Application of casein kinase in regulation and control of flowering and leaf development of medicago sativa

Abstract

The invention discloses application of casein kinase in regulation and control of flowering and leaf development of medicago sativa, and belongs to the technical field of biological genetic engineering. The multifunctionality of MsPPK3 as a key regulatory factor in coordination of vegetative growth and reproductive growth balance of medicago sativa is clarified for the first time, and specifically, MsPPK3 positively regulates plant reproductive growth and negatively regulates plant vegetative growth. Wherein the positive regulation and control of the reproductive growth of the plant is embodied in overexpression early flowering and silence late flowering. The negative regulation and control of plant vegetative growth is characterized in that the overexpressed plant is in a fine stem scattered form, and leaves are miniaturized, thinned and / or weakened form; the silent plants present a thick stem cluster type, a large leaf size, a thick leaf and / or a strengthened leaf. The method has great significance in the aspects of cultivation of different new varieties / new strains of alfalfa and the like.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to the application of a casein kinase in regulating flowering and leaf development in alfalfa. Background Technology

[0002] Alfalfa is the most widely distributed and cultivated perennial leguminous forage grass in the world. It is highly nutritious, rich in protein, carbohydrates, and vitamins, and is known as the "King of Forages." Alfalfa is an important high-quality forage for herbivorous livestock, especially dairy cows, where it is an indispensable high-protein forage, significantly contributing to improved milk quality and yield. Flowering time is a key agronomical trait affecting alfalfa yield and forage quality. Before flowering, alfalfa primarily undergoes vegetative growth, accumulating biomass continuously. After flowering, the growth center shifts to reproductive organs, vegetative growth slows, and stem lignification increases dramatically, leading to a decrease in crude protein content and fiber digestibility, severely impairing its forage quality. Therefore, precise control of flowering time is a core breeding goal for achieving synergistic improvement in alfalfa yield and quality. Although flowering regulation pathways such as photoperiod, vernalization, age, and hormones have been elucidated in the model plant Arabidopsis thaliana, the flowering regulation network of leguminous forage grasses, especially alfalfa, exhibits significant uniqueness, for example, it lacks key... FLOWERING LOCUS C ( FLC )and CONSTANS ( CO Homologous genes and the possibility that the classic photoperiodic output pathway may be reconstructed in alfalfa are among the key genes involved. Currently, the discovery of key genes regulating alfalfa flowering and the elucidation of their molecular mechanisms remain insufficient. Therefore, identifying key genes regulating alfalfa flowering and revealing the molecular mechanisms mediating the transition between vegetative and reproductive growth will not only provide new insights into the evolution and diversity of flowering regulation in leguminous plants, but also hold significant importance for the precise customization of alfalfa flowering time through molecular design breeding, the genetic improvement of forage crops, and the cultivation of high-yielding, high-quality alfalfa varieties. Summary of the Invention

[0003] Based on the aforementioned shortcomings of the existing technology, this invention clarifies for the first time... MsPPK3 As a key regulatory factor, this compound exhibits multifunctionality in coordinating the balance between vegetative and reproductive growth in alfalfa. Specifically, it positively regulates reproductive growth and negatively regulates vegetative growth. Positive regulation of reproductive growth manifests as early flowering (overexpression) and late flowering (silence). Negative regulation of vegetative growth is characterized by overexpressing plants exhibiting a slender, scattered stem type with smaller, thinner, and / or weaker leaves; while silent plants exhibit a thicker, clustered stem type with larger, thicker, and / or stronger leaves. This invention is of great significance in the breeding of different new alfalfa varieties and lines.

[0004] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One of the objectives of this invention is to provide MsPPK3 The application of genes in coordinating the balance between vegetative and reproductive growth in plants, the aforementioned MsPPK3 The CDS sequence of the gene is shown in SEQ ID NO.13.

[0005] Furthermore, the coordination of plant vegetative growth and reproductive growth balance includes positive regulation of plant reproductive growth and negative regulation of plant vegetative growth.

[0006] Furthermore, the positive regulation of plant reproductive growth includes causing the plant to flower earlier; the negative regulation of plant vegetative growth includes causing the plant to have a slender, scattered stem type, and causing the plant leaves to be smaller, thinner, and / or weaker.

