Application of LcCBP60b gene in regulating tiller number and biomass of Leymus chinensis

Abstract

This invention relates to the field of plant genetic engineering technology, and provides sheepgrass. LcCBP60b The application of genes in regulating plant tiller number and biomass yield. This invention is the first to discover... LcCBP60b Genes can increase the number of tillers and biomass of Leymus chinensis, which is of great guiding significance for breeding high-yield and high-quality Leymus chinensis varieties; this invention also clarifies that LcCBP60b Investigating the molecular mechanisms by which genes regulate tiller number and biomass of Leymus chinensis, and further elucidating the molecular modules that regulate plant tiller number and biomass, can enrich the corresponding molecular mechanisms and provide new breeding directions for selecting high-yield and high-quality varieties.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and more specifically, to Leymus chinensis. LcCBP60b Application of genes in regulating plant tiller number and biomass yield. Background Technology

[0002] sheepgrass ( Leymus chinensis L. (Lysimachia) is a perennial herbaceous plant belonging to the genus L. of the Poaceae family. It is widely distributed in the temperate grasslands of Eurasia, especially forming dominant populations in the Mongolian Plateau, Northeast China, and North China, and is an important constructive species in typical grasslands and meadow steppes. It has a well-developed root system, strong resistance to adverse conditions, and is also cold-resistant, drought-resistant, and salt-tolerant, making it a preferred species for ecological restoration and the recovery of degraded grasslands. Tillering is a core agronomic trait for yield formation in Poaceae crops and forage grasses, influenced by the synergistic effects of genetic regulatory networks and external environmental signals. In recent years, with the development of molecular biology techniques, many key genes regulating tillering and their mechanisms of action have been gradually revealed, such as in rice. OsTB1 Corn ZmFC1 Genes such as [the gene] affect the number of tillers by regulating axillary bud growth.

[0003] Calmodulin-binding protein gene family ( Calmodulin-Binding Protein 60, CBP60 α is a family of transcription factors unique to plants that can bind calcium ions. In the model plant Arabidopsis thaliana, it has been shown to participate in plant immunity, growth and development, and stress response. For example, under high temperature stress, overexpression of α is observed. AtCBP60g Compared to the wild type, the Arabidopsis thaliana plants with the gene were shorter and exhibited yellowing; however, they showed significantly improved tolerance to drought stress.

[0004] By comparing and analyzing differentially expressed transcripts in samples before and after treatment, we can reveal the molecular mechanisms of transcriptional regulation in biological processes, thus providing an important pathway for a deeper understanding of related biological processes. However, in Leymus chinensis, an important forage grass species, the functional studies of the CBP60 family genes are relatively limited, and the molecular association mechanism between them and the tillering phenotype has not been reported, which greatly restricts the molecular breeding progress of high-yielding traits in Leymus chinensis. Summary of the Invention

[0005] The purpose of this invention is to provide sheepgrass LcCBP60b Application of genes in regulating plant tiller number and biomass yield.

[0006] This invention utilizes field trials, including CaCl2 spraying experiments conducted during the heading and grain-filling stages of Leymus chinensis. The results showed a significant increase in the number of reproductive branches and seed yield. Transcriptome data analysis revealed the presence of calmodulin-binding proteins. CBP60b Significant gene enrichment, cloning in Leymus chinensis LcCBP60b Genes are extracted and genetically transformed to achieve overexpression. LcCBP60bGenetically modified sheepgrass material. LcCBP60b Analysis of gene promoter cis-regulatory elements, yeast one-hybrid and yeast two-hybrid experiments revealed that the NAC2-CBP60b-THO4B gene module regulates the tiller number and biomass yield of Leymus chinensis plants.

[0007] To achieve the objectives of this invention, in a first aspect, this invention provides sheepgrass. LcCBP60b Application of genes in regulating plant tiller number and biomass yield.

[0008] In this invention, LcCBP60b The gene is the gene that encodes either (a) or (b) the following protein:

[0009] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or

[0010] (b) A protein derived from (a) with the sequence shown in SEQ ID NO:2 substituted, deleted or added with one or more amino acids and having the same function.

[0011] Furthermore, LcCBP60b The genes are:

[0012] i) The nucleotide sequence shown in SEQ ID NO:1;

[0013] 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;

[0014] 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

[0015] iv) Nucleotide sequences that have more than 90% homology with the nucleotide sequences of i), ii) or iii) and express the same functional protein.

[0016] Furthermore, the regulation is positive regulation, which increases plant biomass by promoting tillering.

