
[0004] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present invention is to provide the application of a BPM3 gene in regulating the growth period and yield of plants. This gene can optimize the growth cycle of foxtail millet, help improve the adaptability and yield of foxtail millet, and provide new gene resources and technical means for crop breeding.
[0005] The technical solution for the present invention to solve the above technical problems is as follows: Provide the application of a BPM3 gene in regulating the growth period and yield of plants, wherein the nucleotide sequence of the BPM3 gene is as shown in SEQ ID NO.1.
[0006] Further, the plant is foxtail millet.
[0007] Further, by knocking out the BPM3 gene, the heading date of the plant is prolonged, and the plant height and panicle length of the plant are increased.
[0008] Further, by knocking out the BPM3 gene, the heading date of the plant is prolonged under long-day and short-day light conditions, and the plant height and panicle length of the plant are increased.
[0009] Further, the long-day light is 16 hours of light / 8 hours of darkness; the short-day light is 12 hours of light / 12 hours of darkness.
[0010] Furthermore, overexpression of the BPM3 gene advances the heading date of plants.
[0011] Furthermore, overexpression of the BPM3 gene advances the heading date of plants under long-day light conditions.
[0012] Furthermore, the long-day light is 16 hours of light / 8 hours of darkness.
[0013] A preparation for regulating the growth period and yield of plants, comprising the above-mentioned BPM3 gene or the protein encoded by this gene.
[0014] A method for regulating the growth period and yield of plants, comprising the following steps:
[0015] Regulating the growth period and yield of plants by knocking out or overexpressing the BPM3 gene.
[0016] The present invention has the following beneficial effects:
[0017] (1) Precision in regulating the heading date: By knocking out or overexpressing the BPM3 gene, the present invention can precisely regulate the heading date of foxtail millet. This method can not only adjust the flowering time under different light conditions, but also ensure that the crop enters the reproductive growth stage at an appropriate time, thereby optimizing its growth cycle.
[0018] (2) Improving crop adaptability: By regulating the BPM3 gene, the present invention provides a technical means to improve the adaptability of foxtail millet to different light conditions. The regulation of the heading date of foxtail millet under long-day and short-day light conditions can make it show higher growth stability and adaptability in different environments.
[0019] (3) Increasing yield: Regulating the heading date not only helps optimize the growth cycle of the crop, but also increases the yield of foxtail millet. By delaying or advancing the heading date, foxtail millet can flower and set seeds at an appropriate time, thereby effectively increasing the yield.
[0020] (4) Diversity of gene resources: The present invention provides a new way to regulate the growth period of crops by regulating the BPM3 gene. This technology is not limited to foxtail millet, but can also be applied to the regulation of the heading date of other crops, providing new gene resources and technical means for crop breeding.
[0021] (5) Application value in breeding: Through gene editing technology, foxtail millet lines with excellent heading date regulation characteristics can be quickly screened out, providing more precise technical support for crop breeding and helping to cultivate high-yield and excellent varieties adapted to different climate conditions. Description of the Drawings
[0022] Figure 1Phenotype of the BPM3 gene knockout mutant under short-day conditions in Sanya; among them, Heading date in the figure is the heading stage, Plant height is the plant height, and Panicle length is the panicle length.
[0023] Figure 2 Phenotype of the BPM3 gene knockout mutant under long-day conditions in Beijing; among them, Heading date in the figure is the heading stage, Plant height is the plant height, and Panicle length is the panicle length.
[0024] Figure 3 Phenotype of the BPM3 gene overexpression line under long-day conditions in Beijing; among them, Heading date in the figure is the heading stage, Plant height is the plant height, and Panicle length is the panicle length; Ci846 is the wild-type foxtail millet, sibpm3 is the knockout mutant, and SiBPM3-OE#1 and SiBPM3-OE#2 are overexpression lines. Detailed implementation mode
[0025] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.
[0026] Example 1: Effects of knocking out the BPM3 gene on the growth period and yield of foxtail millet
[0027] I. Preparation of the BPM3 gene knockout mutant
[0028] Select foxtail millet as the experimental material, and use healthy and plump seeds to induce callus for gene editing. When performing gene editing, the CRISPR / Cas9 technology is used to knockout the BPM3 gene. By designing specific primers for the BPM3 gene and combining with the Cas9 nuclease for targeted gene knockout. The designed primer sequences are as follows:
[0029] Upstream primer: 5’-CGGCGTGCACCATTTCGAGACaacacaagcggcagc-3’ (SEQ ID NO.3);
[0030] Downstream primer: 5’-ACAAGGTCGTGCTCGCGACGgttttagagctagaaat-3’ (SEQ ID NO.4).
