CN116200404A - Soybean asparagine synthetase analogous gene and application thereof - Google Patents
Soybean asparagine synthetase analogous gene and application thereof Download PDFInfo
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- CN116200404A CN116200404A CN202310376006.8A CN202310376006A CN116200404A CN 116200404 A CN116200404 A CN 116200404A CN 202310376006 A CN202310376006 A CN 202310376006A CN 116200404 A CN116200404 A CN 116200404A
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Abstract
The invention discloses a soybean asparagine synthetase-like gene and application thereof. The research of the invention shows that the GmASL6 gene shown in SEQ ID NO.1 is a gene which is inhibited by low phosphorus and expressed, the GmASL6 influences the amino acid metabolic process of soybean root nodule, and the over-expression of the GmASL6 gene can promote the growth of soybean root system, increase the nitrogen and phosphorus content of plants and the root nodule number of transgenic composite plants, and reduce the aspartic acid content; gmASL6 was shown to mediate asparagine accumulation or synthesis, ultimately affecting root nodule growth. Meanwhile, the GmASL6 gene and protein can regulate and control the amino acid metabolic process of the transgenic root nodule containing the GmASL6 gene, 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 GmASL6 gene in the synthesis of asparagine in root nodules and the regulation and control effects of the GmASL6 gene 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 is a part of the study that has been pointed out to be involved in regulating soybean nitrogen metabolism, possibly in the adaptation of soybean and nodule to low phosphorus stress mechanisms (Almeida et al, 2000; herna ndez 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 invention aims to overcome the defects and the shortcomings of the problems and provide a soybean asparagine synthetase analogous gene GmASL6 and application thereof.
A first object of the present invention is to provide the use of the soybean asparagine synthetase analogous gene GmASL 6.
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 the GmASL6 gene shown in SEQ ID NO.1 is a gene which is inhibited by low phosphorus and expressed, the GmASL6 gene affects the amino acid metabolic process of soybean nodules, and the excessive expression of the gene can increase the content of soybean nodule asparagine and increase the nodule number of a transgenic composite plant, which indicates that GmASL6 mediates the accumulation or synthesis of asparagine and finally affects the nodule growth. Meanwhile, the GmASL6 gene and protein can regulate the amino acid metabolic process of the transgenic root nodule containing the GmASL6 gene, promote plant growth and increase the nitrogen and phosphorus content of plants.
Thus, the following applications are within the scope of the present invention:
the application of a preparation for over-expressing the GmASL6 gene shown in SEQ ID NO.1 in promoting the growth of soybeans and/or promoting the growth of soybeans under low-phosphorus stress.
Application of preparation for overexpressing GmASL6 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 the GmASL6 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.
The application of the preparation for over-expressing the GmASL6 gene shown in SEQ ID NO.1 in reducing the root nodule asparagine content of soybean plants.
The application of the preparation for over-expressing the GmASL6 gene shown in SEQ ID NO.1 in cultivating transgenic plants of low-phosphorus-resistant plants.
The application of the preparation for over-expressing 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 encoding protein of the gene GmASL6 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 GmASL6 gene 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 GmASL6 gene shown in SEQ ID NO.1 to treat soybean.
The invention also provides a method for cultivating the transgenic plant of the low-phosphorus-resistant plant, which is obtained by introducing the recombinant vector which overexpresses the GmASL6 gene into soybean.
Preferably, the expression vector can be used for constructing a recombinant expression vector containing the GmASL6 gene by using the existing plant expression vector.
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 the GmASL6 gene shown in SEQ ID NO.1 is a gene which is inhibited by low phosphorus and expressed, the GmASL6 influences the amino acid metabolic process of soybean root nodule, and the over-expression of the GmASL6 gene can reduce the asparagine content of the soybean root nodule, increase the nitrogen and phosphorus content of plants and increase the root nodule number of transgenic composite plants; gmASL6 was shown to mediate asparagine accumulation or synthesis, ultimately affecting root nodule growth. Meanwhile, the GmASL6 gene and protein can regulate and control the amino acid metabolic process of the transgenic root nodule containing the GmASL6 gene, 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 GmASL6 gene in the synthesis of asparagine in root nodules and the regulation and control effects of the GmASL6 gene on nitrogen fixation of the root nodules.
