Application of EsPTI2 gene in regulating plant leaf number

Abstract

This invention provides EsPTI2 The application of genes in regulating plant leaf number falls under the field of biotechnology. This invention is the first to discover that overexpression of genes in plants... EsPTI2 The gene can increase the number of leaves in rice and wheat, promote the synthesis of more carbohydrates and other nutrients, provide support for plant growth and development, and thus help biomass accumulation and yield increase. It fills the technical gap in high-yield plant type improvement of this species, provides a theoretical basis and technical reference for molecular design breeding of other grass forage grasses, and promotes the upgrading of forage grass breeding from traditional phenotypic selection to precision molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically... EsPTI2 Application of genes in regulating the number of plant leaves. Background Technology

[0002] Old Mango ( Elymus sibiricus L. Leymus chinensis is a perennial high-quality forage grass belonging to the genus Leymus in the family Poaceae. It possesses extremely strong resistance to cold, drought, and poor soil conditions, and has a very wide range of ecological adaptability. It occupies an irreplaceable position in the sustainable development and ecological security construction system of herbivorous animal husbandry, and its application prospects are very broad.

[0003] The core value of *Lao Mang Mai* (a type of wild wheat) is concentrated in two dimensions: diversified industrial development and ecological-economic synergy. At the industrial level, *Lao Mang Mai* is highly palatable and nutritious, making it a core forage crop in arid and high-altitude (especially the Qinghai-Tibet Plateau) regions. Through variety improvement and optimized cultivation and management techniques, it can continuously provide a stable and sufficient supply of high-quality roughage for livestock such as cattle and sheep, contributing to the large-scale and standardized development of grassland animal husbandry in arid and high-altitude areas. At the ecological level, *Lao Mang Mai* has a well-developed root system and strong soil-fixing ability, possessing excellent water and soil conservation and slope protection properties. It is an indispensable pioneer species in major ecological projects such as degraded grassland ecological restoration, comprehensive water and soil erosion control, mine reclamation, and the construction of ecological barriers in alpine grasslands. It can effectively restore fragile ecosystems, curb grassland degradation and desertification, and achieve a win-win situation for both ecological protection and industrial development.

[0004] With the rapid development of molecular biology and molecular breeding technologies, traditional phenotypic selection breeding is gradually transforming into precision molecular breeding. Breeding new rice and wheat varieties that combine high yield, high quality, strong stress resistance, and wide adaptability has become a core driving force for upgrading these two major crop industries and breaking through production capacity bottlenecks. From a strategic perspective, forage grass farming is an important vehicle for implementing the "grand food concept." Vigorously developing the high-quality forage grass industry, represented by wheat, and efficiently converting forage resources into high-quality meat, eggs, milk, and other animal-derived foods is a key path to broaden food supply channels, optimize dietary structure, and ensure food security. From the perspective of ecological sustainable development, scientifically promoting the planting of high-quality native forage grasses such as wheat can not only restore damaged ecological environments and build a solid ecological security barrier, but also drive farmers and herdsmen to increase their income and become wealthy. From the perspective of actual industrial development needs, a stable and efficient forage grass supply system is the foundation for reducing costs and increasing efficiency in animal husbandry and enhancing core market competitiveness, directly related to improving the livelihoods of people in border areas and rural areas, and the sustainable prosperity of the regional economy.

[0005] Leaves are the core organs of plants for photosynthesis and the synthesis of organic nutrients. Leaf number is a key agronomical trait determining aboveground biomass accumulation, and the two are closely and complexly correlated positively. From a physiological perspective, under suitable environmental conditions such as water, light, and CO2, a greater number of leaves means a larger effective photosynthetic area of ​​the plant canopy, higher light capture and utilization efficiency, and the ability to drive stronger photosynthesis. This results in the continuous synthesis of more carbohydrates such as sugars and starches, providing sufficient photosynthetic products for the growth and development of aboveground organs such as stems and leaves, directly promoting the rapid accumulation of aboveground biomass. In the long-term breeding and cultivation practices of major crops such as rice, wheat, corn, and soybeans, precisely controlling leaf number and constructing a reasonable and compact canopy structure have always been core breeding goals for improving crop photosynthetic efficiency, optimizing population structure, and increasing economic and biological yields. Related trait improvements have yielded significant increases in production and efficiency.

