Application of LcCBP60b-LcALY2 molecular module in regulation and control of growth and development and maturing rate of leymus chinensis

Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses application of an LcCBP60b-LcALY2 molecular module in regulation and control of growth and development and maturing rate of leymus chinensis. The invention discovers that the calmodulin binding protein coding gene LcCBP60b negatively regulates the growth and development and the maturing rate of the leymus chinensis for the first time, and a yeast double-impurity experiment discovers that the LcCBP60b interacts with the LcALY2 protein to regulate the related characters of the maturing rate of the leymus chinensis. The LcCBP60b negatively regulates the activity of the LcALY2 protein and inhibits the regulation effect of the LcALY2 coded THO protein complex 4B in the flowering and ear forming process of leymus chinensis. The LcCBP60b-LcALY2 molecular module verified by the invention provides a new perspective for deeply understanding a Leymus chinensis fruiting regulation mechanism, and provides a direct molecular target for directionally creating new varieties of high-fruiting-rate pasture and cereal crops.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically, it relates to the application of the LcCBP60b-LcALY2 molecular module in regulating the growth, development and seed setting rate of Leymus chinensis. Background Technology

[0002] Leymus chinensis, a perennial rhizomatous forage grass belonging to the genus Leymus in the Poaceae family, is a dominant grass species in the Eurasian steppes. It is widely distributed in the grasslands of Northeast and Inner Mongolia, as well as North and Northwest my country, and boasts advantages such as high yield, high protein content, good palatability, strong regeneration ability, and wide adaptability. As a major dominant species in temperate grasslands, Leymus chinensis achieves rapid growth and reproduction thanks to its extensive root system, and can live for up to 20 years, making it an excellent forage variety for desertification control and ecological protection. While Leymus chinensis has advantages such as long lifespan and strong asexual reproduction ability, its sexual reproduction ability is relatively poor, mainly manifested in low heading rate, low seed setting rate, and low germination rate, which seriously restricts its application and promotion. Exploring the molecular mechanisms of seed setting traits in Leymus chinensis and cultivating Leymus chinensis varieties with high seed setting rates is the primary task for achieving a leap forward in the production and application of Leymus chinensis.

[0003] Current research focuses on the main factors influencing the seed setting traits of Leymus chinensis. Studies show that seed setting traits are affected by germplasm type and cultivation conditions; different Leymus chinensis germplasms exhibit variations in inflorescence location, opening time, spike development conditions, and pollen formation. Furthermore, different Leymus chinensis germplasms are affected by varying ecological environments, resulting in significant differences in heading and seed setting rates, with variations ranging from 2.1% to 71.3% and 7.8% to 73.3%, respectively. A study investigating the flowering sequence, flowering period length, and floret opening time of five Leymus chinensis germplasms revealed substantial differences in several traits among different germplasms, with the longest floret opening time lasting up to 2 hours. Simultaneously, spraying gibberellin and kinetin during the greening and tillering stages of Leymus chinensis significantly increases the number of spikelets, florets, seed setting rate, and spike weight; exogenous CO2 application can enhance photosynthesis, influence phenological stages, and promote heading. However, this approach of investigating the fruit setting trait of Leymus chinensis based on a single factor is insufficient to maintain the stability of the high fruit setting rate trait, and is also limited by equipment and cost. Therefore, exploring the molecular mechanisms of Leymus chinensis fruit setting traits holds promise for developing new, high-fruit-setting, stable, and high-quality Leymus chinensis varieties through molecular breeding.

[0004] The calmodulin-binding protein 60 family is an atypical transcription factor family unique to plants. Previous studies have identified eight family members distributed in the Arabidopsis genome, namely... CBP60a-g and SARD1 ,in CBP60g and SARD1Calmodulin plays a crucial role in plant immune responses, primarily by positively regulating the salicylic acid pathway to enhance plant resistance to pathogens. Increasing research indicates that calmodulin-binding proteins and their pathway-related genes play important functions in plant growth and development. Under short-day conditions, increased gibberellin activity and calcium ion content in chrysanthemum directly inhibit the formation of a complex between calmodulin CmCAM7, a key factor in calcium signal transduction, and CmGAI, a key repressor of gibberellin signaling, thereby relieving the inhibition of CmGAST1 and promoting chrysanthemum flowering. However, the specific mechanisms by which calmodulin-binding proteins and their related pathways regulate the seed setting trait of Leymus chinensis remain unclear. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the LcCBP60b-LcALY2 molecular module in regulating the growth, development, and seed setting rate of Leymus chinensis.

[0006] To achieve the objectives of this invention, in a first aspect, this invention provides... LcCBP60b Application of genes in regulating the growth, development, and seed setting rate of Leymus chinensis.

[0007] The LcCBP60b The genes are: i) The nucleotide sequence shown in SEQ ID NO:1; ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:1 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or, iv) Nucleotide sequences that have more than 90% homology with the nucleotide sequences of i), ii) or iii) and express the same functional protein.

[0008] Furthermore, the regulation is negative regulation.

[0009] Specifically, the application includes: by inhibiting the LcCBP60b The expression of genes or the activity of their encoded proteins can increase the seed setting rate of Leymus chinensis and / or increase spike length and spikelet number.

[0010] Secondly, the present invention provides a method for improving the seed setting rate, increasing spike length, and increasing the number of spikelets of Leymus chinensis, the method comprising reducing the content of Leymus chinensis in Leymus chinensis LcCBP60b The expression level of a gene or the activity of its encoded protein.

[0011] Used to reduce the concentration in sheepgrassLcCBP60b The expression level of a gene or the activity of its encoded protein can be selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, antibodies, etc.

[0012] Furthermore, RNA interference (RNAi) was used to reduce the... LcCBP60b Gene expression levels.

[0013] Furthermore, LcCBP60b Gene silencing expression vectors were introduced into Leymus chinensis, resulting in transgenic Leymus chinensis plants... LcCBP60b Gene expression levels were lower compared to non-transgenic wild-type sheepgrass; Among them, the LcCBP60b Gene silencing expression vectors contain those that can specifically inhibit Leymus chinensis. LcCBP60b A nucleic acid molecule for gene expression, said nucleic acid molecule comprising a sequence complementary to the nucleotide sequence shown in SEQ ID NO:1 or a fragment thereof.