[0017] In this invention, the plant includes herbaceous plants of the genus Leymus in the family Poaceae, with Leymus chinensis being the preferred species.

[0018] Secondly, the present invention provides a method for increasing tillering and improving biomass yield of Leymus chinensis, the method comprising: using genetic engineering techniques to overexpress the [specific ingredient] in Leymus chinensis. LcCBP60b Gene.

[0019] Furthermore, the overexpression method can be selected from the following 1) to 5), or any combination thereof:

[0020] 1) By importing a plasmid containing the gene;

[0021] 2) By increasing the copy number of the aforementioned genes on plant chromosomes;

[0022] 3) By altering the promoter sequence of the aforementioned genes on plant chromosomes;

[0023] 4) By operatively linking a strong promoter (such as the CaMV35S promoter) to the gene;

[0024] 5) By importing enhancers.

[0025] For example, genes LcCBP60b It can be successfully ligated into the UBI3301 GFP-Flag vector, and the Flag tag on the vector does not affect the function and properties of the target protein, nor does it interact with the target protein. It is mainly used as a tool for detection. This fusion protein can be used to further study the downstream functions of the target protein.

[0026] Among them, the UBI promoter can be efficiently expressed in monocotyledonous plant tissues, making... LcCBP60b The gene is overexpressed in sheepgrass plants, fully exerting its function.

[0027] Expression vectors carrying the target gene can be introduced into plant cells using conventional biotechnological methods such as Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, and electroporation (Weissbach, 1998, Method for Plant Molecular Biology VIII, Academy Press, New York, pp. 411-463; Geiserson and Corey, 1998, Plant Molecular Biology, 2). nd Edition).

[0028] Thirdly, the present invention provides the application of the method in the cultivation of high-yield and high-quality sheepgrass varieties.

[0029] Fourthly, the present invention provides the application of transgenic sheepgrass obtained according to the method in plant breeding.

[0030] Breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0031] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0032] This invention is the first to clone a gene that can induce Leymus chinensis to exhibit a multi-tillering phenotype. LcCBP60b The application of this gene can increase the biomass yield of Leymus chinensis, which has important guiding significance for breeding high-yielding Leymus chinensis varieties; this high-yielding line has practical guiding significance for production in Leymus chinensis areas and forage improvement breeding. Meanwhile, this invention clarifies... LcCBP60b Investigating the molecular mechanisms by which genes regulate tillering in Leymus chinensis and further elucidating the molecular modules that regulate tiller number and biomass yield can enrich the corresponding molecular mechanisms and provide new breeding directions for selecting high-yielding Leymus chinensis varieties. Attached Figure Description

[0033] Figure 1 This diagram illustrates the field yield characteristics of Leymus chinensis under CaCl2 treatment conditions in a preferred embodiment of the present invention. A represents the number of reproductive branches, and B represents seed yield. express P <0.05.

[0034] Figure 2 This is a Venn diagram analysis of differentially expressed genes in the transcriptome under CaCl2 treatment conditions, as described in a preferred embodiment of the present invention.

[0035] Figure 3 This is an enrichment map of differentially expressed gene regulatory pathways in the transcriptome under CaCl2 treatment conditions in a preferred embodiment of the present invention.

[0036] Figure 4 This is a gene enrichment map of the LcCBP60 family under CaCl2 treatment conditions in a preferred embodiment of the present invention.

[0037] Figure 5 In the preferred embodiment of the present invention LcCBP60b Structure diagram of the UBI3301 GFP-Flag vector.

[0038] Figure 6 Overexpression in a preferred embodiment of the present invention LcCBP60b Measurement of the height of genetically modified Leymus chinensis plants. Among them, express P <0.05.

[0039] Figure 7 Overexpression in a preferred embodiment of the present invention LcCBP60b Measurement of tiller number in genetically modified Leymus chinensis. Among them, express P <0.05.

[0040] Figure 8 In a preferred embodiment of the present invention LcBP60b Analysis of gene expression levels in different tissues of Leymus chinensis.

[0041] Figure 9 In a preferred embodiment of the present invention LcBP60b Analysis of gene promoter regulatory elements.

[0042] Figure 10 In a preferred embodiment of the present invention LcBP60b Yeast single-hybrid assay analysis of gene promoter.

[0043] Figure 11 This is a yeast two-hybrid analysis of the LcBP60b interacting protein in a preferred embodiment of the present invention.