[0006] Currently, there has been some progress in gene regulation research on traits such as leaf morphology, leaf angle, and leaf area in rice. However, the discovery, functional analysis, and breeding application of key genes for precise regulation of leaf number still need to be deepened. As an important native forage grass, the genetic breeding research of wheatgrass mainly focuses on conventional traits such as stress resistance, grain shattering, and yield. The molecular breeding foundation is relatively weak. In particular, the key regulatory genes controlling leaf number have not been systematically discovered, cloned, and applied. There is a lack of effective technical pathways to improve leaf number traits and increase biomass through molecular means.

[0007] In summary, identifying and efficiently utilizing key functional genes regulating leaf number in rice and wheatgrass, and using molecular methods to directionally regulate leaf development and optimize leaf number configuration, can further optimize plant architecture and canopy structure, improve photosynthetic efficiency and grain yield in rice, thus contributing to high-yield and stable grain crop breeding. For wheatgrass, it can precisely increase aboveground biomass and forage yield while preserving its excellent stress resistance, strengthening its dual value in ecological restoration and forage production, and promoting the high-quality development of the forage industry. Therefore, conducting research on the discovery, functional verification, and breeding applications of genes regulating leaf number in rice and wheatgrass has both significant theoretical research value and strong industrial practical significance. It can provide core gene resources and technical support for the molecular breeding improvement of these two crops, making up for the shortcomings of existing breeding technologies. Summary of the Invention

[0008] This application aims to solve the problems existing in the above-mentioned background art, and the present invention provides... EsPTI2 The application of genes in regulating the number of plant leaves is achieved through overexpression in plants. EsPTI2 Genes are used to increase the number of leaves;

[0009] in, EsPTI2The nucleotide sequence is shown in SEQ ID NO.1;

[0010] The plant in question is either rice or old wheat.

[0011] Of course, including EsPTI2 Gene-related biological materials also have the function of regulating the number of plant leaves, which is achieved by overexpressing gene-related genes in plants. EsPTI2 This is achieved by using genes to increase the number of leaves;

[0012] The biomaterial is any one of B1)-B5):

[0013] B2) contains the contents of claim 1 EsPTI2 Gene recombination vectors;

[0014] B3) contains the contents of claim 1 EsPTI2 Recombinant microorganisms;

[0015] B4) A recombinant vector containing the expression cassette described in B1);

[0016] B5) Recombinant microorganisms containing the recombinant vector described in B2);

[0017] in, EsPTI2 The nucleotide sequence is shown in SEQ ID NO.1;

[0018] The plant in question is either rice or old wheat.

[0019] Furthermore, the present invention also provides a method for preparing plants with high leaf counts, the method comprising overexpressing in the plant EsPTI2 The steps of gene generation;

[0020] in, EsPTI2 The nucleotide sequence is shown in SEQ ID NO.1;

[0021] The plant in question is either rice or old wheat.

[0022] Furthermore, constructing a system containing the aforementioned... EsPTI2 Gene recombination vectors are used to store the gene. EsPTI2 The gene was inserted into the P1300-35S-C-Myc overexpression vector plasmid using primers P1300-35S-C-Myc-F and P1300-35S-C-Myc-R as shown in SEQ ID NO. 3-4. X baIand K Obtained between pnI sites.

