CN116200404B - Soybean asparagine synthetase analogous gene and application thereof - Google Patents
Soybean asparagine synthetase analogous gene and application thereof Download PDFInfo
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- CN116200404B CN116200404B CN202310376006.8A CN202310376006A CN116200404B CN 116200404 B CN116200404 B CN 116200404B CN 202310376006 A CN202310376006 A CN 202310376006A CN 116200404 B CN116200404 B CN 116200404B
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/05—Carbon-nitrogen ligases with glutamine as amido-N-donor (6.3.5)
- C12Y603/05004—Asparagine synthase (glutamine-hydrolyzing) (6.3.5.4)
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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
Technical Field
The invention belongs to the technical field of plant gene breeding. More particularly, it relates to a soybean asparagine synthetase-like gene and application thereof.
Background
Soybean (Glycine max) is an important grain and oil crop, is also a main source of vegetable protein, and has important economic value. Besides rich fat, protein and carbohydrate, soybean also contains various unique active substances such as soybean isoflavone, soybean saponin, soybean polypeptide and the like, and has higher application value in medical care. Unlike other traditional crops, such as rice, wheat, etc., soybean can intergrowth with rhizobia to form a nodule structure. The root nodule can utilize nitrogen element in soil, can fix nitrogen in air into ammonia, and provides nitrogen source for leguminous crops. The symbiotic nitrogen fixation effect of the soybean is not only provided for nitrogen nutrition of the soybean plant, but also can reduce the application of nitrogen fertilizer in agricultural production. According to statistics, soybeans can convey nitrogen elements to an agricultural ecological system by symbiotic nitrogen fixation to reach 1.6 millions of tons each year, and the method has important significance for developing environment-friendly agriculture and reducing nitrogen fertilizer application and environmental pollution.
In leguminous plants, asparagine is a product of nitrogen metabolism of the root nodule, and is involved in the transport process of nitrogen assimilation and nitrogen fixation of the root nodule. Many studies have found that root, root nodule, xylem sap, etc. of legumes are present in higher concentrations of asparagine. Under adverse stress conditions, the accumulation of free amino acids, particularly asparagine, occurs in root systems and root nodule sites of legumes, indicating that the accumulation of asparagine may be involved in regulating the nitrogen metabolism of nodules, but the specific mechanism is not yet known.
Phosphorus element is one of a great number of nutrient elements necessary for the growth and development of crops, and low effective phosphorus concentration in cultivated land soil has become a main limiting factor for crop yield and quality in the global scope. Most of the south areas in China are acid lands, and the problem of low phosphorus availability of the soil generally influences the yield and quality of crops in the south areas. During long-term evolution, plants evolved a range of physiological and molecular mechanisms that accommodate low-phosphorus stress (Vance et al, 2003; liang et al, 2010; wang et al, 2010;Oldroyd and Leyser,2020;Zhu et al, 2020). In legumes such as soybean, alfalfa, clover, low phosphorus stress causes the accumulation of asparagine at root and nodule sites, which has been studied to point out that this portion of the accumulated asparagine is involved in regulating soybean nitrogen metabolism, possibly in the adaptation of soybean and nodule to low phosphorus stress mechanisms (ALMEIDA ET al, 2000; hern hendez et al, 2009;Sulieman et al, 2010,2013;Xue et al, 2018). At present, although an asparagine synthetase family is cloned and reported in Arabidopsis, wheat and rape, the biological function of an asparagine synthetase similar gene participating in the synthesis of asparagine in root nodules and the regulation and control effect of the asparagine synthetase similar gene on root nodule nitrogen fixation are not clear. Therefore, in order to reveal the function of asparagine synthetase-like genes in nodules, intensive research into mechanisms of nitrogen metabolism and transport in leguminous crop nodules is necessary.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects and shortcomings of the problems, and provide a soybean asparagine synthetase analogous gene GmASL and application thereof.
A first object of the present invention is to provide the use of soybean asparagine synthetase-like gene GmASL.
It is a second object of the present invention to provide a product that promotes soybean growth and/or soybean growth under phosphorus stress.
