CN116716311A - Bermudagrass aluminum response gene and primer and cloning method thereof - Google Patents

Bermudagrass aluminum response gene and primer and cloning method thereof Download PDF

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CN116716311A
CN116716311A CN202310535665.1A CN202310535665A CN116716311A CN 116716311 A CN116716311 A CN 116716311A CN 202310535665 A CN202310535665 A CN 202310535665A CN 116716311 A CN116716311 A CN 116716311A
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aluminum
cdmate1
cdstar1
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黄春琼
段宏利
陈志坚
严琳玲
郇恒福
董荣书
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Tropical Crops Genetic Resources Institute CATAS
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Abstract

The invention discloses a bermudagrass aluminum response gene, a primer and a cloning method thereof, belonging to the technical field of plant genetic engineering. The bermuda grass aluminum response gene is CdMATE1 and CdSTAR1, the nucleotide sequence of the CdMATE1 is shown as SEQ ID NO. 1, and the nucleotide sequence of the CdSTAR1 is shown as SEQ ID NO. 2. The nucleotide sequence of an upstream primer for amplifying the CdMATE1 gene is shown as SEQ ID NO. 3, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 4; the nucleotide sequence of the upstream primer for amplifying the CdSTAR1 gene is shown as SEQ ID NO. 5, and the amino acid sequence of the downstream primer is shown as SEQ ID NO. 6. The cloning method comprises aluminum stress treatment, full-length sequence amplification, addition of A tail to PCR products, ligation reaction, escherichia coli transformation and colony PCR verification. The invention resolves the aluminum-resistant mechanism of the bermudagrass from the gene level by combining transcriptome information, which is beneficial to the research, breeding and application of the aluminum-resistant mechanism of the bermudagrass.

Description

Bermudagrass aluminum response gene and primer and cloning method thereof
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a bermuda grass aluminum response gene, a primer and a cloning method thereof.
Background
Bermuda grass (Cynodondactylon (Linnaeus) persoson) is a perennial herb of the Gramineae family, and is one of the most common grasses on natural grasslands in the southern area of China. The bermudagrass has strong trampling resistance and reproduction and regeneration capability, and can be used for highway slope protection, courtyard greening, golf courses and the like; the leaf quantity is rich, the feeding mouth is good, and the feed can also be used as high-quality pasture to feed herbivores such as cattle and sheep.
The existing research on the aluminum stress of the bermudagrass shows that along with the increase of the concentration of the aluminum stress, the hematoxylin staining range of the root tip of the bermudagrass is enlarged from the root tip, the staining degree is deepened, and in addition, the aluminum content of the root system is also increased, so that the root tip is the initial part of the plant under the aluminum stress. Chlorophyll content of the bermuda grass is reduced, leaf color is lightened, and soluble total sugar content is reduced under the stress of aluminum; in addition, enhancement of antioxidant enzyme activity can reduce the toxic effect of aluminum stress on bermuda grass, manifested by increased levels of active oxygen, oxidase and free proline. These studies remain on the aspects of germplasm evaluation and physiological mechanism, and cannot further explain the self advantages of aluminum tolerance of the bermuda grass, and limit the further popularization and utilization of the bermuda grass, so that the bermuda grass aluminum response genes are urgently needed to be excavated, and the aluminum tolerance mechanism of the bermuda grass is studied from the gene layer.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a bermuda grass aluminum response gene, a primer and a cloning method thereof.
The aim of the invention is achieved by the following technical scheme: the bermuda grass aluminum response gene is CdMATE1 and CdSTAR1, the nucleotide sequence of the CdMATE1 is shown as SEQ ID NO. 1, and the nucleotide sequence of the CdSTAR1 is shown as SEQ ID NO. 2.
The primer for amplifying the bermuda grass aluminum response gene is characterized in that the nucleotide sequence of an upstream primer for amplifying the CdMATE1 gene is shown as SEQ ID NO. 3, and the nucleotide sequence of a downstream primer is shown as SEQ ID NO. 4; the nucleotide sequence of the upstream primer for amplifying the CdSTAR1 gene is shown as SEQ ID NO. 5, and the amino acid sequence of the downstream primer is shown as SEQ ID NO. 6.