[0023] Furthermore, the above EsPTI2 Genes, biomaterials, and methods for producing high-yield plants can be applied to the following areas:

[0024] Through overexpression EsPTI2 This is achieved through gene expression levels:

[0025] A1) Applications in the targeted regulation of canopy structure of monocotyledonous plants and improvement of canopy light energy utilization efficiency;

[0026] A2) Application as a functional molecular marker in marker-assisted breeding of traits related to leaf number and biomass in monocotyledonous plants;

[0027] A3) Application in cultivating new monocotyledonous plant varieties with both high biomass and strong stress resistance in high-altitude, arid, and adverse environments;

[0028] A4) Applications in constructing core parental materials for high light efficiency breeding of monocotyledonous plants, improving varieties, and broadening the genetic basis of high-yield breeding germplasm resources;

[0029] A5) Applications in breeding new varieties of compact monocotyledonous food crops and forage crops suitable for high-density planting and improving planting efficiency per unit area;

[0030] The plant in question is either rice or old wheat.

[0031] In summary, this technical solution exhibits several important and beneficial effects: This invention is the first to discover that through precise control... EsPTI2 The expression level of this gene can significantly increase the number of rice leaves, prompting the rice to synthesize more carbohydrates and other nutrients, providing support for plant growth and development, and thus contributing to biomass accumulation and yield increase. This technology can also be applied to wheatgrass, which can effectively improve the plant structure of wheatgrass, further increase the yield per plant, achieve efficient accumulation of aboveground biomass, fill the technical gap in high-yield plant structure improvement of this species, provide a theoretical basis and technical reference for molecular design breeding of other gramineous forage grasses, and promote the upgrading of forage grass breeding from traditional phenotypic selection to precision molecular breeding. Attached Figure Description

[0032] Figure 1 In Example 1 EsPTI2 The relative expression levels of genes in different tissues at different stages of *Oryza sativa* (a type of wheat);

[0033] Figure 2 In Example 2 EsPTI2 Structural diagram of gene overexpression vector;

[0034] Figure 3 In Example 2 EsPTI2 Image showing the results of RNA level identification in gene-overexpressing plants;

[0035] Figure 4 In Example 3 EsPTI2Phenotypic results of transgenic rice plants with overexpressed genes. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The bioinformatics software and products used in the present invention are all commercially available. Various processes and methods not described in detail use conventional methods known to those skilled in the art. The source of materials used, trade names, and components that need to be listed are indicated when they first appear. Unless otherwise specified, the same reagents used thereafter are the same as those initially indicated.

[0037] Example 1 EsPTI2 Acquisition of genes

[0038] This invention has discovered a new gene. EsPTI2 This was obtained by analyzing and comparing the phenotypic differences in the number of blades in extreme materials, as detailed below:

[0039] We planted a population of collected and preserved wild germplasm resources of *Eriocaulon buergerianum*. Based on field data analysis of leaf number, we identified two extreme materials with significantly different leaf numbers: YS-039 (high leaf number) and YS-258 (low leaf number). Transcriptome analysis was performed on the flag leaves of these two materials. After data quality control analysis, DEGs and DEPs screening, GO enrichment, and KEGG pathway analysis, we found 17 genes with significantly different expression, which were significantly correlated with meristematic tissue development, cell division activity, and leaf primordium initiation. Finally, we selected the gene with the most significant expression differences. EsPTI2 Gene.

[0040] The EsPTI2 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the specific sequence is as follows:

[0041] ATGGAGCACCTAGCTAAGTGCAGGGGCATCCCAGCTTGGGCGGCGGCGTACACCTGCGATCCGTCTTCGTCCTCATGGGAGGAGCAGGCCTTCGCACGCGATGCCGCGGCTGACCTGCTGGTGTGGCCGCCGCGCTCCTACTCCTGCACCTTCTGCCGCCGCGAGTTCCGCTCGGCCCAGGCGCTCGGCGGCCACATGAATGTGCACCGCCGAGACAGGGCACGTCTCCGTCATGGCGACGACGACCAAGCCCAACAAGAACAAGCTGATGAACCGCACCAGGACGACTACTCTACCAGTACCATGTACAAGCAGCTAGAGCTTTTCCGTAACCCTAGTACCACCACTCCTTCCTGCCTATCAACCATCAGCAAGGAGAGGAATAACAACAAGGTCGTCGTCTCCATAGCGGCGAGAGATCAAGAAGGTACAGATCACCAGTACGATGACAGCCAGGATTTGGAAATGTCGGAGAGGAGGAAGAGGAGGCGTGTAGATCAGGCGCCGTGGGTGGCCGCTGCCTCGCCATACGAACAAGGGGCATTCCTTGATTATTCAAAGGTAAATATAAAACCAATCACAAACCGTAGTAGCTCTAGTCTTAATCCCCATGTGGATCAAGGAGAAGTAGATCTGTTCCTCAAGAAACAAGGACTGGTAGATCTAGAGCTTAGGCTTGGGACTAATCCAAATGTTGCATCACACACAACTTAA。[[ID=‎1]] [[ID=‎2]]

[0042] [[ID=‎3]] EsPTI2 [[ID=‎4]]The encoded protein sequence is shown in SEQ ID NO.2: [[ID=‎5]] [[ID=‎6]]

[0043] MEHLAKCRGIPAWAAAYTCDPSSSSWEEQAFARDAAADLLVWPPRSYSCTFCREFRSAQALGGHMNVHRRDRALRHGDDDQAQQEQADEPHQDDYSTSTMYKQLELFRNPSTTTPSC LSTISKERNNNKVVVSIAARDQEGTDHQYDDSQDLEMSERRKRRRVDQAPWVAAASPYEQGAFLDYSKVNIKPITNRSSSLNPHVDQGEVDLFLKKQGLVDLELRLGTNPNVASHTT.

[0044] The inventors selected the expression differences that were most significant. EsPTI2 The genes have undergone extensive analysis, experiments, and research. The following are representative examples that clearly demonstrate their effectiveness. EsPTI2 Part of the research experiment on the key role of old wheat in the growth process.

[0045] Example 2: Spatiotemporal Expression Pattern Analysis Experiment of Old Mango (Ophiopogon japonicus) Genes

[0046] 1. Obtaining cDNA from different tissue samples of *Eriocheir sinensis*

[0047] Seeds of variety "YS-039" were sown in seedling trays and cultured in a greenhouse. Multiple tissue samples were collected from *Eriobotrya stenoptera* from the seedling stage to the milk stage, including the seedling stage, early tillering stage, mid- and late tillering stage, early and late jointing stage, booting stage, heading stage, flowering stage, and milk stage. These tissue samples included roots, stems, leaves, and ears. All collected tissue samples were flash-frozen in liquid nitrogen and stored at -80℃ for later use. RNA was extracted from these tissues using the AFT Spin Universal Plant Fast RNA Extraction Kit (Wuhan Aibotek Biotechnology Co., Ltd.). RNA quality was assessed by agarose gel electrophoresis, and RNA quantity was determined by Nanodrop. cDNA was then synthesized using the EX RT kit reverse transcription kit (Wuhan Aibotek Biotechnology Co., Ltd.). The reverse transcription cDNA system is shown in Table 1. The reverse transcription conditions were 45℃ for 2 min, 50℃ for 15 min, and 85℃ for 2 min, inactivating the reverse transcriptase. This cDNA was diluted 2-fold to serve as a template, yielding cDNA samples from each of the aforementioned tissue stages.

