CN116200404B - A soybean asparagine synthetase-like gene and its application - Google Patents

Abstract

The invention discloses a soybean asparagine synthetase-like gene and application thereof. The research of the invention shows that GmASL gene shown in SEQ ID NO. 1 is a gene which is inhibited by low phosphorus and expressed, gmASL affects the metabolic process of soybean root nodule amino acid, and excessive expression of GmASL gene can promote the growth of soybean root system, increase nitrogen and phosphorus content of plants and the root nodule number of transgenic composite plants, and reduce aspartic acid content; gmASL6 is shown to mediate asparagine accumulation or synthesis, ultimately affecting nodule growth. Meanwhile, gmASL genes and proteins can regulate the amino acid metabolic process of the transgenic root nodule containing the GmASL genes and proteins, promote plant growth and promote soybean growth under low phosphorus stress. The invention provides more effective ways for cultivating transgenic plants of low-phosphorus-resistant plants, and defines the biological functions of GmASL genes involved in the synthesis of asparagine in root nodules and the regulation and control effects of the GmASL genes on nitrogen fixation of the root nodules.

Description

A soybean asparagine synthetase-like gene and its application

Technical Field

The present invention belongs to the technical field of plant gene breeding, and more specifically, relates to a soybean asparagine synthetase-like gene and its application.

Background technique

Soybean (Glycine max) is an important grain and oil crop and a major source of plant protein with important economic value. In addition to rich fat, protein and carbohydrates, soybean also contains a variety of unique active substances, such as soybean isoflavones, soybean saponins, soybean peptides, etc., which have high application value in health care. Different from other traditional crops such as rice and wheat, soybean can mutually benefit from symbiosis with rhizobia to form nodule structures. Nodules can utilize nitrogen in the soil and fix nitrogen in the air into ammonia, providing a nitrogen source for leguminous crops. The symbiotic nitrogen fixation of soybeans not only provides nitrogen nutrition for soybean plants themselves, but also reduces the application of nitrogen fertilizers in agricultural production. According to statistics, soybeans transport 16 million tons of nitrogen to agricultural ecosystems through symbiotic nitrogen fixation every year, which is of great significance to the development of environmentally friendly agriculture, the reduction of nitrogen fertilizer application and environmental pollution.

In legumes, asparagine is a product of nodule nitrogen metabolism and participates in the transport of nitrogen assimilates. It also participates in the metabolic process of nodule symbiotic nitrogen fixation. Many studies have found that asparagine is present in high concentrations in the roots, nodules, and xylem sap of legumes. Under adverse stress conditions, free amino acids, especially asparagine, accumulate in the roots and nodules of legumes, indicating that the accumulation of asparagine may be involved in regulating nodule nitrogen metabolism, but the specific mechanism is still unclear.

Phosphorus is one of the essential macronutrients for crop growth and development. Low available phosphorus concentration in cultivated soil has become the main limiting factor for crop yield and quality worldwide. Most of the southern regions of my country are acidic land, and the soil generally has the problem of low phosphorus availability, which affects the yield and quality of crops in southern China. In the long-term evolutionary process, plants have evolved a series of physiological and molecular mechanisms to adapt to low phosphorus stress (Vance et al., 2003; Liang et al., 2010; Wang et al., 2010; Oldroyd and Leyser, 2020; Zhu et al., 2020). In leguminous crops, such as soybeans, alfalfa, and clover, low phosphorus stress can cause the accumulation of asparagine in the root system and nodules. Studies have shown that this part of the accumulated asparagine is involved in regulating soybean nitrogen metabolism, which may be related to the adaptation mechanism of soybean and nodules to low phosphorus stress (Almeida et al., 2000; Hernández et al., 2009; Sulieman et al., 2010, 2013; Xue et al., 2018). At present, although the asparagine synthetase family has been cloned and reported in Arabidopsis, wheat, and rapeseed, the biological function of asparagine synthetase-like genes in asparagine synthesis in nodules and their regulatory role in nodule nitrogen fixation have not yet been clarified. Therefore, in order to reveal the function of asparagine synthetase-like genes in nodules, it is necessary to conduct in-depth research on the mechanism of nitrogen metabolism and transport in nodules of leguminous crops.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the above-mentioned problems and provide a soybean asparagine synthetase-like gene GmASL6 and its application.

The first object of the present invention is to provide the application of soybean asparagine synthetase-like gene GmASL6.

A second object of the present invention is to provide a product for promoting soybean growth and/or promoting soybean growth under phosphorus stress.

The third object of the present invention is to provide a method for increasing the nitrogen and phosphorus content of soybean plants or promoting the growth of root nodules of soybean plants.