[0031] The designed CRISPR / Cas9 vector was introduced into foxtail millet plants by the Agrobacterium transformation method. The transformed foxtail millet seedlings were screened for resistance, and the knockout effect of the BPM3 gene was confirmed by PCR and gene sequencing.
[0032] After knocking out the BPM3 gene by CRISPR / Cas9 technology, the BPM3 gene knockout mutants were successfully obtained. The knockout effect of the target gene was confirmed by PCR and gene sequencing analysis.
[0033] II. Phenotype of BPM3 gene knockout mutants
[0034] The BPM3 gene knockout mutants and wild-type foxtail millet (Ci846) were planted in the same environment respectively. The experiment was divided into two different light conditions: natural long-day light (16 hours of light / 8 hours of darkness, Beijing) and short-day light (12 hours of light / 12 hours of darkness, Sanya). The phenotypes such as heading date, plant height, and panicle length of the plants were observed, and the results are shown in Figure 1 and Figure 2 .
[0035] As can be seen from Figure 1 and Figure 2 , for the heading date, the heading dates of the BPM3 gene knockout mutants were significantly delayed under both long-day light and short-day light conditions. Especially under short-day light conditions, the heading date was postponed by about 12 days compared with the wild type. For plant height and panicle length, compared with the wild type, the plant height and panicle length of the BPM3 gene knockout mutants were significantly increased. The plant height of the mutants increased by about 7 cm, and the panicle length increased by 3 cm.
[0036] In summary, the knockout of the BPM3 gene significantly delayed the heading date and at the same time increased the plant height and panicle length. Through statistical analysis, the changes in various phenotypes of the BPM3 gene knockout mutants had significant differences, indicating that the BPM3 gene plays an important role in regulating the heading date, plant morphology, and yield traits.
[0037] Example 2: Effects of overexpressing the BPM3 gene on the growth period and yield of foxtail millet
[0038] I. Cloning of the BPM3 gene
[0039] Healthy and plump foxtail millet seed leaves were used as materials for gene cloning. RNA extraction and cDNA synthesis were carried out on them. Specifically: Total RNA was extracted from foxtail millet leaves using TRIzol reagent, and mRNA was transcribed into cDNA through a reverse transcription reaction. The reverse transcription reagent used was 5×PrimeScript RT Master Mix.
[0040] According to the known BPM3 gene sequence, specific primers were designed and PCR amplification was carried out. The primer sequences are as follows:
[0041] Forward primer: 5’-ATGGCGTCGTCGACGTGCA-3’ (SEQ ID NO.5);
[0042] Reverse primer: 5’-TCCTTCTAATCCCAGAGACA-3’ (SEQ ID NO.6).
[0043] The full-length sequence of the BPM3 gene was successfully cloned from the cDNA of foxtail millet using the above primers. The PCR products were separated and purified by agarose gel electrophoresis, followed by gene sequencing to confirm the integrity and correctness of the BPM3 gene. The nucleotide sequence of the cloned BPM3 gene is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2:
[0044] ATGGCGTCGTCGACGTGCACGGCGCCGCCGCCCACCGTCCGCGGCGTGCACCATTTCGAGATCATCGGCTACAGACTCATCAAAGGCCTCTCCCCGGGCGAGTACGTCCGGTCCGGCGCCTTCGTCGTCGGCGGCTACCGGTGGTCCGTGCGCTTCTACCCCGGCGGCTTCTCGCCGCCGCACCGCGCGCACGTGTCGGCGTTCCTCAAGATCACGACCAACAGCGCGAGGGCGTGGGCGCGCTTCGACCTCCGCCTGCTCGACCGCGCCACGGGGCTGTCGCGCTCCGTGCACCGCGCTGCGGCGCCCGTCGTCTTCGACTACTCGGCGGCGCACAAGAAGTGCAAGGGCAAGCGCGGCGCCCGCGCGTTCATGCCGCGGGCCGAGCTCGAGGCGTCGCCGTACCTGCGCGGCGACCGCCTCACGATCGAGTGCGTCGTCGATGTCGTCGTCGCCGCCGGCGAGGCCGTCGCTCCGCCTCCGCGCCGGCTCAGGGCGCCGCCGCCGGACCTGTCGAAGCATCTCGGCGACCTGCTGGATCAGCAGGACCTGGCGGACATCGCGTTCCACGTCGGAGGAGAGGTGTTCCGTGCACACAAGGTCGTGCTCGCGACGCGGTCGCCGGTGTTCATGGCGGAGCTCTACGGAGGGATGAAGGAGAAGGGGATGGAGCGCATCGCCATCGACGACGTGCAACCGTTGGTGTTCGGAGCTCTGATCCGTTTCGTGTACACGGATGTGTTGGTGCTCCCTGGTGACCTGGAAGGAGATGACTACAAGGAGATGGTGAGGCATCTCCTTGAGGCGGCGGATCGATACGGCGTGGAGCGGCTGAAGCTGGTATGTGAGAGCATCCTGTGCAGGAGCCTCGACGGGAACACGGTGGAGACGACGCTCGCCTTGGCTGATCAACACTACTGCAAGGCACTGAAAGATGTCTGCGTTCAGTTTATGTCTCTGGGATTAGAAGGATGA(SEQ ID NO.1);
