Application of plant low-phosphorus-stress-resistant gene GmLNG5

Abstract

The invention discloses an application of a low phosphorus stress resistant gene GmLNG5 of a plant. The research on the soybean GmLNG5 gene shows that the GmLNG5 positively regulates the soybean root growth and phosphorus efficiency, is mainly expressed in the soybean root, and enhances the expression under the stress of low phosphorus; the GmLNG5 regulates and controls the growth of the soybean root system by mediating the longitudinal extension of root tip cells, the overexpression of the GmLNG5 gene increases the cell lengths of the extension region and the mature region of the soybean in-vitro hair root tip, the biomass of the in-vitro hair root and the phosphorus content, the plant growth is promoted under low phosphorus stress, and the GmLNG5 gene can be used for cultivating low-phosphorus-resistant plant varieties. The GmLNG5 gene provided by the invention plays an important role in promoting soybean root growth and improving soybean phosphorus efficiency.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology and plant breeding technology, and more specifically, relates to the application of a plant low-phosphorus stress tolerance gene GmLNG5. Background Art

[0002] Phosphorus (P) is one of the mineral nutrients necessary for plant development and growth. It is the main structural component of plants and plays a direct and indirect role in a series of biological and metabolic processes such as energy transfer, membrane and nucleotide synthesis, photosynthesis, and signal transduction. The phosphorus nutrition required by plants mainly depends on the absorption of the roots from the soil, but the phosphorus element in the soil is easily fixed to form compounds that are difficult to be directly intercepted and absorbed by the plant roots. Most of the applied phosphorus fertilizers cannot be absorbed by plants and are lost to the environment, resulting in a waste of phosphorus resources and corresponding environmental pollution. Therefore, increasing the absorption and utilization of phosphorus in the soil is of great significance to improving the phosphorus absorption efficiency of crops.

[0003] Soybean (Glycine max) is rich in nutrients such as protein, unsaturated fatty acids, dietary fiber, lecithin, isoflavones, etc. It is an important grain and oil crop in China. Phosphorus is an important nutrient element for soybean growth. Phosphorus deficiency will inhibit soybean growth and reduce soybean yield. The root system is an organ that directly interacts with the soil. The root system structure has been shown to be important for plant phosphorus acquisition. Genes involved in regulating the elongation or proliferation of root tip cells, such as EXPA, EXPB and EXLB family genes, have been reported to regulate the growth and development of the root system under low phosphorus stress. For example, the soybean GmEXPB2 gene is upregulated by low phosphorus stress. Overexpression of GmEXPB2 promotes the growth of the taproot by increasing the number and size of cortical cells in the soybean root tip meristem and elongation zone, indicating that GmEXPB2 positively regulates the division and elongation of soybean taproot cells and thus regulates the adaptation of soybean taproot to low phosphorus stress (Yang et al., GmPTF1 modifies root architecture responses to phosphate starvation primarily through regulating GmEXPB2 expression in soybean. 2021. The Plant Journal.). However, there are few reports on other genes involved in regulating soybean to adapt to low phosphorus stress.

[0004] Longifolia (LNG) subfamily genes belong to the TONNEAU1 Recruiting Motif (TRM) gene family. Existing studies have shown that LNG subfamily members are involved in the longitudinal elongation of plant leaves and fruit cells. For example, LONGIFOLIA (LNG1) and LONGIFOLA2 (LNG2) genes regulate leaf morphology by promoting longitudinal cell elongation in Arabidopsis. Although LNGs genes are expressed in various tissues of the plant, the functional role of this gene in soybean is unknown; among them, although the GmLNG5 gene has been cloned and reported in Arabidopsis and rice, its biological function in the phosphorus signaling network of leguminous crops is not clear, and there is a large gap in the research on its involvement in soybean development, and whether it is related to phosphorus stress is also unknown. Therefore, a systematic functional analysis of soybean LNG-related genes is of great significance to the growth and metabolic regulation mechanism of soybean. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies of the existing soybean LNG gene and the gene adapting to low phosphorus stress, and to provide the application of the plant low phosphorus stress tolerance gene GmLNG5.

[0006] The purpose of the present invention is to provide application of plant low-phosphorus stress tolerance gene GmLNG5.