Drawings
Fig. 1: analysis of root and nodule expression patterns of GmASL6 under normal and Low phosphorus conditions (after soybean germination, rhizobium was inoculated, and after normal phosphorus (HP: 250. Mu.M KH) 2 PO 4 ) And phosphorus deficiency (LP: 5 mu M KH 2 PO 4 ) Culturing seedlings by water culture under the condition; the data in the figure are mean and standard error of 4 replicates. ". Times". Shows that the difference between normal phosphorus and phosphorus deficiency treatment is significant (Student's t-test, 0.01.ltoreq.P)<0.05 "X" indicates that the difference between normal phosphorus and phosphorus deficient treatments is extremely significant (Student's t-test, P<0.01))。
Fig. 2: gmASL6 promoter driveGUS histochemical localization results in vitro at hairy roots (A: normal phosphorus treatment conditions (HP: 250. Mu.M KH) 2 PO 4 ) An in-vitro hair root GUS dyeing result; b: root elongation zone under normal phosphorus treatment conditions; c: root tip under normal phosphorus conditions; d: lateral root primordia under normal phosphorus treatment conditions; e: under low phosphorus treatment conditions (LP: 5. Mu.M KH) 2 PO 4 ) An in-vitro hair root GUS dyeing result; f: root elongation zone under low phosphorus treatment conditions; g: root tip under low phosphorus treatment conditions; h: lateral root primordia under low phosphorus treatment conditions. The A and E scales are 5mm; other scales are 1 mm).
Fig. 3: subcellular localization results of GmASL6 (GFP for GFP channel, BF for bright field, merge for GFP overlap with BF. GFP fluorescence was observed by laser confocal microscopy. Scale 20 microns in the figure).
Fig. 4: effect of overexpression GmASL6 on large ex vivo hair root growth results graph (A: overexpression of GmASL6 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 overexpression of GmASL 6. Ex vivo hair root is expressed in normal phosphorus (HP: 250. Mu.M KH respectively) 2 PO 4 ) And phosphorus deficiency (LP: 5 mu M KH 2 PO 4 ) After 25 days growth on medium, samples were harvested).
Fig. 5: the result of the effect of over-expression GmASL6 on the growth of soybean composite plant is shown (A: over-expression GmASL6 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 composite plant of transformation no-load control, over-expression GmASL6 transgenic composite plant after root nodule bacteria, composite plant is treated with normal phosphorus (HP: 250. Mu.M KH 2 PO 4 ) And phosphorus deficiency (LP: 5 mu M KH 2 PO 4 ) After 31 days of growth under the conditions, samples were collected. The data in the figures are the mean and standard error of 8 biological replicates. ". Times" indicate significant differences between OX and CK composite plants (Student's t-test, 0.01)<P.ltoreq.0.05), "" indicates that the difference between OX composite plants and CK composite plants was very significant (Student's t-test, 0.001)<P.ltoreq.0.01). The scale of the composite plants and root systems in the figure is 10 cm).
Fig. 6: effect of over-expression GmASL6 on soybean composite plant nodule (A: excessive 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 composite plant transformed with no-load control, OX means excessive GmASL6 transgenic composite plant. After root nodule bacteria is planted, composite plants are treated with normal phosphorus (HP: 250. Mu.M KH 2 PO 4 ) And phosphorus deficiency (LP: 5 mu M KH 2 PO 4 ) After 31 days of growth under conditions, root nodule samples were harvested. The data in the figures are the mean and standard error of 8 biological replicates. ". Times" indicate significant differences between OX and CK composite plants (Student's t-test, 0.01)<P.ltoreq.0.05), "" indicates that the difference between OX composite plants and CK composite plants was very significant (Student's t-test, 0.001)<P.ltoreq.0.01). In the figure, the scale of the root system partial enlarged graph is 1cm, and the scale of the root nodule is 1 cm).
