Application of Medicago polymorpha MtKNOX5 gene in reducing the plant height of leguminous forage

Abstract

This invention discloses alfalfa tribulus. MtKNOX5 The application of genes in reducing the height of leguminous forage plants falls under the field of genetic engineering technology. This invention provides isolated... MtKNOX5 The gene, whose nucleotide sequence is shown in SEQ ID No. 1, was described in this invention. The invention involved constructing a recombinant vector containing the above-mentioned gene expression cassette, transforming it into leguminous forage grasses, and obtaining overexpression lines. Experimental results showed that overexpression... MtKNOX5 The gene can significantly reduce the plant height of alfalfa truncatum (by approximately 46.3%) and significantly shorten the length of each internode. This invention also provides corresponding recombinant expression vectors, engineered bacteria, inoculants, and screening media, which can be applied to dwarfing and high-density planting and lodging-resistant breeding of alfalfa and other leguminous forage crops, and have significant industrial application value.

Description

Technical Field

[0001] This invention relates to a gene for improving the plant height of leguminous forage grasses and its application, particularly to alfalfa. MtKNOX5 The application of genes in reducing plant height falls under the field of genetic engineering technology. Background Technology

[0002] In the new era, the dietary structure of Chinese residents continues to upgrade, and the demand for high-quality protein foods continues to increase, leading to a rigid increase in the demand for forage. China's forage market is one of the world's largest consumer markets, and its growth rate is higher than the global average. Alfalfa, as the world's most widely cultivated legume forage, is a high-quality feed for dairy cows, increasing milk production and improving the quality of fresh milk. It is favored by farmers due to its high fiber content, high protein content, high yield, and ease of cultivation. However, traditional alfalfa varieties are tall, have limited planting density, and are prone to lodging, which seriously restricts mechanized harvesting and yield per unit area.

[0003] Alfalfa plant height and stem strength directly affect its lodging resistance and planting density: excessive plant height can lead to weak stems, increased risk of lodging, and affect forage quality and harvesting efficiency; appropriate dwarfing can enhance stem mechanical strength, improve ventilation and light penetration of the plant population, thereby supporting higher planting density, increasing dry matter yield per unit area, and effectively reducing lodging losses.

[0004] Currently, research on alfalfa plant architecture improvement mainly focuses on adjusting traditional cultivation practices (such as regulating water, fertilizer, and sowing density) and screening for a few morphological traits (such as the number of leaflets and branching angle). However, traditional cultivation adjustments struggle to balance yield and lodging resistance; while breeding based on morphological traits suffers from low variation frequency, phenotypic instability, and significant environmental influences, hindering commercialization. Currently, there are no studies on improving alfalfa plant architecture by overexpressing KNOX family transcription factors (such as...). MtKNOX5 Reports have been made on the use of dwarfing and shortening of stem internode length in leguminous forages (especially alfalfa).

[0005] Therefore, exploring genetic resources that can precisely regulate the height of leguminous forage grasses, shorten the length of stem nodes, and enhance lodging resistance from a genetic perspective has important industrial application value. Summary of the Invention

[0006] To address the shortcomings of existing alfalfa and leguminous forage cultivation, such as poor lodging resistance due to excessive plant height, limited planting density, and difficulty in increasing yield per unit area, as well as the instability of traits and high susceptibility to environmental influences in existing plant type improvement methods, this invention provides a novel application of the MtKNOX5 gene in alfalfa and its supporting products. By overexpressing this gene, this invention can significantly reduce the plant height and shorten the internode length of leguminous forages from a genetic perspective, thereby achieving the goal of dwarfing and dense planting, enhancing lodging resistance, and providing specific gene resources, engineered bacteria, inoculants, and transformation tools for high-yield breeding of alfalfa and leguminous forages.

[0007] The technical solution adopted in this invention is as follows: In a first aspect, the present invention provides an isolated alfalfa ( ) Medicago truncatula ) MtKNOX5 The application of the gene in the preparation of products for reducing the height of leguminous forage plants, the MtKNOX5 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0008] Furthermore, the application is achieved by overexpressing the [specific ingredient] in the legume forage. MtKNOX5 It is achieved through genes.

[0009] Furthermore, the product contains MtKNOX5 Recombinant expression vector of gene expression cassette, or engineered bacteria of Agrobacterium rhizogenes or Agrobacterium tumefaciens containing the recombinant expression vector, or bacterial agent containing the engineered bacteria, or screening medium required for legume forage conversion using the engineered bacteria.

[0010] Furthermore, the legume forage is alfalfa ( Medicago sativa ), Tribulus terrestris ( Medicago truncatula ) or clover ( Trifolium ).

[0011] Secondly, the present invention also provides alfalfa containing tribulus terrestris. MtKNOX5 A recombinant expression vector for the gene. Preferably, the recombinant expression vector is pEarlyGate103-MtKNOX5.