[0048] Table 1. Reverse transcription cDNA system

[0049]

[0050] 2. Old Mango Wheat EsPTI2 Gene expression pattern analysis

[0051] Based on the old mangoes already obtained EsPTI2 Gene sequence information was used to design quantitative real-time primers EsPTI2-RT-F and EsPTI2-RT-R using AlleleID 6.0. Using cDNA from different tissues of *Evodia rutaecarpa* obtained in step 1 as templates, qRT-PCR amplification was performed using 2×HQSYBR qPCR Mix (LoW ROX) reagents and an ABI Q7 quantitative real-time PCR instrument. The quantitative real-time PCR amplification program was: 95℃, 30s, 40 cycles, each cycle consisting of 95℃, 10s, 60℃, 30s, and 72℃, 30s. Each experiment contained three biological replicates. The relative expression level of genes is calculated using this method. Method calculation EsPTI2 The relative expression levels of genes in different tissues of *Leymus chinensis* are analyzed as follows: Figure 1 As shown in Table 2, the quantitative fluorescence system is as follows. The sequence of EsPTI2-RT-F is ACGACCAAGCCCAACAAGAA, and the sequence of EsPTI2-RT-R is GATCTCTCGCCGCTATGGAG.

[0052] according to Figure 1 The analysis results show that EsPTI2 Significant tissue-specific expression was observed at different developmental stages, exhibiting marked dynamic changes throughout the growth process. It peaked during the late tillering stage, then gradually declined from the jointing to the milk stage. Regarding tissue specificity, the expression differences were most significant during the late tillering stage, with the highest relative expression level in roots, followed by leaves, while the expression levels in stems and panicles were lower. As the growth process entered the reproductive growth stage (heading stage to milk stage), the expression differences among tissues gradually decreased, but roots and leaves maintained relatively high expression levels throughout. These results indicate that... EsPTI2 Genes may play an important role in the regulation of leaf physiological functions, especially during the vigorous growth stage of crops.

[0053] Table 2. Quantitative Fluorescence System

[0054]

[0055] Example 3 EsPTI2 Construction of gene overexpression vectors

[0056] 1. Old Mango Wheat EsPTI2 Cloning of genes

[0057] According to the old mangoes obtained EsPTI2RNA was extracted from *Gnaphalium affine* material “YS-039” using CDS-F and CDS-R primers designed based on gene sequence information. PCR amplification was performed using reverse-transcribed cDNA as a template. The PCR reaction system is shown in Table 3. After amplification, agarose gel electrophoresis was performed, and the amplification products were recovered and sequenced. EsPTI2 The genomic sequence and cDNA sequence of the gene were obtained. The CDS-F sequence is ATGGAGCACCTAGCTAAGTG, and the CDS-R sequence is TTAAGTTGTGTGTGATGCAA. PCR amplification program: 97℃, 3 min; 97℃, 15 s; 58℃, 30 s; 72℃, 1 min 30 s, 35 cycles; extension at 72℃ for 5 min.

[0058] PCR products were subjected to 1% agarose gel electrophoresis at 180V for 10 min. This was based on whole-genome data from *Leymus chinensis*. EsPTI2 Gene sequence was obtained, and gene-specific primers were designed. Using *Gnaphalium affine* cDNA as a template, PCR amplification was performed using a high-fidelity enzyme. After TA cloning and colony PCR detection, the colony PCR bands were verified to be consistent with the expected size. Sequencing of the positive colonies showed that... EsPTI2 The CDS region is 714 bp in length, and the sequence is correct, indicating that the correct sequence was obtained. EsPTI2 TA plasmid.

[0059] Table 3 PCR reaction system

[0060]

[0061] 2. Overexpression EsPTI2 Construction of transgenic vectors

[0062] Use restriction endonucleases Xba I and Kpn The plant overexpression vector P1300-35S-C-Myc (purchased from Beijing Coollab Technology Co., Ltd., product number: VT172) was double-digested with enzymes, and simultaneously amplified using primers P1300-35S-C-Myc-F and P1300-35S-C-Myc-R. EsPTI2- TA plasmids, to obtain those containing vector arms EsPTI2 The CDS region of the gene was extracted, and the vector and fragment were recombined using the SESeamless Cloning and Assembly Kit. The recombinant product was transformed into DH5α competent E. coli cells, and overexpression was confirmed by sequencing. EsPTI2 The transgenic vector P1300-35S-C-Myc- EsPTI2 Its structure is as follows Figure 2 As shown.