[0007] Another object of the present invention is to provide a formulation for promoting the expression of the soybean low-phosphorus stress tolerance GmLNG5 gene as shown in SEQ ID NO:1.

[0008] Another object of the present invention is to provide a method for promoting plant growth and / or increasing the phosphorus content of plants.

[0009] Another object of the present invention is to provide a method for constructing a transgenic plant resistant to low phosphorus stress.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention studies the soybean GmLNG5 gene, the GmLNG5 gene sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2, showing that GmLNG5 positively regulates soybean root growth and phosphorus efficiency, is mainly expressed in the soybean root system, and the GmLNG5 protein is mainly located in the nucleus and cell membrane, and is enhanced by low phosphorus stress; GmLNG5 regulates the growth of the soybean root system by mediating the longitudinal elongation of root tip cells, and overexpression of the GmLNG5 gene increases the cell length of the root tip elongation zone and the mature zone of the soybean hairy root, the biomass of the in vitro hairy root and the phosphorus content, showing that the GmLNG5 gene has a significant effect on the growth and total phosphorus content of the soybean hairy root in vitro, promotes plant growth under low phosphorus stress, and can be used to cultivate low phosphorus tolerant plant varieties. The GmLNG5 gene provided by the present invention plays an important role in promoting soybean root growth and improving soybean phosphorus efficiency, which is of great significance for clarifying the biological function of the LNG gene in leguminous crops to adapt to low phosphorus stress regulation.

[0012] Therefore, the present invention provides the following applications of the soybean low-phosphorus stress-tolerant GmLNG5 gene:

[0013] Application in positive regulation of soybean root growth and / or phosphorus efficiency.

[0014] Application in promoting plant growth and / or promoting plant growth under low phosphorus conditions.

[0015] Application in increasing the phosphorus content of plants and / or in preparing a preparation for increasing the phosphorus content of plants.

[0016] Application in preparing products for promoting plant growth and / or improving plant tolerance to low phosphorus stress.

[0017] Application in constructing plants tolerant to low phosphorus stress.

[0018] At the same time, the present invention provides a preparation for promoting the expression of the soybean low-phosphorus stress-resistant GmLNG5 gene for use in promoting plant growth and / or promoting plant growth under phosphorus stress, or in increasing the phosphorus content of plants and / or in preparing products for increasing the phosphorus content of plants, and in cultivating low-phosphorus-resistant plant varieties.

[0019] Preferably, the preparation for promoting the expression of the soybean low-phosphorus stress tolerance GmLNG5 gene is an overexpression vector of the GmLNG5 gene.

[0020] Preferably, the plant is a dicotyledonous plant, in particular a leguminous plant.

[0021] More preferably, the plant is bean or soybean.

[0022] The present invention provides a method for promoting plant growth and / or increasing plant phosphorus content, which comprises overexpressing the plant GmLNG5 gene, or treating the plant with a preparation promoting the expression of the soybean low-phosphorus stress-resistant GmLNG5 gene as shown in SEQ ID NO:1.

[0023] The present invention also provides a method for constructing a low-phosphorus stress-resistant plant, wherein a recombinant expression vector of a soybean low-phosphorus stress-resistant GmLNG5 gene is introduced into a plant, thereby obtaining a low-phosphorus stress-resistant plant.

[0024] Preferably, the recombinant expression vector of the GmLNG5 gene is an expression vector containing the GmLNG5 gene, which can be used in existing plant expression vectors to construct a recombinant expression vector containing the gene GmLNG5; the plant expression vector includes, for example, an overexpression vector or other derived plant expression vectors.

[0025] The present invention also relates to a cell comprising a GmLNG5 gene or a recombinant vector. The cell may be a plant cell, such as a leguminous plant cell, or a microbial cell, such as a bacterial or fungal cell, such as a yeast cell; the cell may be isolated, in vitro, cultured, or a part of a plant.

[0026] Furthermore, the GmLNG5 gene can be introduced into the recipient soybean ex vivo hairy roots via a recombinant expression vector.

[0027] Furthermore, the GmLNG5 gene can be introduced into a recipient soybean plant via the recombinant expression vector.

[0028] Preferably, the plant expression vector carrying the GmLNG5 gene can be transformed into soybean by, for example, Agrobacterium-mediated transformation.