[0012] Thirdly, the present invention also provides alfalfa truncatum. MtKNOX5 Application of genes in lodging resistance breeding of leguminous forage grasses.

[0013] Fourthly, the present invention also provides a method for reducing the height of leguminous forage plants, comprising: containing... MtKNOX5 The gene overexpression vector was introduced into the legume forage grass to obtain overexpression. MtKNOX5 Genetically modified plants.

[0014] Furthermore, the overexpression vector is pEarlyGate103-MtKNOX5.

[0015] Furthermore, the legume forage grass is alfalfa, clover, or trifoliate grass.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention discovers and verifies the presence of alfalfa tribulus. MtKNOX5 A novel function of a gene in regulating plant height in leguminous forage grasses. By constructing an overexpression vector and transforming it into alfalfa, the resulting transgenic plants showed a significant reduction in plant height (approximately 46.3%), a marked shortening of stem internode length, and a compact plant type, facilitating mechanized harvesting and field management.

[0017] 2. This invention closely integrates gene application with the preparation of specific products (recombinant vectors, engineered bacteria, bacterial agents, screening media), which not only protects the new uses of genes, but also comprehensively protects the supporting tools for realizing these uses, providing practical and feasible material support for industrial transformation.

[0018] 3. This invention alters plant structure through gene overexpression, resulting in dwarfing traits that are less affected by environmental factors and genetically stable. Dwarfing lowers the plant's center of gravity and enhances stem mechanical strength, effectively reducing the risk of lodging caused by excessive plant height.

[0019] 4. The gene resources, engineered bacteria, and supporting methods provided by this invention can be widely applied to the molecular design improvement of alfalfa and other leguminous forage crops. This invention directly utilizes gene overexpression to alter plant architecture, without relying on traditional cultivation adjustments (such as water and fertilizer control or thinning), effectively increasing forage yield per unit area while simultaneously increasing planting density, providing a highly efficient and stable new strategy for my country's forage production. Attached Figure Description

[0020] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0021] Figure 1 Example 1: Overexpression of Alfalfa Tribulus terrestris MtKNOX5 ( OX-MtKNOX5 Plant height phenotype of ) A. . OX- MtKNOX5 Expression level detection. B. 8-week wild-type and OX-MtKNOX5 The plant phenotype.

[0022] Figure 2 Example 1: Overexpression of Alfalfa Tribulus terrestris MtKNOX5 ( OX-MtKNOX5 Plant height determination.

[0023] Figure 3 Example 2: Overexpression of Alfalfa Tribulus terrestris MtKNOX5 ( OX-MtKNOX5 Determination of different intersegment lengths. Detailed Implementation

[0024] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0025] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0026] Example 1: Obtaining and measuring the plant height of alfalfa MtKNOX5 gene overexpression lines. 1. Experimental Materials alfalfa (tribulus) Medicago truncatula The R108 ecotype wild-type plant was used as the genetic transformation recipient. The plant expression vector used was pEarlyGate 103, which contains a kanamycin resistance selection marker gene and the Gateway recombination system. Overexpression vector... pEarlyGate103-MtKNOX5 The MtKNOX5 gene coding sequence (CDS) was previously constructed and preserved in our laboratory and has been inserted downstream of the 35S promoter using Gateway recombination technology.

[0027] 2. Agrobacterium-mediated genetic transformation Recombinant plasmid pEarlyGate103-MtKNOX5 Transformation of Agrobacterium tumefaciens (Gastrobacterium tumefaciens) using freeze-thaw method Agrobacterium tumefaciens EHA105 competent cells were plated on YEP solid medium containing rifampicin (75 mg / L) and kanamycin (50 mg / L) and incubated in the dark at 28°C for 48 h. Single colonies were picked for colony PCR identification, and positive colonies were used for subsequent infection.