[0007] The second objective of this invention is to provide a method for regulating the aforementioned MsPPK3 Primer combinations for gene expression, including overexpression of the aforementioned MsPPK3 The primers and / or silencers of the gene described MsPPK3 The primers for the gene have nucleotide sequences as shown in SEQ ID NO.7~SEQ ID NO.8 and SEQ ID NO.9~SEQ ID NO.12, respectively.

[0008] A third objective of this invention is to provide a kit containing the aforementioned primer combination.

[0009] A fourth objective of this invention is to provide a recombinant expression vector containing the aforementioned... MsPPK3 Gene.

[0010] The fifth objective of this invention is to provide a host bacterium containing the aforementioned... MsPPK3 The gene may contain the recombinant expression vector.

[0011] The sixth objective of this invention is to provide the application of the primer combination, the kit, the recombinant expression vector, and / or the host bacterium in coordinating the balance between vegetative and reproductive growth in plants.

[0012] Furthermore, the application includes its use in the breeding of new plant varieties and / or new lines, specifically including any of the following types: (1) Early flowering type; (2) Late-flowering type; (3) Slender, scattered stems; (4) Thick-stemmed, clustered type; (5) Types of blade miniaturization, thinning, and / or weakening; (6) Larger, thicker and / or reinforced blade types.

[0013] Furthermore, the plant includes alfalfa.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention clarifies for the first time MsPPK3 As a key regulatory factor, this compound exhibits multifunctionality in coordinating the balance between vegetative and reproductive growth in alfalfa. Specifically, it positively regulates reproductive growth and negatively regulates vegetative growth. Positive regulation of reproductive growth manifests as early flowering (overexpression) and late flowering (silence). Negative regulation of vegetative growth is characterized by overexpressing plants exhibiting a slender, scattered stem type with smaller, thinner, and / or weaker leaves; while silent plants exhibit a thicker, clustered stem type with larger, thicker, and / or stronger leaves. This invention is of great significance in the breeding of different new alfalfa varieties and lines. Attached Figure Description

[0015] Picture 1 In Embodiment 1 of the present invention MsPPK3 The cloning results, where: A. MsPPK3 B. CDS amplification results; C. Positive identification of single-clone colonies by PCR.

[0016] Picture 2 As in Embodiment 1 of the present invention MsPPK3 Bioinformatics analysis results, including: A. MsPPK3 Gene structure; B. MsPPK3 protein structure; C. MsPPK3 Promoter structure; D. PPKs phylogenetic analysis.

[0017] Picture 3 As in Embodiment 1 of the present invention PPKs Multiple sequence alignment results. Note: Dark blue arrow 1 and dark blue arrow 2 represent the start and stop base positions of the conserved CASEIN KINASE-RELATED (CK) domain, respectively; the amino acid fragments under the red rectangles represent ATP binding sites, the yellow rectangles represent kinase catalytic loops, the blue rectangles represent substrate recognition regions, and the green rectangles represent predicted nuclear localization signal regions.

[0018] Picture 4 As in Embodiment 1 of the present invention MsPPK3 The expression pattern analysis results include: A. MsPPK3 Tissue variability; B. Drought stress and ABA exogenous hormone induction MsPPK3 Expression analysis; C. IAA, SA and MeJA under exogenous hormone induction MsPPK3 Analysis of the expression; D. MsPPK3Subcellular localization analysis. Note: In Figure A, Main roots represent taproots, Lateralroots represent lateral roots, Mature stem represents mature stem, Young stem represents young stem, Mature leaf represents mature leaf, Young leaf represents young leaf, Mature node represents mature stem node, Young node represents young stem node, 1 Bud represents flower bud, 2 Flower bud represents flower bud, 3 Fully opened flower represents fully opened flower, 4 Wilted flower represents withered flower, 5 Young pod represents young fruit, 6 Full-size pod represents large fruit; In Figure D, Fluorescense, Chloroplast, Bright, and Merged represent fluorescence channels, chloroplast channels, bright field, and overlay maps, respectively.