The fourth object of the present invention is to provide a method for cultivating transgenic plants tolerant to low phosphorus.

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

The present invention shows that the GmASL6 gene shown in SEQ ID NO: 1 is a gene whose expression is inhibited by low phosphorus. The GmASL6 gene affects the amino acid metabolism process of soybean nodules. Overexpression of the gene can increase the asparagine content of soybean nodules and increase the number of nodules of transgenic composite plants, indicating that GmASL6 mediates the accumulation or synthesis of asparagine and ultimately affects nodule growth. At the same time, the GmASL6 gene and protein can regulate the amino acid metabolism process of transgenic nodules containing it, promote plant growth, and increase the nitrogen and phosphorus content of plants.

Therefore, the following applications are all within the protection scope of the present invention:

Use of a preparation overexpressing the GmASL6 gene shown in SEQ ID NO: 1 in promoting soybean growth and/or promoting soybean growth under low phosphorus stress.

Use of a preparation overexpressing the GmASL6 gene shown in SEQ ID NO: 1 in preparing a product for promoting soybean growth and/or promoting soybean growth under low phosphorus stress

Use of a preparation overexpressing the GmASL6 gene shown in SEQ ID NO: 1 in increasing the nitrogen and phosphorus content of soybean plants and/or in preparing a product for increasing the nitrogen and phosphorus content of soybean plants.

Use of a preparation overexpressing the GmASL6 gene shown in SEQ ID NO: 1 in reducing the asparagine content in soybean plant root nodules.

Use of a preparation overexpressing the GmASL6 gene shown in SEQ ID NO: 1 in cultivating transgenic plants tolerant to low phosphorus.

Use of a preparation overexpressing the GmASL6 gene shown in SEQ ID NO: 1 in promoting the growth of soybean plant nodules or increasing the number of soybean plant nodules.

Preferably, the amino acid sequence of the protein encoded by the above gene GmASL6 is as shown in SEQ ID NO.2.

The present invention provides a product for promoting soybean growth and/or promoting soybean growth under phosphorus stress, comprising a preparation for overexpressing the GmASL6 gene shown in SEQ ID NO:1.

The present invention provides a method for increasing the nitrogen and phosphorus content of soybean plants or promoting the growth of root nodules of soybean plants, and the soybeans are treated with the preparation for overexpressing the GmASL6 gene shown in SEQ ID NO:1.

The present invention also provides a method for cultivating low-phosphorus tolerant transgenic plants, which is obtained by introducing a recombinant vector for overexpressing the GmASL6 gene into soybeans.

Preferably, the expression vector can be constructed using an existing plant expression vector to form a recombinant expression vector containing the GmASL6 gene.

More preferably, the plant expression vector comprises a binary Agrobacterium vector, such as pTF101s or other derived plant expression vectors.

The present invention has the following beneficial effects:

The present invention shows that the GmASL6 gene shown in SEQ ID NO: 1 is a gene whose expression is inhibited by low phosphorus. GmASL6 affects the amino acid metabolism process of soybean nodules. Overexpression of the GmASL6 gene can reduce the asparagine content of soybean nodules, increase the nitrogen and phosphorus content of the plant, and increase the number of nodules of transgenic composite plants; it shows that GmASL6 mediates the accumulation or synthesis of asparagine, and ultimately affects nodule growth. At the same time, the GmASL6 gene and protein can regulate the amino acid metabolism process of transgenic nodules containing it, promote plant growth, and promote soybean growth under low phosphorus stress. The present invention provides more effective ways to cultivate transgenic plants that are tolerant to low phosphorus, and clarifies the biological function of the GmASL6 gene in participating in the synthesis of asparagine in nodules and its regulatory effect on nodule nitrogen fixation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Analysis results of the expression pattern of GmASL6 in roots and nodules under normal and low phosphorus conditions (soybean was inoculated with rhizobia after germination, and seedlings were grown in hydroponics under normal phosphorus (HP: 250μM KH ₂ PO ₄ ) and phosphorus-deficient (LP: 5μM KH ₂ PO ₄ ) conditions; the data in the figure are the mean and standard error of 4 replicates. "*" indicates a significant difference between normal phosphorus and phosphorus-deficient treatments (Student's t-test, 0.01≤P<0.05), and "**" indicates an extremely significant difference between normal phosphorus and phosphorus-deficient treatments (Student's t-test, P<0.01)).