[0028] Referring to previously reported genetic transformation methods for alfalfa, the explant transformation procedure is as follows: Extracts were taken from 4–6 week old plants in a greenhouse, selecting young, fully expanded leaves with minimal leaf hairs. The leaves were placed in 50 mL capped centrifuge tubes, and a sodium hypochlorite solution containing 0.1% Tween-20 (effective chlorine concentration ≥10%) was added. After inverting and mixing, the tubes were placed on a horizontal shaker for low-speed sterilization for 15 min, with the solution replaced with fresh sterilization solution every 7 minutes. After sterilization, the sodium hypochlorite solution was discarded, and the tubes were quickly rinsed 3–4 times with sterile water (total rinsing time should not exceed 5 min). The leaves were then placed on sterile filter paper to absorb excess water, and the petri dish lid was lightly covered to prevent drying. The leaf margins were cut off with a sterile, sharp blade to form square leaf discs, which were then immersed in Agrobacterium SH3a resuspension and gently shaken to disperse the leaf discs without stacking. The tubes were then treated under vacuum at 650 psi for 10 minutes. After aspirating the bacterial solution, the leaf disc was placed on sterile filter paper to remove excess bacterial solution and then transferred to antibiotic-free SH3a solid medium, ensuring the lower surface of the leaf disc was in contact with the medium. It was then incubated in the dark at 24°C for 24 h. After co-culture, the leaf disc was rinsed three times with sterile water for 1 min each time, followed by rinsing with sterile water containing 1000 mg / L Cefotaxime for 1 min. The leaf disc was then transferred to SH3a solid medium containing the corresponding screening antibiotic and 500 mg / L Cefotaxime, and incubated in the dark at 24°C for another 4 weeks, replacing the medium with fresh medium every 2 weeks. During this period, contamination was checked regularly (every 2–3 days), and uncontaminated material was transferred promptly. After dark culture, the resistant callus was transferred to a medium containing the screening antibiotic and 500 mg / L Cefotaxime. Cefotaxime was cultured in SH9BK differentiation medium under photoperiod for 2 weeks, then transferred to SH9 medium (containing screening antibiotics) for further culture. Subculture was performed every 2 weeks. After normal green shoots differentiated and the shoots elongated, well-grown shoots (about 2–3 cm) were cut and transferred to 1 / 2 MS medium for rooting culture. If rooting was difficult, 1 mg / L IBA could be added to promote rooting. In the later stage of seedling establishment, the agar concentration could be appropriately reduced to facilitate root extension. After the regenerated plants with well-developed roots were transplanted to the greenhouse, and after survival, DNA was extracted from the leaves and positive transgene identification was performed by PCR or qPCR.

[0029] The Agrobacterium tumefaciens engineered strain can be prepared as a liquid inoculum or a freeze-dried powder for genetic transformation of leguminous forage grasses; the screening medium used in the transformation process is SH3a or SH9BK solid medium containing kanamycin (final concentration of 50 mg / L) and cefotaxime sodium (final concentration of 500 mg / L).

[0030] 3. Molecular identification of transgenic positive lines To identify positive transgenic plants, genomic DNA was extracted from T0 generation regenerated plants for the target gene.MtKNOX5 Sequence primers were designed for PCR amplification. Simultaneously, total RNA was extracted from plant leaves, reverse transcribed into cDNA, and detected by real-time quantitative PCR (qRT-PCR). MtKNOX5 The relative expression level of the gene at the transcriptional level. The qRT-PCR reaction system was as follows: FastStart Essential DNA Green Master: 5 μL, primer F+R: 2 μL, cDNA template: 3 μL. The amplification program was as follows: pre-incubation: 95 °C, 10 min; denaturation: 95 °C, 10 s; annealing: 60 °C, 10 s; extension: 72 °C, 10 s; 40 cycles.

[0031] alfalfa MtUBQ The gene is used as an internal reference, and the specific primer sequences are as follows: MtKNOX5-F: 5′-GGTCAATGGCTCTCTCGTCCTA-3′ (SEQ ID No. 2); MtKNOX5-R: 5′-GGGTACGTCGTCGATGACTTC-3′ (SEQ ID No. 3); MtUBQ-F: 5′-CTGACAGCCCACTGAATTGTGA-3′ (SEQ ID No. 4); MtUBQ-R: 5′-TTTTGGCATTGCTGCAAGC-3′ (SEQ ID No. 5).

[0032] Use 2 -ΔΔCT The relative expression level was calculated. Positive plants with significantly higher expression levels than wild-type were selected for self-pollination and screened generation by generation until T3 homozygous overexpression lines were obtained.

[0033] 4. Plant height measurement Seeds of wild-type (WT) and MtKNOX5 overexpression T3 homozygous lines obtained through screening were sown in nutrient soil after sanding to remove hard seeds, and then cultured in a light incubator. The culture conditions were set as follows: temperature 22±2℃, photoperiod 16 h light / 8 h dark, light intensity 200 μmol·m⁻¹. -2 ·s -1 After 8 weeks of growth, 9-10 representative plants were randomly selected from each line, and the vertical height from the base of the plant (i.e., the soil surface) to the top of the main stem (growing point) was measured using a ruler, and the data were recorded. Results are expressed as mean ± standard deviation (Mean ± SD). Student's t-test was used to analyze the significance of differences between transgenic lines and wild-type lines. P <0.05 is considered a significant difference.

