CN102876641B - Soybean purple acid phosphatase GmPAP4, and coding gene and application thereof - Google Patents
Soybean purple acid phosphatase GmPAP4, and coding gene and application thereof Download PDFInfo
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Abstract
The invention relates to soybean purple acid phosphatase GmPAP4 and a coding gene and application thereof. The amino acid sequence of the soybean purple acid phosphatase is shown as SEQ ID NO.2 or the amino acid sequence which is obtained by replacing, deleting or adding one or more amino acids and has the same function and is derived from the SEQ ID NO. 2. The soybean purple acid phosphatase gene GmPAP4 has a nucleotide sequence shown in SEQ ID No. 1. The invention discovers the soybean purple acid phosphatase GmPAP4 and the coding gene thereof for the first time, provides a new candidate gene for improving the utilization rate of phytate phosphorus of plants, and can further improve the utilization rate of phytate phosphorus of plants by using a genetic engineering means.
Description
Technical field
The present invention relates to the genetically engineered field, particularly relate to soybean purple acid phosphatase GmPAP4 and encoding gene thereof and application.
Background technology
Phosphorus in soil element is most to be existed with the organic form, and over half be phytic acid and its esters, can not directly be absorbed by plant.Acid phosphatase is that a class can be decomposed the enzyme general name of utilizing soil organic phosphorus, release inorganic phosphorus to absorb for plant, and this enzyme has the various biological function in plant growth and development process, wherein with involved in plant phosphorus element, absorbs the most important.Therefore, excavate and utilize acid phosphatase gene, for decomposing organic phosphorus in soil, improve that the plant phosphorus element absorbs efficiency, the problems such as the current soil available phosphorus is under-supply, the plain shortage of plant phosphorus that solve have important theory and using value.
Purple acid phosphatase (Purple acid phosphatase, PAP) is a kind of acid phosphatase enzyme extensively be present in the animal and plant body.According to its molecular weight of albumen size, purple acid phosphatase can be divided into two subfamilies, the purple acid phosphatase of the lower molecular weight of encoding (about 35kD) and coding high molecular (about 55kD).Wherein, plant lower molecular weight purple acid phosphatase is more similar to the Phosphoric acid esterase in animal body, and the high molecular purple acid phosphatase is occupied larger proportion in plant.Purple acid phosphatase generally have 5 conservative motifs and 7 high conservatives amino-acid residue (
dxG/G
dxX
y/ G
nh(D/E)/VXX
h/ G
hx
h: the underscore font represents conservative amino acid residues) and the double-core center of a metal ion.PAP in animal body is generally by Fe
3+-Fe
2+form the double-core metal center, and the PAP in plant materials is generally by Fe
3+-Zn
2+or Fe
3+-Mn
2+form the double-core metal center.
About clone and the applied research of purple acid phosphatase gene, currently reported.Li etc. separate and obtain 29 PAPs genes from Arabidopis thaliana, and the sxemiquantitative pcr analysis finds that 7 PAPs genes can be at abduction delivering under low-phosphorus stress; Xiao etc. have cloned the MtPAP1 gene in the clover, through arabidopsis thaliana transformation, prove that this gene has the function of decomposing organophosphorus.
Arabidopis thaliana is lacked to the result of study discovery of AtPAP26 mutant, AtPAP26 utilizes important role at the Arabidopis thaliana organophosphorus; Wang etc. proceed to soybean by Arabidopis thaliana AtPAP15, at phytate, are that under the condition of unique phosphorus source, 3 transgenic line phosphorus utilizations improve respectively 117.8%, 56.5%, 57.8%.
Liang etc. have obtained the PvPAP3 gene from Kidney bean, and Real Time-PCR analyzes discovery, under low-phosphorus stress, this gene in the expression of phosphorus efficiency kind G19833 higher than phosphorus poor efficiency kind DOR364.As can be seen here, the purple acid phosphatase gene in the plants such as Arabidopis thaliana, clover, Kidney bean has the organic phosphorus in activation plant rhizosphere surrounding soil, the function of the interior phosphorus element of promotion plant body recycling.
Yet, about clone and the rhizosphere organophosphorus utilization on every side research of the purple acid phosphatase gene in soybean, report is very few at present.
Summary of the invention
The object of the invention is to provide a kind of soybean purple acid phosphatase and encoding gene thereof, for the organic phosphorus that activates plant rhizosphere surrounding soil, promote phosphorus element recycling in the plant body.