[0019] Picture 5 The indicators for measuring flowering phenotype and growth and development of gene overexpression and RNA interference lines in Example 1 of this invention are as follows: A. Transgenic plants with overexpression and RNA interference MsPPK3 A. Expression level; B. Flowering time of alfalfa; C. Flowering phenotype of alfalfa after 60 days of growth; D. MsPPK3 Overexpression of the number of stem nodes at the flowering stage of alfalfa; E. MsPPK3 F. Plant height of alfalfa at flowering; G. Flowering phenotype of alfalfa at 91 days of growth; H. Number of nodes on the main stem of alfalfa at 82 days of growth; I & J. Plant height changes of alfalfa during the vegetative growth period (0-45 days) and reproductive growth period (50-80 days), using the average flowering time of OE10 as the dividing point. Note: The arrows in Figures C & F have different meanings: red represents fully open flowers, blue represents flower buds, and yellow represents withered flowers.

[0020] Picture 6The diagram shows the overall morphogenesis and organ development of the gene overexpression and RNA interference lines in Example 1 of this invention, including: A. Overall plant structure of alfalfa; B. Leaf morphology and characteristics of alfalfa; C. Stem diameter of alfalfa after 91 days of growth; D. Leaf area of ​​alfalfa after 91 days of growth; E. Number of alfalfa leaves; F. Comparison of alfalfa leaves; G. Midrib thickness of alfalfa leaves; H. Midrib thickness of alfalfa leaves; I. Anatomical structure of alfalfa leaf tissue. Note: In Figure A, the yellow arrow represents a leaf with multiple leaves; in Figure B, number 1 represents multiple leaves, and numbers 2 and 3 represent the petiole and rachis lengths of the same leaf position in each lineage; the section of ① in Figure I is taken from the longitudinal section at the blue dot in Figure F, and the section of ② is taken from the longitudinal section at the red dot in Figure F. tmv represents the midrib center thickness, lt represents the leaf thickness, ue and le represent the upper epidermis and lower epidermis, respectively, and pt and st represent the palisade tissue and spongy tissue, respectively. Detailed Implementation

[0021] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. The reagents, kits, and instruments used in the following examples are commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0022] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0023] Example 1 1 MsPPK3 Gene cloning, bioinformatics analysis, and gene expression pattern analysis 1.1 Materials and Methods 1.1.1 Test Materials "Zhongmu No. 4" alfalfa ( Medicago sativa L.Zhongmu No.4) comes from the Forage Breeding and Cultivation Innovation Team of the Beijing Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0024] 1.1.2 Analysis of gene structure and basic physicochemical properties Cloning from alfalfa MsPPK3The full-length cDNA of the gene was analyzed for gene structure using pfam online software. Multiple sequence alignment was performed using DNAMAN software; cis-regulatory elements of the target gene promoter (2000 bp upstream of ATG) were analyzed using Plantcare; and subcellular protein localization was predicted using WOLF PSORT online software.

[0025] 1.1.3 Different biological stress treatments on alfalfa This invention uses 14 tissue samples from wild-type alfalfa during the flowering period, including the taproot, lateral roots, mature stems (intervals 7 to 9 from top to bottom), young stems (intervals 1 to 2), mature stem nodes, young stem nodes, mature leaves, young leaves, different stages of flowering, and fruit, for analysis. MsPPK3 Differences in gene expression were observed. Alfalfa seeds were placed in petri dishes and germinated under light for 5 days. They were then transplanted into a hydroponic system containing 1 / 2 Hoagland nutrient solution and cultured at approximately 25°C for 4 weeks. Seedlings with consistent growth were subjected to abiotic stress treatments: drought stress treatment and ABA hormone treatment were simulated using 15% PEG and 100 μmol·L⁻¹, respectively. -1 The concentration applied for SA, IAA, and MeJA hormone treatments was 200 μmol·L⁻¹. -1 A blank control was set up. Samples were collected at eight time points after stress: 0, 3, 5, 8, 12, 24, 48, and 72 h (above-ground parts were collected for drought and ABA treatments, and mature leaves were collected for SA, IAA, and MeJA treatments) for analysis. MsPPK3 Expression patterns under abiotic stress. All test samples were stored in liquid nitrogen at -80°C.