[0044] Figure 12 In a preferred embodiment of the present invention NAC2 - CBP60b - THO4B A schematic diagram illustrating the regulation of Leymus chinensis growth and development by molecular modules. Detailed Implementation

[0045] This invention is the first to discover the sheepgrass gene. LcCBP60b Genes can increase the number of tillers in Leymus chinensis, thereby regulating plant biomass.

[0046] First, experimental fields of Leymus chinensis were established. In the spring of the second year, Leymus chinensis plots with uniform growth were selected. CaCl2 spraying experiments were conducted during the heading and grain-filling stages of Leymus chinensis. Leaf and spikelet tissues during the heading and grain-filling stages were frozen with liquid nitrogen for transcriptome analysis. During the seed maturity stage of Leymus chinensis, the number of reproductive branches and yield traits were investigated.

[0047] Transcriptome data analysis revealed the *Leymus chinensis* gene. LcCBP60b It can increase the number of tillers in sheepgrass, thereby regulating biomass yield.

[0048] The present invention also provides a type of sheepgrass. LcCBP60b Methods for cloning genes and constructing expression vectors to further obtain Leymus chinensis. LcCBP60b Overexpression of transgenic lines for the study of Leymus chinensis LcCBP60b Gene function. Through yeast one-hybrid and yeast two-hybrid experiments, the transcriptional regulatory mechanisms affecting the tillering phenotype of Leymus chinensis were further clarified. Application of this gene is beneficial for improving the tillering and biomass yield of Leymus chinensis.

[0049] The present invention adopts the following technical solution:

[0050] This invention provides sheepgrass LcCBP60b The gene, the nucleotide sequence of which is shown in SEQ ID NO:1, and the amino acid sequence of the protein it encodes, are shown in SEQ ID NO:2. LcCBP60b The gene's mRNA sequence has 1959 bases and can encode a protein with 652 amino acids.

[0051] The present invention also provides an expression vector comprising the above-mentioned Leymus chinensis. LcCBP60b Gene.

[0052] This invention also provides a method for constructing the expression vector. First, total RNA is extracted from *Leymus chinensis*, and then rapidly reverse transcribed into cDNA for later use. Specific primers are designed, and a full-length 1959 bp gene is amplified using a PCR instrument. After agarose gel electrophoresis, the PCR product is excised and recovered. The recovered product is ligated into the pEASy-Blunt Zero cloning vector. After transformation, the plasmid is extracted and subjected to batch PCR verification. Bacterial solutions with bands are sent to a sequencing company for sequencing. Solutions with correct sequencing are preserved for plasmid extraction. The UBI3301 GFP-Flag vector is digested with the restriction endonuclease BamHI. After gel recovery, the target plasmid is extracted and ligated into the plant expression vector. After transformation, batch PCR verification is performed. Bacterial solutions with bands are again sent to a sequencing company for sequencing. Solutions with correct sequencing are preserved. The extracted plasmid is the recombinant vector containing the target gene.

[0053] This invention provides transcriptome data analysis to analyze regulation LcCBP60b Potential genes. The specific steps are as follows:

[0054] 1. After RNA extraction, a library was constructed, and preliminary quantification was performed using a Qubit 3.0 real-time fluorescence instrument (concentration >1 ng / μL). Subsequently, the insert fragments in the library were detected using a Qsep400 high-throughput analysis system. After the insert fragments met the expectations, the effective concentration of the library (effective concentration of the library >2 nM) was accurately quantified using the Q-PCR method to ensure the quality of the library.

[0055] 2. After the library passed quality control, PE150 sequencing was performed using the Illumina NovaSeq 6000 sequencing platform. The cDNA library was sequenced using the Illumina high-throughput sequencing platform, producing a large amount of high-quality data, referred to as raw data. Transcript assembly and merging were performed using StringTie v2.2.1 software. The raw sequencing volume, effective sequencing volume, and other metrics were statistically analyzed and then comprehensively evaluated. Subsequent high-quality analysis was based on the clean data.

[0056] 3. Download the annotation files for the *Leymus chinensis* reference genome and gene model from the *Leymus chinensis* genome website (https: / / figshare.com / ). Use HISAT2 v2.0.5 software to compare the sequenced clean reads with the reference genome sequence. Gene function was annotated based on sequence alignment using the following databases: Nr (NCBI non-redundant proteinsequences); Pfam (Protein family); KOG / COG (Clusters of Orthologous Groups of proteins); Swiss-Prot (A manually annotated and reviewed protein sequence database); KO (KEGG Ortholog database); GO (Gene Ontology). Homologous genes of calmodulin-binding protein 60B, which is related to the calcium signaling pathway, were finally enriched, and differential expression was found upon CaCl2 treatment.