Figure 2: Histochemical localization results of GUS driven by GmASL6 promoter in ex vivo hairy roots (A: GUS staining results of ex vivo hairy roots under normal phosphorus treatment conditions (HP: _{250μM KH2PO4} ); B: root elongation zone under normal phosphorus treatment conditions; C: root tip under normal phosphorus treatment conditions; D: lateral root primordium under normal phosphorus treatment conditions; E: GUS staining results of ex vivo hairy roots under low phosphorus treatment conditions (LP: _{5μM KH2PO4} ); F: root elongation zone under low phosphorus treatment conditions; G: root tip under low phosphorus treatment conditions; H: lateral root primordium under low phosphorus treatment conditions. The scale bars in A and E are 5 mm; the other scale bars are 1 mm).

Figure 3: Subcellular localization results of GmASL6 (GFP represents GFP channel, BF represents bright field, Merge represents the overlap of GFP and BF. GFP fluorescence was observed by laser confocal microscopy. The scale bar in the figure is 20 μm).

Figure 4: Effect of overexpression of GmASL6 on the growth of large in vitro hairy roots (A: phenotype of transgenic in vitro hairy roots overexpressing GmASL6 and CK hairy roots; B: dry weight of hairy roots; C: total phosphorus content of hairy roots; D: asparagine concentration of hairy roots. CK refers to in vitro hairy roots transferred with OX empty vector, and OX refers to hairy roots overexpressing GmASL6. Samples were collected after the in vitro hairy roots were grown on normal phosphorus (HP: _{250μM KH2PO4} ) and phosphorus-deficient (LP: _{5μM KH2PO4} ) culture media for 25 days).

Figure 5: Effect of overexpression of GmASL6 on the growth of soybean composite plants (A: phenotype of composite plants overexpressing GmASL6; B: dry weight of composite plants; C: total nitrogen content of composite plants; D: total phosphorus content of composite plants; E: total root length; F: root nitrogen content; G: root phosphorus content. CK refers to composite plants transformed with empty vector control, and overexpression refers to composite plants overexpressing GmASL6 transgenic. After inoculation with rhizobia, composite plants were grown under normal phosphorus (HP: 250 μM KH ₂ PO ₄ ) and phosphorus-deficient (LP: 5 μM KH ₂ PO ₄ ) conditions for 31 days, and samples were collected. The data in the figure are the mean and standard error of 8 biological replicates. "*" indicates that there is a significant difference between OX composite plants and CK composite plants (Student's t-test, 0.01<P≤0.05), and "**" indicates that there is an extremely significant difference between OX composite plants and CK composite plants (Student's t-test, 0.001<P≤0.01). The scale of the composite plants and roots in the figure is 10 cm.

Figure 6: Effect of overexpression of GmASL6 on root nodules of soybean composite plants (A: nodule phenotype of overexpression of GmASL6 composite plants; B: nodule fresh weight; C: nodule number; D: nodule asparagine concentration; E: aspartic acid concentration; F: glutamine concentration. CK refers to composite plants transformed with empty vector control, and OX refers to composite plants with overexpression of GmASL6 transgenic plants. After inoculation of rhizobia, composite plants were grown under normal phosphorus (HP: 250 μM KH ₂ PO ₄ ) and phosphorus-deficient (LP: 5 μM KH ₂ PO ₄ ) conditions for 31 days, and nodule samples were collected. The data in the figure are the mean and standard error of 8 biological replicates. "*" indicates that there is a significant difference between OX composite plants and CK composite plants (Student's t-test, 0.01<P≤0.05), and "**" indicates that there is an extremely significant difference between OX composite plants and CK composite plants (Student's t-test, 0.001<P≤0.01). The scale of the magnified root system in the figure is 1 cm, and the scale of the nodule is 1 cm).

Figure 7: Heterologous expression of GmASL6-GST recombinant protein and enzyme activity analysis results (A: Western blot analysis results of GmASL6-GST protein, lane I is the whole protein after the Escherichia coli culture containing GST empty vector was disrupted, lane II is the GST empty protein after purification by GST magnetic beads, lane III is the whole protein after the Escherichia coli culture containing GmASL6-GST was disrupted, lane IV is the GmASL6-GST protein lane after purification by GST magnetic beads; B: GmASL6-GST asparagine synthetase enzyme activity analysis, "***" indicates that the difference in asparagine synthetase enzyme activity between GmAS6-GST and GST empty vector is extremely significant (Student’s t-test, 0.001<P≤0.01)).

Detailed ways

The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples 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 art.