A third object of the present invention is to provide a method for increasing nitrogen and phosphorus content of soybean plants or promoting root nodule growth of soybean plants.
A fourth object of the present invention is to provide a method for growing transgenic plants that are tolerant to low phosphorus plants.
The above object of the present invention is achieved by the following technical scheme:
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 gene affects the amino acid metabolic process of soybean nodule, and excessive expression of the gene can increase the asparagine content of soybean nodule and increase the nodule number of transgenic composite plants, which indicates that GmASL6 mediates asparagine accumulation or synthesis and finally affects 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 increase the nitrogen and phosphorus content of plants.
Thus, the following applications are within the scope of the present invention:
Use of a preparation for overexpressing GmASL gene shown in SEQ ID No.1 to promote soybean growth and/or promote soybean growth under low-phosphorus stress.
Application of preparation for over-expressing GmASL gene shown in SEQ ID NO.1 in preparation of product for promoting soybean growth and/or promoting soybean growth under low-phosphorus stress
The application of the preparation for over-expressing GmASL gene 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.
Use of a preparation for overexpressing GmASL gene shown in SEQ ID No.1 to reduce root nodule asparagine content in soybean plants.
The application of the preparation for over-expressing GmASL gene shown in SEQ ID NO. 1 in culturing transgenic plants with low phosphorus tolerance.
The application of the preparation for over-expressing GmASL gene shown in SEQ ID NO.1 in promoting the growth of soybean plant root nodule or increasing the number of soybean plant root nodule.
Preferably, the amino acid sequence of the coding protein of the gene GmASL is shown as SEQ ID NO. 2.
The invention provides a product for promoting soybean growth and/or promoting soybean growth under phosphorus stress, which contains a preparation for over-expressing GmASL genes shown in SEQ ID NO. 1.
The invention provides a method for improving nitrogen and phosphorus content of soybean plants or promoting root nodule growth of soybean plants, which adopts the preparation for over-expressing GmASL gene shown in SEQ ID NO. 1 to treat soybean.
The invention also provides a method for cultivating transgenic plants of low-phosphorus-resistant plants, which is obtained by introducing recombinant vectors which overexpress GmASL genes into soybeans.
Preferably, the expression vector can be constructed by using the existing plant expression vector to construct a recombinant expression vector containing GmASL genes.
More preferably, the plant expression vector comprises a binary agrobacterium vector or the like, such as pTF101s or other derived plant expression vectors.
The invention has the following beneficial effects:
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 nodule amino acid, and excessive expression of GmASL gene can reduce the asparagine content of soybean nodule, increase the nitrogen and phosphorus content of plants and increase the nodule number of transgenic composite plants; 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.
Drawings
Fig. 1: gmASL 6A graph of analysis results of root system and root nodule expression patterns under normal phosphorus and low phosphorus conditions (rhizobium is inoculated after soybean germination, water culture seedling is carried out under normal phosphorus (HP: 250 mu M KH 2PO4) and phosphorus deficiency (LP: 5 mu M KH 2PO4) conditions), wherein data in the graph are average value and standard error of 4 times of repetition, which indicate that the difference between normal phosphorus and phosphorus deficiency treatment is obvious (Student's t-test, P <0.05 less than or equal to 0.01), and which indicate that the difference between normal phosphorus and phosphorus deficiency treatment is extremely obvious (Student's t-test, P < 0.01)).
Fig. 2: gmASL6 promoter-driven GUS was shown in the results of histochemical localization of isolated hair roots (A: the results of GUS staining of isolated hair roots under normal phosphorus treatment conditions (HP: 250. Mu.M KH 2PO4), B: the root elongation zone under normal phosphorus treatment conditions, C: the root tip under normal phosphorus treatment conditions, D: the lateral root primordium under normal phosphorus treatment conditions, E: the results of GUS staining of isolated hair roots under low phosphorus treatment conditions (LP: 5. Mu.M KH 2PO4), F: the root elongation zone under low phosphorus treatment conditions, G: the root tip under low phosphorus treatment conditions, H: the lateral root primordium under low phosphorus treatment conditions, both scale A and E are 5mm, and the other scale is 1 mm).