[0057] The present invention provides LcCBP60b The gene and its application can promote an increase in the number of tillers and plant height of Leymus chinensis, laying the foundation for the subsequent construction of the gene vector and obtaining high-yielding transgenic plants; at the same time, the gene involved in this invention can affect the biological yield of Leymus chinensis.

[0058] 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.

[0059] Example 1: Effects of CaCl2 treatment on seed yield and yield composition of Leymus chinensis in the field

[0060] A randomized block design was used for CaCl2 spraying experiments: plots were 2.5 m × 3 m in size, with 0.5 m protective rows, and four replicates per group. CaCl2 was sprayed at the heading and flowering stages of Leymus chinensis at a concentration of 0.8 g / L, and a control group was included. Through investigation and statistical analysis of field yield traits, the results showed that CaCl2 significantly affected the number of reproductive branches of Leymus chinensis (…). Figure 1 A), and further increase the field seed yield of Leymus chinensis (A) Figure 1 B), which increased by 71.71% and 68.94% respectively compared to the control.

[0061] Example 2: Differential Gene Analysis of Leymus chinensis Transcriptome under CaCl2 Treatment

[0062] Using young spikelets of Leymus chinensis at the heading and grain-filling stages treated with CaCl2 as material, and with a control group, transcriptome analysis was conducted to screen for common or unique differentially expressed genes among the treatment combinations, indicating differences in gene expression among each combination. Statistical analysis of differentially expressed genes using Venn diagrams among different sample combinations was performed. Figure 2 A total of 4 differentially expressed genes were identified across all combinations. The number of differentially expressed genes unique to each combination were 1953, 2304, 9332, and 475, respectively. Among them, 993 genes were upregulated and 960 genes were downregulated in the control vs. CaCl2 treatment combination at the heading stage; and 239 genes were upregulated and 236 genes were downregulated in the control vs. CaCl2 treatment combination at the grain-filling stage.

[0063] KEGG functional enrichment showed that differentially expressed genes in the different combinations at the heading stage were mainly involved in the biosynthesis of amino acids such as unsaturated fatty acids and phenylalanine, as well as the metabolism of various fatty acids, polysaccharides such as glycosamines, chlorophyll, and starch and sucrose. Figure 3 A). The differentially expressed genes in the different combinations during the grain-filling stage mainly involve glycosphingolipid biosynthesis, metabolism of starch, sucrose, sphingolipids, galactose, and glycerides, and polysaccharide degradation. This indicates that CaCl2 treatment during the growth and development of Leymus chinensis affects signal transduction, sugar and lipid metabolism, and other processes in its reproductive growth, thereby affecting seed yield. Figure 3 B). Further analysis of differentially expressed genes enriched six genes related to calmodulin-binding protein function. All of these genes are regulated by CaCl2 and showed differential expression between treatment and control, resulting in different seed yield phenotypes in different samples. Among them, Lc6Xm081419 and Lc6Ns029448... LcCBP60b The sequence similarity is over 98%, indicating that LcCBP60b In Leymus chinensis, calcium ion signaling regulates the plant's tiller number and biomass. Figure 4 ).

[0064] Example 3: Calmodulin-binding protein from sheepgrass LcCBP60b Gene cloning

[0065] 1.1 Experimental Materials:

[0066] Leymus chinensis, Escherichia coli competent strain DH5α; Agrobacterium tumefaciens competent strain EHA105; cloning vector pEASy-Blunt Zero and plant expression vector pCAMBIA3301GFP-Flag (both cloning and plant expression vectors were purchased from Shanghai Yuanmu Biotechnology Co., Ltd.), reverse transcription kit, restriction endonuclease BamHI enzyme, homologous recombinase, plasmid extraction kit, gel recovery kit, etc.

[0067] 1.2 Experimental Methods:

[0068] 1.2.1 Extraction of total RNA from Leymus chinensis:

[0069] 0.1 g of Leymus chinensis leaves were ground in liquid nitrogen and then total RNA was extracted using the Huayueyang RNA Extraction Kit, following the instructions. RNA concentration and purity were determined using a Denovix micro spectrophotometer, and RNA quality was assessed by agarose gel electrophoresis.