[0029] More preferably, the plant expression vector carrying the GmLNG5 gene can be transformed into tobacco epidermal cells by, for example, Agrobacterium-mediated transformation.

[0030] The present invention also relates to a method for producing a plant, the method comprising: regenerating a transgenic plant from the plant cell of the present invention.

[0031] A preferred embodiment provided by the present invention is to introduce the recombinant vector containing the above-mentioned gene GmLNG5 into soybeans, and the growth and phosphorus content of the obtained soybean plants will change significantly, thereby obtaining soybean plants resistant to low phosphorus stress.

[0032] The present invention has the following beneficial effects:

[0033] The present invention studies the gene of soybean GmLNG5, and shows that GmLNG5 positively regulates soybean root growth and phosphorus efficiency, is mainly expressed in soybean roots, and GmLNG5 protein is mainly located in the nucleus and cell membrane, and is enhanced by low phosphorus stress; GmLNG5 regulates the growth of soybean roots by mediating the longitudinal elongation of root tip cells, and overexpression of the GmLNG5 gene increases the cell length of the root tip elongation zone and the mature zone of soybean hairy roots, the biomass of the in vitro hairy roots and the phosphorus content, showing that the GmLNG5 gene has a significant effect on the growth of soybean hairy roots in vitro and the total phosphorus content, promotes plant growth under low phosphorus stress, and can be used to cultivate low phosphorus tolerant plant varieties. The GmLNG5 gene plays an important role in promoting soybean root growth and improving soybean phosphorus efficiency, which is of great significance for clarifying the biological function of the LNG gene in leguminous crops to adapt to low phosphorus stress regulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the histochemical localization analysis of GmLNG5 fused to GUS protein in soybean hairy roots in vitro (the scale bar in the figure is 1 cm, and the scale bar in the enlarged image of the local root system is 0.2 mm).

[0035] Figure 2 Subcellular localization analysis of GmLNG5 fused to GFP protein in tobacco leaves (the figure shows the contents captured under a laser confocal microscope in the green fluorescence channel (GFP), bright field (Bright field) and overlapped images (Merge), with a scale bar of 20 μm).

[0036] Figure 3 Effects of overexpression of GmLNG5 on the response of transgenic soybean hairy roots to phosphorus availability in vitro (A is the phenotype of empty control (CK) and transgenic hairy roots overexpressing GmLNG5 (OX) under normal phosphorus and low phosphorus conditions; B is the relative expression level of the GmLNG5 gene; C is the fresh weight of hairy roots; D is the total phosphorus content; OX represents the overexpression line, and CK represents the transgenic line transformed with an empty vector; the scale bar is 1.2 cm, and the data are the mean and standard error of 7 replicates; asterisks (*) indicate significant differences between the overexpression line OX and the control line CK Student's t-test; *: P < 0.05, **: 0.001 <P<0.01,***:P<0.001)。

[0037] Figure 4Effect of overexpressing GmLNG5 on the elongation of transgenic in vitro hairy root tip cells of soybean (A and B are the cell phenotypes of the elongation zone and mature zone of the hairy root tip cells of GmLNG5; C and D are the cell lengths of the elongation zone and mature zone; E and F are the cell widths of the elongation zone and mature zone; the data in the figure are the lengths and widths of 60 cells, and the asterisks (*) indicate significant differences between CK and OX *: P<0.05, **: 0.001<P<0.01, ***: P<0.001, scale bar = 50 μm). Detailed implementation manners

[0038] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0040] Example 1: Cloning of GmLNG5 gene and construction of vector

[0041] 1. Construction of overexpression vector pTF101s-GmLNG5-OX

[0042] Using the cDNA of soybean YC03-3 (from the Root Biology Research Center of South China Agricultural University) as a template, specific primers GmLNG5-OX-F / R (SEQ ID NO:3: 5’-CCCGGGATGTCTAAAAAGGTTTTGACATCAATG-3’; SEQ ID NO:4: 5’-CTACTTGGAAAGCAGCAGTTGTCTGCCCGGG-3’) were designed according to the full-length CDS sequence of GmLNG5 (the GmLNG5 gene sequence is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO:2), and Phanta Max Super Fidelity DNA Polymerase (Novoprotein, China) was used for PCR to amplify the gene fragment. The 50 μL reaction system was: 2×phata max buffer 25 μL, forward and reverse primers (10 mM) 2 μL each, 2 mM dNTPs 1 μL, Phanta Max Super Fidelity DNA Polymerase 1 μL, cDNA template 2 μL, ddH2O 18 μL. The PCR system was pre-denatured at 95°C for 3 min, denatured at 95°C for 15 sec, annealed at 58°C for 30 s, extended at 72°C for 1 min, and finally extended at 72°C for 5 min, with the denaturation to extension repeated 30 times.