Fig. 3: gmASL6 (GFP represents GFP channel, BF represents bright field, merge represents GFP overlap with BF.) the GFP fluorescence is observed by confocal laser microscopy (scale 20 μm).
Fig. 4: effect of over-expression GmASL on large ex vivo hair root growth results (A: over-expression GmASL transgenic ex vivo hair root and CK hair root phenotype; B: hair root dry weight; C: mao Genquan phosphorus content; D: hair root asparagine concentration. CK means ex vivo hair root transferred to OX empty, OX means over-expression GmASL hair root. Ex vivo hair root was grown for 25 days on normal phosphorus (HP: 250. Mu.M KH 2PO4) and phosphorus deficiency (LP: 5. Mu.M KH 2PO4) medium, respectively, and samples were collected).
Fig. 5: the effect of over-expression GmASL on the growth of soybean composite plants is shown in the graph (A: over-expression GmASL composite plant phenotype; B: composite plant dry weight; C: composite plant total nitrogen content; D: composite plant total phosphorus content; E: total root length; F: root nitrogen content; G: root phosphorus content. CK means a composite plant transformed with no-load control, and over-expression GmASL transgenic composite plant after root nodule bacteria, the composite plant grows for 31 days under the conditions of normal phosphorus (HP: 250. Mu.M KH 2PO4) and phosphorus deficiency (LP: 5. Mu.M KH 2PO4), respectively, and samples are collected.
Fig. 6: effect of over-expression GmASL on soybean composite plant nodule (A: over-expression GmASL6 composite plant nodule phenotype; B: nodule fresh weight; C: nodule number; D: nodule asparagine concentration; E: aspartic acid concentration; F: glutamine concentration. CK means conversion of empty control composite plants, OX means over-expression GmASL transgenic composite plants after nodule bacteria growth under normal phosphorus (HP: 250 μm KH 2PO4) and phosphorus deficiency (LP: 5 μm KH 2PO4) respectively for 31 days, nodule samples are collected, data in the graph are mean value and standard error of 8 biological replicates ". A". Ex "indicates significant difference between OX composite plants and CK composite plants (Student's t-test,0.01< P.ltoreq.0.05), and". A ". Extra" indicates extremely significant difference between OX composite plants (Student's t-test,0.001< P.01) root system is 1cm in the graph, scale of the root system map is 1 cm.
Fig. 7: FIG. GmASL shows the result of heterologous expression and enzyme activity analysis of recombinant GST-recombinant protein (A: gmASL-GST protein, western blot analysis, lane I is total protein after crushing of E.coli bacterial liquid containing GST no load, lane II is GST no load protein after purification by GST magnetic beads, lane III is total protein after crushing of E.coli bacterial liquid containing GmASL-GST, lane IV is GmASL-GST protein after purification by GST magnetic beads; B: gmASL-GST asparagine synthetase enzyme activity analysis, ". Times." showsthat the difference between GmAS-GST and GST no load asparagine synthetase enzyme activity is very remarkable (Student's t-test,0.001< P.ltoreq.0.01)).
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Nucleotide sequence shown in SEQ ID NO.1 ：ATGTTGGGAATTTTCAAGCAGAAGTTGGTTAATGCACCCAAGGAGCTGAACAGTCCAGCTTCTTTGAATTCATGCATTAAGCCTAAGCTAAGTCATGAAATCCTGAAGGATTTCATGTCCTGCAATTCCTCCAATGCTTTCTCAATGTGCTTTGGGAATGATGCTTTGCTAGCCTATTCCACTTCATACAAGCCCTCCATTAATCATAGGTTATTCTCTGGATTGGATAACATATACTGTGTTTTCCTGGGTGGCCTGCACAACCTTAGCATGCTCAACAAGCAGTATGGACTATCAAAGGGAACAAATGAGGCCATGTTTATCATTGAAGCATATCGTACACTTCGCGACAGGGGTCCATACCCTGCTGATCAAGTCCTCAAAGAACTTGAAGGCAGTTTTGCATTTGTGATCTATGACAACAAGGATGGAACAGTTTTTGTTGCATCTGGTTCTAATGGCCATATTGAGCTCTACTGGGGTATTGCAGGTGATGGTTCTGTTATAATTTCTGAAAATCTGGAGCTTATAAAAGCAAGTTGTGCTAAATCATTTGCACCATTTCCAGCTGGGTGTATGTTTCATAGTGAACACGGTCTCATGAACTTTGAGCATCCAACACAGAAGATGAAAGCAATGCCTCGGATTGACAGCGAGGGGGTTATGTGCGGGGCCAACTTCAATGTTGACTCTCAGTCAAAGATCCAGGTGATGCCACGTGTTGGAAGTGAAGCTAATTGGGCAACTTGGGGCTAA.