[0070] 1.2.2 cDNA Synthesis

[0071] cDNA synthesis was performed using the Novozymes First Strand cDNA Synthesis Kit (HiScript III 1st Strand cDNA Synthesis Kit) according to the following system and reaction procedure:

[0072] (1) RNA template denaturation:

[0073] Prepare 2 μg total RNA and 2 μL 5×g DNA wiper mix in an RNase-free centrifuge tube, and bring the volume to 10 μL with RNase-free ddH2O. Gently mix by pipetting and briefly centrifuge. Incubate at 42 °C for 2 min on a PCR instrument.

[0074] (2) First-chain synthesis reaction

[0075] Add 2 μL of 10 × RTMix, 2 μL of HiScript III EnzymeMix, and 1 μL of Oligo (dT)20VN sequentially to the premixed solution from the previous step. Make up the remaining solution with sterile ddH2O to a total of 20 μL. Then, place the mixture in a PCR instrument, gently mix the reaction solution, and centrifuge briefly. Incubate the mixture in the PCR instrument at 37°C for 45 min, then at 85°C for 5 s. Store the obtained product at -20°C.

[0076] 1.2.3 LcCBP60b Gene cloning and vector ligation

[0077] Design specific primers, with primer sequences for adding BamHI restriction sites as follows:

[0078] Upstream primer: 5'-ggatcttccagagatATGCAGCGGCCTGGGCGG-3'

[0079] Downstream primer: 5'-ctgccgttcgacgatCTAGTCATCTAGCTCAACGAGCTGA-3'

[0080] Using the reverse-transcribed cDNA as a template, the total volume was 50 μL, consisting of 25 μL of 2×Phanta Max Master Mix (DyePlus), 2 μL each of forward and reverse primers, 2 μL of cDNA, and 19 μL of ddH2O. PCR amplification was performed according to the following program: 95 ℃ for 5 min; 95 ℃ for 15 s; 60 ℃ for 15 s; 72 ℃ for 5 min for 40 cycles; 72 ℃ for 10 min; and the reaction was terminated at 12 ℃. The PCR products were electrophoresed on a 1% agarose gel in 1×TAE electrophoresis buffer at 170 V for 25 min. The band corresponding to the target gene size was excised and weighed in a 2 ml centrifuge tube. The target gene was recovered using the Tiangen Agarose Gel DNA Recovery Kit (DP209) according to the manufacturer's instructions.

[0081] The pEASy-Blunt Zero vector was then ligated and transformed into DH5α E. coli. Batch PCR was used to screen for banded bacterial solutions, which were then sent to the company for sequencing. Positive colonies were preserved for later use, and plasmids were extracted from the bacterial solutions.

[0082] The specific steps for homologous recombination are as follows:

[0083] 0.5-4 μL of PCR amplification product

[0084] pEASy-Blunt Zero cloning vector 1 μL

[0085] 2 × ClonExpress Mix 5μL

[0086] Add ddH2O to a final volume of 10 μL.

[0087] Gently pipette to mix, briefly centrifuge to collect the liquid at the bottom of the tube, and incubate at 50 °C for 10 min. After the reaction is complete, place the centrifuge tube on ice.

[0088] The specific steps for E. coli transformation are as follows:

[0089] (1) Transformation of Escherichia coli competent cells with recombinant plasmid

[0090] DH5α competent cells were thawed on ice. After complete thawing, the product from the previous step was added to the competent cells, the tube was gently shaken, and then incubated on ice for 30 min. Subsequently, the competent cells were heat-shocked in a 42 °C water bath for 30 s, and then quickly placed on ice for 2 min. Next, 900 μl of antibiotic-free LB liquid medium was added. The bacterial suspension was placed in a shaker at 37 °C and thawed at 200 rpm for 1 h. The thawed bacterial suspension was centrifuged at 5000 rpm for 5 min, the bacterial pellet was collected, and 800 μl of the supernatant was discarded. The remaining bacterial suspension was resuspended and spread onto solid LB agar plates containing kanamycin (50 mg / L). After the plates showed no significant liquid accumulation, they were incubated upside down in a 37 °C incubator for 16 h.

[0091] (2) Identification of positive clones

[0092] After bacterial colonies grow on the plate, single clones are randomly selected for PCR identification and sequencing verification. The sequencing results are then spliced ​​together and compared with the target gene sequence. Clones with the correct sequence are considered positive clones.

[0093] (3) Store the correctly sequenced bacterial culture at -80 ℃ using 60% glycerol.