[0026] 1.1.4 Real-time quantitative PCR (qRT-PCR) determination of expression levels Specific primers qMsPPK3-F / R were designed using the NCBI Primer-BLAST online tool (Table 1) to... MsActin As an internal reference gene, total RNA was extracted from the samples described in 1.1.3 and reverse transcribed into cDNA. The resulting cDNA was diluted 10-fold, and three biological replicates were set up for each sample. The expression level of the target gene was detected by real-time quantitative PCR (qRT-PCR). The total reaction volume was 20 μL, containing 6 μL of RNase-free ddH2O, 1 μL each of qMsPPK3-F / R, 10 μL of 2×SYBR premixEx Taq II, and 2 μL of cDNA. The PCR amplification program was set as follows: 95℃ for 30 s, 95℃ for 5 s, 60℃ for 31 s, 95℃ for 15 s, 40 cycles, 60℃ for 1 min, 95℃ for 15 s, and 60℃ for 15 s. Using 2...-ΔΔCt The relative expression level is calculated using this method.

[0027] Table 1. Details of gene cloning and quantitative primers

[0028] Note: (1) Lowercase letters in the sequence represent sequences that are homologous to the ends of the corresponding linearized vectors (pCmbia3301 overexpression vector and psmRYCE:shRNA-YFP silencing vector), while uppercase letters represent sequence fragments on the MsPPK3 gene.

[0029] (2) RNA interference requires four primers. The same target slice needs to be first reverse-linked to the vector, and then forward-linked to the vector that has already been reverse-linked, so that the vector sequence forms a hairpin structure.

[0030] 1.1.5 Subcellular localization experiment To complete the Agrobacterium-mediated transient transformation experiment in tobacco leaves, a buffer solution (1 mol·L⁻¹) for resuspending Agrobacterium was first prepared. -1 MES 1mL, 1 mol·L -1 MgCl 1 mL, 1 mol·L -1 10 μL of acetylsyleugenone was added, and the volume was adjusted to 100 mL with ddH2O. Wild-type Nicotiana benthamiana seeds were sown and cultured under light conditions (12 h light / 12 h dark) for one month at 22℃. Agrobacterium tumefaciens containing the pBWA(V)HS-MsPPK3-GLosgfp overexpression vector and the pBWA(V)HS-GLosgfp empty vector recombinant plasmid (see DOI: 10.1371 / journal.pone.0003647 and DOI: 10.1038 / nmeth.1318) were inoculated into LB liquid medium (containing Kan and Rif) and cultured at 28℃ with shaking until OD... 600 The OD value reached 0.6. Centrifuge at 5,000 rpm for 5 minutes, discard the supernatant, resuspend the bacteria in the prepared resuspension solution, and adjust the OD value. 600 The concentration was increased to 0.4, and then incubated at room temperature for 2-3 hours. Healthy tobacco plants were selected, and the treated Agrobacterium-mediated bacterial solution was injected into the lower epidermis of the leaves using a 1 mL syringe (without the needle). The injection site was marked. The treated plants were then cultured under low light conditions for 48 hours before observation. Leaf tissue from the marked area was excised to prepare temporary slides, and the subcellular localization signals and corresponding images of the pBWA(V)HS-MsPPK3-GLosgfp fusion protein were observed and acquired under a laser confocal microscope.

[0031] 1.2 Results and Analysis 1.2.1 Analysis of gene structure and basic physicochemical properties Using the F1 population from a cross between a late-flowering phenotype alfalfa plant ('Zhongmu 1' alfalfa) and an early-flowering phenotype alfalfa plant (Cangzhou alfalfa), a flowering-related QTL was located using genetic linkage mapping. A candidate gene related to flowering time regulation was identified by comparing the QTL interval with a reference genome. The amino acid sequence encoded by this gene was then compared with the Arabidopsis protein database using BLAST, revealing that this gene is associated with Arabidopsis flowering time regulation genes. Photoregulatory Protein Kinase 3 ( PPK3 Since they are highly homologous, they are named as follows: MsPPK3 .

