Application of GmbetaCA11 protein and / or GmbetaCA11 gene in promoting soybean growth and / or increasing soybean yield

Abstract

The invention provides an application of a GmbetaCA11 protein and / or a GmbetaCA11 gene in promoting soybean growth and / or increasing soybean yield, and belongs to the technical field of plant genetic engineering. The Gm beta CA11 gene is a gene for coding soybean carbonic anhydrase. By enhancing the expression level of the GmbetaCA11 gene and increasing the accumulation amount of the GmbetaCA11 protein in soybean bodies, the soybean growth and development process can be effectively regulated and controlled, the soybean plant growth can be remarkably promoted, and the soybean yield related traits can be improved. Through overexpression of the GmbetaCA11 gene in soybeans, the plant height and yield can be significantly increased; the plant height and single plant yield of the transgenic plant in the mature period are obviously higher than those of a control plant, and the growth situation in the seedling period is also better than that of the control plant. According to the invention, the important function of the GmbetaCA11 gene in regulation and control of soybean growth and yield formation is defined, and the GmbetaCA11 gene has important theoretical value and application prospect in cultivation of new high-yield soybean varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to GmβCA11 protein and / or GmβCA11 Application of genes in promoting soybean growth and / or increasing soybean yield. Background Technology

[0002] Soybeans are one of the most widely cultivated oilseed crops globally, rich in high-quality protein and oil, and are an important source of edible oils and plant protein for humans. In recent years, through agronomic management and genetic breeding measures such as optimizing planting density, improving fertilization techniques, and selecting stress-resistant and high-yielding varieties, soybean yields have steadily increased. To further tap the potential of soybean yields and achieve even higher yield targets, it is still urgent to explore key gene resources that regulate soybean yield formation at the molecular level, and to cultivate new high-yielding and high-quality soybean varieties using modern biotechnology methods such as genetic engineering and molecular design breeding, thereby continuously improving soybean yields.

[0003] Crop production is essentially a process of photosynthetic material production and transformation. The products of photosynthesis are the material basis for crop yield; approximately 90% to 95% of crop biomass originates from photosynthesis, and the level of photosynthetic efficiency directly determines the crop's production potential. However, in long-term breeding practices, the improvement of photosynthetic efficiency-related traits has not received sufficient attention. To date, yield increases achieved through optimizing photosynthetic efficiency remain very limited, which means that improving photosynthetic efficiency is a crucial potential breakthrough for increasing crop yields. Numerous studies have confirmed that regulating key aspects of photosynthesis through molecular biology techniques can significantly improve crop yields. For example, enhancing crops' CO2 fixation capacity using Rubisco biogenesis and chloroplast bioengineering techniques can effectively increase crop biomass and yield. In C3 crops such as tobacco and rice, designing novel photorespiration pathways can significantly improve photosynthetic rate, biomass accumulation, and grain yield under field conditions, with increases ranging from 10% to 40%. Furthermore, genetic modifications to proteins in the photosynthetic reaction center and genes for key carbon assimilation enzymes have been shown to improve photosynthetic efficiency and promote growth in crops such as Arabidopsis and wheat. Notably, research on high-efficiency photosynthesis breeding in soybeans has clearly indicated that optimizing the expression regulation patterns of functional genes related to carbon metabolism and energy conversion in C3 crops may be a key direction for improving photosynthetic efficiency, but functional analyses of such genes in soybeans remain relatively scarce. Therefore, by combining genetics, molecular biology and genetic engineering technologies, we can systematically reveal the molecular mechanisms of energy absorption, transfer and conversion in photosynthesis, and discover key genes that regulate photosynthetic efficiency. This will promote the efficiency of light energy conversion and utilization in crops and reduce excitation energy dissipation, providing new theoretical basis, technical ideas and genetic resources for increasing soybean yield.