[0094] Plasmid extraction (the following reagents are from Tiangen Biotech Co., Ltd. plasmid mini-prep kit) The specific steps are as follows:

[0095] First, incubate 10 ml of the bacterial suspension containing the positive clone on a shaker. Use the Tiangen plasmid extraction kit (model DP103) according to the instruction manual. Take 5 ml of the bacterial suspension and centrifuge at 12000 rpm for 1 min, then discard the supernatant. Add 0.25 ml of P1 lysis buffer to the centrifuge tube containing the bacteria, and vortex to completely suspend the bacteria. Next, add 0.25 ml of P2 solution, and gently invert the tube several times to ensure complete lysis of the bacteria. Quickly add 350 μL of P3 solution, and after gently shaking the tube, a white flocculent precipitate will form. Centrifuge at 12000 rpm for 10 min. Transfer the supernatant to an adsorption column and centrifuge at 12000 rpm for 1 min. Add 0.5 ml of protein removal solution to the adsorption column, centrifuge, and discard the waste liquid in the bottom collection tube. The adsorption column was washed twice with 0.7 ml of washing buffer, the waste liquid was discarded, and the column was centrifuged for 2 min. It was then placed in a well-ventilated area and allowed to air dry at room temperature for 5 min to ensure complete evaporation of the ethanol. Finally, 0.03 ml of eluent was added to the column, and it was allowed to stand at room temperature for 3 min, followed by centrifugation at 12000 rpm for 2 min to collect the plasmid.

[0096] The gene is 1959 bp in length (SEQ ID NO:1), and the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2. This gene is named... LcCBP60b .

[0097] Example 4 LcCBP60b Construction of plant gene expression vectors

[0098] The vector was digested with BamHI alone. The digestion system consisted of 50 μL of BamHI, 5 μL of 10× buffer, 2 μg of vector plasmid, and ddH2O to a final volume of 50 μL. The mixture was incubated at 37 °C for 2 h, followed by incubation at 65 °C for 20 min to inactivate the restriction enzyme. After agarose gel electrophoresis, the gel was excised and recovered using a gel extraction kit, and stored at -20 °C for later use.

[0099] Design specific primers (forward primer: 5′-ATGCAGCGGCCTGGGCGG-3′; reverse primer: 5′-GTCATCTAGCTCAACGAGC-3′). Using the above plasmid as a template, the total volume was 50 μL, i.e., 25 μL of 2×Fly Super Mix; 2 μL each of the forward and reverse primers; 2 μL of cDNA; and 19 μL of ddH2O. The amplification program was as follows: 94 ℃ for 3 min; 94 ℃ for 20 s; 55 ℃ for 20 s; 72 ℃ for 1 min for 35 cycles; 72 ℃ for 5 min; and the reaction was terminated at 16 ℃. The PCR reaction was then performed. The PCR products were detected by agarose gel electrophoresis, and the gel was excised and recovered using a gel extraction kit. The products were stored at -20 ℃ for later use.

[0100] The homologous recombination procedure was performed as follows: the solution (3.5 μL of PCR product, 5 μL of homologous recombinase, and 1.5 μL of vector) was incubated in a PCR chamber at 50°C for 30 min. Subsequently, the homologous recombination transformation product was transformed into E. coli using the same method described above, and sequencing was performed using the universal primers for the vector itself.

[0101] UBI3301 F:5'-GCTCTAGAACTAGTGGATCCACTAGTCC-3'

[0102] UBI3301 R:5'-CGGGATCCTCTAGAGGTCACG-3'

[0103] The bacterial culture with correct sequencing was preserved, and plasmid was extracted and transformed into Agrobacterium EHA105 competent cells. Positive clones were screened, and the plasmid was extracted and verified by restriction enzyme digestion, confirming successful transformation into Agrobacterium. This expression vector ( LcCBP60b The structure of the pCAMBIA3301 GFP-Flag vector is shown below. Figure 5 It can be directly used for the conversion of plants such as alfalfa, Arabidopsis thaliana, and tobacco.