[0032] MsPPK3 The gene was cloned from alfalfa, and its genome number is [genome number missing]. Msa1118850 ( Picture 1 ) 。 Sequence analysis showed that the gene was 6761 bp in length, containing 16 exons and 15 introns, with a predicted maximum open reading frame length of 2127 bp. Picture 2 A). Analysis of the predicted protein indicates that it encodes a polypeptide of 709 amino acids, containing a conserved CASEIN KINASE-RELATED domain (CK domain) between amino acids 147 and 679, with InterPro accession number IPR050235. Picture 2 B). Using PlantCARE for MsPPK3 Cis-acting element analysis of the upstream 2000bp promoter revealed 10 light-responsive elements, including Box4, GT1-motif, and I-box; two drought-induced MYB binding sites (MBS); and two TCA-elements, three CGTCA-motifs, three ABRE elements, and one TGA-element. These elements are cis-acting elements involved in salicylic acid (SA), jasmonic acid (MeJA), abscisic acid (ABA), and auxin (IAA) responses, respectively. Picture 2 C). On plants PPKs Analysis of protein evolutionary relationships indicates that plants PPKs Divided into two main branches (Branch I and II), MsPPK3 belongs to Class II along with AtPPK3, AtPPK2, and OsHD16, which have undergone functional verification, and is relatively far removed from Class I AtPPK1 and AtPPK4. Picture 2 D). Multiple sequence alignment indicates that, MsPPK3 Amino acid sequence and alfalfa ( Medicago truncatulaThe predicted protein gene 42063 showed the highest homology (99.72%), followed by pea protein. Pisum sativum Predicted protein Psat3g088920 and soybean ( Glycine max Predicted protein GLYMA_16G041500 (all numbers are from the EnsemblPlants website). MsPPK3 It possesses conserved CK functional domains, including a substrate recognition region, a kinase catalytic loop, an ATP binding site, and a predicted nuclear localization signal. Picture 3 ).

[0033] A MsPPK3 CDS sequence >MsaPPK3-CDS (Msa1118850) SEQ ID NO.13: B MsPPK3 protein sequence >MsaPPK3-AA (Msa1118850) SEQ ID NO.14: MPELRSGARRSKRLGDLQPGPQPVDQGENWAEPAQNRTRRRVGGGRGRGGNATGLGKGSSPAVPTRRTAAGRGRGARLIDLDPQPCDLLPEPVALRAQEPVYNNVEVVANNNIAMEGGSGDKGVAAEEDASTTPVPERVQVGNSPVYKTERKLGKGGFGQVYVGRRVSGGSDRVGPD AIEVALKFEHRNSKGCNYGPPYEWQVYSNLNGCYGIPGVHYKGRQGDFYILVMDILGPSLWDVWNSLGQSMSPSMAACIAVEAISILEKLHLKGFVHGDVKPENFLLGQPGTADDKKLYLIDLGLASKWKDASSGQHVEYDQRPDIFRGTIRYASVHAHLGRTGSRRDDLESLAYTL IFLIKGRLPWQGYQGDNKSFLVCKKKMSTSAELMCCFCPAPFKLFLEAVTNMRFDEEPNYSKLISLFDSLIEPCTPLRPIRIDGALKVGQKRGRMLINLEEDEQPKKKVRLGSPATQWISVYNARRPMKQRYHYNVADNRLRQHVDKGIEDGLYISCVASSANLWALIMDAGTGFSS QVYELSPAFLHKDWIMEQWEKNYYISSIAGAVNGSSLVVMSKGTPYTQQSYKVSESFPFKWINKKWKEGFHVTSMTTAGSRWGVVMSRNAGYSDQVVELDFLYPSEGIHRRWENNYRITSMAATNDQAAFILSIPKRKLLDETQETLRTSAFPSTHVKEKWSKNLYIASICYGRTVC 1.2.2 MsPPK3 Gene expression pattern analysis qRT-PCR was used to detect the presence of [agents] in 14 tissues of 3-month-old alfalfa. MsPPK3 The expression levels of the gene were analyzed, and the results showed that the gene was expressed in all tested tissues, exhibiting significant tissue specificity: the highest expression level was found in fully open flowers, followed by flower buds, and the lowest expression level was found in withered flowers. Picture 4A). Detection results using cDNA from fully open flowers as a template showed that the internal reference gene... MsActin The average Ct value is 22.50, while MsPPK3 The Ct value was 24.76, indicating that the gene maintained a high basal expression level in the dominant expression tissue.