[0004] As a core crop group in global food and oilseed production, C3 crops have long been a hot topic in plant and breeding research due to their photosynthetic mechanisms and molecular regulatory networks for yield formation. In model crops such as rice, wheat, and Arabidopsis thaliana, as well as major food crops, numerous genes involved in the regulation of photosynthesis, carbon metabolism, and growth and development have been cloned and functionally verified. Some of these genes have been successfully applied in breeding practices, achieving a synergistic improvement in yield and quality. For example, key photosynthetic genes such as OsPPDKB and OsNADP-ME, cloned in rice, can significantly improve photosynthetic rates through heterologous expression or overexpression. In wheat, optimized expression of the gene TaSUT1, which regulates the transport of photosynthetic products, can effectively improve grain filling efficiency and thousand-grain weight. However, compared with C3 food crops such as rice and wheat, research on the molecular mechanisms related to photosynthetic regulation and yield formation in soybean, an important C3 oilseed crop, is relatively lagging. On the one hand, the complex soybean genome and polyploid characteristics make gene function analysis difficult. On the other hand, systematic mining and functional verification of genes related to photosynthetic efficiency and yield in soybean are severely lacking. The expression patterns, biological functions, and yield effects of many genes related to carbon assimilation, energy conversion, and growth and development in soybean remain unclear. Crucially, soybean differs significantly from other C3 crops in photosynthetic characteristics, carbon metabolism pathways, and growth and development patterns. The applicability of high-efficiency genes validated in other crops to soybean still needs verification. There is an urgent need to discover yield-regulating genes unique to soybean or functionally conserved in soybean. Therefore, conducting research on functional genes related to photosynthesis and growth and development in soybean, and systematically analyzing their molecular mechanisms regulating photosynthetic efficiency, biomass accumulation, and yield formation, will not only fill the research gap in soybean molecular breeding but also provide an important theoretical foundation and core gene resources for the breeding of high-yielding and high-quality soybean varieties, possessing significant scientific value and application prospects. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide GmβCA11 protein and / or GmβCA11 Application of genes in promoting soybean growth and / or increasing soybean yield. By enhancing the expression level of the GmβCA11 gene and increasing the accumulation of GmβCA11 protein in soybeans, the growth and development process of soybeans can be effectively regulated, significantly promoting soybean growth and thus improving soybean yield traits, providing new gene resources and technical approaches for high-yield molecular breeding of soybeans.

[0006] The objective of this invention is achieved through the following technical solution: This invention provides GmβCA11 protein and / or GmβCA11 Application of the gene in promoting soybean growth and / or increasing soybean yield; the amino acid sequence of the GmβCA11 protein is shown in SEQ ID NO.2.

[0007] Preferably, through overexpression GmβCA11 Genes promote soybean growth and / or increase soybean yield.

[0008] Preferably, the GmβCA11 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0009] This invention provides a biomaterial, the biomaterial comprising: GmβCA11 Gene overexpression vector; Gm βCA11 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0010] Preferably, the biomaterial includes engineered bacteria and / or plant cells.

[0011] This invention provides a soybean intermediate material, the soybean intermediate material comprising: GmβCA11 Gene overexpression vector.

[0012] This invention provides a method for promoting soybean growth and / or increasing soybean yield, comprising: Increase the content of GmβCA11 protein in soybeans.

[0013] Preferably, the method for increasing the content of GmβCA11 protein in soybeans includes increasing the content of GmβCA11 protein in soybeans. GmβCA11 Gene expression levels; the improvement of soybean expression levels GmβCA11 Methods for measuring gene expression include: transferring the biological material of claim 4 into soybean.

[0014] Preferably, the soybeans include soybean jack.

[0015] The present invention provides a primer set for identifying whether soybean growth traits have been improved, comprising: primer F as described in SEQ ID NO.3 and primer R as shown in SEQ ID NO.4.