[0104] The specific steps for Agrobacterium EHA105 transformation are as follows:

[0105] GV3101 Agrobacterium competent cells were extracted from a -80°C frozen environment and thawed on ice. After thawing to a partially liquid state, the recombinant expression vector plasmid was added, and the tube was gently agitated to mix thoroughly. The Agrobacterium competent cells were then incubated on ice for 5 min, followed by treatment in liquid nitrogen for 5 min, then heat-shocked in a 37°C water bath for 5 min, and finally cooled on ice for 5 min. Next, 700 μL of LB medium (antibiotic-free) was added to the tube containing the competent cells, and the cells were cultured at 28°C and 200 rpm for 3 h with shaking to promote recovery. After recovery, the cells were centrifuged at 5000 rpm for 5 min to pellet the cells, reserving 200 μL of supernatant, which was used to resuspend the Agrobacterium competent cells. These cells were then plated on agar plates containing kanamycin (50 mg / L) and rifampin (50 mg / L) and allowed to stand until no visible liquid remained on the plate surface. After sealing the plate with Parafilm M, it was placed in an incubator at 28°C and incubated for another 2 days.

[0106] After colonies grew, five single clones were selected from each gene-transformed plate for PCR positive identification. Homologous recombination primers for the expression vector were used for identification. The PCR reaction system and procedure were consistent with the E. coli positive identification method in Example 3. The positive clones were then propagated in culture and stored for subsequent experiments.

[0107] Example 5 LcCBP60b Genetic transformation of sheepgrass seeds

[0108] (1) Obtaining Leymus chinensis callus

[0109] The viability of Leymus chinensis seeds was detected using the TTC staining method. The seeds were then sterilized with 40% NaOH, air-dried, and the lemma was removed. The explants were then sterilized by soaking in 75% alcohol for 5 minutes and then sterilized with 0.2% mercuric chloride, followed by rinsing with sterile water five times for 1 minute each time, and then air-drying. Young spikelets at the heading stage were collected, soaked in 75% alcohol for 5 minutes, and then air-dried. The sterilized seeds and young spikelets were placed separately on induction medium SM5, with contaminated medium removed and transferred during the process. The medium was changed after 15-21 days of subculturing. The firm, pale yellow callus that grew on the induction medium was transferred to propagation medium SM2, and nitrogen was added to regulate the callus state. A portion of the callus was transferred to differentiation medium SMDT, and the differentiation success rate was observed. The medium was changed every 5-21 days of subculturing. Finally, a large amount of firm, pale yellow callus was obtained for infection.

[0110] (2) Agrobacterium infection

[0111] Select a single clone of Agrobacterium, incubate overnight at 28°C with shaking, and when the OD value of the bacterial suspension reaches 0.8, add 50 μl of 1 mg / ml AS and shake for another 0.5 h. Pour the bacterial suspension into a 50 ml centrifuge tube, centrifuge at 3500 rpm for 15 min, discard the supernatant, retain the precipitate, add resuspension SM3, sonicate for 5 min, apply vacuum for 10 min (pressure 0.4-0.5 kPa), soak for 5 min, discard the upper bacterial suspension, remove the infected callus tissue, place it on filter paper and air dry, turning the callus tissue continuously during drying until completely dry. Co-culture the infected callus tissue for 3-5 days.

[0112] (3) Screening of positive callus from Leymus chinensis

[0113] After co-culture, the callus tissue was transferred to SM-20P selection medium and cultured in the dark for 1-2 months, with the medium being changed every 15 days. The selected pale yellow callus tissue was then used for differentiation experiments.

[0114] (4) Callus differentiation

[0115] After dark culture and screening, the callus tissue was transferred to SMDT-5P selection and differentiation medium under a light intensity of 2000 lx, and the medium was changed every 15-21 days. During this period, the callus tissue was observed and its condition was adjusted. After the positive callus tissue differentiated, when the differentiated shoots grew to 4-6 cm in the culture bottle, the differentiated shoots were transferred to 1 / 2 SM-5P rooting medium and rooted under light.

[0116] (5) Culture medium composition

[0117] ①SM2 solid propagation medium: 4.74 g / L MS powder, 30 g / L sucrose, 2.0 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid), 0.5 mg / L 6-BA, 7.6-7.8 g / L agar (pH 5.8-6.0).

[0118] ②SM3 infection solution: 4.74 g / L MS powder, 30 g / L sucrose, 3.0 mg / L 2,4-D, 0.5 mg / L 6-BA (pH 5.8-6.0);

[0119] ③SM5 solid induction medium: 4.74 g / L MS powder, 30 g / L sucrose, 5.0 mg / L 2,4-D, 0.5 mg / L 6-BA, 7.6-7.8 g / L agar (pH 5.8-6.0).

[0120] ④MSDT solid differentiation medium: 4.74 g / L MS powder, 30 g / L sucrose, 2.0 mg / L 2,4-D, 1.0 mg / L KT, 7.6-7.8 g / L agar (pH 5.8-6.0).