A method for cloning an aluminum response gene of bermuda grass, comprising the steps of:
s1, aluminum stress treatment: carrying out water planting rooting on aluminum-resistant bermuda grass germplasm A22, selecting seedlings with root length of about 5cm and consistent growth vigor, putting the seedlings into aluminum treatment liquid for aluminum stress treatment, and collecting root systems after 24 hours of treatment; wherein the aluminum treatment solution is a mixed solution of calcium chloride and aluminum chloride, the concentration of the calcium chloride in the mixed solution is 450-550 mu M, the concentration of the aluminum chloride is 450-550 mu M, and the pH value of the aluminum treatment solution is 3.5-4.5;
s2, amplifying the full-length sequence: taking root cDNA of the bermuda grass after aluminum stress treatment for 24 hours in the step S1 as a template, adopting the primers, respectively carrying out PCR amplification on target fragments by using high-fidelity enzyme GXL, carrying out electrophoresis detection after the PCR amplification is finished, and recovering CdMATE1 and CdSTAR1 fragments;
s3, adding tail A into PCR products: adding a poly (A) tail to the 3' -end of the PCR product by using a DNAA-Tailing Kit to obtain a CdMATE1 gene added with the A tail and a CdSTAR1 gene added with the A tail respectively;
s4, connection reaction: performing enzyme digestion reaction on plasmid pCXSN-Myc by using restriction enzyme XcmI, detecting enzyme digestion products by electrophoresis, recovering corresponding fragments, respectively connecting CdMATE1 plasmid with A tail and CdSTAR1 gene with A tail to linearized plant expression vector pCXSN-Myc by using T4 ligase, and obtaining products which are recombinant plasmids pCXSN-Myc-CdMATE1 and pCXSN-Myc-CdSTAR1;
s5, escherichia coli transformation: respectively adding recombinant plasmids pCXSN-Myc-CdMATE1 and pCXSN-Myc-CdSTAR1 into competent cells of escherichia coli Trans T1, adding an LB culture medium for culturing for 50-70 min, coating bacterial liquid on an LB solid culture medium containing kanamycin, and culturing for 12-16h in an inverted mode;
s6, colony verification by PCR: and (3) detecting colony products cultured by the solid culture medium in the step (S5), selecting positive clone colonies, placing the positive clone colonies into an LB liquid culture medium containing kanamycin for amplification culture, and selecting positive clone bacterial liquid for sequencing analysis to obtain CdMATE1 and CdSTAR1 full-length cDNA sequences.
Further, the specific operations of the hydroponic rooting in step S1 are as follows: cutting creeping stem segments of aluminum-resistant bermuda grass germplasm A22, immersing stem segments in a nutrient solution for water culture rooting, wherein the nutrient solution is 1/2Hoagland nutrient solution, and the pH value is 5.8.
Further, the PCR amplification system in step S2 is: 5 XPS GXLBuffer:10 μL,2.5mM dNTP mix:1.6 μL, forward primer:0.5 μl, reverse primer: 0.5. Mu.L, 20-fold dilution of cDNA:1 μl, GXL Enzyme:0.4 mu L ddH 2 O：12μL，Total：20μL；
The PCR reaction parameters are as follows:
further, the reaction system for adding the tail A in the step S3 is as follows: 10 XA-Tailing Buffer:5 μL,2.5mM dNTPMix: 4. Mu.L, A-tagging Enzyme: 0.5. Mu.L, 0.5-5. Mu.g of end smooth DNA fragment: 10 mu L ddH 2 O:30.5 μL, total: 50. Mu.L; reaction conditions: 72 ℃ for 20min; placing on ice for 2min; preserving at 4 ℃.
Further, the reaction system of the cleavage reaction in step S4 is: plasmid:3 μg (xμL), 10 XNEB Buffer:3 μL, xcmI:1 mu L ddH 2 O: (26-x) μL, total: 30. Mu.L; reaction conditions: the reaction is carried out for more than 3 hours at 37 ℃.