Another purpose of the present invention is to provide the carrier that contains said gene.
Another object of the present invention is to provide the host cell that contains said gene or carrier.
Still a further object of the present invention is to provide soybean purple acid phosphatase and the application of encoding gene in improving plant phytate phosphorus utilization ratio thereof.
Open reading frame (ORF) sequence of soybean purple acid phosphatase GmPAP4 gene, as shown in SEQ ID NO.1, length is 1329bp.The protein sequence of soybean purple acid phosphatase gene GmPAP4 coding, its aminoacid sequence as shown in SEQ ID NO.2 or this sequence through replacing one or several amino acids formed aminoacid sequence with same function, SEQID NO.2 length is 442 amino-acid residues.
Should be appreciated that the degeneracy of considering codon, for example can be in its coding region, under the condition that does not change aminoacid sequence, or at its non-coding region under the condition that does not affect protein expression, the gene order of the above-mentioned albumen of encoding is modified.Therefore, the present invention also comprises replacement, the interpolation that the gene order of the above-mentioned albumen of encoding is carried out and/or lacks one or more Nucleotide, has the nucleotide sequence with above-mentioned encoding gene identical function.The present invention also comprises just sequence or the antisense sequences based on described gene, comprise cloning vector or the expression vector that contains described nucleotide sequence or its fragment, the host cell that contains described carrier, utilize described host cell to prepare method of soybean purple acid phosphatase etc.
Described this sequence is through replacing one or several amino acids formed aminoacid sequence with same function, refers to that site outside the active function territory of this sequence carries out the sequence that limited amino acid whose protection replaces gained and still can keep original activity.The active function territory of aminoacid sequence of the present invention is 135 ~ 430, and outside this site, replaceable one or several amino acid obtains having the aminoacid sequence of same function.Such as, at nonactive section, the Ala of the 123rd is replaced with to Val, or the Leu of the 440th is replaced with to Thr, and there is same functional effect.
It is material that phosphorus efficiency soybean varieties " middle yellow 15 " is take in the present invention, take Arabidopis thaliana AtPAP15 sequence as basis, utilizes the homologous gene clone technology, separates open reading frame (ORF) sequence that obtains the purple acid phosphatase gene GmPAP4 in soybean.Adopt the fluorescence real-time quantitative PCR technology, detect the differential expression of GmPAP4 gene in phosphorus efficiency (middle yellow 15) and phosphorus poor efficiency (ox hair Huang) soybean varieties, found that, at phytate, be under the condition of unique phosphorus source, phytate phosphorus is processed 15d ~ 70d, the expression amount of GmPAP4 gene in phosphorus efficiency soybean varieties (middle yellow 15) is far away higher than the expression amount in phosphorus poor efficiency kind (ox hair Huang), and maximum difference can reach 8 times (phytate phosphorus is processed 56d ~ 63d).
On this basis, by the structure of prokaryotic expression carrier, realized the prokaryotic expression of GmPAP4 gene in e. coli bl21, utilized the His label, separation and purification has obtained the GmPAP4 proteins encoded after the abduction delivering; Through enzyme biopsy cls analysis, find, the GmPAP4 proteins encoded has activity of acid phosphatase, and the activity of acid phosphatase during with pH=5.0 is the highest.Simultaneously by building the GFP fusion expression vector of GmPAP4, adopt particle gun bombardment technology that the pCamE-GmPAP4::GFP fusion gene built is imported to onion entocuticle cell, utilize the distribution situation of the expressed albumen of this fusion gene of fluorescence microscope in onion entocuticle cell, result shows that there is obvious fluorescence at the cytolemma position of the onion entocuticle cell that transforms the pCamE-GmPAP4::GFP plasmid, illustrates that the GmPAP4 proteins encoded mainly is distributed in cytolemma.Utilize the plant overexpression vector pCamE-GmPAP4 of the constructed GmPAP4 gene completed, adopt the agrobacterium mediation converted technology to proceed in Columbia type Arabidopis thaliana, phytate phosphorus decomposition through transgenic arabidopsis utilizes evidence, GmPAP4 to have the decomposition plant rhizosphere function of phytic acid state phosphorus, raising phytate phosphorus utilising efficiency on every side.