[0034] 5. Experimental Results (1) Validation of gene overexpression level qRT-PCR test results show (e.g.) Figure 1 As shown in Figure A), three independent MtKNOX5 overexpression lines (OX-MtKNOX5-1, OX-MtKNOX5-2, OX-MtKNOX5-3) were expressed. MtKNOX5 The relative expression levels of the gene reached 41, 27, and 23 times that of the wild type, respectively. Statistical analysis showed that the relative expression level of MtKNOX5 in the overexpression lines was significantly higher than that in the wild type. P <0.001). This result confirms that the obtained dwarf phenotype is indeed due to the overexpression of the MtKNOX5 gene, excluding the influence of somatic variation or T-DNA position effects.

[0035] (2) Plant height phenotype determination The plant height measurement results showed that, under the same growth conditions, the average plant height of the wild type was 31.3 ± 2.5 cm. MtKNOX5 The average plant height of the overexpression lines was 16.8 ± 2.2 cm. Compared with the wild-type control, MtKNOX5 The plant height of the overexpression lines was significantly reduced. P <0.05), the decrease was approximately 46.3%, such as Figure 1 China B and Figure 2 As shown. This indicates... MtKNOX5 Overexpression of the gene can significantly inhibit the plant height of alfalfa.

[0036] Example 2: Determination of stem node height in Alfalfa MtKNOX5 gene overexpression lines 1. Experimental materials and culture conditions The plant material used in this embodiment is the T3 generation obtained in Example 1. MtKNOX5 Overexpression homozygous lines and corresponding wild-type (WT)R108 plants were obtained. Seed sowing, germination, and plant culture conditions were completely consistent with those in Example 1 (22±2℃, 16 h / 8 h photoperiod, 200 μmol·m⁻¹). -2 s -1 (Light intensity). After the plants have grown for 8 weeks, samples were taken from plants of consistent growth status from each strain for testing.

[0037] 2. Methods for determining and measuring intersegmental position Referring to relevant plant internode length measurement standards, the internode counting method for this experiment was determined as follows: starting from the cotyledon node (node ​​0), the nodes on the main stem were counted sequentially upwards. During measurement, an electronic vernier caliper (accuracy 0.01 mm) was used to measure the length of each adjacent internode from the first node (node ​​1, i.e., the first stem node above the cotyledon node) to the seventh node (node ​​7). The length of each internode was the shortest distance between the base and the two node scars at the top of that internode. Nine to ten plants were randomly selected from each line for measurement, and the length data of each internode were recorded.

[0038] 3. Data Statistics and Significance Analysis All measurement data were summarized, and the mean and standard deviation (Mean ± SD) of internode lengths (internodes 1 to 7) for each line were calculated. To compare the differences in internode length between overexpressing lines and wild-type lines, Student's t-test (two-tailed) was used for significance analysis between the two groups. P <0.05 is used as the standard for judging whether the difference is statistically significant.

[0039] 4. Experimental Results Intersegment length measurement results are as follows Figure 3 As shown. After systematic measurement and statistical analysis of the lengths of internodes 1 to 7, it was found that under normal growth conditions, the internode length of wild-type plants showed a normal elongation trend as the node position increased. However... MtKNOX5 The overexpression lines had significantly shorter average internode lengths than the corresponding wild-type internodes. P <0.05). Furthermore, MtKNOX5 Overexpression lines showed a high degree of consistency in the trend toward internode shortening. This result indicates that overexpression... MtKNOX5 This resulted in overall plant dwarfing by inhibiting the elongation of each stem node. This confirms that the shortening of internode length is one of the main reasons for the reduced plant height in the overexpression lines of Example 1.

[0040] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An isolated species of alfalfa. MtKNOX5 The application of genes in the preparation of products for reducing the height of leguminous forage plants is characterized by, The MtKNOX5 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. The application according to claim 1, characterized in that, The application is achieved by overexpressing the [specific ingredient] in the legume forage. MtKNOX5 It is achieved through genes.

3. The application according to claim 1, characterized in that, The product contains MtKNOX5 Recombinant expression vector of gene expression cassette, or engineered bacteria of Agrobacterium rhizogenes or Agrobacterium tumefaciens containing the recombinant expression vector, or bacterial agent containing the engineered bacteria, or screening medium required for legume forage conversion using the engineered bacteria.

4. The application according to claim 1, characterized in that, The legume forage grasses mentioned are alfalfa, trifle alfalfa, or clover.

5. Contains alfalfa. MtKNOX5 Recombinant gene expression vectors.

6. Tribulus terrestris alfalfa MtKNOX5 Application of genes in lodging resistance breeding of leguminous forage grasses.

7. A method for reducing the height of leguminous forage plants, characterized in that, include: Will contain MtKNOX5 The gene overexpression vector was introduced into the legume forage grass to obtain overexpression. MtKNOX5 Genetically modified plants.

8. The method according to claim 7, characterized in that, The overexpression vector was pEarlyGate103-MtKNOX5.

9. The method according to claim 7, characterized in that, The legume forage grasses mentioned are alfalfa, trifle alfalfa, or clover.

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