SEQ ID NO.2 shows the amino acid sequence ：MLGIFKQKLVNAPKELNSPASLNSCIKPKLSHEILKDFMSCNSSNAFSMCFGNDALLAYSTSYKPSINHRLFSGLDNIYCVFLGGLHNLSMLNKQYGLSKGTNEAMF IIEAYRTLRDRGPYPADQVLKELEGSFAFVIYDNKDGTVFVASGSNGHIELYWGIAGDGSVIISENLELIKASCAKSFAPFPAGCMFHSEHGLMNFEHPTQKMKAMPRIDSEGVMCGANFNVDSQSKIQVMPRVGSEANWATWG*.
EXAMPLE 1 construction of vectors
The cDNA nucleotide sequence of the gene cloned by the early experiment is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO. 2. According to the invention, through functional research on the gene, the asparagine synthetase-like gene of soybean is identified, which is named GmASL < 6 >, and further through research on soybean transgenic composite plants, systematic functional analysis is performed on GmASL < 6 > expressed by root nodules.
1. Overexpression vector: gmASL6-pTF101s vector
Using soybean cDNA as template, adopting GmASL-pTF 101s gene forward and reverse specificity primer F:5'-TTCGCGAGCTCGGTACCCGGGATGTTGGGAATTTTCAAGCAGA-3' and R:5'-CGACTCTAGAGGATCCCCGGGTTAGCCCCAAGTTGCCC-3' performing PCR reaction to amplify the full length of CDS sequence of GmASL gene.
The PCR reaction system is as follows: vazyme phata Buffer. Mu.L, 10mM dNTP 1. Mu.L, forward and reverse primers 2. Mu.L, soybean cDNA 3. Mu.L, vazyme phata Hi-Fi enzyme (Mejibio, china) 1. Mu.L, ddH 2 O16. Mu.L.
The PCR procedure was set as follows: 94℃for 3min and 30 repeated cycles (specifically comprising 94℃for 30 seconds, 60℃for 30 seconds, 72℃for 1 min) and 72℃for 10min, the amplified product was stored at 16 ℃.
The target band is recovered and purified after amplification. The pTF101s plasmid was single digested with SmaI restriction enzyme, and the single digested product was recovered and purified, and the digested product and the desired gene fragment were ligated by a one-step cloning ligation method. GmASL6-pTF101s vector was obtained for subsequent experiments.
2. Subcellular localization vector construction: gmASL6-pEGAD vector
Using soybean cDNA as template, using GmASL-pEGAD gene forward and reverse specific primer F:5'-CTAGCGCTACCGGTATGTTGGGAATTTTCAAGCAGAAG-3' and R:5'-TGGTGGCGACCGGTAGGCCCCAAGTTGCCCAATT-3' performing PCR reaction to amplify the full length of CDS sequence of GmASL gene.
The PCR amplification method and conditions were the same as above, and the target band was recovered and purified by gel electrophoresis of the amplified product using agarose gel kit. The pEGAD plasmid was single digested with an AgeI restriction enzyme, and the single digested product was recovered and purified, and the digested product and the desired gene fragment were ligated by a one-step cloning ligation method. The one-step cloning connection product is directly transferred into escherichia coli competence, and after sequencing and comparison, plasmids are extracted and transferred into agrobacterium GV3101, and the obtained product is stored at the temperature of minus 80 ℃ for standby. The 35S GmASL6-GFP vector was obtained for subsequent experiments.