[0063] The sequence of P1300-35S-C-Myc-F is gagaacacgggggatctagaATGGAGCACCTAGCTAAGTG (SEQ ID NO.3), and the sequence of P1300-35S-C-Myc-R is acaggcctttcgaaggtaccTTAAGTTGTGTGTGATGCAA (SEQ ID NO.4).

[0064] 3. Overexpression EsPTI2 Construction of transgenic lines

[0065] The above-mentioned carrier P1300-35S-C-Myc- EsPTI2 Electroporation transformation of EHA105 Agrobacterium competent cells was performed, and the cells were plated on LB medium containing 25 mg / L Rifampicin and 50 mg / L Kanamycin, and cultured at 28°C for 2 days. Colonies positive by colony PCR were stored in glycerol at −80°C. Mature rice seeds (Nipponbare) were used... Rice L. spp. japonicaAfter dehulling, the callus was soaked in 70% ethanol for 1 min, then in 30% NaClO + 2 drops of Tween-20 for 20 min, rinsed with sterile water, and inoculated onto N6 medium (Beijing Cooler Master Technology Co., Ltd.) containing 2 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D). It was incubated in the dark at 28°C for 21 days. Naturally dividing callus tissue was transferred to fresh induction medium and subcultured for 14 days before transformation. Agrobacterium was resuspended in AAM liquid medium (Beijing Cooler Master Technology Co., Ltd.) containing 100 μM acetosyringone (AS), and the OD600 was adjusted to 0.08-0.1. After soaking the callus tissue in the bacterial suspension for 15 min, the bacterial suspension was discarded, and the callus tissue was placed on sterile filter paper to absorb excess bacterial suspension. It was then transferred to N6D-2 solid co-medium containing 100 μM AS and lined with sterile filter paper, and incubated in the dark at 25°C for 3 days. After 3 days of co-culture, callus tissue was washed more than 5 times with clean water, then rinsed once with sterile water containing 400 mg / L carbenicillin. After the callus was dried, it was transferred to N6D-2 medium containing 50 mg / L Hyg (Hygromycin) and 200 mg / L Timentin, and cultured at 28°C under light (16h / 8h photoperiod, 2000 lx light intensity) for 4-5 weeks, changing the medium every two weeks. Resistant callus was selected and placed on N6 differentiation medium containing 0.5 mg / L NAA (α-Naphthaleneacetic acid), and cultured at 28°C under light (16h / 8h photoperiod, 2000 lx light intensity) for 2-3 weeks until green shoots appeared. Then, it was transferred to 1 / 2 MS medium containing 10 mg / L Hyg and cultured until rooting (7-10 days). After 3 days of hardening off, the seedlings were transplanted to a greenhouse for further culture to obtain overexpressed... EsPTI2 Transgenic strains.

[0066] 4. Genetically modified rice EsPTI2 Phenotypic Validation

[0067] Leaf tissues were collected from seedlings of the transgenic rice plants obtained in step 3, and extracted using the AFT Spin Universal Plant Fast RNA Extraction Kit. EsPTI2RNA from overexpressing plants was analyzed by agarose gel electrophoresis to determine RNA quality and by Nanodrop ultraviolet spectrophotometer (Thermo Fisher Scientific, MA, USA) to determine RNA quantity. cDNA was then synthesized using the EXRT kit reverse transcription assay. This cDNA was diluted two-fold and used as a template. qRT-PCR amplification was performed using primers EsPTI2-RT-F and EsPTI2-RT-R, the Hieff UNICON qPCRSYBR GREEN Master Mix quantitative reagent, and an ABI Q7 real-time PCR instrument. The RNA was then detected in positive plants. EsPTI2 The expression level, using 2 -ΔΔCt The relative expression level of the target gene was calculated using the method. The sequence of EsPTI2-RT-F is ACGACCAAGCCCAACAAGAA, and the sequence of EsPTI2-RT-R is GATCTCTCGCCGCTATGGAG.