[0034] Given MsPPK3 The promoter region contains multiple cis-acting elements for abiotic stress responses. Further analysis was conducted to determine its response characteristics to ABA and drought (simulated with 15% PEG). The results showed that... MsPPK3 Expression levels showed significant dynamic changes over treatment time: the ABA-treated group reached its peak expression at 5 h, while the PEG-simulated drought-treated group reached its peak at 8 h. Both treatments showed significant induction effects, with subsequent expression levels slightly decreasing but remaining at a high level. In contrast, the control group (CK) remained at a low expression level throughout. Picture 4 B). The above results indicate that MsPPK3 Its expression can be induced by drought and ABA, and the response to ABA signaling is more rapid, suggesting that it may be involved in the early stress resistance response process of alfalfa. Further analysis MsPPK3 The results of responses to different hormones (IAA, SA, MeJA) showed that, compared with the CK group, the IAA treatment group peaked at 72 h and was significantly higher than other treatment groups; the SA treatment group showed only a slight upregulation at 5 h; and the MeJA treatment group showed no significant change in expression. Picture 4 C). Indicates MsPPK3 The induced response to IAA was significantly stronger than that to SA and MeJA, while the response to the latter two was weaker.

[0035] To clarify MsPPK3 Subcellular localization of the protein was investigated by transiently expressing the constructed pBWA(V)HS-MsPPK3-GLosgfp fusion expression vector in the epidermal cells of *Nicotiana benthamiana* leaves. Fluorescence signal observation revealed that the fluorescence signal of the pBWA(V)HS-MsPPK3-GLosgfp fusion protein was concentrated in the nucleus region; while the fluorescence signal of the empty vector pBWA(V)HS-GLosgfp was widely distributed throughout the cell. Picture 4 D). This positioning result is consistent with the prediction result of the WOLF PSORT online software, suggesting... MsPPK3 It may perform biological functions within the cell nucleus.

[0036] 2 MsPPK3 Functional Analysis 2.1 Materials and Methods 2.1.1 Test Materials "Zhongmu No. 4" alfalfa.

[0037] 2.1.2 Constructing a Carrier alfalfa MsPPK3 The gene was amplified from a cDNA sample by PCR and cloned into the pENTR / D-TOPO cloning vector. Positive clones with correct sequencing results were transformed and transferred into the pCmbia3301 overexpression vector to obtain the desired gene. 3 The 5Spro::MsCIB2 constructor. MsPPK3 A 500 bp non-conserved fragment from the gene (amplified as a silencing fragment) was inserted into the modified shRNA-YFP silencing vector psmRYCE:shRNA-YFP, yielding the 35Spro::500bp-MsPPK3 construct. The vector construction method can be found in Zhang Lili's article on alfalfa. MsCIB2 Functional analysis of flowering regulation [D]. Chinese Academy of Agricultural Sciences, 2024. DOI:10.27630 / d.cnki.gznky.2024.000201. (The methods mentioned in the original text are not translated.)

[0038] 2.1.3 Genetic transformation of alfalfa Alfalfa seeds were surface-sterilized in a 10% (v / v) sodium hypochlorite solution for 6 minutes, then rinsed five times with pure water and germinated in an aeroponic incubator (16 hours light / 8 hours dark, 60% humidity). After 7 days, uniform seedlings were transplanted into Hoagland nutrient solution, with the pH maintained at 5.95. The solution was changed weekly. After 3 weeks of hydroponics, the alfalfa seedlings were planted in soil. The constructed overexpression vector plasmid (35Spro::MsPPK3) and low-expression vector plasmid (35Spro::500bp-MsPPK3) were introduced into Agrobacterium EHA105. Alfalfa leaves after 2 weeks of soil cultivation were collected using Agrobacterium-mediated transformation for alfalfa genetic transformation experiments.

[0039] 2.2 Results and Analysis 2.2.1 MsPPK3 Functional analysis of regulating macroscopic growth and development of alfalfa To clarify MsPPK3 This invention relates to the regulatory role of alfalfa in the growth and development of alfalfa, and obtained alfalfa through genetic transformation. MsPPK3 Gene overexpression lines (OE10, OE30) and RNA interference lines (Ri16, Ri17). qRT-PCR analysis confirmed that OE10 and OE30... MsPPK3 The expression levels were 2.04-fold and 1.91-fold higher than those in wild-type (WT), respectively, while the expression levels in Ri16 and Ri17 were significantly downregulated. Picture 5 A).

