Application of BPM3 gene in regulation and control of plant drought resistance

Abstract

The invention discloses application of a BPM3 gene in regulation and control of plant drought resistance, and belongs to the technical field of plant molecular biology. According to the invention, the BPM3 gene is knocked out, so that the important function of the BPM3 gene in regulating the drought resistance of the millet is disclosed. The BPM3 gene knockout significantly reduces the drought resistance of millet, and especially under seedling stage drought and PEG simulated drought conditions, the mutant shows more obvious drought sensitivity than a wild type. The invention provides the method for optimizing the drought resistance of the millet by regulating the BPM3 gene, and the method has a wide breeding application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of plant molecular biology, and specifically relates to the application of the BPM3 gene in regulating plant drought resistance. Background Art

[0002] Drought is one of the main environmental factors leading to crop yield reduction globally. Especially in arid and semi-arid regions, the drought resistance of crops is crucial. The study of drought resistance involves the genetic mechanisms, environmental adaptability, and physiological regulation processes of plants. In recent years, through genome-wide association analysis and gene editing technologies, multiple genes related to drought resistance have been gradually revealed, but the functions of many genes are still not fully understood.

[0003] Currently, multiple genes related to plant drought resistance have been discovered. For example, the ABA (abscisic acid) signaling pathway plays an important role in the drought response of plants. ABA helps plants cope with drought stress by regulating the opening and closing of stomata, the accumulation of osmolytes, and the activity of antioxidant enzymes.

[0004] Foxtail millet (Setaria italica) is a C4 plant of the Gramineae family. As a drought-tolerant and barren-tolerant crop, it has important agricultural value in arid and semi-arid regions of China and the world. Foxtail millet has a short growth cycle, is drought-tolerant, and has strong stress resistance. It is suitable for planting in arid environments and is an important food and feed crop globally. In recent years, with the intensification of global climate change, the demand for foxtail millet in arid regions has gradually increased. Therefore, improving the drought resistance of foxtail millet has become an important goal in modern agricultural breeding.

[0005] The BPM3 gene belongs to the BTB-MATH (Broad Complex, Tramtrack, Bric-à-Brac-MATH) protein family. This protein family is widely present in plants and animals and is involved in the regulation of various biological processes, such as plant development, stress response, and metabolic regulation. The typical characteristics of BTB-MATH family members are the possession of 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 plant drought resistance. Summary of the Invention

[0006] To solve the above-mentioned deficiencies existing in the prior art, the purpose of the present invention is to provide the application of the BPM3 gene in regulating plant drought resistance.

[0007] The technical solution of the present invention to solve the above technical problems is as follows: Provide the application of the BPM3 gene in regulating plant drought resistance, wherein the nucleotide sequence of the BPM3 gene is as shown in SEQ ID NO.1.

[0008] Further, the plant is millet.

[0009] Furthermore, the drought resistance of plants was reduced by knocking out the BPM3 gene.

[0010] Furthermore, the drought resistance of plants at the seedling stage was reduced by knocking out the BPM3 gene.

[0011] Furthermore, reduced drought resistance of plants at the seedling stage is manifested in wilting and drying of plant leaves and reduced plant survival rate.

[0012] Furthermore, the drought resistance of plants throughout their growth period was reduced by knocking out the BPM3 gene.

[0013] Furthermore, reducing the drought resistance of the plant throughout its growth period is manifested in reduced plant height, shorter ear length, and reduced leaf length, leaf width, aboveground fresh weight, aboveground dry weight, and single plant yield.

[0014] A preparation for regulating plant drought resistance comprises the above-mentioned BPM3 gene or the protein encoded by the gene.

[0015] A method for regulating plant drought resistance comprises the following steps:

[0016] Regulating plant drought resistance by knocking out or overexpressing the BPM3 gene.

[0017] The present invention has the following beneficial effects:

[0018] (1) Precise regulation of drought resistance: The present invention can precisely regulate the drought resistance of millet by knocking out the BPM3 gene. The knockout mutant shows drought sensitivity and can be used to study the important role of the BPM3 gene in regulating plant drought resistance.

[0019] (2) Research on improving crop adaptability: By knocking out the BPM3 gene, the present invention provides a technical means to study the adaptability of millet to drought environments. After knocking out the BPM3 gene, millet showed obvious drought sensitivity, providing an experimental basis for in-depth exploration of the adaptation mechanism of plants under drought stress.

[0020] (3) Research value for improving yield: By studying the effect of the BPM3 gene on drought tolerance, it helps to understand how to improve the drought resistance of crops through gene regulation, thereby providing a theoretical basis for crop breeding and increasing yield in arid environments.

[0021] (4) Diversity of genetic resources: The present invention provides a new approach to study crop drought resistance by regulating the BPM3 gene. This technology is not limited to millet, but can also be applied to drought resistance research of other crops, providing new genetic resources and technical means for crop breeding.