Beneficial effect of the present invention:
(1) the present invention has found soybean purple acid phosphatase GmPAP4 and encoding gene thereof first, for improving plant phytate phosphorus utilization ratio, provides new candidate gene.
(2) open reading frame of soybean purple acid phosphatase gene GmPAP4 of the present invention and proteins encoded thereof can decompose and utilize phytic acid state phosphorus around the plant rhizosphere, improve transgenic arabidopsis phytate phosphorus utilising efficiency, can utilize genetic engineering means further to improve plant phytate phosphorus utilising efficiency.
The accompanying drawing explanation
Fig. 1 is the figure as a result of the pcr amplification of " middle yellow 15 " low-phosphorous processing GmPAP4 gene cDNA; Wherein M is DNA Marker DL 2000; The 1 pcr amplification result that is " middle yellow 15 " low-phosphorous processing cDNA.
Fig. 2 is that phytate phosphorus is processed the differential expression of lower GmPAP4 gene in " middle yellow 15 " and " ox hair Huang " root system figure as a result; Wherein, X-coordinate means the number of days that phytate phosphorus is processed, and ordinate zou means the ratio of GmPAP4 at " middle yellow 15 " and " ox hair Huang " root system expression amount.
Fig. 3 is the SDS-PAGE electrophoresis detection result of GmPAP4 gene prokaryotic albumen; BL21 F-strain to the pET32a-GmPAP4 Plasmid Transformation carries out the abduction delivering test, and wherein arrow is depicted as and expresses purpose fragment 61.2KDa; M is the protein standard molecular weight; 1 is not contain the empty bacterial strain of recombinant plasmid; 2 is the bacterial strain that contains unloaded plasmid; 3 is the bacterial strain that contains goal gene GmPAP4; The 4 GmPAP4 differential proteins for the separation and purification acquisition.
Fig. 4 is the activity of acid phosphatase detected result figure of GmPAP4 gene coded protein; Wherein, pET-32a is empty carrier, and GmPAP4 is recombinant plasmid.
Fig. 5 is the activity of acid phosphatase detected result figure of GmPAP4 gene coded protein under condition of different pH.
Fig. 6 is the onion epidermis observation of subcellular localization result of GmPAP4 proteins encoded; Wherein, A figure: the empty carrier pCamE-GFP under ultraviolet excitation transforms the Fluirescence observation result of onion cell; B figure: the recombinant plasmid pCamE-GmPAP4::GFP under ultraviolet excitation transforms the Fluirescence observation result of onion cell.
Fig. 7 is T
3pCR detected result figure for transgenic arabidopsis goal gene GmPAP4; Wherein, 1-9 is T
3the transgenic arabidopsis plant, the Arabidopis thaliana positive plant that contains goal gene that 5-9 obtains for screening, M is DNA Marker DL2000, and 10 is the wild-type contrast, and 11 is blank, and 12 is the plasmid positive control.
Fig. 8 is T
3turn the RT-PCR detected result figure of GmPAP4 gene Arabidopis thaliana; Wherein, M is DNA Marker DL2000; 1 ~ 4 is T
3for transgenic arabidopsis; 5 is the wild-type contrast; 6 is blank; 7 is the plasmid positive control.
Fig. 9 is under phytate phosphorus and 2 kinds of processing of suitable phosphorus, T
3overexpression turns the growing state of GmPAP4 gene Arabidopis thaliana; Wherein ,+Pi processes for suitable phosphorus, and+Po is that phytate phosphorus is processed; WT is wild-type, and G4 is T
3for Arabidopis thaliana.
Figure 10 is T
3overexpression turns the biological anharmonic ratio of GmPAP4 gene Arabidopis thaliana and wild-type plant overground part figure as a result; Wherein ,+Pi processes for suitable phosphorus, and+Po is that phytate phosphorus is processed; WT is wild-type, and G4 is T
3for Arabidopis thaliana.
Embodiment
Following examples are used for the present invention is described, but are not used for limiting the scope of the invention.Without departing from the spirit and substance of the case in the present invention, the modification that the inventive method, step or condition are done or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that in embodiment, technique means used is well known to those skilled in the art.