[0068] The results are as follows Figure 3 As shown, in the transgenic lines EsPTI2 The expression level was 1.0 to 1841.4 times that of the control plants, with the highest levels observed in the three transgenic lines OE-4, OE-23, and OE-24. EsPTI2 The expression levels were 931.45 times, 1149.40 times, and 1841.50 times that of the control, respectively, with all differences reaching highly significant levels. P The value is less than 0.001, indicating that the constructed expression vector has been successfully expressed.

[0069] It should be noted that existing technologies can be used to build it. EsPTI Other overexpression vectors for gene 2, such as expression cassettes and recombinant vectors containing these cassettes, can be constructed using existing technologies. For example, cDNA obtained by reverse transcription of total RNA from rice or wheatgrass leaves can be used as a template. Specific amplification primers are designed based on the complete CDS sequence of the target gene, and the full-length coding region of the target gene is amplified using high-fidelity PCR. Next, a plant expression cassette of the target gene is constructed. The amplified target gene fragment is inserted into an expression frame carrying a monocot constitutive strong promoter and a NOS terminator via homologous recombination or restriction endonuclease double digestion and ligation. The complete expression cassette fragment is then inserted into the multiple cloning site of plant binary expression vectors such as the pCAMBIA series via double enzyme ligation or seamless cloning. After PCR identification, restriction enzyme digestion verification, and Sanger sequencing confirmation of sequence accuracy, a recombinant plant expression vector containing the target gene expression cassette is obtained. This recombinant vector can be directly transformed into Agrobacterium competent cells for subsequent stable genetic transformation of rice or wheatgrass. ,EsPTIThe gene overexpression vector also has the function of regulating the number of plant leaves. Since the construction of other vectors is based on existing technology, they will not be described one by one to reduce unnecessary repetition.

[0070] Example 4 EsPTI2 Verification of the function of gene overexpression in plants to increase leaf number

[0071] Using the method of Example 2, we obtained EsPTI2 Transgenic rice overexpression lines OE, transplanted wild-type (WT), and overexpression lines EsPTI2 Transgenic rice line OE was selected, and plants with consistent growth stages were observed for phenotypic development. Results showed that overexpression... EsPTI2 The number of leaves in the plant was significantly higher than that in the wild type, increasing by 88.2% ( Figure 4 This result confirms... EsPTI2 Genes play an important role in the yield trait of old awn wheat by positively regulating leaf number.

[0072] In summary, it can be concluded that the [source] comes from the old mango plant. EsPTI2 After the gene was overexpressed in monocotyledonous rice model plants, the number of leaves increased significantly. Based on the theory of biological genetics, this gene can be homologously applied to wheat plants. EsPTI2 Gene overexpression can regulate the number of leaves of *Strombococcus variabilis*, increase the yield of *Strombococcus variabilis* forage, and achieve efficient accumulation of aboveground biomass.

[0073] Example 5 EsPTI2 Industrial applications of gene overexpression in plants to increase leaf number