Fig. 7: the result of heterologous expression and enzyme activity analysis of GmASL6-GST recombinant protein is shown in the graph (A: the result of western blot analysis of GmASL6-GST protein, lane I is total protein obtained by crushing a GST-containing empty E.coli bacterial liquid, lane II is GST empty protein obtained by purifying by using GST magnetic beads, lane III is total protein obtained by crushing a GmASL 6-GST-containing E.coli bacterial liquid, lane IV is GmASL6-GST protein obtained by purifying by using GST magnetic beads; B: gmASL6-GST asparagine synthetase enzyme activity analysis, ". Times." indicates that the difference of the enzyme activities of GmAS6-GST and GST empty inter-asparagine synthetase is very remarkable (Student's t-test,0.001< P is less than or equal to 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.
The nucleotide sequence shown in SEQ ID NO. 1: ATGTTGGGAATTTTCAAGCAGAAGTTGGTTAATGCACCCAAGGAGCTGAACAGTCCAGCTTCTTTGAATTCATGCATTAAGCCTAAGCTAAGTCATGAAATCCTGAAGGATTTCATGTCCTGCAATTCCTCCAATGCTTTCTCAATGTGCTTTGGGAATGATGCTTTGCTAGCCTATTCCACTTCATACAAGCCCTCCATTAATCATAGGTTATTCTCTGGATTGGATAACATATACTGTGTTTTCCTGGGTGGCCTGCACAACCTTAGCATGCTCAACAAGCAGTATGGACTATCAAAGGGAACAAATGAGGCCATGTTTATCATTGAAGCATATCGTACACTTCGCGACAGGGGTCCATACCCTGCTGATCAAGTCCTCAAAGAACTTGAAGGCAGTTTTGCATTTGTGATCTATGACAACAAGGATGGAACAGTTTTTGTTGCATCTGGTTCTAATGGCCATATTGAGCTCTACTGGGGTATTGCAGGTGATGGTTCTGTTATAATTTCTGAAAATCTGGAGCTTATAAAAGCAAGTTGTGCTAAATCATTTGCACCATTTCCAGCTGGGTGTATGTTTCATAGTGAACACGGTCTCATGAACTTTGAGCATCCAACACAGAAGATGAAAGCAATGCCTCGGATTGACAGCGAGGGGGTTATGTGCGGGGCCAACTTCAATGTTGACTCTCAGTCAAAGATCCAGGTGATGCCACGTGTTGGAAGTGAAGCTAATTGGGCAACTTGGGGCTAA.
The amino acid sequence shown in SEQ ID NO. 2: 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 analogous gene of soybean is identified, and is named as GmASL6, and further through research on soybean transgenic composite plants, systematic functional analysis is performed on GmASL6 expressed by root nodules.
1. Overexpression vector: gmASL6-pTF101s vector
The soybean cDNA is used as a template, and a forward and reverse specific primer F of GmASL6-pTF101s gene is adopted: 5'-TTCGCGAGCTCGGTACCCGGGATGTTGGGAATTTTCAAGCAGA-3' and R:5'-CGACTCTAGAGGATCCCCGGGTTAGCCCCAAGTTGCCC-3', PCR was performed to amplify the full length of CDS sequence of GmASL6 gene.
The PCR reaction system is as follows: vazyme phata Buffer 25. Mu.L, 10mM dNTP 1. Mu.L, forward and reverse primers 2. Mu.L, soybean cDNA 3. Mu.L, vazyme phasa high-fidelity enzyme (Mejibio, china) 1. Mu.L, ddH 2 O 16μL。
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. The GmASL6-pTF101s vector was obtained for the subsequent experiments.