[0043] After the PCR product was determined by gel electrophoresis, an agarose gel kit (Meiji Biotechnology, China) was used to recover and purify the target band according to the instructions. The fragment was then inserted into the Sam I restriction site of the linearized pTF101s plasmid using the Clone Express II One Step Cloning Kit 29 Recombination Exchange Kit (Novozyme, China) and transformed into E. coli DH5α. After culturing at 37°C for 12 h, the positive clones were shaken and sent to the company for sequencing. After successful sequencing, the plasmid was extracted from the sample, and the target gene fragment was connected to the pTF101s vector using the one-step cloning method.

[0044] 2. Construction of subcellular localization expression vector pEGAD-GmLNG5-GFP

[0045] According to the GmLNG5 gene, the forward specific primer (SEQ ID NO: 5: 5'-GGGGACTCTAGCGCTACCGGTATGTCTAAAAAGGTTTTGACATCAATG-3') and the reverse specific primer (SEQ ID NO: 6: 5'-CTCACCATGGTGGCGACCGGTGC CTTGGAAAGCAGCAGTTGTCTG-3') of the GmLNG5-GFP gene were designed, and the GmLNG5-OX plasmid (synthesized by Qingke Bio: using soybean YC03-3 root cDNA as a template, primers were designed in the CDS sequence of GmLNG5 for amplification) was used as a template to amplify the full-length CDS sequence of the gene. The PCR amplification system was: 25 μL 2×Vazyme phata buffer, 1 μL Vazyme phata enzyme, 1 μL dNTP, 1 μL of forward and reverse primers, 3 μL cDNA template, and finally 16 μL of ddH2O was added to make up to 50 μL. The PCR amplification procedure was as follows: 94°C pre-denaturation for 2 min, 94°C denaturation for 30 s, 58°C annealing for 40 s, 72°C extension for 30 s, 30 cycles from denaturation to extension, and storage of the PCR product at 16°C. The obtained amplified product was detected by gel electrophoresis. If the band size was correct, the target fragment was further recovered and purified using an agarose gel DNA recovery kit (Meiji Bio, China).

[0046] AgeΙ was selected as the single restriction site of the pEGAD vector, and the target gene fragment was connected to the pEGAD vector using the one-step cloning method. The connection reaction system was the same as above, and the connection product was then transferred into the competent Escherichia coli DH5α. After culturing at 37°C for 12 hours, the positive clones were shaken and sent to the company for successful sequencing, and the positive strains were stored in a -80°C refrigerator.

[0047] 3. Construction of GmLNG5 fusion GUS expression vector pTF102-GmLNG5-GUS

[0048] According to the GmLNG5 gene, the forward specific primer (SEQ ID NO:7:5'-CTATGACATGATTACGAATTCCGAATACGAGAGCTTTGCCACTAG-3') and the reverse specific primer (SEQ ID NO:8:5'-GACTGACCTACCCGGGGATCCGTG TTCACAAGGCCTCAGTTATG-3') of the GmLNG5-GUS gene were designed, and the promoter sequence of the gene was amplified using the DNA of soybean YC03-3 as a template. The PCR amplification system was: 25 μL 2×Vazyme phata buffer, 1 μL Va zymephata enzyme, 1 μL dNTP, 1 μL of forward and reverse primers, 3 μL cDNA template, and finally 16 μL of ddH2O was added to make up to 50 μL. The PCR amplification procedure was as follows: 94°C pre-denaturation for 2 min, 94°C denaturation for 30 s, 58°C annealing for 3 min, 72°C extension for 3 min, 30 cycles from denaturation to extension, and storage of the PCR product at 16°C. The obtained amplified product was detected by gel electrophoresis. If the band size was correct, the target fragment was further recovered and purified using an agarose gel DNA recovery kit (Meiji Bio, China).