3. GmASL construction of a recombinant protein heterologous expression vector of GmASL-GST: gmASL6-pGEX
Using soybean cDNA as template, using GmASL-pGEX gene forward and reverse specific primer F:5'-GGGGCCCCTGGGATCCATGTTGGCTATATTCCACAAAGC-3' and R:5'-GGGAATTCGGGGATCCCTAATGTTGGTCCCATTCCATC-3' performing PCR reaction to amplify the full length of CDS sequence of GmASL gene.
The PCR amplification method and conditions were the same as above, and the target band was recovered and purified by gel electrophoresis of the amplified product using agarose gel kit. The pGEX plasmid was single digested with BamHI restriction enzymes, and the single digested product was recovered and purified, and the digested product and the target gene fragment were ligated by a one-step cloning ligation method. The one-step cloning connection product is directly transferred into escherichia coli competence, and after sequencing and comparison, plasmids are extracted and transferred into escherichia coli BL21 for storage at-80 ℃ for standby. GmASL6 of the 6-pGEX vector was obtained for subsequent experiments.
4. Construction of a tissue localization analysis vector: gmASL6-pTF102 vector
Using soybean genomic DNA as a template, forward and reverse specific primers F of GmASL-pTF 102 gene: 5'-CTATGACATGATTACGAATTC CTCGGAAGTCCGAGTGTC-3' and R:5'-GACTGACCTACCCGGGGATCCCAATAATCAGCAATCCAAATAGCTG-3' PCR was performed to amplify a 2000bp promoter fragment of GmASL.
The PCR reaction system comprises: vazyme phata Buffer. Mu.L, 10mM dNTP 1. Mu.L, forward and reverse primers 2. Mu.L, soybean genomic DNA 3. Mu.L, vazyme phata high-fidelity enzyme (Mejibio, china) 1. Mu.L, ddH 2 O16. Mu.L.
The PCR procedure was set as follows: 94℃for 3min and 30 repeated cycles (specifically comprising 94℃for 30 seconds, 60℃for 30 seconds, 72℃for 1 min) and 72℃for 10min, the amplified product was stored at 16 ℃.
Recovering and purifying the amplified target fragment: after gel electrophoresis of the amplified product, the target band was recovered and purified using agarose gel kit (metagee, china). Ligation of promoter fragments and vectors: the pTF102 plasmid was double digested with EcoRI and BamHI, and the double digested product was recovered and purified, and the digested product and the promoter fragment were ligated by a one-step cloning ligation method. The reaction system comprises: assembly mix Buffer. Mu.L (Quan Shi gold, china), 1. Mu.L of vector double cleavage product and 4. Mu.L of promoter fragment. The one-step cloning connection method temperature system comprises the following steps: 15min at 50℃and 5 seconds at 4 ℃. The one-step cloning connection product is directly transferred into escherichia coli competence, and after sequencing and comparison, plasmids are extracted and transferred into agrobacterium K599 for standby. GmASL6-pTF102 vector was obtained for subsequent experiments.
Example 2GmASL expression profiling, tissue localization and subcellular localization
1. Tissue-specific analysis of GmASL expression
After the soybean seedlings are inoculated with rhizobium, water culture treatment is carried out on two groups, one group is normal phosphorus concentration treatment (HP: 250 mu M KH 2PO4) and the other group is low phosphorus treatment (LP: 5 mu M KH 2PO4), root systems and root nodule samples of different days are collected, RNA is reversely transcribed into cDNA by using a Promega company reverse transcription kit, and the expression mode of GmASL is further detected by quantitative PCR. The reaction system for quantitative PCR was 20. Mu.l, including 2X SYBR GREEN PCR MASTER mix 10. Mu.l, nucleic-FREE WATER. Mu.l, forward and reverse primers at a concentration of 10. Mu.mol/l each of 0.5. Mu.l, and 2. Mu.l of diluted cDNA template. The reaction procedure is that denaturation is carried out for 1 minute at 95 ℃; then, 40 cycles of 94℃for 15 seconds, 60℃for 15 seconds, and 72℃for 30 seconds were performed.