2. Subcellular localization vector construction: gmASL6-pEGAD vector
Using soybean cDNA as template, using GmASL6-pEGAD gene forward and backward specific primer F:5'-CTAGCGCTACCGGTATGTTGGGAATTTTCAAGCAGAAG-3' and R:5'-TGGTGGCGACCGGTAGGCCCCAAGTTGCCCAATT-3', PCR was performed to amplify the full length of CDS sequence of GmASL6 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. Construction of GmASL6-GST recombinant protein heterologous expression vector: gmASL6-pGEX
Using soybean cDNA as template, using GmASL6-pGEX gene forward and reverse specific primer F:5'-GGGGCCCCTGGGATCCATGTTGGCTATATTCCACAAAGC-3' and R:5'-GGGAATTCGGGGATCCCTAATGTTGGTCCCATTCCATC-3', PCR was performed to amplify the full length of CDS sequence of GmASL6 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. The GmASL6-pGEX vector was obtained for the subsequent experiments.
4. Construction of a tissue localization analysis vector: gmASL6-pTF102 vector
The soybean genome DNA is used as a template, and a forward and reverse specific primer F of the GmASL6-pTF102 gene is used: 5'-CTATGACATGATTACGAATTC CTCGGAAGTCCGAGTGTC-3' and R:5'-GACTGACCTACCCGGGGATCCCAATAATCAGCAATCCAAATAGCTG-3', a PCR reaction was performed to amplify a 2000bp promoter fragment of GmASL 6.
The PCR reaction system comprises: vazyme phata Buffer 25. Mu.L, 10mM dNTP 1. Mu.L, forward and reverse primers 2. Mu.L, soybean genomic DNA 3. Mu.L, vazyme phasa high-fidelity enzyme (Megaku, china) 1. Mu.L, ddH 2 O 16μL。
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 5. Mu.L (Quan Shi gold, china), 1. Mu.L of vector double cleavage product, 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. The GmASL6-pTF102 vector was obtained for subsequent experiments.
Example 2GmASL6 expression Pattern analysis, tissue localization and subcellular localization
1. Tissue-specific analysis of GmASL6 expression
After soybean seedlings are inoculated with rhizobia, water planting treatment is carried out on the soybean seedlings in two groups, wherein one group has normal phosphorus concentrationTreatment (HP: 250. Mu.M KH) 2 PO 4 ) Another group is low-phosphorous treatment (LP: 5 mu M KH 2 PO 4 ) Root systems and root nodule samples of different days were harvested, the RNA was reverse transcribed into cDNA using Promega reverse transcription kit, and the expression pattern of GmASL6 was further detected by quantitative PCR. The reaction system for quantitative PCR was 20. Mu.l, including 2X SYBR Green PCR master mix. Mu.l, nucleic-free water 7. Mu.l, forward and reverse primers at a concentration of 10. Mu.m, each 0.5. Mu.l, and diluted cDNA template 2. Mu.l. 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:
as a result, as shown in FIG. 1, it can be seen that GmASL6 was down-regulated by low phosphorus at 31 days. 31 days after rhizobium inoculation, gmASL6 is greatly down-regulated in the rhizobium, and the down-regulating amplitude reaches 89%.
2. Histochemical localization analysis of GmASL6
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 the GmASL6-pTF102 plasmid was inoculated into liquid YEP medium and cultured for 24h. Dipping activated K599 bacteria liquid transferred into GmASL6-pTF102 plasmid with a scalpel, cutting multiple wounds on cotyledon and cotyledon node parts of germinated soybean, placing on sterilized filter paper wetted with water in advance, sealing with fresh-keeping film, culturing for 4 days in dark place, transferring bean paste into a liquid containing 200 mu g L -1 Glufosinate (Sigma, USA) and 50mg L -1 The cells were cultured in MS medium of timentin (Ding Guo Bio, guangzhou) for 15 days in the absence of light.