Nucleotide sequence shown in SEQ ID NO.1: ATGTTGGGAATTTTCAAGCAGAAGTTGGTTAATGCACCCAAGGAGCTGAACAGTCCAGCTTCTTTGAATTCATGCATTAAGCCTAAGCTAAGTCATGAAATCCTGAAGGATTTCATGTCCTGCAATTCCTCCAATGCTTTCTCAATGTGCTTTGGGAATGATGCTTTGCTAGCCTATTCCACTTCATACAAGCCCTCCATTAATCATAGGTTATTCTCTGGATTGGATAACATATACTGTGTTTTCCTGGGTGGCCTGCACAACCTTAGCATGCTCAACAAGCAGTATGGACTATCAAAGGGAACAAATGAGGCCATGTTTATCATTGAAGCATATCGTACACTTCGCGACAGGGGTCCATACCCTGCTGAT CAAGTCCTCAAAGAACTTGAAGGCAGTTTTGCATTTGTGATCTATGACAACAAGGATGGAACAGTTTTTGTTGCATCTGGTTCTAATGGCCATATTGAGCTCTACTGGGGTATTGCAGGTGATGGTTCTGTTATAATTTCTGAAAATCTGGAGCTTATAAAAGCAAGTTGTGCTAAATCATTTGCACCATTTCCAGCTGGGTATGTTTCATAGTGAACACGGTCTCATGAACTTTGAGCATCCAACACAGAAGATGAAAGCAATGCCTCGGATTGACAGCGAGGGGGTTATGTGCGGGGCCAACTTCAATGTTGACTCTCAGTCAAAGATCCAGGTGATGCCACGTGTTGGAAGTGAAGCTAATTGGGCAACTTGGGGCTAA.

Amino acid sequence shown in SEQ ID NO.2: MLGIFKQKLVNAPKELNSPASLNSCIKPKLSHEILKDFMSCNSSNAFSMCFGNDALLAYSTSYKPSINHRLFSGLDNIYCVFLGGLHNLSMLNKQYGLSKGTNEAMFIIEAYRTLRDRGPYPADQVLKELEGSFAFVIYDNKDGTVFVASGSNGHIELYWGIAGDGSVIISENLELIKASCAKSFAPFPAGCMFHSEHGLMNFEHPTQKMKAMPRIDSEGVMCGANFNVDSQSKIQVMPRVGSEANWATWG*.

Example 1 Construction of vector

A gene was cloned through preliminary experiments of the present invention, and its cDNA 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. The present invention has studied the function of the gene and identified the gene as a soybean asparagine synthetase-like gene, named GmASL6. Further, through the study of soybean transgenic composite plants, a systematic functional analysis of GmASL6 expressed in nodules was conducted.

1. Overexpression vector: GmASL6-pTF101s vector

Using soybean cDNA as a template, PCR reaction was carried out using the forward and reverse specific primers F: 5’-TTCGCGAGCTCGGTACCCGGGATGTTGGGAATTTTCAAGCAGA-3’ and R: 5’-CGACTCTAGAGGATCCCCGGGTTAGCCCCAAGTTGCCC-3’ of the GmASL6-pTF101s gene to amplify the full-length CDS sequence of the GmASL6 gene.

The PCR reaction system was as follows: Vazyme phata Buffer 25 μL, 10 mM dNTP 1 μL, forward and reverse primers 2 μL, soybean cDNA 3 μL, Vazyme phata high-fidelity enzyme (Meiji Biotechnology, China) 1 μL, and ddH ₂ O 16 μL.

The PCR program was set as: 94°C for 3 min, 30 repeated cycles (specifically including: 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min), 72°C for 10 min, and the amplified product was stored at 16°C.

After amplification, the target band was recovered and purified. The pTF101s plasmid was digested with SmaI restriction endonuclease, the digested product was recovered and purified, and the digested product and the target gene fragment were connected by one-step cloning and connection method. The GmASL6-pTF101s vector was obtained for subsequent experiments.

2. Construction of subcellular localization vector: GmASL6-pEGAD vector

Using soybean cDNA as a template, PCR reaction was carried out with the forward and reverse specific primers F: 5’-CTAGCGCTACCGGTATGTTGGGAATTTTCAAGCAGAAG-3’ and R: 5’-TGGTGGCGACCGGTAGGCCCCAAGTTGCCCAATT-3’ of the GmASL6-pEGAD gene to amplify the full-length CDS sequence of the GmASL6 gene.

The PCR amplification method and conditions are the same as above. After the amplified product is subjected to gel electrophoresis, the target band is recovered and purified using an agarose gel kit. The pEGAD plasmid is digested with AgeI restriction endonuclease, and the single digestion product is recovered and purified. The digestion product and the target gene fragment are connected by a one-step cloning and ligation method. The one-step cloning and ligation product is directly transferred into the competent state of Escherichia coli. After sequencing and alignment, the plasmid is extracted and transferred into Agrobacterium GV3101 and stored at -80°C for use. The 35S:GmASL6-GFP vector is obtained for subsequent experiments.