Further, the linkage system of the linearized plant expression vector pCXSN-Myc in step S4 is: 50ng Vector:1 μL,50ng Insert DNA:2 mu L,10 xT 4 ligase Buffer：1μL，5U/L T4 ligase：1μL，ddH 2 O:6 μL, total: 10. Mu.L; reaction conditions: the reaction was carried out at 4℃overnight.
Further, in step S6, colony PCR products are detected by 1% agarose gel electrophoresis, and the reaction system of colony PCR is: 2×Green r Taq mix: 10. Mu.L of the solution,
forward primer:0.5 μl: reverse primer:0.5 μl: and (3) a template: 2 mu L, ddH 2 O:7 μL, total: 20. Mu.L; colony PCR reaction parameters were as follows:
the invention has the following advantages: the genes CdMATE1 and CdSTAR1 disclosed by the invention are the genes which are differentially up-regulated in aluminum-resistant materials and aluminum-intolerant materials under aluminum stress and are compared in NCBI to determine the full-length genes according to the sequencing result of transcriptomes under the aluminum stress condition. The invention further discloses a cloning method of the aluminum response gene of the bermudagrass, and the aluminum-resistant mechanism of the bermudagrass is analyzed from the gene level by combining transcriptome information, so that the method is beneficial to research, breeding and application of the aluminum-resistant mechanism of the bermudagrass.
Drawings
FIG. 1 is a full length clone map of Cynodon dactylon CdMATE1 (A) and CdSTAR1 (B).
FIG. 2 is a graph showing the predictive analysis of the secondary structures of CdMATE1 (A) and CdSTAR1 (B) proteins.
FIG. 3 is a graph showing the prediction of the tertiary structure of CdMATE1 (A) and CdSTAR1 (B) proteins.
FIG. 4 is a graph of the transmembrane domain analysis of CdMATE1 (A) and CdSTAR1 (B) proteins.
FIG. 5 is a phylogenetic tree analysis of the homologous proteins of CdMATE1 and MAEs.
FIG. 6 is a graph of phylogenetic tree analysis of the homologous proteins of CdTAR 1 and TAR1 s.
Detailed Description
The invention will be further described with reference to the following examples of embodiments of the accompanying drawings, to which the scope of the invention is not limited: example 1:
1 materials and methods
1.1 Pre-culture of Bermuda grass
The stolon sections with 2 sections of aluminum-resistant bermuda grass germplasm A22 with consistent size and growth are cut from a lawn grass germplasm nursery, inserted on KT plates with holes punched on the surfaces, plants can be fixed on holes on the plates, foam plates are arranged on a turnover box containing 10L 1/2Hoagland nutrient solution (pH value is 5.8), the nutrient solution submerges the stem sections, and water culture rooting is carried out in a greenhouse.
1.2 aluminum stress treatment of bermuda grass
After the preculture is finished, selecting seedlings with the root length of about 5cm and consistent growth vigor, inserting the seedlings into a KT plate with holes on the surface, and placing the seedlings into a tray containing 1L of 500 mu M CaCl 2 Aluminum treatment fluid (500 mu MAlCl) 3 ) In the beaker (shading treatment is carried out), aluminum stress treatment is carried out in a greenhouse, the pH value is 4.0, and root systems are collected after 24 hours of treatment.
1.3 cloning of aluminium response candidate genes and bioinformatics analysis
1.3.1 cloning of CdMATE1 and CdSTAR1
(1) And (5) designing a primer. Full length primers for the CdMATE1 and CdSTAR1 genes were designed using Oligo7, and the sequences are shown in Table 1.
(2) Amplification of the full-Length sequence of CdMATE1 and CdSTAR1. The target fragment was amplified with high fidelity enzyme GXL using root cDNA of Cynodon dactylon A22 as template after 24h of aluminum treatment, and the amplification system is shown in Table 2. And (5) performing electrophoresis detection after the PCR amplification is finished.