The soybean GmEF 1-alpha is adopted as an internal reference, and the primers for quantitative PCR detection of the gene expression level are respectively as follows:
The results are shown in FIG. 1, and it can be seen that GmASL6 was down-regulated by low phosphorus at day 31. At 31 days after rhizobia inoculation, gmASL.sup.6 was greatly down-regulated in the rhizomes, with a down-regulation amplitude of 89%.
2. GmASL6 histochemical localization analysis of 6
Seeds with consistent sizes and complete seed coats are selected, and after 13h of chlorine disinfection (100 mL sodium hypochlorite is added to 4.2mL hydrochloric acid to react to generate chlorine), soybean seed germs are germinated downwards in an MS culture medium, and are subjected to illumination culture for 5d at 25 ℃. Agrobacterium K599 containing GmASL-pTF 102 plasmid was inoculated into liquid YEP medium and cultured for 24h. The activated K599 bacteria liquid transferred into GmASL-pTF 102 plasmid is dipped by a scalpel, a plurality of wounds are cut horizontally at cotyledon and cotyledon node parts of germinated soybean, then the soybean is placed on sterilized filter paper which is wetted by water in advance, the soybean is sealed by a preservative film and cultivated for 4 days in a dark place, and then the soybean is transferred into an MS culture medium containing 200 mu g L -1 glufosinate (Sigma, USA) and 50mg L -1 timetin (Ding national organism, guangzhou) and cultivated for 15 days in a dark place.
Transgenic soybean in-vitro hairy root treatment: MS liquid solid culture media (50 mg L -1 Temeitin bacteria inhibition) of normal phosphorus treatment (HP: 250 mu M KH 2PO4) and low phosphorus treatment (LP: 5 mu M KH 2PO4) are respectively prepared, white tender in vitro hair roots with better growth vigor are selected and transferred to the MS culture medium for normal phosphorus treatment and low phosphorus treatment, and after light-shielding culture for 14d, the in vitro hair root samples are collected.
Taking out the isolated hair roots after normal phosphorus and low phosphorus treatment, washing the hair roots with secondary water for 2 times, respectively placing the hair roots in GUS staining solution for staining (the GUS staining solution contains 0.1M Na 2HPO4/NaH2PO4, 1mM X-Gluc, and the pH is 7.2), synchronously vacuumizing the hair roots for 30min, and transferring the hair roots to the dark place at 37 ℃ for staining overnight. The stained roots were transferred to 75% ethanol for storage, GUS staining was observed using a stereomicroscope (Leica, germany), and photographed.
As a result, as shown in FIG. 2, under normal phosphorus treatment conditions, GUS staining of GUS transgenic soybean in vitro hair roots was mainly distributed in vascular tissue, lateral root primordia, root tips and the like of the root elongation zone (FIGS. 2A,2B,2C, 2D). However, in the low-phosphorus treatment, GUS staining was significantly reduced in the vascular tissue, lateral root primordia, root tip, etc. in the root elongation zone, and in particular, little staining was observed in the root tip (FIGS. 2E,2F,2G, 2H).
3. GmASL6 subcellular localization analysis of 6
The GmASL-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 3-4 weeks of age. The GV3101 strain into which GmASL-pEGAD plasmid had been transferred was taken out from the ultra-low temperature refrigerator and inoculated into YEP medium, and shake-cultured at 28℃for 24 hours. Centrifuge at 5000rpm for 10min, discard supernatant, then re-suspend GV3101 with an infiltration solution (infiltration solution: 10mM MgCl2, 100. Mu.M acetosyringone, 10mM MES), adjust the OD of the infiltration solution to 0.5 and incubate at 22℃for 4h in the dark. GmASL6-pEGAD dip was injected into tobacco lamina from the back of the lamina using a syringe. After 2 days, the tobacco leaves were subjected to GFP fluorescence observation under a laser confocal microscope, and photographed.
As a result, as shown in FIG. 3, it can be seen that GmASL6:GFP protein has green fluorescent signals on the cell membrane, cytoplasm and cell membrane, and that the green fluorescent signals of 3835:GFP protein are mainly present on the cell membrane and cell nucleus, revealing GmASL as a cell membrane and cell nucleus localization protein.