[0016] The beneficial effects of this invention are: This invention provides GmβCA11 protein and / or GmβCA11 The application of genes in promoting soybean growth and / or increasing soybean yield. GmβCA11 The gene encodes soybean carbonic anhydrase. This invention, by enhancing the expression level of the GmβCA11 gene and increasing the accumulation of GmβCA11 protein in soybeans, can effectively regulate the growth and development process of soybeans, significantly promote soybean plant growth, and thus improve soybean yield-related traits. Phenotypic identification results of this invention show that overexpression of the GmβCA11 gene in soybeans... GmβCA11The gene significantly increases plant height and yield; the mature transgenic plants exhibit significantly higher plant height and yield per plant than the control plants, and their seedling growth is also superior to the control. In summary, this invention clarifies... GmβCA11 The important functions of genes in regulating soybean growth and yield formation, and related findings can be applied to regulate soybean growth and yield through biotechnology. Gmβ CA11 Gene expression to improve soybean yield traits has important theoretical value and application prospects for breeding new high-yield soybean varieties.

[0017] The plant expression vector carrying the GmβCA11 gene described in this invention can transform plant cells or tissues via conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, and Agrobacterium-mediated transformation, and then regenerate plants using plant tissue culture techniques. The transformed hosts can be monocotyledonous plants such as rice, wheat, and corn, or dicotyledonous plants such as soybean, cucumber, tomato, and alfalfa. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1 for GmβCA11 Relative expression levels in different tissues of Jack soybean; using cDNA from roots, stems, leaves, flowers, pods at 15 days, and seeds at 42 days as templates, the expression pattern of GmβCA11 in different plant tissues was verified by real-time quantitative qRT-PCR. Figure 2 pBA002- GmβCA11 Overexpression vector map; Figure 3 This image shows the PCR validation results of T1 generation positive transgenic soybean plants; the top row of gel images represents the Bar detection bands, and the bottom row represents the target fragment specific detection bands. M1 represents the 5000 bp Marker, with band composition (from bottom to top): 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 2000 bp, 3000 bp, 5000 bp; M2 represents the 2000 bp Marker, with band composition (from bottom to top): 100 bp, 250 bp, 500 bp, 750 bp, 1000 bp, 2000 bp; lanes 1-3 represent line 1, and lanes 4-6 represent line 2. Figure 4 for GmβCA11Phenotypic diagram of overexpressing transgenic plants; Jack represents the recipient control material, and OE-1 and OE-2 represent GmβCA11 overexpressing transgenic plants; Figure 5 This is a graph showing the plant height and yield per plant of GmβCA11 overexpressing transgenic plants; Jack represents the recipient control material, and OE-1 and OE-2 represent... GmβCA11 Overexpression of transgenic plants. Detailed Implementation

[0020] This invention provides GmβCA11 protein and / or GmβCA11 Application of genes in promoting soybean growth and / or increasing soybean yield. As an optional embodiment of this invention, overexpression can be used... GmβCA11 The gene promotes soybean growth and / or increases soybean yield. As an optional embodiment of the invention, the amino acid sequence of the GmβCA11 protein is shown in SEQ ID NO. 2. In this invention, GmβCA11 The CDS sequence of the gene is shown in SEQ ID NO.1. As an optional embodiment of the present invention, the soybean includes soybean Jack.

[0021] This invention relates to a gene encoding soybean carbonic anhydrase. GmβCA11 Its coding region (CDS) nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2. This invention utilizes Agrobacterium tumefaciens-mediated soybean cotyledonary node transformation to successfully construct and obtain... GmβCA11 Transgenic soybean plants with overexpression of the gene. Experimental results showed that, compared with wild-type control plants, overexpression... GmβCA11 The gene can significantly promote the growth of transgenic soybean plants, and significantly increase plant height and yield per plant. The present invention provides… GmβCA11 Genes can be introduced into plants as target genes to effectively regulate the growth and development of transgenic plants, and have important application value in high-yield molecular breeding of soybeans and the cultivation of new high-yield soybean varieties.

[0022] This invention provides a biomaterial, the biomaterial comprising: GmβCA11 Gene overexpression vector; Gm βCA11 The CDS sequence of the gene is shown in SEQ ID NO.1. As an optional embodiment of the present invention, the biomaterial comprises engineered bacteria and / or plant cells. In this invention, the biomaterial can improve the quality of soybeans... GmβCA11 Gene expression increases the content of GmβCA11 protein.