TABLE 1 full-length cloning primers for CdMATE1 and CdSTAR1
TABLE 2 target fragment amplification System
The PCR parameters were as follows:
(3) Fragment recovery. After the completion of the electrophoresis detection, cdMATE1 and cdtar 1 fragments were recovered using Extraction Mini Kit gel recovery kit (nuuzan, china).
(4) The PCR product was added with tail A. The 3' -end of the PCR product was tagged with poly (A) tail using DNAA-Tailing Kit, and the reaction system is shown in Table 3.
TABLE 3 reaction System for fragments with A tail
Reaction conditions: 72 ℃ for 20min; placing on ice for 2min; preserving at 4 ℃.
(5) Obtaining linearization carrier. Plasmid pCXSN-Myc was subjected to cleavage reaction (see Table 4) using restriction enzyme XcmI (BioLabs), and the cleavage products were detected by running gel using 1% agarose gel electrophoresis, and the corresponding fragments were recovered.
TABLE 4XcmI cleavage vector reaction System
Reaction conditions: the reaction is carried out for more than 3 hours at 37 ℃.
(6) And (3) connection reaction. The cloned CdMATE1 and CdSTAR1 genes were ligated to the linearized plant expression vector pCXSN-Myc using T4 ligase (BioLabs), the ligation reaction system is shown in Table 5, and the resulting products were recombinant plasmids pCXSN-Myc-CdMATE1 and pCXSN-Myc-CdSTAR1.
TABLE 5 fragments of interest and linearized vector ligation systems
Reaction conditions: the reaction was carried out at 4℃overnight.
(7) E.coli transformation. Melting competent cells Trans T1 of Escherichia coli stored at-80deg.C on ice, adding 5 μl of the ligation product of 2.3.5 (1) (6) into 50 μl of T1, and standing on ice for 30min; placing on ice at 42 ℃ for 45s for 2min; 200. Mu.L of LB medium was added; culturing at 37 ℃ and 200rpm for 1h; 100. Mu.L of the bacterial liquid is smeared on LB solid medium (containing kanamycin) and is cultured for 12-16 hours in an inverted mode at 37 ℃.
(8) Colonies were verified by PCR. Positive single colonies were cultured in 10. Mu.L of LB medium, and this bacterial liquid was used as a template for PCR identification (see Table 6). Colony PCR products are detected by 1% agarose gel electrophoresis, positive clones are placed in LB liquid culture medium (containing kanamycin) for amplification culture, and bacterial liquid of the positive clones is selected for sequencing analysis, so that CdMATE1 and CdSTAR1 full-length cDNA sequences are obtained.
TABLE 6 reaction System for colony PCR
Colony PCR reaction parameters were as follows:
1.3.2 bioinformatics analyses of CdMATE1 and CdTAR 1
Physicochemical properties of the CdMATE1 and CdSTAR1 genes were analyzed using ExpASYProt software; the tertiary structure is predicted by SWISS-MODEL software; the conserved domain is analyzed by NCBI website; the transmembrane domain was analyzed using TMHMM 2.0 online website; the phylogenetic tree was analyzed using MEGA7 software.
2 results and analysis
2.1 cloning of CdMATE1 and CdSTAR1
The homology of the protein and MATE family genes is extremely high through NCBIblast homology comparison, so the gene is named as CdMATE1; protein STAR1 has very high homology with STAR1 family genes and is therefore designated CdSTAR1. The experiment results are shown in fig. 1, which further uses the root system cDNA of the aluminum-resistant germplasm A22 of the bermuda grass under the treatment of 500 mu M aluminum as a template to amplify the full-length cDNA sequences of CdMATE1 and CdSTAR1. The size of the CdMATE1 gene is 1,800bp (figure 1A), the size of the CdSTAR1 gene is 549bp (figure 1B), and the sequence of the CdMATE1 gene and the CdSTAR1 gene are consistent with the sequencing result of a transcriptome through sequencing analysis.