[0049] Select EcoRΙ as the single restriction site of pTF102 vector, use one-step cloning method to connect the target gene fragment to pTF102 vector, transfer the ligation product into competent Escherichia coli DH5α, culture at 37℃ for 12h, shake the positive clones, send them to the company for sequencing, and store the positive strains in a -80℃ refrigerator after successful sequencing.

[0050] Example 2 Obtaining transgenic material

[0051] 1. Obtaining in vitro hairy root materials of transgenic soybean

[0052] (1) Seed germination: Select clean and plump soybean seeds (WT) of uniform size and place them in a drying dish and sterilize them with chlorine. The chlorine production reaction is as follows: add 4.2 mL of HCl to 100 mL of sodium hypochlorite solution. After sterilization for 12 hours, take them out of the drying dish and place them on a clean bench to blow off the residual chlorine. Then plant the seeds in sterile MS germination medium and place them in a constant temperature and light incubator at 24°C.

[0053] (2) Shaking bacteria: During the germination period, take K599 Agrobacterium carrying the GmLNG5 overexpression vector and the pTF101s empty vector stored in glycerol at -80°C and streak the activated strain on the YEP solid medium containing streptomycin and spectacle resistance. After streaking, culture in a 28°C incubator in the dark. After 2 days, pick a single clone for detection, and use the positive strains for later use. On the night before infection, inoculate the positive strains into a 50mL centrifuge tube containing 5mL YEP liquid medium and place it in a 28°C constant temperature shaking incubator for dark culture for 12 to 16 hours.

[0054] (3) Infection and co-cultivation: In a clean bench, use a scalpel to cut the germinated seeds at the hypocotyl region approximately 0.5 cm away from the cotyledon node, peel off the seed coat, cut the seeds in half along the hypocotyl, remove the young shoots at the cotyledon node, and use a scalpel blade to cut the bean petals in half along the hypocotyl. Then, use a scalpel blade to dip into the bacterial solution and cut wounds at the cotyledon node and hypocotyl. Place the explants with wounds in a glass culture dish filled with moist filter paper, seal with sterile plastic wrap, and place in a 24°C constant temperature and light incubator for 5 to 7 days. Then transfer to a culture dish containing timentin (final concentration 50 mg L -1 ) and glufosinate (final concentration 20 μg L -1 ) on MS solid medium and cultured at 24°C in the dark.

[0055] (4) Treatment: When root hairs are visible after 7-10 days of cultivation on MS solid medium, select root hairs of uniform size and transplant them into rooting medium with different phosphorus contents. After 12 days of cultivation at 24°C in the dark, the root hairs are harvested.

[0056] 2. In vitro detection of transgenic soybean hairy roots

[0057] The total RNA of the transgenic root hairs was extracted and reverse transcribed into cDNA for later use. Real-time fluorescence quantitative PCR analysis was performed using the Applied Biosystems StepOnePlus Real-Time PCR system, and quantitative PCR was performed using the quantitative kit of Promega. The soybean housekeeping gene GmEF1-a (Glyma17g23900) was used as the reference gene, and the GmLNG5 quantitative primers were designed and synthesized. The specific primers used were:

[0058] GmEF1-aF: SEQ ID NO:9:5'-TGCAAAGGAGGCTGCTAACT-3';

[0059] GmEF1-aR: SEQ ID NO:10:5'-CAGCATCACCGTTCTTCAAA-3';

[0060] GmLNG5-RT-F: SEQ ID NO:11:5'-ATCCAGGAAAGGAGGTTTTGT-3';

[0061] GmLNG5-RT-R: SEQ ID NO:12:5'-AGCCCCGATGCTAACAGTAT-3';

[0062] Fluorescence quantitative detection was performed, and the 20μL reaction system was: 10μL 2×Go Tap qPCR Master Mix, 2μL cDNA template, 0.4μL upstream and downstream primers, 7.2μL Nuclease-free water; the reaction conditions were: 95℃ pre-denaturation for 1min, 95℃ denaturation for 15s, 60℃ renaturation for 15s, 72℃ extension for 30s, and 40 cycles from denaturation to extension. The relative expression level is the ratio of the expression level of the target gene GmLNG5 to the expression level of the housekeeping gene GmEF1-a.