[0040] 2.2.1.1 MsPPK3Positive regulation of alfalfa flowering time Under standard growth conditions (16 h light / 8 h dark, 25℃ and 60% humidity), gene expression levels were positively correlated with flowering time. The flowering time of the overexpression lines (OE10 52.3 days, OE30 56.7 days) was significantly earlier than that of the WT lines (75.71 days) by 19.01–23.41 days, while neither of the two interference lines flowered during the 130-day growth period. Picture 5 B&C&F). This indicates MsPPK3 It is a positive regulator of flowering induction in alfalfa.

[0041] 2.2.1.2 MsPPK3 Controlling vegetative growth and developmental transition in alfalfa Further phenotypic analysis revealed that this gene primarily regulates developmental processes by controlling the number of stem nodes. At flowering (appearance of the first small purple flower), the overexpressing lines had significantly fewer stem nodes than the WT line, but no significant difference in plant height. Picture 5 D&E). In the standardized assay at day 82, the overexpressing lines still had fewer stem nodes than the WT lines, while the interference lines had significantly more stem nodes than the WT lines. Picture 5 G). The above results indicate that improving MsPPK3 Expression of OE10 shortens the vegetative growth phase, prompting plants to shift to reproductive growth after accumulating fewer stem nodes, thus causing alfalfa to flower earlier; loss of OE10 function leads to an abnormally prolonged vegetative growth period. In the later stages of growth (91 days), the number of primary branches increased in OE10 overexpressing lines, while there was no difference between the interference lines and the WT lines. Picture 5 E). This indicates that an increase in the number of branches is not necessarily... MsPPK3 The direct regulatory effect of the gene is more likely an indirect consequence of its promotion of early flowering, leading to premature loss of apical dominance. In conclusion, MsPPK3 It may have the function of regulating the duration of vegetative growth in alfalfa.

[0042] 2.2.1.3 MsPPK3 Coordinating the transition between nutrient and reproductive growth in alfalfa Continuous monitoring of plant height revealed that during the vegetative growth period (20-50 days), the OE10 plant height increased significantly faster than the WT plant, laying the foundation for early flowering; however, growth was inhibited in the interference lines. Picture 5 I). After entering the reproductive growth stage, the early-flowering line (OE10) maintained its vegetative growth advantage, while the interference line that never flowered showed compensatory growth, and its plant height eventually caught up with the WT (Wt). Picture 5 J). This indicates MsPPK3 It may coordinate the processes of nutrition and reproductive growth.

[0043] 2.2.2 MsPPK3Functional analysis of regulating the microscopic growth and development of alfalfa On the 91st day of plant growth, the plants were harvested for observation and index measurement. Different transgenic lines showed significant differences in overall plant type, stem structure, leaf morphology and anatomical structure.

[0044] 2.2.2.1 MsPPK3 Affecting the overall plant type and stem structure of alfalfa In terms of overall plant type, the overexpression lines (OE10, OE30) exhibited a "slender-stemmed, scattered" type with thin, weak stems and fine, outward-spreading branches, resulting in a loose plant type; the wild type (WT) was a "balanced, upright" type with evenly distributed stems and upright growth; while the RNA interference line (Ri17) showed a "thick-stemmed, clustered" type with robust stems and compact, clustered branches. Picture 6 A). Vernier caliper measurements of stem diameter revealed that the average stem diameter of the overexpression lines (OE10: 1.55 mm, OE30: 1.59 mm) was significantly thinner than that of the WT line (1.81 mm), while the stem diameter of Ri17 (2.07 mm) was significantly thicker than that of the WT line (…). Picture 6 C). This indicates that MsPPK3 Overexpression may induce early flowering, but it may also weaken the secondary growth or lignification process of the stem, leading to a decrease in mechanical support; conversely, functional inhibition may enhance the construction of vegetative organs.