2.2 analysis of CdMATE1 and CdTAR 1 proteins
(1) Physicochemical property analysis of CdMATE1 and CdSTAR1 proteins
Analysis by ExPASyProt software shows that CdMATE1 gene codes 598 amino acid residues, the molecular weight of the protein is 62.56kD, the theoretical isoelectric point is 7.62, and the protein is hydrophilic protein; the CdSTAR1 gene codes 182 amino acid residues, the molecular weight of the protein is 20.24kD, the theoretical isoelectric point is 9.96, and the protein is hydrophilic protein (see Table 7).
TABLE 7 analysis of protein information for CdMATE1 and CdSTAR1
(2) Analysis of the secondary Structure of CdMATE1 and CdSTAR1 proteins
Analysis of CdMATE1 and CdSTAR1 by SOPMA software revealed that CdMATE1 protein contained, as shown in fig. 2, an α -helix of 328, 55.97, an extended chain of 49, 8.36, a β -turn of 19, 6.17% and a random coil of 190, 32.42% (fig. 2A); cdSTAR1 protein contains 75 alpha-helices, accounting for 41.21%, and extends to 27, 14.84%; beta-turns were 12, 6.59% and random coil 68, 37.36% (fig. 2B).
(3) Prediction of the tertiary structure of CdMATE1 and CdSTAR1 proteins
The three-level structure of CdMATE1 and CdSTAR1 is predicted by SWISS-MODEL software, as shown in figure 3, the result shows that the coverage rate of CdMATE1 protein and the template is 100%, and the sequence consistency is 79.58% (figure 3A); cdSTAR1 had 100% coverage with template with sequence identity 70.99% (FIG. 3B)
(4) Conserved domain analysis of CdMATE1 and CdSTAR1
To further understand the structure of the CdMATE1 and CdSTAR1 genes, the domains were analyzed using CDD Tools software. The experimental results show that: cdMATE1 belongs to a MATE family member, and contains a conserved sequence of MATE family; cdSTAR1 contains a conserved sequence of the P-loopNTPase family, possessing a highly conserved adenosine triphosphate binding cassette (ABC).
(5) Transmembrane structural analysis of CdMATE1 and CdSTAR1
The transmembrane domains of CdMATE1 and CdSTAR1 were analyzed using TMHMM servervv2.0 software, as shown in fig. 4, and the results indicated that CdMATE1 contained 10 transmembrane domains (fig. 4A) while CdSTAR1 protein did not have a transmembrane domain (fig. 4B). Analysis by SignalP 5.0server software revealed that CdMATE1 and CdTAR 1 were free of signal peptides, not secreted proteins. The WoLF PSORT predictive analysis found that CdMATE1 was localized on the intima system (endomembrane system), suggesting that it is a membrane protein; cdSTAR1 is localized on the nucleus (nucleolus), suggesting that it is a nucleoprotein.
2.3CdMATE1 and CdTAR 1 phylogenetic tree analysis
(1) Cdmate1 phylogenetic tree analysis
From the phylogenetic tree, it can be seen that different plant MATE proteins can be divided into 4 groups, as shown in fig. 5. Wherein, the bermuda grass CdMATE1 belongs to 1 branch with Arabidopsis thaliana AtMATE-11, atMATE-10, rice OsMATE-20, osMATE-19, corn ZmMATE-2 and wheat TaMATE-26, taMATE-27 in class 1, and has highest homology. (2) CdSTAR1 phylogenetic tree analysis
As can be seen from FIG. 6, different plant STAR1 proteins can be divided into 4 groups. Wherein, the bermuda grass CdSTAR1 belongs to 1 branch with the same genus as the Arabidopsis thaliana AtSTAR1-12, the rice OsSTAR1-18, the corn ZmSTAR1-11, the wheat TaSTAR1-26, the TaSTAR1-24 and the TaSTAR1-25 in class 3, and has the highest homology.