[0063] The results of GmLNG5 expression detection in transgenic hairy roots showed that the expression level of OX-GmLNG5 in transgenic hairy roots was significantly higher than that in the control group, regardless of high or low phosphorus conditions, and the upregulation fold in low phosphorus treatment was higher than that in high phosphorus treatment, indicating that GmLNG5 expression was upregulated by low phosphorus.

[0064] Example 3 Analysis of GmLNG5 gene expression pattern and protein subcellular localization

[0065] 1. Histochemical localization analysis of GmLNG5

[0066] The transgenic soybean hairy roots in which the GUS reporter gene was driven by the constructed pTF102-GmLNG5-GUS promoter were used as materials. The transgenic soybean hairy roots were simultaneously treated with different phosphorus levels: normal phosphorus (+P: 1250μM KH2PO4) and low phosphorus (-P: 12.5μM KH2PO4) by GUS staining to analyze the histochemical localization of GmLNG5 in soybean hairy roots.

[0067] The results are as follows Figure 1 As shown in the results, obvious GUS staining could be observed in the main rhizome, main root tip, lateral rhizome and lateral root tip under normal or low phosphorus conditions, and low phosphorus stress enhanced the GUS staining intensity, indicating that low phosphorus enhanced the expression of GmLNG5 in soybean roots.

[0068] 2. Subcellular localization analysis of GmLNG5

[0069] The pEGAD-GmLNG5-GFP vector and the pEGAD empty vector were introduced into tobacco epidermal cells by Agrobacterium infection for transient expression, and then the GFP fluorescence signal of tobacco epidermal cells was observed using a laser confocal microscope.

[0070] The results are as follows Figure 2 As shown, compared with the 35S::GFP protein expressed in the cytoplasm, cell membrane and cell nucleus, the GFP signal of the 35S::GmLNG5-GFP fusion protein existed in the cell nucleus and cell membrane, indicating that the GmLNG5 protein was mainly localized in the cell nucleus and cell membrane.

[0071] Example 4 GmLNG5 Functional Analysis

[0072] 1. Effect of overexpression of GmLNG5 on phosphorus availability in transgenic soybean hairy roots in vitro

[0073] To explore the effect of GmLNG5 on the response of transgenic soybean hairy roots to phosphorus availability, about 0.1 g of transgenic hairy roots were inoculated in solid MS medium with normal phosphorus (+P: 1250 μM KH2PO4) and low phosphorus (-P: 12.5 μM KH2PO4) concentrations and cultured for 12 days. At the same time, the control (CK) was set as the above soybean YC03-3 infected with pTF101s-K599 bacterial solution. Each treatment was set up with 7 independent biological replicates, and the fresh weight and total phosphorus content of the hairy roots were measured after harvest. Real-time fluorescence quantitative PCR detection was also performed, and the detection method and conditions were the same as above.

[0074] The results are as follows Figure 3 As shown in Figure 2, under +P conditions, overexpression of GmLNG5 promoted the growth of soybean hairy roots in vitro ( Figure 3 A). Real-time fluorescence quantitative PCR results showed that under normal phosphorus supply and low phosphorus conditions, the expression level of GmLNG5 in transgenic hairy roots overexpressing GmLNG5 was significantly higher than that in the control (CK), with the expression levels increased by 42 times and 555 times, respectively ( Figure 3 B). Compared with CK, the fresh weight of transgenic hairy roots of excess GmLNG5 increased by 30.9% ( Figure 3 C), the total phosphorus content increased by 60.1% ( Figure 3 D). Under -P conditions, the expression of excess GmLNG5 also significantly promoted the growth of soybean hairy roots in vitro. Compared with CK, the fresh weight of the transgenic hairy roots of excess GmLNG5 increased by 21.9% and the total phosphorus content increased by 62.9%. These results indicate that the soybean GmLNG5 gene positively regulates soybean root growth and phosphorus efficiency.

[0075] 2. Effect of overexpression of GmLNG5 on the elongation of root tip cells of transgenic soybean hairy roots in vitro

[0076] To further analyze the effect of overexpression of GmLNG5 on the elongation of root tip cells in soybean hairy roots in vitro, the roots 1 cm from the root tips of the overexpressing transgenic plants were selected and pressed into slices. Cell photographs were taken using an inverted microscope, and the cell length and width in the elongation zone and maturation zone of the root tip were measured using Image J software, and the results were statistically analyzed.