[0045] 2.2.2.2 MsPPK3 Influence on leaf morphology and characteristics of alfalfa Regarding leaf characteristics, MsPPK3 The impact on gene expression was also significant. The disrupted lines exhibited the trait of multiple leaves (Ri16 average 6.3 leaves, Ri17 average 7.7 leaves), with Ri17 having the largest leaf area (3.48 cm²). 2 ), significantly larger than WT (2.39 cm). 2 ) and OE10 (1.24 cm) 2 () Picture 6 B&D&E). The petiole and rachis lengths at the same leaf position differed significantly among WT, OE10, and Ri17, with Ri17 having the longest, followed by WT, and OE10 the shortest, consistent with the trend in leaf size. Picture 6 B). In terms of leaf structure, Ri17 leaves were significantly thicker (203.98 μm), dark green in color, with strong veins and distinct serrations on the leaf margins; WT indicators were moderate; while the overexpression lines had significantly thinner leaves, lighter green in color, weaker veins, and rounded leaf margins. Picture 6 F&H). These gradient differences indicate that suppression MsPPK3 Expression may promote cell expansion, tissue differentiation, and potential accumulation of pigments (such as chlorophyll) in leaves, thereby enhancing vegetative growth; while overexpression has the opposite inhibitory effect.

[0046] Microscopic anatomical structures supported the aforementioned macroscopic phenotypes. Sectional observation revealed that the midrib thickness and total leaf thickness of the overexpressing lines were significantly thinner than the WT lines, while the interference lines were significantly thicker than the WT lines. Picture 6 G). Further observation showed that the mesophyll tissue (palisade and spongy tissue) of Ri17 was thicker, and its epidermal cells were also significantly larger than those of WT and OE10. Picture 6 G&I). This is confirmed at the cellular and tissue levels. MsPPK3 Genes negatively regulate the swelling of leaf cells and the differentiation and development of tissues.

[0047] In summary, during the later stages of vegetative growth, MsPPK3 Genes play a crucial switching role in allocating growth and development resources: high expression of genes drives plant resources towards reproductive growth, leading to weakened development of vegetative organs; low expression, on the other hand, ensures continuous investment of resources in vegetative growth, resulting in a more robust plant structure. This systematic morphological and anatomical evidence, corroborated by previously observed developmental phenotypes such as flowering time and number of stem nodes, collectively elucidates... MsPPK3 As a key regulatory factor, it plays a multifunctional role in coordinating the balance between vegetative and reproductive growth in alfalfa.

[0048] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. MsPPK3 The application of genes in coordinating the balance between vegetative and reproductive growth in plants is characterized by, The MsPPK3 The CDS sequence of the gene is shown in SEQ ID NO.

13.

2. The application according to claim 1, characterized in that, The coordination of plant vegetative growth and reproductive growth balance includes positive regulation of plant reproductive growth and negative regulation of plant vegetative growth.

3. The application according to claim 2, characterized in that, The positive regulation of plant reproductive growth includes making the plant flower earlier; the negative regulation of plant vegetative growth includes making the plant shape slender and scattered, and making the plant leaves smaller, thinner and / or weaker.

4. A device for regulating the control described in claim 1 MsPPK3 Primer combinations for gene expression, characterized in that, Including overexpression of the above MsPPK3 The primers and / or silencers of the gene described MsPPK3 The primers for the gene have nucleotide sequences as shown in SEQ ID NO.7~SEQ ID NO.8 and SEQ ID NO.9~SEQ ID NO.12, respectively.

5. A reagent kit, characterized in that, It contains the primer combination described in claim 4.

6. A recombinant expression vector, characterized in that, It contains the contents of claim 1 MsPPK3 Gene.

7. A host bacterium, characterized in that, It contains the contents of claim 1 MsPPK3 The gene or contains the recombinant expression vector as described in claim 6.

8. The application of the primer combination of claim 4, the kit of claim 5, the recombinant expression vector of claim 6, and / or the host bacterium of claim 7 in coordinating the balance between vegetative and reproductive growth in plants.

9. The application according to claim 8, characterized in that, The application includes the development of new plant varieties and / or new lines, specifically including any of the following types: (1) Early flowering type; (2) Late-flowering type; (3) Slender, scattered stems; (4) Thick-stemmed, clustered type; (5) Types of blade miniaturization, thinning, and / or weakening; (6) Larger, thicker and / or reinforced blade types.

10. The application according to claim 9, characterized in that, The plants mentioned include alfalfa.

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