[0014] Preferably, the nucleic acid molecule consists of 1-250 bp of the nucleotide sequence shown in SEQ ID NO:1. Preferably, it is used for constructing... LcCBP60b The original vector for the gene silencing expression vector was pFGC1008.

[0015] Thirdly, this invention provides the application of the LcCBP60b-LcALY2 molecular module in regulating the growth, development, and seed setting rate of Leymus chinensis. It regulates the growth, development, and seed setting rate of Leymus chinensis by controlling the interaction between the LcCBP60b and LcALY2 proteins; specifically, the gene... LcALY2 The encoded THO protein complex 4B, as a THO / TREX complex, participates in the transcriptional regulation of Leymus chinensis growth and development genes, as well as the regulation of signal transduction related to flowering, spike formation and seed formation, thereby affecting the growth, development and seed setting rate of Leymus chinensis.

[0016] The LcCBP60b protein is composed of the LcCBP60b Gene encoding; The LcALY2 protein is composed of LcALY2 Gene encoding, the LcALY2 The genes are: I) The nucleotide sequence shown in SEQ ID NO:2; II) A nucleotide sequence of SEQ ID NO:2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; III) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:2 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or, A nucleotide sequence that has more than 90% homology with the nucleotide sequences of I), II) or III) and expresses the same functional protein.

[0017] Furthermore, by reducing the expression or activity of the LcCBP60b protein, its interaction with the... LcALY2 The binding of the encoded THO protein complex 4B (i.e., LcALY2 protein) relieves transcriptional repression of downstream flowering and embryonic development-related genes, thereby increasing the seed setting rate of Leymus chinensis and / or increasing spike length and spikelet number.

[0018] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (I) This invention analyzes the effect of gene LcCBP60b on spike formation of Leymus chinensis, and clarifies that LcCBP60b interacts with LcALY2 to inhibit the signal transduction of THO complex 4B encoded by LcALY2 in the processes of flowering, spike formation and seed development of Leymus chinensis, laying a theoretical foundation for elucidating the regulatory mechanism of seed setting rate of Leymus chinensis.

[0019] (ii) The invention is the first discovery LcCBP60b The gene has a negative regulatory function on the growth, development, and seed setting rate of Leymus chinensis.

[0020] Previous studies have shown that CBP60b responds to plant immune processes, primarily by directly binding to specific cis-acting elements (such as the core sequence (A / T)AATT) in the promoter regions of target genes to activate their transcription, thereby regulating an immune signaling pathway that includes important genes such as SARD1, EDS1, PAD4, and SID2. When CBP60b functions normally, it inhibits an unknown negative regulator of immunity. Once its function is lost due to attack by pathogen effector proteins, it triggers the plant's own "guard" proteins (such as TNL-like disease resistance proteins) to activate a strong immune response (ETI). This design allows plants to more sensitively perceive interference from pathogens. In Leymus chinensis, by constructing... LcCBP60b- Phenotypic identification of overexpressed and silenced transgenic lines revealed that this gene significantly responded to the formation of traits related to seed setting rate in Leymus chinensis. This invention explores... LcCBP60b Interacting proteins that regulate the gene-fruit setting rate trait; investigation LcCBP60bThe molecular mechanism by which the gene and the LcCBP60b-LcALY2 molecular module regulate the seed setting rate of Leymus chinensis provides a new perspective for elucidating the molecular mechanism of seed setting rate in Leymus chinensis. Attached Figure Description

[0021] Figure 1 In a preferred embodiment of the present invention, sheepgrass LcCBP60b Comparison diagram of phylogenetic tree analysis of CBPs in different species.

[0022] Figure 2 This is a schematic diagram of the construction of the transgenic vector in a preferred embodiment of the present invention, wherein the upper part is... LcCBP60b A schematic diagram of overexpression vector construction is shown below. LcCBP60b Schematic diagram of silent carrier construction.

[0023] Figure 3 In a preferred embodiment of the present invention LcCBP60b Analysis of transgenic overexpression and silenced transgenic DNA and RNA expression levels, where A represents... LcCBP60b DNA level identification of overexpressing transgenic plants, B is LcCBP60b DNA level identification of silent transgenic plants, C is LcCBP60b RNA expression level analysis of overexpressed and silenced transgenic plants.

[0024] Figure 4 In a preferred embodiment of the present invention LcCBP60b Phenotypic analysis of overexpression and silenced transgenic plants, where A represents the wild-type WT plant type; B represents... LcCBP60b Overexpression transgenic strain; C is LcCBP60b Silent transgenic strains; D represents wild-type and LcCBP60b The spike type of the transgenic plant; E represents wild type and LcCBP60b The spikelets of the transgenic plants; F1 represents the wild type and... LcCBP60b Distribution of the number of florets on different spikelets of transgenic plants.

[0025] Figure 5 In a preferred embodiment of the present invention LcCBP60b Statistics on agronomic traits of overexpressed and silenced transgenic lines, where A represents the wild-type (WT) at the grain-filling stage. LcCBP60b Overexpression plants and LcCBP60b Plant height; B is wild type (WT). LcCBP60b Overexpression plants and LcCBP60b Ear length; C indicates wild type (WT) LcCBP60b Overexpression plants and LcCBP60b Spikelet number; D represents wild type (control). LcCBP60b Overexpression plants and LcCBP60b Average spikelet length; E represents wild type (control).LcCBP60b Overexpression plants and LcCBP60b Total number of florets; F represents wild type (control). LcCBP60b Overexpression plants and LcCBP60b Fruit set rate.

[0026] Figure 6 In a preferred embodiment of the present invention LcCBP60b Transcriptome data analysis of silent strains: A shows statistical analysis of differentially expressed genes in RNAi-LcCBP60b vs. WT; B shows the volcano distribution map of differentially expressed genes in RNAi-LcCBP60b vs. WT; and C shows the GO enrichment analysis of differentially expressed genes in RNAi-LcCBP60b vs. WT.