[0023] The present invention relates to the above. GmβCA11There are no particular limitations on the construction method of the gene overexpression vector; any conventional construction method in the art can be used. This invention relates to the... GmβCA11 There are no specific limitations on the backbone vector for gene overexpression vectors; any conventional backbone vector in the field can be used, as long as it can achieve the desired result. GmβCA11 Gene overexpression is acceptable. As an optional embodiment of the present invention, the backbone vector may include the pBA002 expression vector plasmid.

[0024] As an optional embodiment of the present invention, when the biomaterial is engineered bacteria, the engineered bacteria can be *Escherichia coli* and / or *Agrobacterium*; the *Agrobacterium* can be *Agrobacterium tumefaciens*; the *Agrobacterium tumefaciens* can be *Agrobacterium tumefaciens* strain EHA105. The present invention relates to bacteria containing... GmβCA11 There are no specific limitations on the preparation method of the engineered bacteria for gene overexpression vectors; any conventional preparation method in this field may be used.

[0025] As an optional embodiment of the present invention, when the biological material is plant cells, the plant cells may be soybean cells.

[0026] This invention provides a soybean intermediate material, the soybean intermediate material comprising: GmβCA11 Gene overexpression vector.

[0027] The present invention relates to the above. GmβCA11 There are no particular limitations on the construction method of the gene overexpression vector; any conventional construction method in the art can be used. This invention relates to the... GmβCA11 There are no specific limitations on the backbone vector for gene overexpression vectors; any conventional backbone vector in the field can be used, as long as it can achieve the desired result. GmβCA11 Gene overexpression is acceptable. As an optional embodiment of the present invention, the backbone vector may include the pBA002 expression vector plasmid. In the present invention, the... GmβCA11 The gene overexpression vector was ultimately highly expressed in the target soybean. GmβCA11 Genes that increase the content of GmβCA11 protein.

[0028] This invention does not specifically limit the preparation method of the soybean intermediate material; any conventional preparation method in the art can be used. As an optional embodiment of this invention, the preparation method of the soybean intermediate material includes: […]. GmβCA11 The gene overexpression vector is introduced into soybean plants. This invention does not specifically limit the introduction method; any conventional introduction method in the art can be used. As an optional embodiment of this invention, the introduction method can be achieved by... GmβCA11 The gene overexpression vector is transferred into Agrobacterium, and then the resulting Agrobacterium is introduced into soybean cells. This invention does not specifically limit the methods used in the above steps; any method conventional in the art can be used. As an optional embodiment of this invention, the... Gmβ CA11 The method for transferring the gene overexpression vector into Agrobacterium can be the freeze-thaw method; the method for introducing Agrobacterium into soybean cells can be the cotyledon node transformation method.

[0029] This invention provides a method for promoting soybean growth and / or increasing soybean yield, comprising: increasing the content of GmβCA11 protein in soybeans. This invention does not specifically limit the method for increasing the content of GmβCA11 protein in soybeans; any method conventional in the art can be used. As an optional embodiment of this invention, the method for increasing the content of GmβCA11 protein in soybeans includes increasing the content of GmβCA11 protein in soybeans... GmβCA11 Gene expression; the improvement of soybean... GmβCA11 The method for gene expression includes transferring the biological material described in the above-described technical solution into soybean. As an optional embodiment of the present invention, the soybean includes soybean jack.

[0030] This invention provides a primer set for identifying whether soybean growth traits have been improved, comprising: primer F as shown in SEQ ID NO.3 and primer R as shown in SEQ ID NO.4. Using this primer set, real-time quantitative PCR can be performed on cDNA from soybean leaves to determine the soybean plant's growth characteristics. GmβCA11 Gene expression levels. If soybean plants... GmβCA11 A significant increase in gene expression levels indicates improved soybean growth performance, resulting in a corresponding increase in soybean yield.