[0045] MASSTCTAPPPTVRGVHHFEIIGYRLIKGLSPGEYVRSGAFVVGGYRWSVRFYPGGFSPPHRAHVSAFLKITTNSARAWARFDLRLLDRATGLSRSVHRAAAPVVFDYSAAHKKCKGKRGARAFMPRAELEASPYLRGDRLTIECVVDVVVAAGEAVAPPPRRLRAPPPDLSKHLGDLLDQQDLADIAFHVGGEVFRAHKVVLATRSPVFMAELYGGMKEKGMERIAIDDVQPLVFGALIRFVYTDVLVLPGDLEGDDYKEMVRHLLEAADRYGVERLKLVCESILCRSLDGNTVETTLALADQHYCKALKDVCVQFMSLGL(SEQ ID NO.2).
[0046] II. Construction of the BPM3 gene overexpression vector
[0047] The full-length sequence of the cloned BPM3 gene was inserted into the pCAMBIA1390.1 plasmid to construct the BPM3 gene overexpression vector. The vector contains the CaMV 35S promoter to drive the high expression of the BPM3 gene in foxtail millet. The constructed BPM3 overexpression vector was transformed into Agrobacterium tumefaciens GV3101, and the transformed plasmid was introduced into foxtail millet plants using the Agrobacterium-mediated method. The transformed foxtail millet plants were subjected to resistance screening, and the overexpression of the BPM3 gene was verified by PCR and RT-PCR techniques.
[0048] Through Western blot and RT-PCR analysis, foxtail millet plants with overexpression of the BPM3 gene were successfully obtained, confirming the high expression of the BPM3 gene in transgenic plants.
[0049] III. Phenotypic analysis of BPM3 gene overexpression lines
[0050] The BPM3 gene overexpression lines and wild-type foxtail millet (Ci846) were planted under the same conditions, specifically: growing under natural long-daylight (16 hours of light / 8 hours of darkness, Beijing) conditions, and observing phenotypic traits such as the heading date, plant height, and panicle length of the plants. The results are shown in Figure 3 .
[0051] As Figure 3 can be seen, for the heading date, the BPM3 gene overexpression lines had an earlier heading date under long-daylight conditions, and the heading date was about 3 days earlier than that of the wild type. For plant height and panicle length, there were no significant differences between the BPM3 gene overexpression lines and the wild type.
[0052] In summary, the overexpression of the BPM3 gene can advance the heading stage of foxtail millet, with little effect on plant height and panicle length.
[0053] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
Claims
1. Application of the BPM3 gene in regulating the growth period and yield of plants, wherein, The nucleotide sequence of the BPM3 gene is shown in SEQ ID NO.
1.
2. The application according to claim 1, wherein The plant is foxtail millet.
3. The application according to claim 1 or 2, characterized in that, Knocking out the BPM3 gene prolongs the heading stage of the plant, and increases the plant height and panicle length.
4. The application according to claim 3, wherein Knocking out the BPM3 gene prolongs the heading stage of the plant under long-day and short-day conditions, and increases the plant height and panicle length.
5. The application according to claim 4, characterized in that, Long-day condition is 16 hours of light / 8 hours of darkness; short-day condition is 12 hours of light / 12 hours of darkness.
6. The application according to claim 1 or 2, characterized in that, Overexpressing the BPM3 gene advances the heading stage of the plant.
7. The application according to claim 6, characterized in that, Overexpressing the BPM3 gene advances the heading stage of the plant under long-day conditions.
8. The application according to claim 7, wherein Long-day condition is 16 hours of light / 8 hours of darkness.
9. A preparation for regulating the growth period and yield of plants, characterized in that, It includes the BPM3 gene described in claim 1 or the protein encoded by this gene.
10. A method for regulating the growth period and yield of plants, characterized in that, Regulating the growth period and yield of plants by knocking out or overexpressing the BPM3 gene described in claim 1.