[0074] As can be seen from Examples 2 to 4, EsPTI2Overexpression of this gene in plants can increase leaf number. Therefore, precise regulation of leaf number can be used to optimize the canopy structure of monocots, effectively expanding the photosynthetic area and improving the efficiency of light capture and utilization in the plant population. This provides a core functional target for the synergistic improvement of grain yield in food crops and forage yield in forage crops. Simultaneously, this gene is directly related to leaf number and biomass traits, and can be developed into a functional molecular marker for marker-assisted breeding of high-yield traits in monocots, significantly shortening the breeding cycle and improving the accuracy of target trait screening. For planting needs in high-altitude, arid, and other adverse environments, this gene can increase leaf photosynthetic area to ensure the accumulation of photosynthetic products under adverse conditions, achieving a synergistic improvement of high biomass and strong stress resistance. This provides a key gene resource for cultivating new monocot varieties with excellent adaptability in ecologically fragile areas. Furthermore, this gene can be used to construct core parental materials for high-light-efficiency breeding of monocots, directionally improving the plant architecture and photosynthetic efficiency of existing main varieties, effectively broadening the genetic basis of high-yield breeding germplasm resources, and overcoming the bottleneck of germplasm homogenization in existing high-yield breeding. This gene can also support the breeding of new varieties of compact monocotyledonous grain crops and forage crops. By regulating the number of leaves, it can optimize the plant type to adapt to high-density planting patterns, alleviate the problem of shading under high-density planting, and significantly improve the planting efficiency per unit area. It provides a brand-new breeding idea and gene support for intensive grain production and forage planting. These applications have been well applied, especially in rice and old awn wheat plants.

[0075] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-described technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. EsPTI2 The application of genes in regulating the number of plant leaves is characterized by, by overexpressing in plants EsPTI2 genes to increase leaf number; in, EsPTI2 The nucleotide sequence is shown in SEQ ID NO.1; The plant in question is either rice or old wheat.

2. EsPTI2 The application of gene-related biomaterials in regulating plant leaf number is characterized by, It works by overexpressing in plants EsPTI2 Genes are used to increase the number of leaves; The biomaterial is any one of B1)-B5): B1) Contains the contents of claim 1 EsPTI2 Gene expression cassettes; B2) contains the contents of claim 1 EsPTI2 Gene recombination vectors; B3) contains the contents of claim 1 EsPTI2 Recombinant microorganisms; B4) A recombinant vector containing the expression cassette described in B1); B5) Recombinant microorganisms containing the recombinant vector described in B2); in, EsPTI2 The nucleotide sequence is shown in SEQ ID NO.1; The plant in question is either rice or old wheat.

3. A method for preparing plants with a high leaf count, characterized in that, The method involves overexpression in plants. EsPTI2 The steps of gene generation; in, EsPTI2 The nucleotide sequence is shown in SEQ ID NO.1; The plant in question is either rice or old wheat.

4. The claim 1 EsPTI2 The application of genes, the biomaterials described in claim 2, or the method described in claim 3 in the targeted regulation of monocotyledonous plant canopy structure and improvement of canopy light energy utilization efficiency is characterized in that... Through overexpression EsPTI2 Gene expression levels are achieved. The monocotyledonous plant is rice or wheat.

5. The claim 1 EsPTI2 The application of genes, the biological material described in claim 2, or the method described in claim 3 as functional molecular markers in marker-assisted breeding of traits related to leaf number and biomass in monocotyledonous plants is characterized by, Through overexpression EsPTI2 This is achieved through gene expression levels. The plant in question is either rice or old wheat.

6. The claim 1 EsPTI2 The application of genes, the biological materials described in claim 2, or the method described in claim 3 in cultivating new monocotyledonous plant varieties with both high biomass and strong stress resistance in high-altitude, arid, and adverse environments is characterized by the following: Through overexpression EsPTI2 This is achieved through gene expression levels, and the plant in question is either rice or wheat.

7. The claim 1 EsPTI2 The application of genes, the biological materials described in claim 2, or the method described in claim 3 in constructing core parental materials for high light-efficiency breeding of monocotyledonous plants, improving varieties, and broadening the genetic basis of high-yield breeding germplasm resources is characterized by the following: Through overexpression EsPTI2 This is achieved through gene expression levels. The plant in question is either rice or old wheat.

8. The claim 1 EsPTI2 The application of genes, the biological materials described in claim 2, or the method described in claim 3 in breeding new varieties of compact monocotyledonous food crops and forage crops suitable for high-density planting and improving planting efficiency per unit area is characterized by the following: Through overexpression EsPTI2 This is achieved through gene expression levels. The monocotyledonous plant is rice or wheat.

Images