[0077] The results are as follows Figure 4 The results showed that the length of cells in the elongation zone and mature zone of the root tips of the transgenic plants overexpressing OX-GmLNG5 were significantly longer than those in CK ( Figure 4 A and B). Under +P and -P conditions, compared with CK, the length of cells in the elongation zone of the root tip of OX-GmLNG5 hairy roots increased by 14.1% and 20.6%, respectively, and the length of cells in the maturation zone increased by 25.2% and 28.5%, respectively ( Figure 4 C and D). In addition, there was no significant difference in the cell widths of the elongation zone and the maturation zone in the root tips between CK and OX-GmLNG5 hairy roots ( Figure 4 E and F).

[0078] In summary, the present invention studies the gene of soybean GmLNG5, showing that GmLNG5 positively regulates soybean root growth and phosphorus efficiency, is mainly expressed in soybean roots, and GmLNG5 protein is mainly located in the nucleus and cell membrane, and is enhanced by low phosphorus stress; GmLNG5 regulates the growth of soybean roots by mediating the longitudinal elongation of root tip cells, and overexpression of the GmLNG5 gene increases the length of cells in the elongation zone and mature zone of the root tip of soybean hairy roots, the biomass of the in vitro hairy roots, and the phosphorus content, showing that the GmLNG5 gene has a significant effect on the growth and total phosphorus content of soybean hairy roots in vitro, promotes plant growth under low phosphorus stress, and can be used to cultivate low-phosphorus tolerant plant varieties. The GmLNG5 gene plays an important role in promoting soybean root growth and improving soybean phosphorus efficiency, which is of great significance for clarifying the biological function of the LNG gene in leguminous crops to adapt to low phosphorus stress regulation.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. The soybean low-phosphorus stress tolerance GmLNG5 gene as shown in SEQ ID NO: 1 is used in positively regulating soybean root growth and / or phosphorus efficiency.

2. Use of the soybean low-phosphorus stress tolerance GmLNG5 gene as shown in SEQ ID NO: 1 in promoting plant growth and / or promoting plant growth under low-phosphorus conditions.

3. Use of the soybean low-phosphorus stress tolerance GmLNG5 gene as shown in SEQ ID NO: 1 in increasing the phosphorus content of plants and / or in preparing a preparation for increasing the phosphorus content of plants.

4. Use of the soybean low-phosphorus stress-tolerant GmLNG5 gene as shown in SEQ ID NO: 1 in the preparation of a product for promoting plant growth and / or improving plant tolerance to low-phosphorus stress.

5. Use of a formulation for promoting the expression of the soybean low-phosphorus stress-tolerant GmLNG5 gene as shown in SEQ ID NO: 1 in promoting plant growth and / or promoting plant growth under phosphorus stress.

6. Use of a preparation for promoting the expression of the soybean low-phosphorus stress tolerance GmLNG5 gene as shown in SEQ ID NO: 1 in increasing the phosphorus content of plants and / or in preparing a product for increasing the phosphorus content of plants.

7. Use of the soybean low-phosphorus stress-tolerant GmLNG5 gene as shown in SEQ ID NO: 1 in constructing plants tolerant to low-phosphorus stress.

8. Use of a preparation for promoting the expression of the soybean low-phosphorus stress-tolerant GmLNG5 gene as shown in SEQ ID NO: 1 in breeding low-phosphorus-tolerant plant varieties.

9. A method for promoting plant growth and / or increasing plant phosphorus content, characterized in that: The plant's GmLNG5 gene is overexpressed, or the plant is treated with a formulation that promotes the expression of the soybean low-phosphorus stress-tolerant GmLNG5 gene as shown in SEQ ID NO:

1.

10. A method for constructing a plant tolerant to low phosphorus stress, characterized in that: The recombinant expression vector of soybean low-phosphorus stress-tolerant GmLNG5 gene is introduced into plants to obtain low-phosphorus stress-tolerant plants; the sequence of the GmLNG5 gene is shown in SEQ ID NO:1.

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