[0022] (5) Application value in breeding: Through gene editing technology, foxtail millet lines with excellent drought resistance characteristics are screened out, providing precise technical support for crop breeding and contributing to the cultivation of high-yield and excellent varieties adapted to arid environments. Description of the Drawings

[0023] Figure 1 It is the result of the drought phenotype of the BPM3 gene knockout mutant in soil culture at the seedling stage.

[0024] Figure 2 It is the result of the drought phenotype of the BPM3 gene knockout mutant in hydroponics at the seedling stage.

[0025] Figure 3 It is the result of the drought phenotype of the BPM3 gene knockout mutant during the whole growth period. Detailed Implementation Modes

[0026] 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.

[0027] Example 1:

[0028] I. Preparation of BPM3 gene knockout mutant

[0029] Select healthy and plump foxtail millet seeds, and obtain plant materials for gene editing through cultivation and callus induction steps.

[0030] When performing gene editing, the CRISPR / Cas9 technology is used to knockout the BPM3 gene. The specific process is as follows:

[0031] First, design specific primers for the BPM3 gene and synthesize gRNA; among them, the nucleotide sequence of the BPM3 gene is shown in SEQ ID NO.1, and the amino acid sequence encoded by it is shown in SEQ ID NO.2. The primer sequences are as follows:

[0032] ATGGCGTCGTCGACGTGCACGGCGCCGCCGCCCACCGTCCGCGGCGTGCACCATTTCGAGATCATCGGCTACAGACTCATCAAAGGCCTCTCCCCGGGCGAGTACGTCCGGTCCGGCGCCTTCGTCGTCGGCGGCTACCGGTGGTCCGTGCGCTTCTACCCCGGCGGCTTCTCGCCGCCGCACCGCGCGCACGTGTCGGCGTTCCTCAAGATCACGACCAACAGCGCGAGGGCGTGGGCGCGCTTCGACCTCCGCCTGCTCGACCGCGCCACGGGGCTGTCGCGCTCCGTGCACCGCGCTGCGGCGCCCGTCGTCTTCGACTACTCGGCGGCGCACAAGAAGTGCAAGGGCAAGCGCGGCGCCCGCGCGTTCATGCCGCGGGCCGAGCTCGAGGCGTCGCCGTACCTGCGCGGCGACCGCCTCACGATCGAGTGCGTCGTCGATGTCGTCGTCGCCGCCGGCGAGGCCGTCGCTCCGCCTCCGCGCCGGCTCAGGGCGCCGCCGCCGGACCTGTCGAAGCATCTCGGCGACCTGCTGGATCAGCAGGACCTGGCGGACATCGCGTTCCACGTCGGAGGAGAGGTGTTCCGTGCACACAAGGTCGTGCTCGCGACGCGGTCGCCGGTGTTCATGGCGGAGCTCTACGGAGGGATGAAGGAGAAGGGGATGGAGCGCATCGCCATCGACGACGTGCAACCGTTGGTGTTCGGAGCTCTGATCCGTTTCGTGTACACGGATGTGTTGGTGCTCCCTGGTGACCTGGAAGGAGATGACTACAAGGAGATGGTGAGGCATCTCCTTGAGGCGGCGGATCGATACGGCGTGGAGCGGCTGAAGCTGGTATGTGAGAGCATCCTGTGCAGGAGCCTCGACGGGAACACGGTGGAGACGACGCTCGCCTTGGCTGATCAACACTACTGCAAGGCACTGAAAGATGTCTGCGTTCAGTTTATGTCTCTGGGATTAGAAGGATGA(SEQ ID NO.1);

[0033] MASSTCTAPPPTVRGVHHFEIIGYRLIKGLSPGEYVRSGAFVVGGYRWSVRFYPGGFSPPHRAHVSAFLKITTNSARAWARFDLRLLDRATGLSRSVHRAAAPVVFDYSAAHKKCKGKRGARAFMPRAELEASPYLRGDRLTIECVVDVVVAAGEAVAPPPRRLRAPPPDLSKHLGDLLDQQDLADIAFHVGGEVFRAHKVVLATRSPVFMAELYGGMKEKGMERIAIDDVQPLVFGALIRFVYTDVLVLPGDLEGDDYKEMVRHLLEAADRYGVERLKLVCESILCRSLDGNTVETTLALADQHYCKALKDVCVQFMSLGL(SEQ ID NO.2);

[0034] Forward primer: 5'-CGGCGTGCACCATTTCGAGAC-3' (SEQ ID NO.3);

[0035] Reverse primer: 5'-ACAAGGTCGTGCTCGCGACG-3' (SEQ ID NO.4).