[0027] Figure 7 In a preferred embodiment of the present invention, the interaction between LcCBP60b and LcALY2 regulates the fruit set of Leymus chinensis. Specifically, A represents the gradient verification results on SD / -TL plates after co-transferring yeast strain Y2H with the pGADT7-LcCBP60b and pGBKT7-LcALY2 fusion plasmid; B represents the gradient verification results on SD / -TLHA plates after co-transferring yeast strain Y2H with the pGADT7-LcCBP60b and pGBKT7-LcALY2 fusion plasmid; and C represents the BFIC experiment based on a tobacco transient expression system to verify the interaction between LcCBP60b and LcALY2.

[0028] Figure 8 The subcellular localization results of LcCBP60b and LcALY2 in a preferred embodiment of the present invention are shown. Detailed Implementation

[0029] This invention provides the application of the LcCBP60b-LcALY2 molecular module in the seed setting rate trait of Leymus chinensis. LcCBP60b Genes regulate plant height, reproductive branch length, spikelet number, and seed setting rate in Leymus chinensis during its reproductive growth phase. (Construction) LcCBP60b Gene silencing in transgenic plants resulted in longer spikelets and an increased number of spikelets during the reproductive growth stage, effectively increasing the seed setting rate of Leymus chinensis; overexpression in transgenic plants exhibited the completely opposite traits. LcCBP60b The study investigated the mechanisms regulating seed setting traits in Leymus chinensis, laying a theoretical foundation for elucidating the regulatory mechanisms of seed setting rate in this grass. It also opened up new avenues for transforming fundamental theoretical findings into practical applications that enhance forage productivity and ecological benefits, thereby increasing Leymus chinensis seed yield and promoting its resource utilization.

[0030] The present invention adopts the following technical solution: This invention provides the application of the LcCBP60b-LcALY2 molecular module in regulating the seed setting rate of Leymus chinensis.

[0031] Preferably, the regulation of the seed setting rate of Leymus chinensis is achieved by silencing... LcCBP60b Genes promote increased spike length and spikelet number in Leymus chinensis. Conversely, overexpression... LcCBP60b Genes inhibit spikelet formation in Leymus chinensis, resulting in fewer spikelets.

[0032] Preferably, the silencing LcCBP60b is achieved by... LcCBP60b The specific positive fragment and its complementary sequence were constructed into the PFGC1008 vector to obtain a recombinant plasmid, which was then overexpressed. LcCBP60b It is by... LcCBP60b The complete CDS sequence was constructed on the pUbi-GFP vector to obtain recombinant plasmids, which were then transformed into EHA105 Agrobacterium competent cells and then into Leymus chinensis materials.

[0033] Preferably, the gene that interacts with LcCBP60b is LcALY2.

[0034] The LcCBP60b The nucleotide sequence of the gene is shown in SEQ ID NO:1. LcALY2 The nucleotide sequence of the gene is shown in SEQ ID NO:3.

[0035] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0036] The biomaterials used in this invention are as follows: (1) Leymus chinensis: The Leymus chinensis material used in this invention is LC-4 (WT), which was provided by the Heilongjiang Academy of Agricultural Sciences (Leymus chinensis LC-4 can be found in the literature Di Guili, You Jia, Gao Chao, et al. Research on the establishment of tissue culture and regeneration system of Leymus chinensis young spikelets [J]. Heilongjiang Agricultural Sciences, 2021, (06): 119-124).

[0037] (2) Tobacco: The tobacco used in this invention is Tobacco Benzoinus (Benzoinus chinensis). Nicotiana benthamiana L.).

[0038] (3) Strains: The strains used in this invention are mainly Escherichia coli competent cells DH5α (Escherichiacoli), Agrobacterium competent cells EHA105, GV3101 (P19+psoup), and yeast Y2H.

[0039] (4) Vectors: The vectors used in this invention include the overexpression vector pUbi-GFP, the silencing vector pFGC1008, the yeast double-hybrid vectors pGADT7 and pGBKT7, the subcellular localization vector pSuper1300-GFP, and the BFIC vectors pNC-EnYFP and pNC-EcYFP. All of the above vectors were provided by the Forage Seed Building Laboratory of China Agricultural University.

[0040] (5) Antibiotics: 100 mg / mL kanamycin (Kan), 100 mg / mL ampicillin (Amp), 100 mg / mL chloramphenicol (Chl), 100 mg / mL spectinomycin (Spec), 50 mg / mL rifampin (Rif), 200 μg / mL acetosyringone (AS), 10 mg / mL dipropionam, 200 mg / mL termethin (Tim), 200 mg / mL cephalosporin (Cef), 200 mg / mL carbopenicillin sodium (Car).

[0041] (6) Culture medium formula and nutrient solution 1 L of lysozyme (LB) medium mainly consists of: 5 g of yeast extract, 10 g of tryptone and 10 g of sodium chloride. Add 15g of microbial-specific agar to 1 L of LB solid medium.

[0042] 1 L of yeast extract peptone glucose (YPD) medium mainly consists of: 10 g yeast extract, 20 g tryptone, 20 g glucose, and 15 g agar.

[0043] All nutrient-deficient culture media used in the yeast hybrid experiment were purchased from Beijing Pangeno Biotechnology Co., Ltd.

[0044] The experimental procedures involved in this invention are carried out as follows: one, TransZol Total RNA extracted from Leymus chinensis using the Up method All RNA samples involved in this invention were extracted using a high-purity RNA extraction kit from Beijing TransGen Biotech Co., Ltd.

[0045] II. Reverse Transcription of cDNA Synthesis The cDNA used as a template in this invention is reverse transcribed using a two-step kit, wherein the reverse transcription kit is purchased from TransScript One-Step GDNA Removal and First-Strand cDNA Synthesis Kit from Beijing TransGen Biotech Co., Ltd.

[0046] III. Vector Construction and E. coli Transformation The vector construction method involved in this invention is homologous recombination, and the main steps include PCR primer design, amplification of the target fragment, recovery of the target fragment, ligation of the target fragment with the enzyme digestion vector, heat shock transformation of Escherichia coli DH5α, identification of positive colonies and sequencing verification.