[0031] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0032] Example 1 soybeans GmβCA11 Organized expression of genes in Jack material (1) Design primers to extract total RNA from plant tissues and reverse it into cDNA: Using cultivated soybean (Jack) as material, roots, stems, and leaves were collected at the seedling stage (V4 stage), fully opened flowers at the flowering stage (R2 stage), pods were collected 15 days after flowering, and seeds were collected 42 days after flowering. All samples were flash-frozen in liquid nitrogen and stored at -80°C. Total RNA was then extracted from each tissue using a plant total RNA extraction kit (DP419, Tiangen), and RNA integrity was assessed by 1% agarose gel electrophoresis. cDNA synthesis was performed according to the instructions of the HiScript® 1st Strand cDNA Synthesis Kit. Primers were designed as follows: GmβCA11The qPCR primer F is shown in SEQ ID NO.3, specifically: '5- AATTGGCTTGCCTGCAAAGG -3'; GmβCA11 The qPCR primer R is shown in SEQ ID NO.4, specifically: '5- GGTGGCCACATCTTTAACGG -3'; Tubulin The qPCR primer F is shown in SEQ ID NO.5, specifically: '5- GGAGTTCACAGAGGCAGAG -3'; Tubulin The qPCR primer R is shown in SEQ ID NO.6, specifically: '5- CACTTACGCATCACATAGCA -3'.

[0033] (2) Real-time quantitative PCR, the specific steps are as follows: Step 1: Dilute the cDNA samples from each tissue obtained in (1) 10 times for qPCR reaction. Prepare the reaction solution (20 μL system) according to the instructions of ChamQUniversal SYBR qPCR Master Mix: 10 μL 2×ChamQUniversal SYBR qPCR Master Mix, 0.4 μL each of primers F and R, 5 μL cDNA template, and 4.2 μL ddH2O to make up to 20 μL.

[0034] Step 2: The reaction was carried out on a Bio-RAD CFX96TM Real-Time System fluorescence quantitative analyzer. The reaction program was set as follows: 95℃ pre-denaturation for 30 sec; then 95℃ for 10 sec, 60℃ for 30 sec, for a total of 40 cycles; then melting curves were collected at 95℃ for 15 sec, 60℃ for 60 sec, and 95℃ for 15 sec.

[0035] Step 3: Analyze the data using Excel. The relative expression levels of candidate genes in soybean leaves were analyzed by comparing CT (Cycle Threshold) values. The formula is: relative expression level = 2... -△△CT (△△CT=(CT) Target -CT Tubulin ) genotype -(CT Target - CT Tubulin ) calibrator )calculate GmβCA11 Relative expression levels in various organizations. Figure 1 The results show that: GmβCA11It is expressed in roots, stems, leaves, flowers, and pods, with the highest expression level in leaves, followed by pod pericarps, and no expression in seeds.

[0036] Example 2 soybeans GmβCA11 Gene cloning and construction of plant overexpression vectors (1) Using the cDNA from soybean Jack leaves obtained in Example 1 as a template, the CDS fragment was amplified by high-fidelity polymerase chain reaction. The primers were designed as follows: SEQ ID NO.7: Primer F '5-GGATCTTCCAGAGATACACCCCATTGCTACGAAGG-3' SEQ ID NO.8: Primer R '5-CTGCCGTTCGACGATTCATCTCTCTCGGTGTTGCG-3' (2) PCR amplification, the specific steps are as follows: Step 1: Prepare the PCR reaction solution (50 μL system) according to the following component order: 25 μL 2×Planta MaxBuffer, 2 μL each of primers F and R, 1 μL dNTP Mix, 2 μL leaf cDNA template, 1 μL Super-Fidelity DNA Polymerase, and 17 μL ddH2O to bring the volume to 50 μL.

[0037] Step 2: The reaction was performed on a BIO-RAD PTC-200 PCR instrument. The reaction program was set as follows: 95℃ pre-denaturation for 3 min; then 95℃ for 15 sec, 58℃ for 15 sec, 72℃ for 2 min 15 sec, for a total of 35 cycles; then 72℃ for complete extension for 5 min; and stored at 4℃.