The molecular cloning of embodiment 1GmPAP4 gene
(1) soybean seedling is cultivated and is lacked the phosphorus processing: choose full, neat and consistent soybean varieties " middle yellow 15 " seed, the consistent material of selecting after vernalization to sprout is seeded in quartz sand, after 7d, move in nutritive medium, after living true leaf is launched, lack phosphorus and coerce (0mmol/L) processing, contrast phosphorus concentration for the treatment of is 1.0mmol/L.Get plant root after phosphorus Stress treatment 21d, after liquid nitrogen flash freezer ,-80 ℃ of Ultralow Temperature Freezers are preserved stand-by.
(2) the Real-time PCR Analysis material therefor is processed: choose soybean varieties " middle yellow 15 " and " ox hair Huang " seed (purchased from Chinese Academy of Agricultural Sciences's country's Soybean Germplasm storehouse) of full neat and consistent, be seeded in vermiculite.Emerge after 7d and get plant root and (be designated as 0d) in contrast, then 2 different phosphate treatment group (suitable phosphorus, phytate phosphorus are set, phosphorus concentration is 1.0mmol/L), process 7d, 14d, 21d, 28d, 35d, 42d, 49d, 56d, 63d, 70d at phosphorus subsequently, get respectively the plant root sample, liquid nitrogen flash freezer ,-80 ℃ of Ultralow Temperature Freezers are preserved stand-by.
(3) total RNA extracts: the total RNA(of soybean plant strain lacks phosphorus, suitable phosphorus, phytate phosphorus processing) extract and carry out with reference to TRNzol Total RNA Reagent operational guidance.
(4) the cDNA reverse transcription is synthetic: the total RNA of plant obtains cDNA after reverse transcription, and building-up process is with reference to PrimeScript
tM1st Strand cDNA Synthesis Kit operational guidance carries out.
(5) design of pcr amplification the primer: utilize the AtPAP15 sequence, at NCBI website comparison soybean EST, obtain the E-serial of goal gene by the splicing of electronic cloning technology.According to this E-serial design primer, sequence is as follows:
F1：5’-GGTACCATGGAACTCAAACAACAAAAACTCC-3’
R1：5’-CTCGAGTTAGGGCGTCAAAAGTGTACTTCTG-3’
(6) molecular cloning of GmPAP4 gene open reading frame (ORF): utilize designed F1 and R1 primer, the reverse transcription cDNA lacked under the phosphorus processing of take carries out pcr amplification as template, clones the open reading frame sequence of GmPAP4 gene.PCR reaction system and program are as follows:
The PCR program:
95℃?10min
72℃?10min
10℃?hold
Pcr amplification product detects through agarose gel electrophoresis, reclaims the purpose fragment, and is connected in the pGM-T carrier.Reaction system is 10 * Ligation Buffer, 1 μ L, pGM-T Vector 1 μ L, PCR Product 7 μ L, T
4dNA Ligase 1 μ L, mix rear 16 ℃ of connections and spend the night.As shown in Figure 1,1 swimming lane obtains a specific band of about 1.3kb left and right to electrophoresis result.
(7) transform intestinal bacteria, obtain positive colony: connect product and adopt the thermal shock method to transform competent escherichia coli cell, the picking positive colony, carry out plasmid extraction, enzyme is cut and is detected and order-checking, and the open reading frame sequence that obtains the GmPAP4 gene is shown in the long 1329bp of SEQ ID NO.1().
The Differential expression analysis of embodiment 2GmPAP4 gene in different soybean varieties
Take GmPAP4 as target gene, and soybean constitutive expression Actin11 gene is the internal reference contrast, and design Realtime-PCR primers F 2 and R2, adopt
green I fluorescence dye method is carried out fluorescence real-time quantitative PCR, by comparing C
tcarry out the relative quantitative assay of GmPAP4 gene expression dose.