[0047] In this invention LcCBP60b The primer sequences used for constructing the overexpression vector are shown in SEQ ID NO:3 and SEQ ID NO:4, and are ligated into the pUbi-GFP vector; LcCBP60b The primer sequences used to construct the silencing vector are shown in SEQ ID NO:5-8 and were ligated into the pFGC1008 vector. LcCBP60b The primer sequences used to construct the subcellular localization vector are shown in SEQ ID NO:9 and SEQ ID NO:10; LcALY2 The primer sequences used to construct the subcellular localization vector are shown in SEQ ID NO:11 and SEQ ID NO:12, and were respectively inserted into the pSuper1300-GFP vector; LcCBP60b The primer sequences used to construct the yeast dual hybrid vector are shown in SEQ ID NO:13 and SEQ ID NO:14, and were inserted into the vector pGADT7. LcALY2 The primer sequences used to construct the yeast dual hybrid vector are shown in SEQ ID NO:15 and SEQ ID NO:16, and were inserted into the vector pGBKT7. LcCBP60b The primer sequences used to construct the colocalization experimental vector are shown in SEQ ID NO:17 and SEQ ID NO:18, and were inserted into the vector pNC-EnYFP. LcALY2 The primer sequences used to construct the colocalization experimental vector are shown in SEQ ID NO:19 and SEQ ID NO:20, and are inserted into the vector pNC-EcYFP; the primer sequences used in this invention are shown in Table 1.

[0048] Table 1 Primers related to vector construction

[0049] Cloning of the target gene fragment: cDNA was synthesized using whole-plant RNA of Leymus chinensis as a template, and the target gene fragment was cloned. The amplification system and amplification procedure are as follows: (1) Amplification system: 25 μL of 2×Phanta Max MasterMix (Dye plus), 1 μL of template, 2 μL of forward primer, 2 μL of reverse primer, and ddH2O to make up to 50 μL. The concentration of all primers used was 10 uM.

[0050] (2) Amplification program: 95℃ for 3 min; 95℃ for 15 sec, 56℃ for 30 sec, 72℃ for 1 min, 40 cycles; 72℃ for 5 min.

[0051] (3) Gel electrophoresis: Electrophoresis was performed using 1% agarose gel in 1×TAE electrophoresis buffer at 180V for 15min.

[0052] (4) Gel recovery of target fragment: The gel after electrophoresis was cut under UV light to recover the solid. The recovery method was performed according to the kit instructions. The gel recovery kit was purchased from Novizan DNA Recovery and Purification Kit.

[0053] (5) Homologous recombination: The concentration of the recovered target fragment was determined using a micro-nucleic acid analyzer. Homologous recombination was then performed between the target gene and the linear vector at a ratio of 3:1. The specific reaction system was as follows: the total amount of the target gene fragment was approximately 90 ng, the linear vector was approximately 30 ng, and an equal volume of 2×clonExpress Mix (purchased from Novizan ClonExpress Ultra One Step Cloning Kit) was added. The reaction conditions were 50℃ for 15-30 min.

[0054] (6) Transformation of colonic competent cells: The colonic competent cells were dissolved on ice, and the recombinant product was completely transferred into the competent cells. The cells were gently mixed, incubated on ice for 15 min, heat-shocked at 42°C for 90 sec, and then incubated on ice for 3 min. 500 μL of antibiotic-free LB liquid was added to the colonic competent cells, and the cells were recovered at 37°C for 1 hour before being plated with the corresponding antibiotic plate.

[0055] IV. Agrobacterium-mediated transformation The Agrobacterium competent cells involved in this invention were all purchased from Beijing Coollab Technology Co., Ltd. The transformation of Agrobacterium competent cells was carried out using the freeze-thaw method, and the main steps are as follows: Dissolve Agrobacterium competent cells on ice. Transfer 200 ng of the correctly sequenced plasmid into the Agrobacterium competent cells, mix gently, incubate on ice for 5 min, flash freeze in liquid nitrogen for 5 min, heat shock for 5 min, and incubate on ice for 5 min. Add 500 μL of antibiotic-free LB liquid to the Agrobacterium competent cells, thaw at 28°C for 2 hours, and then plate with the corresponding antibiotic.

[0056] V. Yeast Dual-Mix Screening Library The gene modules involved in this invention were mainly obtained through yeast double-hybrid screening libraries. The pGBKT7-LcCBP60b fusion vector was constructed and transformed into Y2H yeast. A yeast double-hybrid screening library was then performed using a matting method. After further screening on four-deficient plates, colony PCR was performed on the grown yeast clones. The PCR products were sequenced using the relevant vector primers, and the primer sequences are shown in SEQ ID NO:21 and SEQ ID NO:22 (Table 2). After sequencing alignment, candidate genes were initially obtained, and subsequent one-to-one experimental verification was conducted.

[0057] Table 2. PCR primers for yeast double-hybrid screening library

[0058] VI. Transient Expression of Tobacco The subcellular localization assays and BFIC co-localization assays involved in this invention were both performed using a transient expression system in tobacco. The main procedure involved shaking the Agrobacterium tumefaciens plasmid corresponding to the experiment, centrifuging, and resuspending it in tobacco resuspension. Tobacco seedlings with 4-6 leaves were selected for the experiment, and after injection onto the back of the leaves, they were cultured for 2-3 days before the relevant experiments were conducted.

[0059] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional technical means well known to those skilled in the art, and the raw materials involved are all commercially available products.

[0060] Example 1: Sheepgrass LcCBP60b Phylogenetic tree analysis of genes To verify the evolutionary origin of the gene described in this invention and its differences from existing genes, a phylogenetic tree analysis was performed on LcCBP60b and its homologous gene sequences in different species. The gene sequences were obtained from the GenBank database, and homologous sequences from different species were selected as references. Multiple sequence alignment was performed using MEGA11 software, and the phylogenetic tree was constructed using the maximum likelihood method. Figure 1 Sequence alignment results show that the gene of this invention contains *Leymus chinensis*. LcCBP60b It is highly homologous to the CBP60 sequence in common wheat and perennial ryegrass, with the LcCBP60b gene of Leymus chinensis showing a similarity of up to 97.45% to the LpCBP60D gene of perennial ryegrass.