Example 3 acquisition and analysis of transgenic Material
1. Obtaining transgenic Soy roots
Soybean seeds with consistent size and complete seed coats are selected, and after 13h of chlorine disinfection (100 mL sodium hypochlorite is added into 4.2mL hydrochloric acid to react to generate chlorine), soybean seeds with germs are germinated downwards in an MS culture medium, and are cultivated for 5d under illumination at 25 ℃. The activated agrobacterium K599 bacteria liquid transferred into the target carrier is dipped by a scalpel, a plurality of wounds are cut horizontally at cotyledon and cotyledon node parts of germinated soybeans, then the soybeans are placed on sterilized filter paper which is wetted by water in advance, the sterilized filter paper is sealed by a preservative film, after 4 days of light-proof culture, the soybean is transferred into an MS culture medium containing 200 mu g L -1 glufosinate (Sigma, U.S.) and 50mg L-1 timentin (Ding national biology, guangzhou) and is cultivated for 15 days in light-proof.
2. Obtaining of Soybean composite plants
Selecting soybean seeds with uniform and full size, sterilizing with 10% H 2O2 for 10min, and cleaning with sterile water for 5 times. Sowing soybean seeds on the surface of quartz sand, adding secondary water, covering quartz sand with the thickness of 2cm on the seeds, and culturing for 5 days under the illumination condition. Agrobacterium K599, which had been transformed into GmASL-pTF 101s, was spread on the surface of solid YEP medium and incubated at 30℃for 24h, with no load as corresponding no load control. The activated K599 is dipped on the needle head of the injector, the hypocotyl of the soybean seedling is perforated, then the wound part is covered with K599 thalli, the seedling is continuously cultivated by covering moist sand, the seedling is covered with a layer of preservative film, and water is sprayed for a plurality of times every day to keep the wound part moist. And growing hair roots at the wound position for about 15 days, taking a part of samples, and detecting to leave only positive hair roots. And after the positive result is Mao Genchang to about 10cm, cutting off main roots of the soybean plants, and inoculating rhizobium. After rhizobia is planted, the composite plants are transferred into sand culture and 200mL of normal phosphorus (HP: 250. Mu.M KH 2PO4) and low phosphorus (LP: 5. Mu.M KH 2PO4) nutrient solution are applied once a week.
3. Detection of transgenic Material
(1) Detection of transgenic soybean in-vitro hairy root and hypocotyl composite plant transgenic hairy root
The total RNA of the obtained transgenic hairy root is extracted, reversely transcribed into cDNA, and the expression quantity of GmASL is detected by quantitative PCR. The soybean EF1-a was used as a reference gene, and the primer and the method were the same as in example 2, and the relative expression level was the ratio of the expression level of the target gene GmASL to the expression level of the housekeeping gene.
Example 4GmASL functional analysis
1. Effect of over-expression GmASL on soybean transgenic in vitro hairy root growth
After the transgenes and the empty control hair roots grow to 15d, the isolated hair roots with good growth vigor, similar hair root morphology and fresh weight of about 0.1g are selected, transferred to an MS liquid solid culture medium (containing 50mg L -1 timentin bacteria inhibition) with normal phosphorus treatment (HP: 250 mu M KH 2PO4) and low phosphorus treatment (LP: 5 mu M KH 2PO4), and continuously cultivated for 14d in a dark place, and then a hair root sample is collected. The asparagine concentration, the dry weight of the hair root and the total phosphorus content were measured separately. Each treatment of this experiment was set up with 6 independent biological replicates and no load controls.
The results are shown in fig. 4, over-expression GmASL6 promotes the growth of soybean in vitro hair roots and significantly increases their phosphorus content (fig. 4A). Under normal phosphorus treatment conditions, the dry weight of the transgenic in-vitro hair roots is increased by 58.7% and the phosphorus content is increased by 45.8% compared with CK. Under low phosphorus treatment conditions, the dry weight of transgenic ex vivo hair roots was increased by 110% compared to CK (fig. 4B), while their phosphorus content was increased by 31% (fig. 4C). Moreover, under normal and low phosphorus conditions, the asparagine concentration of transgenic hair roots increased by 30.8% and 25.8%, respectively (fig. 4D), revealing that excess GmASL6 enhanced the synthesis of asparagine in isolated hair roots.