[0038] Step 3: PCR product recovery GmβCA11 The target gene was then ligated into the linearized pCE-Zero vector (purchased from Novizan) via recombination. EcoR The target gene was inserted into the pCE-Zero vector by restriction endonuclease digestion to obtain the recombinant vector. The recombinant vector was transformed into *E. coli* TOP10, screened with ampicillin (Amp), cultured, and sequenced. GmβCA11 The nucleotide sequence of the target gene is shown in SEQ ID NO.1. Confirmation was obtained containing... GmβCA11 T-vectors for gene fragments.

[0039] SEQ ID NO.1:

[0040] The amino acid sequence of GmβCA11 is shown in SEQ ID NO.2, specifically: MSTSSINGWCLSSISPAKTSLKKATLGPSVFATVSTPSSPSSSSSFPSLIQDRPVFAAPAPIITPTVREDMAKEYEQAIEELQKLLREKSELKATAAEKVEQITASLGTSSSDGIPSSEASDRIKAGFIHFKKEKYDKNPALYGELAKGQSPKFMVFACSDSRVCPSH VLDFQPGEAFVVRNVANIVPPYDQSKYAGTGAAVEYAVLHLKVSEIVVIGHSACGGIKGLLSFPYDGTYSTDFIEEWVKIGLPAKAKVKTQHGDAPFAELCSHCEKEAVNVSLGNLLTYPFVRDGLVNKTLSLKGGYYDFVKGSFELWGLQFGLASSFSVKDVATILHWKL .

[0041] (3) Construction of plant overexpression vectors Design homologous recombination adapter primers, with the following sequences: SEQ ID NO.9: Primer F '5-GGGCCCAGGCCTACGCGTATGTCGACCTCTTCCATT-3' SEQ ID NO.10: Primer R '5-TCGGGGAAATTCGAGCTCCTATAGCTTCCAATGCAGA-3' The contents obtained by (2) above GmβCA11 Using a T-vector containing the gene fragment as a template, amplification was performed to produce a gene with a recombination adapter. Gmβ CA11 CDS fragments, then use Mlu I and SacI. The pBA002 expression vector plasmid was digested with restriction endonucleases (see reference [Yang, Y., Wang, L., Zhang, D., Che, Z., Wang, Q., Cui, R., Zhao, W., Huang, F., Zhang, H., Cheng, H., & Yu, D. (2024). Soybean type-B responseregulator GmRR1 mediates phosphorus uptake and yield by modifying rootarchitecture. Plant physiology, 194(3), 1527–1544. https: / / doi.org / 10.1093 / plphys / kiad570]), and then homologous recombinase was used to digest the pBA002 expression vector plasmid. GmβCA11 Genes were inserted into the expression vector pBA002 to construct pBA002- GmβCA11 Overexpression vectors (such as Figure 2 As shown in the figure, the vector was then transferred into Agrobacterium tumefaciens strain EHA105 using a freeze-thaw method for subsequent soybean genetic transformation. The expression vector also contains the marker gene Bar, which encodes phosphinothricin acetyltransferase (PPT) isolated from Streptomyces. The Bar gene exhibits resistance to the herbicide glufosinate and can be used for positive identification of transgenic plants; it is one of the safe marker genes for application.

[0042] Figure 2 pBA002- GmβCA11 Overexpression vector map. Among them: GmβCA11 CDS: Target gene GmβCA11 The coding region; CaMV 35S promoter: Cacamal vegetable mosaic virus (CaMV) 35S promoter; NOS terminator: Namine synthase gene terminator; NOS promoter: Namine synthase gene promoter; Bar: Phosphinicotin acetyltransferase gene; E9 terminator: 3' end sequence of the small subunit of the ribulose-1,5-bisphosphate carboxylase E9 gene; SacⅠ: SacⅠ restriction endonuclease cleavage site; MluⅠ: MluⅠ restriction endonuclease cleavage site. LB T-DNA repeat: T-DNA left boundary; RB T-DNA repeat: T-DNA right boundary; Primer OE-F: Amplification of the target gene. GmβCA11 Upstream primer; Primer OE-R: for amplifying the target gene GmβCA11 The downstream primer; Primer Bar-F: upstream primer for amplifying the marker gene Bar; PrimerBar-R: downstream primer for amplifying the marker gene Bar.