[0061] Example 2: Sheepgrass LcCBP60b Creation of genetically modified materials 1.1 Cloning of the LcCBP60b gene from Leymus chinensis (a) Extraction of total RNA from Leymus chinensis According to the above experimental procedures TransZolTotal RNA was extracted from Leymus chinensis using the Up method. All reagents used were high-performance RNA extraction kits from Beijing TransGen Biotech Co., Ltd. The specific steps are as follows: (1) Take the tissues of each part of the sheep grass, grind them into powder quickly in liquid nitrogen, and take an appropriate sample (the volume of dry powder is about 0.5 cm of the centrifuge tube).

[0062] (2) Add 1 mL of [unspecified ingredient] to each centrifuge tube. TransZol The extract and 200 μL of RNA ExactionAgent were thoroughly vortexed to form a homogenate, and then centrifuged at 12,000 rpm and 4°C for 15 min.

[0063] (3) Transfer the supernatant to a new centrifuge tube, add an equal volume of anhydrous ethanol, and gently invert to mix.

[0064] (4) Transfer the mixed liquid into the centrifuge column in batches, centrifuge at 12000×g at room temperature for 30 seconds, and discard the waste liquid.

[0065] (5) Add 500 μL of CB9 to each centrifuge column, centrifuge at room temperature for 30 seconds, and discard the waste liquid.

[0066] (6) Repeat step (5) once.

[0067] (7) Add 500uL WB9, 12000×g, centrifuge at room temperature for 30sec, and discard the waste liquid.

[0068] (8) Repeat step (7) once.

[0069] (9) Centrifuge at 12000×g at room temperature for 2min to completely remove residual ethanol.

[0070] (10) Transfer the centrifuge column to a new centrifuge tube, add 50 uL of sterile water (Rnase-free Water) to the center of the column, place at room temperature for 2 min, and centrifuge at 12000×g for 2 min at room temperature.

[0071] (ii) cDNA synthesis The cDNA synthesis was performed according to the method described in the above experimental procedure. The specific system and operation are as follows: (1) Reaction system: Add 1 μg total RNA, 1 μL Anchored Oligo(dT) 2×ES Reaction Mix 10 μL, gDNA Remover 1 μL, and sterile water (Rnase-free Water) to a final volume of 20 μL. All reagents were purchased from Beijing TransGen Biotech Co., Ltd. and are products of the One-Step Reverse Transcription Kit (catalog number AT311).

[0072] (2) Reaction procedure: 42℃ for 30 min, 85℃ for 2 min.

[0073] (III) Gene Cloning Perform the gene cloning method described in the above experimental procedure. LcCBP60b The primer sequences used for cloning the target fragment are shown in SEQ ID NO:3-4.

[0074] (iv) Purification of LcCBP60b target fragment The cloned LcCBP60b target fragment was subjected to electrophoresis. Bands of the correct size and clear shape were selected, and solid recovery was performed according to the gel recovery method for the target fragment described above in this invention. The specific operation is as follows: (1) Cut the clear and correctly sized bands under a UV lamp, transfer them to a 2 ml centrifuge tube, and weigh them; (2) Add an equal weight of GDP to a centrifuge tube and sol-gel at 55°C. Invert the tube several times during the melting process to accelerate the sol-gel process. (3) Transfer all the melted liquid to a centrifuge column, centrifuge at 12000×g at room temperature for 1 min, and discard the waste liquid; (4) Add 300uL GDP to the centrifuge column, incubate at room temperature for 2min, centrifuge at 12000×g at room temperature for 1min, and discard the waste liquid; (5) Add 600 μL LGW to the centrifuge column, centrifuge at room temperature for 1 min, and discard the waste liquid; (6) Repeat step (5); (7) Centrifuge at 12000×g at room temperature for 2 min to completely remove anhydrous ethanol; (8) Transfer the centrifuge column to a new 1.5 mL centrifuge tube, add 30 μL of elution buffer to the center of the centrifuge column, incubate at room temperature for 2 min, and centrifuge at 12000 × g for 2 min at room temperature.

[0075] (V) Construction of transgenic expression vectors (overexpression and silencing expression vectors): LcCBP60b A schematic diagram of the transgenic expression vector is shown below. Figure 2 As shown. LcCBP60b The overexpression vector construction involves measuring the concentration of the purified fragment and then using it to construct the overexpression vector. The vector construction method follows the vector construction method described in the experimental procedures of this invention. LcCBP60b The construction of silencing vectors is mainly based on primer sequences such as SEQ ID NO:5-8, amplification templates, and reaction systems, which are used to construct... LcCBP60bThe forward and reverse fragments of the silencing vector are first constructed by combining the reverse fragment with the pFGC1008 reverse fragment linear vector under the action of Novitamin A recombinase according to the vector construction method of this invention. After subsequent sequencing confirms that the vector is correct, this step is repeated to ligate the forward silencing fragment.

[0076] (vi) Transformation of colonic competence: LcCBP60b The overexpression and silencing recombinant plasmids were used to transform into competent colon cells, and the specific steps were carried out according to the colon competence transformation method in the above-mentioned experimental operation of the present invention.

[0077] (vii) Sequence alignment: Single colonies were picked from the overnight culture plates and added to 600 μL of LB broth with kanamycin (Kan) / chloramphenicol (Chl). The plates were incubated at 37°C with shaking for 4-6 hours. Colony PCR was then performed using the vector primer sequences shown in SEQ ID NO:23-26. The products were detected by electrophoresis. Single colonies with correct bands were sequenced, and the gene sequences were corrected through sequence alignment.

[0078] (viii) Agrobacterium transformation: The successfully sequenced plasmid was transformed with Agrobacterium, following the Agrobacterium transformation method described above in this invention. Single clones of the successfully transformed Agrobacterium were selected, and after a short shake, culture PCR was performed (primers are shown in Table 3). Colonies with correct bands were preserved by shaking for subsequent transgenic construction.