Transgenic soybean in-vitro hairy root treatment: the normal phosphorus treatments (HP: 250. Mu.M KH) were formulated separately 2 PO 4 ) Low phosphorus treatment (LP: 5 mu M KH 2 PO 4 ) MS liquid solid medium (all containing 50mg L) -1 And (3) bacteriostasis of timentin), selecting white and tender in-vitro hair roots with better growth vigor, transferring the in-vitro hair roots into an MS (MS) culture medium for normal phosphorus and low phosphorus treatment, and collecting in-vitro hair root samples after light-shielding culture for 14 d.
Taking out the isolated hair roots after normal phosphorus and low phosphorus treatment, washing with secondary water for 2 times, and respectively placing into GUS staining solution (GUS staining solution contains 0.1M Na) 2 HPO 4 /NaH 2 PO 4 1mM X-Gluc, pH 7.2), vacuum was applied simultaneously for 30min, and the mixture was transferred to 37℃for overnight dyeing in the absence of light. The stained roots were transferred to 75% ethanol for storage, GUS staining was observed using a stereomicroscope (Leica, germany), and photographed.
The results are shown in FIG. 2, pro under normal phosphorus treatment conditions GmASL6 GUS staining of GUS transgenic soybean in vitro hair roots was mainly distributed in vascular tissue, lateral root primordia, root tips, etc. of 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. 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 3-4 weeks of age. The GV3101 strain into which the GmASL6-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. The GmASL6-pEGAD dye liquor 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 shown in FIG. 3, it can be seen that the green fluorescent signal of GmASL6: GFP protein was mainly present in the cell membrane and nucleus, compared with 35S: GFP protein, which revealed that GmASL6 was a cell membrane and a nuclear 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 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 after 4 days of light-proof culture, the soybean is transferred into a container containing 200 mu g L -1 Glufosinate (Sigma, usa) and 50mg L-1 timentin (tripod organisms, guangzhou) were cultured in MS medium for 15 days in the dark.
2. Obtaining of Soybean composite plants
Selecting uniform and full soybean seeds, and using 10% H 2 O 2 After sterilization for 10min, the mixture was washed with sterile water 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 GmASL6-pTF101s, was spread on the surface of the 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 value is Mao Genchang to about 10cm, cutting off main roots of the soybean plants, and inoculating rhizobium. After rhizobium inoculation, the composite plants are transplanted into sand culture, and normal phosphorus (HP: 250 mu M KH) is poured once a week 2 PO 4 ) And low phosphorus (LP: 5 mu M KH 2 PO 4 ) Camping200mL of nutrient solution.
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 GmASL6 is detected by quantitative PCR. The soybean EF1-a is used as a reference gene, the primer and the method are the same as in example 2, and the relative expression amount is the ratio of the expression amount of the target gene GmASL6 to the expression amount of the housekeeping gene.
Example 4GmASL6 functional analysis
1. Influence of overexpression of GmASL6 on soybean transgenic in-vitro hairy root growth
When the transgenic and no-load control hair root grows to 15d, selecting in vitro hair root with good growth vigor, similar hair root morphology and fresh weight of about 0.1g, and transferring to normal phosphorus treatment (HP: 250 μm KH) 2 PO 4 ) Low phosphorus treatment (LP: 5 mu M KH 2 PO 4 ) On MS liquid solid medium (all containing 50mg L) -1 And (3) bacteriostasis of the timentin), and collecting a hair root sample after continuing to culture for 14 days in a dark place. 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.
As shown in fig. 4, over-expression of GmASL6 promoted growth of soybean in vitro hair roots and significantly increased 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 excessive GmASL6 enhanced the synthesis of asparagine in isolated hair roots.