[0043] Example 3 GmβCA11 Creation of Gene Overexpression Transgenic Soybeans Using Agrobacterium tumefaciens-mediated transformation of soybean cotyledonary nodes to transform pBA002- GmβCA11 The overexpression vector was transferred into the receptor material Jack, and the specific method is described below: (1) Select mature, plump, clean seeds without disease spots or hardness, and arrange them in a single layer in a 90×15 mm petri dish. Sterilize the surface of the soybean seeds with chlorine using a dry sterilization method in a fume hood for 6-7 hours. Cover the petri dish and transfer it to a sterile laminar flow hood. Open the lid of the petri dish and blow strong air for 25-40 minutes to remove residual chlorine. Sow the sterilized seeds with the hilum facing down on the germination medium (SG4). Stack the petri dishes, wrap them with plastic wrap, and place them in a biological incubator at 24°C in the dark for 16-24 hours.

[0044] (2) Preparation of Agrobacterium to infect soybean cotyledon nodes. Take 100 μL of pBA002- obtained in Example 2. GmβCA11 Agrobacterium overexpression vector was cultured in 5 mL of YEB liquid medium supplemented with spectinomycin (1 / 1000) at 28°C with shaking at 220 rpm for 24–36 h. Then, 0.2–1 mL of saturated culture was transferred to 250 mL of YEB liquid medium supplemented with spectinomycin for expansion culture until OD500 was reached. 600 =0.85~0.9. Aliquot the bacterial culture into two 50 mL sterile centrifuge tubes, centrifuge (5000 rpm, 10 min, 25℃), collect the colonies, gently pipette with 25~50 mL of liquid co-culture medium (LCCM), resuspend the precipitate for later use.

[0045] (3) Plant tissue culture. First, use a scalpel to make incisions at the cotyledon nodes of the imbibed soybean seeds to obtain explants. Then, place all explants into the prepared infection solution and incubate with shaking at 120-130 rpm for 30 min. Afterward, remove the explants and place them on sterile absorbent paper to absorb any remaining infection solution. Then, place them on solid co-culture medium (CCM) with sterile absorbent paper. All the above operations are performed in a laminar flow hood. Stack the culture dishes, seal them with plastic wrap, and co-culture them in an artificial intelligence incubator at 25°C in the dark for 3-5 days. After sterilization, insert the explants into shoot induction medium (SIM) containing the screening agent glufosinate and culture them at 25°C under light for 14 days. Afterwards, the remaining cotyledons were removed and transferred to bud elongation medium (SEM). The medium was cultured at 25°C under light for 2-4 weeks. During this period, the SEM medium was replaced in a timely manner according to the actual situation. The elongated and regenerated plants were cut off from the root and quickly inserted into solid rooting medium (SM). When the roots were about 2-3 cm long, the rooted seedlings were removed from the medium, the residual medium on the roots was washed off, and the seedlings were transferred to soil and then moved to a greenhouse for further cultivation.

[0046] (4) Positive identification of transgenic plants. After the regenerated plant has grown its third new leaf, the upper, middle and lower layers of leaves are cut off with scissors and mixed together. DNA is extracted according to the instructions of the plant total DNA extraction kit from Shanghai Pudi Biotechnology Co., Ltd. Then, using the DNA sample as a template, PCR amplification is performed using the 2×Rapid Taq Master Mix (Novizan) kit, including the amplification of the target gene. GmβCA11 Identification of the target gene at the DNA level and the selection of the gene bar. GmβCA11 Primers for specific fragment amplification, including: SEQ ID NO.11: 35S-F '5-GACGCACAATCCCACTATCC-3'; SEQ ID NO.12: OE-R '5-GGAAACATTCACAGCTTCCTT-3'. Gene screening. Bar The specific amplification primers are as follows: SEQ ID NO.13: Bar-F '5- CGAGACAAGCACGGTCAACTT-3'; SEQ ID NO.14: Bar-R '5- AAACCCACGTCATGCCAGTTC-3'. The target band was then detected by 1% agarose gel electrophoresis (e.g., ...). Figure 3 If a 967 bp target gene fragment and a 360 bp Bar gene fragment are amplified, the transgenic plant is considered a positive plant. Positive plants are harvested and planted individually until homozygous transgenic plants are identified in the T2 generation, thus obtaining the desired transgenic plant. GmβCA11 Gene overexpression plants.