[0079] Table 3 Primers for detecting overexpression and silencing vectors

[0080] 1.2 LcCBP60b Construction of transgenic plants Young spikelets of Leymus chinensis germplasm LC-4 were sterilized and then cultured as callus. The resulting fluffy callus was used for infection. LcCBP60b After activation, Agrobacterium was shaken vigorously and cultured overnight until the OD600 reached approximately 0.6, at which point callus infection was initiated. Agrobacterium was resuspended in co-culture liquid medium, and the callus was infected with Agrobacterium. After co-culturing in the dark for 2-3 days, the callus was transferred to selection medium for 4-6 weeks of selection culture. After secondary differentiation, the callus was transferred to differentiation medium for further differentiation culture. Once distinct shoots formed, the callus was transferred to rooting medium.

[0081] This invention provides methods for gene cloning, vector construction, and transgenic construction of Leymus chinensis genes, laying the foundation for subsequent functional analysis of Leymus chinensis genes and providing new methods for the breeding of new Leymus chinensis varieties.

[0082] Example 3 LcCBP60b Phenotypic identification of transgenic plants According to Example 2, LcCBP60b overexpressing plants and silenced transgenic plants were obtained in Leymus chinensis. Figure 3 To identify the DNA and RNA levels of transgenic plants, select qualified transgenic plants for subsequent phenotypic identification.

[0083] LcCBP60b The phenotypic and agronomical traits of transgenic plants are statistically analyzed in [the table below]. Figures 4 - 5 The results showed that, based on observations of the heading stage of transgenic plants, overexpression of Leymus chinensis in Leymus chinensis was observed. LcCBP60b After gene expression, plant height and biomass were significantly reduced. The plant height of overexpressing transgenic plants was 70.76% lower than that of wild-type plants. No spikelets were observed in these plants. After separating the plants, extremely shortened spikelets of reproductive branches were visible inside. The spikes of overexpressing plants were thinner and shorter than those of wild-type plants. The spike length of overexpressing plants was 56.55% shorter than that of wild-type plants, and the number of spikelets and spikelet length were reduced by 50% and 27.84%, respectively. The total number of florets in overexpressing plants was significantly lower than that in wild-type plants, accounting for only 22.35% of the total. At the same time, the seed setting rate of overexpressing plants was almost zero. The plant height of the silent transgenic plants was not significantly different from that of the wild type, but the spike length was significantly longer. The spike length of the silent transgenic plants increased by 35.17% compared with the wild type, the spikelet length increased by 28.03% compared with the wild type, and the total number of florets increased by 21.79% compared with the wild type. The seed setting rate of the silent transgenic plants was 2.6 times that of the wild type.

[0084] The above data indicates that in sheepgrass LcCBP60b Genes negatively regulate spike formation and seed setting in Leymus chinensis. Overexpression in Leymus chinensis LcCBP60b The gene significantly inhibited the development of Leymus chinensis plants and spike formation, greatly affecting the seed setting rate; while the phenotype of the silenced transgenic plants showed that silencing the gene promoted spike elongation and increased the seed setting rate.

[0085] Example 4 Transcriptome Sequencing Mining LcCBP60b Molecular mechanisms regulating seed setting rate in Leymus chinensis In order to further explore the genes of Leymus chinensis in this invention LcCBP60b Molecular mechanisms regulating seed setting rate, selecting wild-type and [other] individuals at the heading stage. LcCBP60b Transcriptome sequencing was performed on the spikelets of silent transgenic plants. During transcriptome sampling, samples were collected from different plants in a pooled manner to eliminate differences between different biological systems. Transcriptome data analysis is as follows: Figure 6As shown, 7278 differentially expressed genes were screened from the transcriptome data of wild-type and silent transgenes using |log2FC|>1 as the screening criterion. GO function and KEGG metabolic pathway enrichment analyses were performed on the differentially expressed genes. GO enrichment analysis results showed that the differentially expressed genes were mainly concentrated in seed development-related pathways and flowering-related pathways. Among them, 48 differentially expressed genes were found in the seed development-related pathway GO:0009791 (post-embryonic development), and 23 genes were enriched in the post-embryonic development regulation pathway GO:0048580 (regulation of post-embryonic development).

[0086] Example 5: Yeast dual-hybrid screening for LcCBP60b interacting proteins In order to further explore the molecular mechanism by which the LcCBP60b gene of Leymus chinensis regulates spike length and seed setting rate, this invention uses a yeast library to screen for LcCBP60b interacting proteins. The specific operations mainly include yeast library preparation, yeast double-hybrid screening library, and one-to-one yeast interaction verification.

[0087] Yeast library preparation: Spikes from *Leymus chinensis* at both flowering and grain-filling stages were used for total RNA extraction using a polysaccharide-polyphenol RNA extraction kit from Beijing TransGen Biotech Co., Ltd. Equal volumes of the two RNAs were mixed to form 1 μg of cDNA for initial synthesis. The purified cDNA was homogenized using a TRIMMER DIRECT cDNA homogenization kit (purchased from Shenzhen Newbang Biotechnology Co., Ltd.). The homogenized cDNA was then subjected to PCR amplification. The amplified cDNA was purified using a DNA fragment purification kit and digested with the restriction endonuclease SfiI. The digested cDNA was then column-processed using CHROMA SPIN-1000-TE to remove short fragments. After purification by phenol and chloroform extraction, the cDNA was washed with ethanol and dissolved in water. The pGADT7 three-frame vector was ligated to an appropriate amount of column-processed cDNA using DNA ligase and incubated overnight at 12°C. The resulting ligation solution was purified and dissolved in 20 μL of TE elution buffer to obtain the primary library. Take a small amount of primary library ligation medium and electrotransform it into competent HST08 cells; take an appropriate amount of transformation medium and spread it on an Amp-resistant LB agar plate, incubate overnight at 37°C; calculate the primary library volume based on the number of colonies grown on the plate, ensuring that the volume is greater than 3.0 × 10⁻⁶. 6CFU. Thirty-two clones were randomly selected for culture PCR to check for insert fragments, ensuring the average length of the insert fragment was greater than 1 kb. The primary library was ensured to contain more than 3 million clones. The clones were electroporated into *E. coli* HST08, plated on 15 24.5 cm × 24.5 cm LB agar plates, and incubated overnight at 37°C. The amplified colonies were recovered, and endotoxin-free plasmids were extracted using the NucleoBond Xtra Midi EF kit (MedChemExpress), retaining approximately 40 mL of the amplified library glycerol. 10 μg of the amplified library plasmid was used to transform the plasmid into Y187 yeast using a yeast transformation kit (MedChemExpress). The transformed plasmids were plated on 100 15 cm diameter SD / -Leu agar plates and incubated upside down at 30°C for 3 days. Using freezing medium containing 25% glycerol (purchased from Thermo Fisher Scientific), scrape off 50 mL of the plate to recover the colonies, and dispense 1 mL per tube.