3. Construction of heterologous expression vector of GmASL6-GST recombinant protein: GmASL6-pGEX

Using soybean cDNA as a template, PCR reaction was carried out with the forward and reverse specific primers F: 5’-GGGGCCCCTGGGATCCATGTTGGCTATATTCCACAAAGC-3’ and R: 5’-GGGAATTCGGGGATCCCTAATGTTGGTCCCATTCCATC-3’ of the GmASL6-pGEX gene to amplify the full-length CDS sequence of the GmASL6 gene.

The PCR amplification method and conditions are the same as above. After the amplified product is subjected to gel electrophoresis, the target band is recovered and purified using an agarose gel kit. The pGEX plasmid is digested with BamHI restriction endonuclease, and the single digestion product is recovered and purified. The digestion product and the target gene fragment are connected by a one-step cloning and ligation method. The one-step cloning and ligation product is directly transferred into the competent state of Escherichia coli. After sequencing and alignment, the plasmid is extracted and transferred into Escherichia coli BL21 and stored at -80°C for use. The GmASL6-pGEX vector is obtained for subsequent experiments.

4. Construction of tissue localization analysis vector: GmASL6-pTF102 vector

Using soybean genomic DNA as a template, PCR reaction was carried out with the forward and reverse specific primers F: 5’-CTATGACATGATTACGAATTC CTCGGAAGTCCGAGTGTC-3’ and R: 5’-GACTGACCTACCCGGGGATCCCAATAATCAGCAATCCAAATAGCTG-3’ of the GmASL6-pTF102 gene to amplify the 2000 bp promoter fragment of GmASL6.

The PCR reaction system included: Vazyme phata Buffer 25 μL, 10 mM dNTP 1 μL, forward and reverse primers 2 μL, soybean genomic DNA 3 μL, Vazyme phata high-fidelity enzyme (Meiji Biotechnology, China) 1 μL, and ddH ₂ O 16 μL.

The PCR program was set as: 94°C for 3 min, 30 repeated cycles (specifically including: 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min), 72°C for 10 min, and the amplified product was stored at 16°C.

Recovery and purification of the amplified target fragment: After gel electrophoresis of the amplified product, the target band was recovered and purified using an agarose gel kit (Meiji Bio, China). Connect the promoter fragment to the vector: Use EcoR I and BamH I to double-digest the pTF102 plasmid, recover and purify the double-digestion product, and connect the digestion product and the promoter fragment by one-step cloning and ligation. The reaction system contains: Assembly mix Buffer 5μL (Quanshi Jin, China), vector double-digestion product 1μL, and promoter fragment 4μL. The temperature system of the one-step cloning and ligation method includes: 50℃15min, 4℃ cooling for 5 seconds. The one-step cloning and ligation product was directly transferred to the competent Escherichia coli. After sequencing and alignment, the plasmid was extracted and transferred to Agrobacterium K599 for standby use. The GmASL6-pTF102 vector was obtained for subsequent experiments.

Example 2 Analysis of GmASL6 expression pattern, tissue localization and subcellular localization

1. Tissue-specific analysis of GmASL6 expression

Soybean seedlings were inoculated with rhizobia and then hydroponically treated in two groups, one group was treated with normal phosphorus concentration (HP: 250μM KH ₂ PO ₄ ) and the other group was treated with low phosphorus (LP: 5μM KH ₂ PO ₄ ). Root and nodule samples were collected on different days, and the RNA was reverse transcribed into cDNA using the reverse transcription kit of Promega Company, and the expression pattern of GmASL6 was further detected by quantitative PCR. The reaction system of quantitative PCR was 20 μL, including 10 μL of 2×SYBR Green PCR master mix, 7 μL of Nuclease-free water, 0.5 μL of each forward and reverse primer with a concentration of 10 μmol/L, and 2 μL of diluted cDNA template. The reaction procedure was denaturation at 95℃ for 1 minute; then 40 cycles of 94℃ for 15 seconds, 60℃ for 15 seconds, and 72℃ for 30 seconds.

Soybean GmEF1-α was used as an internal reference, and the primers used for quantitative PCR detection of gene expression were:

The results are shown in Figure 1, which shows that GmASL6 was down-regulated by low phosphorus at day 31. At day 31 after rhizobium inoculation, GmASL6 was significantly down-regulated in nodules, with a down-regulation rate of 89%.