[0036] Secondly, using the pYLCRISPR-Cas9-MH plasmid as a vector, the gRNA and Cas9 nuclease vectors were constructed into a plasmid, which contains the Cas9 protein and a specific gRNA, and can target and cut a specific region of the BPM3 gene, resulting in gene knockout.

[0037] Finally, the designed CRISPR / Cas9 vector was introduced into foxtail millet plants by Agrobacterium-mediated transformation. The transformed foxtail millet seedlings were screened for resistance, and the knockout effect of the BPM3 gene was confirmed by PCR and gene sequencing.

[0038] After knocking out the BPM3 gene by CRISPR / Cas9 technology, the BPM3 gene knockout mutants were successfully obtained. Through PCR and gene sequencing analysis, the knockout effect of the target gene was confirmed, and the knockout of the BPM3 gene was successfully achieved.

[0039] II. Drought resistance phenotype analysis of BPM3 gene knockout mutants at the seedling stage

[0040] 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 drought treatment conditions: natural drought and PEG-simulated drought, and the growth, survival rate and phenotypic changes of plants at the seedling stage were observed.

[0041] Among them, the natural drought condition is as follows: The experimental plants are planted in plug trays. Water is normally applied in the early stage. After the plants grow to the three-leaf stage (two weeks), watering is stopped for drought treatment for one week, and then re-watering is carried out. The survival rate is counted after one week of re-watering.

[0042] The PEG-simulated drought condition is as follows: Under hydroponic conditions, a drought environment is simulated by adding a 15% concentration of PEG solution. Normal nutrient solution is added to the seedlings in the early stage. After normal growth for 14 days, it is changed to a nutrient solution containing 15% PEG for 7 days to observe the growth status, survival rate, and drought-related phenotypes of the plants.

[0043] After the above two different drought treatments, the results are shown in Figure 1 and Figure 2 .

[0044] From Figure 1 and Figure 2 , it can be seen that under drought conditions, the BPM3 gene knockout mutants showed significant growth inhibition, with leaves wilting and drying up, confirming the important role of the BPM3 gene in the regulation of plant drought resistance. In addition, under natural drought and PEG-simulated drought conditions, the survival rate of the BPM3 gene knockout mutants was significantly lower than that of the wild type. Under soil culture conditions, the survival rate of the mutants was almost zero, while the wild type showed better performance, indicating that knocking out the BPM3 gene led to a significant reduction in the drought tolerance of foxtail millet.

[0045] III. Analysis of drought resistance phenotypes of BPM3 gene knockout mutants during the whole growth period

[0046] The experimental plants are planted in the field to simulate natural drought conditions, and the BPM3 gene knockout mutants and wild type are subjected to drought treatment throughout the growth period with continuous drought stress. Physiological indices of the plants are measured to evaluate their drought resistance, and the results are shown in Figure 3 .

[0047] From Figure 3 , it can be seen that under drought stress environment, the BPM3 gene knockout mutants showed obvious drought intolerance compared with the control, with reduced plant height, shorter panicle length, and decreased leaf length, leaf width, aboveground fresh weight, aboveground dry weight, and yield per plant. These experiments all show that the BPM3 gene knockout mutants are drought intolerant.

[0048] In summary, the present invention verifies the key role of the BPM3 gene in regulating the drought resistance of foxtail millet. The BPM3 gene knockout mutants showed obvious drought sensitivity under drought conditions, which provides an experimental basis for further studying the molecular mechanism of the BPM3 gene in plant drought resistance and provides new technical support for the drought resistance breeding of foxtail millet.

[0049] The above are only the preferred embodiments of the present invention and are 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 plant drought resistance, wherein, The nucleotide sequence of the BPM3 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The plant is Setaria italica.

3. The application according to claim 1 or 2, characterized in that, Knocking out the BPM3 gene reduces the drought resistance of the plant.

4. The application according to claim 3, characterized in that, Knocking out the BPM3 gene reduces the drought resistance of the plant at the seedling stage.

5. The application according to claim 4, wherein The reduction of the drought resistance of the plant at the seedling stage is manifested as wilting and drying of the plant leaves and a decrease in the plant survival rate.

6. The application according to claim 1 or 2, characterized in that, Knocking out the BPM3 gene reduces the drought resistance of the plant during the whole growth period.

7. The application according to claim 6, characterized in that, The reduction of the drought resistance of the plant during the whole growth period is manifested as a decrease in plant height, a shortening of the panicle length, and a decrease in leaf length, leaf width, fresh weight of the above-ground part, dry weight of the above-ground part, and yield per plant.

8. A preparation for regulating plant drought resistance, characterized in that, It includes the BPM3 gene described in claim 1 or the protein encoded by this gene.

9. A method for regulating plant drought resistance, characterized in that, It includes the following steps: Regulating the drought resistance of plants by knocking out or overexpressing the BPM3 gene.

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