[0088] Yeast dual-hybrid screening library: The constructed pGBKT7-LcCBP60 plasmid was transformed into yeast strain Y2H, and nutrient deficiency screening was performed using SD / -Tre plates. After culturing at 30℃ for 2-3 days, single yeast clones were picked from the plates and added to 500 μL of SD / -Trp liquid medium for gentle shaking. Colony PCR was performed using the vector primers (Table 4) as shown in SEQ ID NO:27 and SEQ ID NO:28. Yeast strains with correct bands were selected and added to 100 mL of SD / -Trp liquid for shaking until the OD600 reached 0.6. Then, 1 mL of a yeast library was added for yeast conjugation, and the mixture was incubated for 3-6 hours. During this time, the yeast culture was observed under a light microscope. After shaking until half of the yeast cells showed a "Mickey Mouse head" shape under the microscope, the culture was briefly centrifuged, and the yeast cells were resuspended in an appropriate amount of 0.9% sterile NaCl solution. The resuspended yeast cells were then plated onto 15 cm diameter SD / -Leu-Trp-His-Ade solid medium and incubated upside down at 30℃ for 2-3 days. Single yeast clones from the four-deficient plates were then streaked onto fresh four-deficient solid medium for secondary selection. The confluent yeast cells were subjected to PCR using primer sequences shown in SEQ ID NO:27 and SEQ ID NO:28. The PCR products were identified by electrophoresis, and non-empty vector sequences were selected for sequencing.

[0089] Yeast two-hybrid one-to-one verification: In this invention, after screening the yeast two-hybrid library, the candidate interacting gene LcALY2 was obtained by sequencing. The target gene was then cloned using primers shown in SEQ ID NO:15 and SEQ ID NO:16. The target gene fragment was subjected to electrophoresis, DNA fragment purification, homologous recombination, transformation of *E. coli* competent cells, and sequencing to obtain the correct LcALY2 fusion vector for yeast two-hybridization. The specific system and process are as follows: The yeast double-hybrid experimental system consisted of single-stranded salmon sperm DNA solution, 500 ng of pGBKT7-LcCBP60 plasmid, 500 ng of pGADT7-LcALY2 plasmid, and 100 μL of PEG / liAc solution. After gentle inversion and mixing, the mixture was heat-shocked at 30°C for 30 min and then at 42°C for 15 min. During transformation, the mixture was inverted and mixed every 5-10 min. After centrifugation at 10,000 rpm for 15 sec, the yeast was resuspended in 100 μL of sterile 0.9% NaCl solution and plated on 15 cm SD / -Leu-Trp solid medium. The medium was incubated upside down at 30°C for 2-3 days. The salmon sperm DNA and PEG / liAc solution were purchased from Beijing Bomei Biotechnology Co., Ltd. Single yeast clones on the plates were serially diluted with 0.9% NaCl solution, and 10 μL was used for spotting. Positive and negative controls were also included. Results are as follows: Figure 7 As shown in Figures A and B, LcCBP60 and LcALY2 can grow normally on a four-deficient plate, indicating that they have a strict interaction relationship.

[0090] Table 4 pGBKT7 PCR primers

[0091] Example 6: BFIC experiment verifies its interaction and working area. To further verify their interaction and explore their working region, a BFIC experiment was conducted. Gene expression vectors were constructed and corresponding Agrobacterium-mediated transformations were performed according to the vector construction method described in the above experimental procedures of this invention. The main steps are as follows: (1) Cloning of the target fragment: The target gene is cloned according to the reaction system and reaction procedure of the above-mentioned experimental operation of the present invention. The primer sequences used are shown in SEQ ID NO:17-20.

[0092] (2) Purification of target fragment: After electrophoresis, the PCR product is taken and the bands of the appropriate size are recovered as solids. The specific operation method is the same as the purification method in the above experimental operation of this invention.

[0093] (3) Homologous recombination: The purified target gene fragment is homologously recombinated with the linear vector. The specific operation is carried out in accordance with the method mentioned in the above experimental operation of this invention.

[0094] (4) Transformation of competent cells in the large intestine: The obtained recombinant plasmids are transformed into competent cells for subsequent sequencing analysis.

[0095] (5) Bacterial PCR and sequencing: After shaking the single clones obtained in step four, bacterial PCR was performed using the primers shown in SEQ ID NO:29-32. After electrophoresis analysis, the corresponding single clones were selected for first-generation sequencing to verify the gene sequence.

[0096] (6) The obtained positive clones were subjected to transient expression in tobacco after being shaken. After 2-3 days, they were observed under a laser confocal microscope at an excitation wavelength of 561 nm.

[0097] The results are as follows Figure 7 As shown in Figure C, it is evident in the observation field that when a single gene is co-injected with an empty vector, it does not emit light. Only when LcCBP60 and LcALY2 are co-injected, obvious fluorescence can be observed in tobacco cells. After co-injection with the internal reference gene Athook (nuclear localization), the images were fused and observed, revealing that LcCBP60 and LcALY2 directly interact, and the working region of their interaction is in the cell nucleus.

[0098] The PCR primers used in the BFIC experiment are shown in Table 5.

[0099] Table 5. Relevant PCR primers used in the BFIC experiment.