2. Histochemical localization analysis of GmASL6

Seeds of uniform size and intact seed coat were selected. After 13 hours of chlorine disinfection (4.2 mL of hydrochloric acid was added to 100 mL of sodium hypochlorite to react and generate chlorine), the soybean seeds were germinated in MS medium with the embryo facing downwards and cultured at 25°C for 5 days under light. Agrobacterium K599 containing the GmASL6-pTF102 plasmid was inoculated in liquid YEP medium and cultured for 24 hours. The activated K599 bacterial liquid into which the GmASL6-pTF102 plasmid had been transferred was taken with a scalpel, and multiple wounds were cut horizontally in the cotyledons and cotyledon nodes of the germinated soybeans. Then, the soybeans were placed on sterilized filter paper moistened with water in advance, sealed with plastic wrap, and cultured in the dark for 4 days. Then, the soybeans were transferred to MS medium containing 200 μg L ^-1 glufosinate (Sigma, USA) and 50 mg L ^-1 timentin (Dingguo Biology, Guangzhou) and cultured in the dark for 15 days.

Treatment of transgenic soybean in vitro hairy roots: Prepare MS liquid solid culture medium (both containing 50 mg L ^-1 timentin for antibacterial effect) for normal phosphorus treatment (HP: 250 μM KH ₂ PO ₄ ) and low phosphorus treatment (LP: 5 μM KH ₂ PO ₄ ) respectively, select white and tender in vitro hairy roots with good growth and transfer them to MS culture medium for normal phosphorus and low phosphorus treatments, and collect in vitro hairy root samples after culturing in the dark for 14 days.

The in vitro hair roots after normal phosphorus and low phosphorus treatment were taken out, rinsed twice with secondary water, _and placed in GUS staining solution (GUS staining solution contains: 0.1M _Na2HPO4 / _NaH2PO4 , _1mM X-Gluc, pH 7.2) for staining, and vacuumed for 30 minutes, and transferred to 37℃ for staining overnight in the dark. The dyed hair roots were transferred to 75% ethanol for storage, and the GUS staining was observed using a stereo microscope (Leica, Germany) and photographed.

The results are shown in Figure 2. Under normal phosphorus treatment conditions, GUS staining of Pro _GmASL6 : GUS transgenic soybean hairy roots in vitro was mainly distributed in the vascular tissue, lateral root primordium and root tip of the root elongation zone (Figures 2A, 2B, 2C, 2D). However, under low phosphorus treatment conditions, GUS staining in the vascular tissue, lateral root primordium and root tip of the root elongation zone was significantly weakened, especially in the root tip, where almost no staining was observed (Figures 2E, 2F, 2G, 2H).

3. Subcellular localization analysis of GmASL6

The GmASL6-pEGAD vector constructed in Example 1 was transferred into Agrobacterium GV3101, and the lower epidermis infection transformation experiment was further performed on tobacco leaves of 3 to 4 weeks old. The GV3101 strain that had been transferred with the GmASL6-pEGAD plasmid was taken out of the ultra-low temperature refrigerator, inoculated into the YEP medium, and cultured at 28°C for 24 hours. Centrifuged at 5000rpm for 10 minutes, the supernatant was discarded, and then GV3101 was resuspended in the infecting solution (the infecting solution contained: 10mM MgCl2, 100μM acetosyringone, 10mM MES), and the OD value of the infecting solution was adjusted to 0.5 and then cultured at 22°C in the dark for 4 hours. The GmASL6-pEGAD infecting solution was injected into the tobacco leaves from the back of the leaves with a syringe. After 2 days, the tobacco leaves were observed for GFP fluorescence under a laser confocal microscope and photographed.

The results are shown in Figure 3. It can be seen that compared with the 35S:GFP protein which has green fluorescence signals in the cell membrane, cytoplasm and cell membrane, the green fluorescence signal of the GmASL6:GFP protein is mainly present in the cell membrane and cell nucleus, revealing that GmASL6 is a cell membrane and cell nucleus localized protein.

Example 3 Acquisition and analysis of transgenic materials

1. Obtaining transgenic soybean hairy roots

Soybean seeds of uniform size and intact seed coat were selected. After 13 hours of chlorine disinfection (4.2 mL of hydrochloric acid was added to 100 mL of sodium hypochlorite to react and generate chlorine), the soybean seeds were germinated with the embryo facing downward in MS medium and cultured at 25°C for 5 days under light. The activated Agrobacterium K599 bacterial solution transferred into the target vector was taken with a scalpel, and multiple wounds were cut horizontally in the cotyledons and cotyledon nodes of the germinated soybeans. Then, the soybeans were placed on sterilized filter paper moistened with water in advance, sealed with plastic wrap, and cultured in the dark for 4 days. Then, the soybeans were transferred to MS medium containing 200 μg L ^-1 glufosinate (Sigma, USA) and 50 mg L-1 timentin (Dingguo Biological, Guangzhou) and cultured in the dark for 15 days.