[0047] Further analysis of the obtained GmβCA11 Gene-overexpressing plants and wild-type plants were subjected to real-time quantitative PCR using the method described in Example 1, and the results were obtained. GmβCA11 Gene overexpression plants GmβCA11 Gene expression levels were significantly higher than in wild-type plants. GmβCA11 Gene expression levels.

[0048] Example 4 GmβCA11 Phenotypic determination of transgenic plants with overexpressed genes Two stable plants were identified using the plant transgenic method described in Example 3. GmβCA11 The transgenic plants overexpressing the gene were named OE-1 and OE-2, respectively. To determine the growth phenotype and yield-related traits of these overexpressing transgenic plants, the recipient material Jack and the two overexpressing transgenic soybean materials were grown under greenhouse conditions. Photographs were taken and recorded when the plants reached the pod-setting stage (e.g., ...). Figure 4 After maturity, plant height and yield-related traits were measured. The specific measurement method is as follows: Healthy soybean plants were randomly selected, and the length of the main stem (from the cotyledon node to the top) was measured using a soft measuring tape, which is the plant height; the yield of a single plant was measured using an analytical balance, and 3 plants were measured for each line.

[0049] Analysis of variance was performed on the phenotypic data using EXCEL software. Comparison of yield-related traits between transgenic plants and the control material showed that, during the pod-setting stage, the overexpressing plants exhibited significantly better growth than the control material. Furthermore, compared to the control material, both... GmβCA11 The plant height and yield per plant of the transgenic plants overexpressing the gene were significantly increased. Figure 5 ).therefore, Gmβ CA11 It plays an important role in regulating the growth and development of soybeans. GmβCA11 Genes can significantly regulate the growth and development process of soybeans and effectively increase the yield of individual soybean plants, making them important functional genes for high-yield soybean breeding.

[0050] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. GmβCA11 protein and / or GmβCA11 Application of the gene in promoting soybean growth and / or increasing soybean yield; the amino acid sequence of the GmβCA11 protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, Through overexpression GmβCA11 Genes promote soybean growth and / or increase soybean yield.

3. The application according to claim 1 or 2, characterized in that, The GmβCA11 The CDS sequence of the gene is shown in SEQ ID NO.

1.

4. A biomaterial, characterized in that, The biomaterials include GmβCA11 Gene overexpression vector; Gmβ CA11 The CDS sequence of the gene is shown in SEQ ID NO.

1.

5. The biomaterial according to claim 4, characterized in that, The biomaterials include engineered bacteria and / or plant cells.

6. A soybean intermediate material, characterized in that, The soybean intermediate material includes GmβCA11 Gene overexpression vector.

7. A method for promoting soybean growth and / or increasing soybean yield, characterized in that, include: Increase the content of GmβCA11 protein in soybeans.

8. The method according to claim 7, characterized in that, The method for increasing the content of GmβCA11 protein in soybeans includes increasing the content of GmβCA11 protein in soybeans. GmβCA11 Gene expression levels; The improvement of soybeans GmβCA11 Methods for measuring gene expression include: transferring the biological material of claim 4 into soybean.

9. The method according to claim 7 or 8, characterized in that, The soybeans mentioned include soybean Jack.

10. A primer set for identifying whether soybean growth traits have been improved, characterized in that, include: Primer F as described in SEQ ID NO.3 and primer R as described in SEQ ID NO.4.

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