F2：5’-CGACCTCTTCCTCGTAAAACC-3’
R2：5’-GTGCTTGTCTCCTGCCAAAG-3’
The relative expression quantity of GmPAP4 gene in different Root System In Soybean Cultivars=2
– △ △ CT, Δ Δ Ct=(Ct
gmPAP4– Ct
actin11)
middle yellow 15 root systems-(Ct
gmPAP4-Ct
actin11)
ox hair xanthorrhiza systemresult as shown in Figure 2, phytate phosphorus is processed 15d ~ 70d, the GmPAP4 gene at the expression amount of " middle yellow 15 " root system far away higher than the expression amount in " ox hair Huang ", and, when phytate phosphorus is processed 56 ~ 63d, GmPAP4 is 8 times of expression amount in " ox hair Huang " root system at the expression amount of " middle yellow 15 " root system.The prokaryotic expression of embodiment 3GmPAP4 gene
(1) recombinant plasmid pGM-GmPAP4 and prokaryotic expression carrier pET-32a (+) are adopted to the KpnI/XhoI double digestion, and reclaim the purpose fragment, pET-32a (+) expression vector after reclaiming is connected with the GmPAP4 endonuclease bamhi.The endonuclease reaction system is KpnI 1 μ L, XhoI 1 μ L, 10 * L Buffer, 2 μ L, H
2o 11 μ L, Plasmid 5 μ L.The ligation system is GmPAP4Fragment 5 μ L, pET-32a (+) Vector 1 μ L, T
4dNA Ligase 1 μ L, 10 * Ligation Buffer, 1 μ L, ddH
2o 2 μ L, mix rear 16 ℃ of connections and spend the night.
(2) merge the abduction delivering of recombinant protein: above-mentioned connection product is transformed to bacillus coli DH 5 alpha, cut detection through PCR and enzyme, plasmid transformation escherichia coli BL21 bacterial strain by positive colony, the picking positive colony, cut detection, order-checking evaluation through plasmid extraction, enzyme, obtain the positive colony that contains GmPAP4 gene correct coding sequence.
(3) the SDS-PAGE protein electrophoresis is analyzed: utilizing 0.8mmol/L IPTG abduction delivering to turn has the BL21 of prokaryotic expression carrier bacterial strain, the bacterium liquid of collect respectively before inducing, inducing rear 12h.After the bacterium liquid of collecting is centrifugal, pouring liquids, add respectively 200 μ L sample-loading buffers, after vibration mixes, is placed in boiling water 10 minutes, wink from; Configure respectively 12% separation gel, 5% concentrated glue.Respectively get 30 μ L sample loadings; Concentrated glue part is with the 80V constant voltage, and the separation gel part is with 120V constant voltage electrophoresis.The SDS-PAGE electrophorogram as shown in Figure 3, is induced through IPTG, and recombinant plasmid specific band occurred on the position of about 61.2KDa, with the expression target protein in the same size of expection.Wherein, pET-32a (+) carrier background is expressed 20.4KDa albumen; It is 1182bp that the GmPAP4 gene is removed signal peptide, 393 amino acid of encoding, and expressing is the albumen of 40.8KDa in theory, the two is 61.2KDa altogether.
The purifying of embodiment 4GmPAP4 gene coded protein and activity of acid phosphatase detect
(1) isolation and purification of GmPAP4 gene coded protein: the operational guidance according to Ni-Agarose His label protein purification kit carries out.
(2) activity of acid phosphatase of GmPAP4 gene coded protein detects: the standardized solution of at first preparing p-nitrophenol (ρ-NP), colorimetric estimation light absorption value under the 405nm wavelength, take p-nitrophenol content as X-coordinate, and light absorption value is ordinate zou drawing standard curve.Then take ρ-NPP as substrate, reaction system comprises 10mmol/L MgCl
210mmol/L ρ-NPP, 50mmol/L sodium acetate-acetate buffer and 20 μ L GmPAP4, reaction cumulative volume 1.5mL, 37 ℃ of water-baths, reaction 30min, add 0.5mL 1mol/L NaOH solution termination reaction, measure the light absorption value under 405nm, calculate the amount of the ρ-NP of reaction generation according to typical curve, calculate the relative reactivity of GmPAP4.Result as shown in Figure 4, with contrasting (pET-32a), compare, the activity of acid phosphatase of recombinant plasmid pET32a-GmPAP4 has improved 40%, and difference reaches utmost point conspicuous level, illustrate that target protein GmPAP4 can decomposition reaction substrate ρ-NPP, there is the activity of acid phosphatase.
(3) the GmPAP4 gene coded protein detects at the activity of acid phosphatase of condition of different pH: reaction system is selected respectively different damping fluids, pH value 3.5 ~ 5.5 is 50mmol/L sodium acetate acetate buffer, pH value 6.0 ~ 7.0 is the 50mmol/LTris acetate buffer, and pH value 7.5 ~ 9.0 is the 50mmol/LTrisHCl damping fluid; All the other method stepss and GmPAP4 proteins encoded activity of acid phosphatase detect basic identical.Result as shown in Figure 5, by analyzing the activity of acid phosphatase of 7 GmPAP4 albumen under condition of different pH, found that, GmPAP4 albumen activity of acid phosphatase under the condition of pH=5.0 is the highest, meets the feature of acid phosphatase.