2. Obtaining soybean composite plants

Select soybean seeds of uniform size and fullness, sterilize with 10% H ₂ O ₂ for 10 minutes, and wash with sterile water 5 times. Sow soybean seeds on the surface of quartz sand, add secondary water, cover the seeds with 2 cm thick quartz sand, and culture under light conditions for 5 days. Agrobacterium K599 that has been transferred to GmASL6-pTF101s is spread on the surface of solid YEP culture medium, cultured at 30°C for 24 hours, and the empty load is used as the corresponding empty load control. The activated K599 is dipped into the syringe needle, and a hole is made at the hypocotyl position of the soybean seedling. After that, the K599 bacteria are covered at the wound site, and the seedlings are covered with moist sand to continue to be cultured. The seedlings need to be covered with a layer of plastic wrap, and water is sprayed several times a day to keep the wound site moist. After about 15 days, hairy roots begin to grow at the wound site. Take a part of the sample, and after testing, only the positive hairy roots are left. When the positive hairy roots grow to about 10 cm, cut off the main root of the soybean plant and inoculate rhizobia. After the rhizobium was inoculated, the composite plants were moved to sand culture and irrigated with 200 mL of normal phosphorus (HP: 250 μM KH ₂ PO ₄ ) and low phosphorus (LP: 5 μM KH ₂ PO ₄ ) nutrient solutions once a week.

3. Detection of genetically modified materials

(1) Detection of transgenic hairy roots in vitro and hypocotyl composite plants of transgenic soybean

Total RNA of the transgenic hairy roots was extracted and reverse transcribed into cDNA, and then the expression of GmASL6 was detected by quantitative PCR. Soybean EF1-a was used as the reference gene, the primers and methods were the same as in Example 2, and the relative expression was the ratio of the expression of the target gene GmASL6 to the expression of the housekeeping gene.

Example 4 GmASL6 functional analysis

1. Effect of overexpression of GmASL6 on the growth of transgenic soybean hairy roots in vitro

When the transgenic and empty control hairy roots grow to 15 days, select the in vitro hairy roots with good growth, similar hairy root morphology and fresh weight of about 0.1g, and transfer them to MS liquid solid medium (both containing 50mg L ^-1 timentin for antibacterial) with normal phosphorus treatment (HP: 250μM KH ₂ PO ₄ ) and low phosphorus treatment (LP: 5μM KH ₂ PO ₄ ) and continue to culture in the dark for 14 days, and then collect the hairy root samples. The asparagine concentration, hairy root dry weight and total phosphorus content were measured respectively. Six independent biological replicates and an empty control were set for each treatment in this experiment.

The results are shown in Figure 4. Overexpression of GmASL6 promoted the growth of soybean detached hairy roots and significantly increased their phosphorus content (Figure 4A). Under normal phosphorus treatment conditions, the dry weight of transgenic detached hairy roots increased by 58.7% and the phosphorus content increased by 45.8% compared with CK. Under low phosphorus treatment conditions, the dry weight of transgenic detached hairy roots increased by 110% (Figure 4B) and its phosphorus content increased by 31% (Figure 4C) compared with CK. Moreover, under normal phosphorus and low phosphorus conditions, the asparagine concentration of transgenic hairy roots increased by 30.8% and 25.8%, respectively (Figure 4D), revealing that overexpression of GmASL6 enhances the synthesis of asparagine in detached hairy roots.

2. Effects of overexpression of GmASL6 on the growth of transgenic soybean composite plants

After obtaining the transgenic composite soybean plants by the hypocotyl injection method, they were inoculated with rhizobia. After the rhizobia were inoculated, the composite plants were moved to sand culture and irrigated with 200 mL of normal phosphorus (HP: 250 μM KH ₂ PO ₄ ) and low phosphorus (LP: 5 μM KH ₂ PO ₄ ) nutrient solutions once a week.

The results are shown in Figure 5. Overexpression of GmASL6 increased the amount of transgenic in vitro hairy roots (Figure 5A). Overexpression of GmASL6 increased plant dry weight and plant nitrogen and phosphorus content (Figures 5B-D). The promotion of root development was specifically manifested in increased root length (Figure 5E) and increased root nitrogen content (Figure 5F). The results of measuring root asparagine content (Figure 5G) showed that overexpression of GmASL6 reduced root asparagine content.

The effect of overexpression of GmASL6 on the nodules of soybean composite plants is shown in Figure 6. Overexpression of GmASL6 increased the number and weight of nodules (Figure 6A), increased the fresh weight of nodules, and under low phosphorus conditions, the number of nodules increased significantly compared with CK (Figure 6B, 6C). At the same time, overexpression of GmASL6 regulated the amino acid metabolism process of transgenic nodules and significantly changed the concentrations of different amino acids in nodules: asparagine, aspartic acid, and glutamine (Figure 6D, 6E, 6F). By measuring the asparagine content of nodules, the results showed that overexpression of GmASL6 reduced the asparagine content of plant nodules.