[0121] ⑤SM-20P solid screening medium: 4.74 g / L MS powder, 30 g / L sucrose, 2.0 mg / L 2,4-D, 300 mg / L termethin, 10 mg / L hygromycin, 7.6-7.8 g / L agar (pH 5.8-6.0).

[0122] ⑥SMDT-5P solid screening differentiation medium: 4.74 g / L MS powder, 30 g / L sucrose, 1.0 mg / L 2,4-D, 1.0 mg / L KT (kinetin), 300 mg / L termethin, 1 mg / L hygromycin, 7.6-7.8 g / L agar (pH 5.8-6.0).

[0123] ⑦ 1 / 2 SM-5P solid rooting medium: 4.74 g / L MS powder, 30 g / L sucrose, 300 mg / L termethin, 1 mg / L hygromycin, 7.6-7.8 g / L agar (pH 5.8-6.0).

[0124] This invention provides a method for cloning the *Leymus chinensis* gene and an expression vector, which will pave the way for subsequent... LcCBP60b Gene vector transformation lays the foundation for obtaining transgenic plants and plays an important role in the breeding, production and promotion of new varieties.

[0125] Example 6 Overexpression LcCBP60b Obtaining genetically modified sheepgrass material

[0126] According to Example 3, based on the Leymus chinensis genetic transformation system, the following were obtained: LcCBP60b Transgenic materials overexpressing the gene in *Leymus chinensis* were cultured in indoor pots for one month, and plant height and tillering were measured. The results showed that the plant height of the transgenic material was 75.80% of the control. Figure 6 (A and B), overexpression LcCBP60b The number of tillers in the genetically modified Leymus chinensis was significantly higher than that in the wild type, being 2.05 times that of the control. Figure 7 (A and B).

[0127] Example 7: Sheepgrass LcCBP60b Gene function analysis and molecular module analysis

[0128] First, RNA was extracted from the leaves, lower stolons, above-ground stems, and spikelets of Leymus chinensis, and then reverse transcribed into cDNA for verification. LcCBP60b The expression profile of genes in Leymus chinensis showed that... LcCBP60bThe gene is constitutively expressed and is present in the leaves, underground stolons, aboveground stems, and spikelets of Leymus chinensis, with the highest expression level in the leaves. It participates in regulating the growth and development of Leymus chinensis. Figure 8 Based on the sequenced Leymus chinensis genome file, extract... LcCBP60b The 2000bp upstream sequence of the gene start codon was used as the predicted promoter region. Online tools such as TBtools and Plant Care were used to analyze the cis-regulatory elements in the promoter region, obtaining multiple binding sites including NAC transcription factors, MYB binding sites, TCT-motifs (photoperiod response elements), ABREs (ABA response elements), and TC-rich repeats (defense and stress response elements). Figure 9 Yeast one-hybrid assays showed that the LcNAC2 protein (Lc5Xm064842) can bind to... LcCBP60b Gene promoter region, ( Figure 10 Based on yeast two-hybrid experiments, the interaction between LcCBP60b and LcTHO4B was verified. Lc2Ns004092 Protein-protein interactions Figure 11 This invention reveals a molecular model of how the NAC2-CBP60b-THO4B gene module regulates the tiller number and biomass yield of Leymus chinensis plants. Figure 12 ).

[0129] 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. Overexpression of Leymus chinensis LcCBP60b Application of genes in the positive regulation of tiller number in Leymus chinensis; in, LcCBP60b The gene is a gene that encodes a protein with an amino acid sequence as shown in SEQ ID NO:

2.

2. A method for increasing the tillering of Leymus chinensis, characterized in that, The method includes: using genetic engineering techniques to overexpress the expression described in claim 1 in *Leymus chinensis*. LcCBP60b Gene.

3. The method according to claim 2, characterized in that, The overexpression is performed by introducing a plasmid containing the gene.

4. The method according to claim 2, characterized in that, The overexpression method involves increasing the copy number of the gene on the plant chromosome.

5. The method according to claim 2, characterized in that, The overexpression is performed by operatively linking a strong promoter to the gene.

6. The method according to claim 5, characterized in that, The promoter is the CaMV35S promoter.

7. The application of the method according to any one of claims 2-6 in increasing the tiller number of Leymus chinensis.

8. The application of transgenic sheepgrass obtained by the method according to any one of claims 2-6 in sheepgrass breeding.

9. The application according to claim 8, characterized in that, Breeding methods include hybridization, backcrossing, self-pollination, or asexual reproduction.

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