The Subcellular Localization of embodiment 5GmPAP4 proteins encoded
(1) design of PCR the primer: utilize biosoftware DNAMAN design one couple of PCR primers, amplification GmPAP4 removes the open reading frame after terminator codon, carries out the Subcellular Localization of proteins encoded, and primer sequence is as follows:
F3：5'-GTCGACATGGAACTCAAACAACAAAAACTC-3'，
R3：5'-GGTACCGGGCGTCAAAAGTGTACTTC-3'，
(2) amplification of the required open reading frame of Subcellular Localization: take the pGM-GmPAP4 plasmid as template, utilize F3 and R3 primer, amplification does not contain the GmPAP4 gene open reading frame sequence of terminator codon, cut glue after electrophoresis detection and reclaim the purpose band, with the pGM-T carrier, be connected, blue hickie screening, order-checking, pick out the errorless positive colony pGM-PAP4 of open reading frame sequence for follow-up test.
(3) structure of fusion expression vector: intermediate carrier pGM-PAP4 and GFP fusion expression vector pCamE-GFP are used respectively to SalI and KpnI double digestion.Reclaim enzyme and cut product, with carrier, pCamE-GFP is connected.The endonuclease reaction system is SalI 1 μ L, KpnI 1 μ L, 10 * MBuffer, 2 μ L, H
2o 6 μ L, Plasmid 10 μ L.Linked system is PAP4 Fragment 5 μ L, pCamE-GFP Vector 1 μ L, T
4dNA Ligase 1 μ L, 10 * Ligation Buffer1 μ L, H
2o 2 μ L, mix rear 16 ℃ of connections and spend the night.Connecting product adopts the thermal shock method to transform competent escherichia coli cell.The picking positive colony, carry out plasmid extraction, and enzyme is cut and detected and order-checking, obtains the fusion expression vector pCamE-GmPAP4::GFP for Subcellular Localization.PCamE-GFP is identical with expression vector pCamE-GFP in patent CN 101942426B.
(4) fusion expression vector pCamE-GmPAP4::GFP via Particle Bombardment Transformation: adopt particle gun bombardment technology that fusion expression vector pCamE-GmPAP4::GFP is transformed to onion entocuticle cell, step of converting carries out according to the particle gun process specifications, and control group is empty carrier pCamE-GFP.
(5) fluorescence microscope: the onion entocuticle cell after the particle gun bombardment is placed in fluorescence microscopy Microscopic observation green fluorescence in intracellular distribution situation.As shown in Figure 6, result shows that there is obvious fluorescence at the cytolemma position of the onion entocuticle cell that transforms the pCamE-GmPAP4::GFP plasmid to result, and hence one can see that, and the GmPAP4 proteins encoded mainly is distributed in plant cell membrane.
The functional analysis of embodiment 6GmPAP4 genetic expression albumen
(1) structure of the plant overexpression vector pCamE-GmPAP4 of GmPAP4 gene: pGM-GmPAP4 and pCamE plasmid are adopted to Sal I/Kpn I double digestion, reclaim enzyme and cut product, connected.The endonuclease reaction system is SalI 1 μ L, KpnI 1 μ L, 10 * M Buffer, 2 μ L, BSA 2 μ L, H
2o 6 μ L, Plasmid 8 μ L.Linked system is GmPAP4 Fragment5 μ L, pCamE Vector 1 μ L, T
4dNA Ligase 1 μ L, 10 * Ligation, 1 μ L, ddH
2o 2 μ L, mix rear 16 ℃ of connections and spend the night.To connect product and transform bacillus coli DH 5 alpha, and through PCR and enzyme, cut double check, and obtain positive colony and checked order, the correct overexpression vector that obtains checking order carries out follow-up test.
(2) agrobacterium-mediated transformation infects Arabidopis thaliana: the Agrobacterium that adopts flower-dipping method (Floral dip) will carry pCamE-GmPAP4 proceeds in Columbia type Arabidopis thaliana.PCR testing goal gene GmPAP4, result as shown in Figure 7.