2. Influence of overexpression of GmASL6 on growth of soybean transgenic composite plants
And (3) obtaining a soybean transgenic composite plant according to the hypocotyl injection method, and then inoculating rhizobium. Seed root of knotAfter the tumor cells, the composite plants were transplanted into sand cultures and were watered with normal phosphorus (HP: 250. Mu.M KH) once a week 2 PO 4 ) And low phosphorus (LP: 5 mu M KH 2 PO 4 ) 200mL of nutrient solution.
As shown in FIG. 5, the transgenic in vitro Mao Genliang (FIG. 5A) is increased after the expression of GmASL6 is over-expressed, and the plant dry weight and the nitrogen and phosphorus content of the plant are improved after the expression of GmASL6 is over-expressed (FIG. 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) that the content of asparagine in the root system is reduced by over-expressing GmASL 6.
The effect of over-expressed GmASL6 on soybean composite plant nodules, as shown in fig. 6, over-expressed GmASL6 increased the number of nodules and the weight of the nodules (fig. 6A), increased the fresh weight of the nodules, and significantly increased the number of nodules compared with CK under low phosphorus conditions (fig. 6b,6 c). Meanwhile, after the GmASL6 is overexpressed, 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 concentration (fig. 6d,6e,6 f), by measuring the root nodule asparagine content, the results show that over-expression of GmASL6 reduced the plant root nodule asparagine content.
Example 5GmASL6 enzyme Activity assay
1. GmASL6 protein purification
Inoculating BL21 E.coli into which GmASL6-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 a concentration of 100mM respectively, shaking uniformly, split charging into 50mL centrifuge tubes, centrifuging at 5000rpm for 10 minutes at 4 ℃, removing the supernatant, adding 50mM PBS buffer for resuspension, crushing at 35pis high pressure, obtaining clear transparent bacterial liquid, centrifuging at 5000rpm for 10 minutes, taking the supernatant into a new 50mL shake-table centrifuge tube, adding 3mL of GSH magnetic beads, sealing, placing on ice, and combining in a refrigeration house for 5 hours.
After the protein supernatant and the magnetic beads are combined, the pre-cooled PBS buffer solution is used for washing off the impurity proteins, 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 proteins 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 condition that the GmASL6 enzyme solution is free of the impurity proteins is verified (figure 7A), so that the subsequent test can be carried out.
2. Enzyme activity determination of GmASL6 recombinant protein
The purified GmASL6-GST and GST protein are added into coomassie brilliant blue G-250, the protein concentration is measured, the GST protein is used as a blank control for enzyme activity analysis, and GmAS5 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 the GmASL6-GST protein was not significantly changed from that of GST at 37℃and pH 8.0 using aspartic acid and glutamine as substrates, which indicates that the GmASL6-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 (10)
1. The application of a preparation for over-expressing the GmASL6 gene shown in SEQ ID NO.1 in promoting the growth of soybeans and/or promoting the growth of soybeans under low-phosphorus stress.
2. The application of a preparation for over-expressing 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.
3. The application of the preparation for over-expressing the GmASL6 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.
4. The application of the preparation for over-expressing the GmASL6 gene shown in SEQ ID NO.1 in reducing the root nodule asparagine content of soybean plants.
5. The application of the preparation for over-expressing the GmASL6 gene shown in SEQ ID NO.1 in cultivating transgenic plants of low-phosphorus-resistant plants.
6. The application of the preparation for over-expressing 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.
7. The use according to any one of claims 1 to 6, wherein the amino acid sequence of the protein encoded by the GmASL6 gene is shown in SEQ ID No. 2.
8. A product for promoting soybean growth and/or promoting soybean growth under phosphorus stress is characterized by comprising a preparation for over-expressing GmASL6 gene shown in SEQ ID NO. 1.
9. A method for increasing nitrogen and phosphorus content of soybean plants or promoting root nodule growth of soybean plants, comprising treating soybean with the formulation of claim 8.
10. 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 GmASL6 genes into soybeans.
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