2. Effect of over-expression GmASL on growth of transgenic composite plants of soybean
And (3) obtaining a soybean transgenic composite plant according to the hypocotyl injection method, and then inoculating rhizobium. After rhizobia is planted, the composite plants are transferred into sand culture and 200mL of normal phosphorus (HP: 250. Mu.M KH 2PO4) and low phosphorus (LP: 5. Mu.M KH 2PO4) nutrient solution are applied once a week.
As shown in FIG. 5, transgenic in vitro Mao Genliang was added after the expression of GmASL (FIG. 5A), and the expression of GmASL increased the dry weight of the plants and the nitrogen and phosphorus content of the plants (FIGS. 5B-D). And promoting root development specifically shows an increase in root length (fig. 5E), and an increase in root nitrogen content (fig. 5F). The result of measuring the content of asparagine in the root system shows (figure 5G), the over-expression GmASL is carried out, and the content of asparagine in the root system is reduced.
The effect of over-expression GmASL on soybean composite plant nodule as shown in fig. 6, over-expression GmASL increased the number of nodules and the weight of nodules (fig. 6A), increased the fresh weight of nodules, and significantly increased the number of nodules compared to CK under low phosphorus conditions (fig. 6b,6 c). Meanwhile, after GmASL is over-expressed, the amino acid metabolic process of the transgenic nodule is regulated, and different amino acids in the nodule are obviously changed: asparagine, aspartic acid, glutamine concentrations (fig. 6d,6e,6 f), by measuring the root nodule asparagine content, the results show that over-expression GmASL reduces the plant root nodule asparagine content.
Example 5GmASL enzyme Activity assay
1. GmASL6 protein purification
Inoculating BL21 E.coli which has been transferred into GmASL-pGEX plasmid in example 1 into liquid LB medium, shake culturing at 28 ℃, adding IPTG to a final concentration of 1mM when the OD of a spectrophotometer with a wavelength of 600 is measured to be 0.5-0.6, shake-culturing at 28 ℃ for 4 hours, adding 3mL of PMSF and DTT with 100mM concentration respectively, shaking uniformly, split charging into 50mL centrifuge tubes, centrifuging at 5000rpm for 10 minutes at 4 ℃, removing supernatant, adding 50mM PBS buffer for resuspension, crushing at 35pis under high pressure, obtaining clear transparent bacterial liquid, centrifuging at 5000rpm for 10 minutes, taking supernatant into a new 50mL centrifuge tube, adding 3mL GSH magnetic beads, sealing, placing on ice, and combining in a cold storage for 5 hours.
After the combination of the protein supernatant and the magnetic beads is finished, the pre-cooled PBS buffer solution is used for washing off the impurity protein, when the protein content of the washed buffer solution is measured to be 0, 3mL of pre-cooled protein eluent is added for eluting the recombinant protein on the magnetic beads, the protein concentration is measured by a Coomassie brilliant blue method, an appropriate amount of SDS polyacrylamide gel electrophoresis and Western Blot are taken, and the enzyme solution GmASL is verified to be free of the impurity protein (figure 7A), so that the subsequent test can be carried out.
2. Enzyme activity assay of GmASL recombinant proteins
The purified GmASL-GST and GST protein are added into coomassie brilliant blue G-250, the protein concentration is measured, GST protein is used as a blank control for enzyme activity analysis, and GmAS is used as a positive control. According to the following reaction system: 100mM Tris-HCl (pH 8.0), was reacted in a water bath at 100mM NaCl,5mM ATP,10mM MgCl2, 10mM Asp,10mM Gln,37 ℃for 15 minutes, and after stopping the reaction by adding 500. Mu.L of ethanol, the asparagine concentration was measured.
As a result, as shown in FIG. 7B, the asparagine synthetase enzyme activity of GmASL-GST protein was not significantly changed from GST at 37℃and pH 8.0 using aspartic acid and glutamine as substrates, which indicates that GmASL-GST protein did not have asparagine synthetase activity.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments 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.
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