Example 5 GmASL6 enzyme activity analysis

1. GmASL6 protein purification

The BL21 Escherichia coli into which the GmASL6-pGEX plasmid had been transferred in Example 1 was inoculated into liquid LB medium and cultured with shaking at 28°C. When the bacteria were cultured to an OD of 0.5-0.6 as measured by a spectrophotometer at a wavelength of 600, IPTG was added to a final concentration of 1 mM. After induction culture at 28°C on a shaker for 4 h, 3 mL of 100 mM PMSF and DTT were added respectively. After shaking, the mixture was divided into 50 mL centrifuge tubes and centrifuged at 5000 rpm for 10 min in a centrifuge at 4°C. The supernatant was removed and resuspended in 50 mM PBS buffer. The mixture was crushed under high pressure at 35 pis to obtain a clear and transparent bacterial solution, and then centrifuged at 5000 rpm for 10 minutes. The supernatant was taken into a new 50 mL centrifuge tube, and 3 mL of GSH magnetic beads were added. The tube was sealed and placed on ice, and then placed in a cold storage shaker for combination for 5 h.

After the protein supernatant was bound to the magnetic beads, the impurities were washed away with pre-cooled PBS buffer. When the protein content of the washing buffer was determined to be 0, 3 mL of pre-cooled protein elution solution was added to elute the recombinant protein on the magnetic beads, and the protein concentration was determined by the Coomassie Brilliant Blue method. An appropriate amount was taken for SDS polyacrylamide gel electrophoresis and Western Blot to verify that there were no impurities in the GmASL6 enzyme solution (Figure 7A), and subsequent experiments could be carried out.

2. Determination of enzyme activity of GmASL6 recombinant protein

The purified GmASL6-GST and GST proteins were added to Coomassie Brilliant Blue G-250 to determine their protein concentrations. GST protein was used as a blank control for enzyme activity analysis, and GmAS5 was used as a positive control. According to the following reaction system: 100mM Tris-HCl (pH 8.0), 100mM NaCl, 5mM ATP, 10mM MgCl2, 10mM Asp, 10mM Gln, reacted in a 37°C water bath for 15min, 500μL ethanol was added to terminate the reaction, and the asparagine concentration was determined.

The results are shown in Figure 7B. With aspartic acid and glutamine as substrates, at a temperature of 37°C and a pH of 8.0, the asparagine synthetase activity of the GmASL6-GST protein was not significantly different from that of GST, indicating that the GmASL6-GST protein does not have asparagine synthetase activity.

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 (9)

1. Use of a preparation for over-expressing GmASL gene shown in SEQ ID No.1 for promoting soybean growth and/or for promoting soybean growth under low phosphorus stress, characterized in that the preparation is an over-expression vector.

2. Use of a preparation of GmASL gene expressed in SEQ ID No.1 for the preparation of a product for promoting soybean growth and/or for promoting soybean growth under low phosphorus stress, characterized in that the preparation is an overexpression vector.

3. The application of a preparation for over-expressing GmASL genes shown in SEQ ID NO. 1 in improving nitrogen and phosphorus content of soybean plants and/or preparing products for improving nitrogen and phosphorus content of soybean plants is characterized in that the preparation is an over-expression vector.

4. The use of a preparation for over-expressing GmASL gene shown in SEQ ID No. 1 for reducing the root nodule asparagine content of soybean plants, characterized in that the preparation is an over-expression vector.

5. The application of the preparation for over-expressing GmASL gene shown in SEQ ID NO. 1 in culturing transgenic plants of low-phosphorus-tolerant plants is characterized in that the preparation is an over-expression vector.

6. The application of a preparation for over-expressing GmASL genes shown in SEQ ID NO.1 in promoting the growth of soybean plant nodules or increasing the number of soybean plant nodules is characterized in that the preparation is an over-expression vector.

7. A product for promoting soybean growth and/or promoting soybean growth under phosphorus stress is characterized by comprising a preparation which overexpresses GmASL gene shown in SEQ ID NO. 1; the formulation is an over-expression vector.

8. A method for increasing nitrogen and phosphorus content of soybean plants or promoting root nodule growth of soybean plants, comprising treating soybean with the product of claim 7.

9. A method for cultivating transgenic plants of low-phosphorus-resistant plants is characterized in that the method is obtained by introducing a recombinant vector which overexpresses GmASL gene into soybean, and the sequence of GmASL gene is shown as SEQ ID NO. 1.