[0100] Example 7: Subcellular localization observation of LcCBP60 and LcALY2 proteins To further investigate the accuracy of the experiment, subcellular localization of LcCBP60 and LcALY2 proteins was observed. A fusion vector with a GFP tag was constructed, transformed into Agrobacterium, and transiently expressed in tobacco was performed. The specific procedures were as follows: First, the target gene was cloned, purified and recovered by electrophoresis, and the target gene fragment was homologously recombinated with the linear vector. After transformation into competent E. coli, plate selection, and single-clone culture, PCR was performed. After electrophoresis analysis, the corresponding single clones were sequenced. The sequenced bacterial culture was shaken and plasmids were extracted, then transformed with Agrobacterium, screened by plate, and single-clone culture was performed. After PCR, positive single clones were selected for subsequent tobacco expression experiments.

[0101] After the transformed Agrobacterium tumefaciens bacterial suspension was shaken to the appropriate concentration, it was resuspended in tobacco resuspension solution. After standing in the dark for 2-3 hours, tobacco seedlings with 4-6 leaves were selected for injection. After culturing in the dark for 6 hours after injection, the seedlings were transferred to the culture room for normal culture and observed after 48-72 hours.

[0102] The results are as follows Figure 8 As shown, multiple subcellular localization results indicate that, under an excitation wavelength of 488 nm, the LcCBP60b expression region in tobacco cells overlaps with the Athook (nuclear localization) expression region, indicating that it is mainly localized in the nucleus. In contrast, LcALY2 in tobacco cells, in addition to overlapping with the Athook (nuclear localization) expression region, also shows obvious fluorescence in the cytoplasm, indicating that LcALY2 expression is dual localized in both the nucleus and cytoplasm, which is consistent with the results observed in Example 6.

[0103] Based on the above series of results, it can be concluded that... LcCBP60b Genes negatively regulate spike formation and seed setting in Leymus chinensis. Wild-type and... LcCBP60b Transcriptome sequencing was performed on spikelets from silent transgenic plants at the heading stage. Differential gene metabolic pathway enrichment analysis revealed that differentially expressed genes were mainly enriched in pathways related to flowering and post-embryonic development in Leymus chinensis. After screening with a yeast two-hybrid library, interaction between LcCBP60b and LcALY2 was found. LcALY2 The encoded THO complex subunit 4B is a key component of the THO / TREX complex, primarily involved in nucleocytoplasmic transport of messenger RNA (mRNA) and playing a role in the regulation of gene transcriptional elongation. In LcCBP60b-silenced transgenic plants, LcCBP60b expression was significantly reduced, leading to… LcALY2 The THO complex subunit 4B encoded by the gene can serve as a key component of the THO / TREX complex, mediating the production of TAS3 ta-siRNA and thereby regulating signaling communication between ovule cells. In overexpressing plants, the expression level of LcCBP60b increases, forming a complex with LcALY2, which interferes with the function of the THO / TREX complex, thereby affecting the development of Leymus chinensis plants and spike formation, and negatively regulating the seed setting rate of Leymus chinensis.

[0104] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. LcCBP60b Application of genes in regulating the growth, development, and seed setting rate of Leymus chinensis; in, The LcCBP60b The genes are: i) The nucleotide sequence shown in SEQ ID NO:1; ii) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO:1 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; iii) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:1 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or, iv) Nucleotide sequences that have more than 90% homology with the nucleotide sequences of i), ii) or iii) and express the same functional protein.

2. The application according to claim 1, characterized in that, The regulation mentioned is negative regulation.

3. The application according to claim 2, characterized in that, The application includes: by inhibiting the LcCBP60b The expression of genes or the activity of their encoded proteins can increase the seed setting rate of Leymus chinensis and / or increase spike length and spikelet number.

4. A method for improving the seed setting rate, increasing spike length, and increasing the number of spikelets of Leymus chinensis, characterized in that, The method includes reducing the content of sheepgrass. LcCBP60b The expression level of a gene or the activity of its encoded protein; Among them, the LcCBP60b The gene is as described in claim 1.

5. The method according to claim 4, characterized in that, Used to reduce the concentration in sheepgrass LcCBP60b The expression level of a gene or the activity of its encoded protein is selected from at least one of shRNA, siRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, and antibodies.

6. The method according to claim 4, characterized in that, Using RNA interference to reduce the LcCBP60b Gene expression levels.

7. The method according to claim 6, characterized in that, Will LcCBP60b Gene silencing expression vectors were introduced into Leymus chinensis, resulting in transgenic Leymus chinensis plants... LcCBP60b Gene expression levels were lower compared to non-transgenic wild-type sheepgrass; Among them, the LcCBP60b Gene silencing expression vectors contain those that can specifically inhibit Leymus chinensis. LcCBP60b A nucleic acid molecule for gene expression, said nucleic acid molecule comprising a sequence complementary to the nucleotide sequence shown in SEQ ID NO:1 or a fragment thereof.

8. The method according to claim 7, characterized in that, The nucleic acid molecule consists of 1-250 bp of the nucleotide sequence shown in SEQ ID NO:

1.

9. The application of the LcCBP60b-LcALY2 molecular module in regulating the growth, development, and seed setting rate of Leymus chinensis, characterized in that... The growth, development, and seed setting rate of Leymus chinensis are regulated by modulating the interaction between LcCBP60b and LcALY2 proteins. Wherein, the LcCBP60b protein is as described in claim 1 LcCBP60b Gene encoding; The LcALY2 protein is composed of LcALY2 Gene encoding, the LcALY2 The genes are: I) The nucleotide sequence shown in SEQ ID NO:2; II) A nucleotide sequence of SEQ ID NO:2 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function; III) A nucleotide sequence that hybridizes with the sequence shown in SEQ ID NO:2 under stringent conditions and expresses a protein with the same function, wherein the stringent conditions are hybridization at 65°C in 0.1×SSPE containing 0.1% SDS or 0.1×SSC containing 0.1% SDS, followed by washing the membrane with the same solution; or, A nucleotide sequence that has more than 90% homology with the nucleotide sequences of I), II) or III) and expresses the same functional protein.

10. The application according to claim 9, characterized in that, By reducing the expression or activity of the LcCBP60b protein, its binding to the LcALY2 protein is weakened, thereby relieving transcriptional repression of downstream flowering and embryonic development-related genes, and thus increasing the seed setting rate of Leymus chinensis and / or increasing spike length and spikelet number.

Images