(3) the phytate phosphorus utilization of transgenic arabidopsis is analyzed: choose 4 T
3transgenic arabidopsis strain and wild-type contrast are material; Each material is planted respectively 30 basins, and 1 strain/basin carries out 2 kinds of phosphorus and processes (phytate phosphorus is processed, suitable phosphorus is processed, and concentration is 1.0mmol/L).T under processing with phytate phosphorus
3the goal gene GmPAP4cDNA of transgenic arabidopsis is template, carries out the RT-PCR amplification, and the primer is the Totomycin primer on the pCamE-GmPAP4 carrier, and primer sequence is F4:5'-CTATTTCTTTGCCCTCGGAC-3'; R4:5'-ATGAAAAAGCCTGAACTCACC-3 '; Amplification length is 1026bp, and result as shown in Figure 8, amplifies the purpose band as seen.
During plant strain growth, observe the growing state of Arabidopis thaliana plant under 2 kinds of phosphorus are processed, and the weighing overground part is biological heavy, carries out phytate phosphorus utilization analysis.As shown in Figure 9, phytate phosphorus is compared T under processing to the Arabidopis thaliana growing state with wild-type
3for the transgenic arabidopsis plant show that plant leaf is large, the petiole characteristics such as length, leaf look greening, growth potential is strong, the wild-type contrast shows that blade is less, petiole is shorter, the leaf look blue, the phosphate deficiency shape a little less than growth potential, shows T
3can decompose and utilize rhizosphere phytate phosphorus, GmPAP4 on every side to there is the effect that improves the anti-low-phosphorous ability of transgenic arabidopsis under the phytate phosphorus condition for the transgenic arabidopsis plant.
Further measure T
3biological heavy for the overground part of transgenic arabidopsis plant and wild-type, as shown in figure 10, the transgenic arabidopsis under suitable phosphorus condition contrasts biology with wild-type heavily do not have significant difference to result; And biological weight of GmPAP4 Arabidopis thaliana overground part that turn under the phytate phosphorus condition compared with wild-type, improve 39.3%, difference reaches utmost point conspicuous level; T is described
3can decompose and utilize the phytate phosphorus around rhizosphere thereby be subject to low-phosphorous impact less for the transgenic arabidopsis plant, there is higher biology weight; It is larger that the wild-type plant is subject to low-phosphorous impact, has lower biology weight, and GmPAP4 has the effect that improves the anti-low-phosphorous ability of transgenic arabidopsis under the phytate phosphorus condition.
Conclusion: the expressed albumen of soybean purple acid phosphatase GmPAP4 gene has the decomposition plant rhizosphere function of phytate phosphorus, raising transgenic arabidopsis phytate phosphorus utilising efficiency on every side.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (6)
1. a soybean purple acid phosphatase, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO.2.
2. the gene of the described soybean purple acid phosphatase of claim 1 of encoding.
3. gene according to claim 2, is characterized in that, its nucleotide sequence is as SEQ ID No.1.
4. the carrier that contains claim 2 or 3 described genes.
5. the host cell that contains the described gene of claim 2 or 3 or the described carrier of claim 4.
6. the described soybean purple acid phosphatase of claim 1, the application of the described gene of claim 2 or 3 in improving Arabidopis thaliana phytate phosphorus utilization ratio.
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| CN104946684A (en) * | 2015-06-17 | 2015-09-30 | 华南农业大学 | Function of purple acid phosphatase GmPAP33 gene for promoting reuse of phosphorus in soybean mycorrhiza |
| CN105647884B (en) * | 2016-02-02 | 2020-12-15 | 河北农业大学 | Soybean purple acid phosphatase GmPAP36, and coding gene and application thereof |
| CN108048474B (en) * | 2017-11-10 | 2021-02-19 | 华南农业大学 | Acid phosphatase protein gene GmPAP1-like and application thereof |
| CN108588116B (en) * | 2018-05-10 | 2021-02-19 | 华南农业大学 | Application of soybean purple acid phosphatase gene GmPAP35 |
| CN111066593B (en) * | 2020-01-17 | 2020-12-11 | 华中农业大学 | Method for researching functions of acid phosphatase in symbiosis of alfalfa rhizobium and mycorrhiza |
| CN114164222B (en) * | 2021-12-07 | 2023-06-06 | 江苏师范大学 | Garlic purple acid phosphatase AsPAP gene and application thereof in improving alliin content of garlic callus |
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