Note that:
fig. 5: arabidopsis thaliana (AtMATE-10, atMATE-11, atMATE-12, atMATE-13, atMATE-14, atMATE-15, atMATE-16, atMATE-17, atMATE-18); rice (OsMATE-20, osMATE-19, osMATE-21, osMATE-22, osMATE-23, osMATE-24, osMATE-25); corn (ZmMATE-2, zmMATE-5, zmMATE-3, zmMATE-7, zmMATE-9, zmMATE-8, zmMATE-6, zmMATE-4); wheat (TaMATE-26, taMATE-27, taMATE-30, taMATE-29, taMATE-28, taMATE-31, taMATE-33)
Fig. 6: arabidopsis thaliana (AtSTAR 1-12, atSTAR1-17, atSTAR1-13, atSTAR1-12, atSTAR1-16, atSTAR 1-14); rice (OsSTAR 1-18, osSTAR1-22, osSTAR1-20, osSTAR1-23, osSTAR1-19, osSTAR 1-21); corn (ZmSTAR 1-11, zmSTAR1-9, zmSTAR1-3, zmSTAR1-10, zmSTAR1-2, zmSTAR1-7, zmSTAR1-6, zmSTAR1-8, zmSTAR1-5, zmSTAR 1-4); wheat (TaSTAR 1-26, taSTAR1-24, taSTAR1-25, taSTAR1-29, taSTAR1-27, taSTAR1-30, taSTAR 1-28)
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art who is skilled in the art to which the present invention pertains will appreciate that the technical scheme and the inventive concept according to the present invention are equally substituted or changed within the scope of the present invention.

Claims (9)

1. The bermuda grass aluminum response gene is characterized in that the gene is CdMATE1 and CdSTAR1, the nucleotide sequence of the CdMATE1 is shown as SEQ ID NO. 1, and the nucleotide sequence of the CdSTAR1 is shown as SEQ ID NO. 2.
2. The primer for amplifying the aluminum response gene of bermuda grass according to claim 1, wherein the nucleotide sequence of the upstream primer for amplifying the CdMATE1 gene is shown as SEQ ID NO. 3, and the nucleotide sequence of the downstream primer is shown as SEQ ID NO. 4; the nucleotide sequence of the upstream primer for amplifying the CdSTAR1 gene is shown as SEQ ID NO. 5, and the amino acid sequence of the downstream primer is shown as SEQ ID NO. 6.
3. The method for cloning the aluminum response gene of bermuda grass according to claim 1, which comprises the following steps:
s1, aluminum stress treatment: carrying out water planting rooting on aluminum-resistant bermuda grass germplasm A22, selecting seedlings with root length of about 5cm and consistent growth vigor, putting the seedlings into aluminum treatment liquid for aluminum stress treatment, and collecting root systems after 24 hours of treatment; wherein the aluminum treatment solution is a mixed solution of calcium chloride and aluminum chloride, the concentration of the calcium chloride in the mixed solution is 450-550 mu M, the concentration of the aluminum chloride is 450-550 mu M, and the pH value of the aluminum treatment solution is 3.5-4.5;
s2, amplifying the full-length sequence: taking root system cDNA of the bermuda grass after aluminum stress treatment for 24 hours in the step S1 as a template, respectively carrying out PCR amplification on target fragments by using the primer of claim 2 and using high-fidelity enzyme GXL, and carrying out electrophoresis detection and recovering CdMATE1 and CdSTAR1 fragments after the PCR amplification is finished;
s3, adding tail A into PCR products: adding a poly (A) tail to the 3' -end of the PCR product by using a DNAA-Tailing Kit to obtain a CdMATE1 gene added with the A tail and a CdSTAR1 gene added with the A tail respectively;
s4, connection reaction: performing enzyme digestion reaction on plasmid pCXSN-Myc by using restriction enzyme XcmI, detecting enzyme digestion products by electrophoresis, recovering corresponding fragments, respectively connecting CdMATE1 plasmid with A tail and CdSTAR1 gene with A tail to linearized plant expression vector pCXSN-Myc by using T4 ligase, and obtaining products which are recombinant plasmids pCXSN-Myc-CdMATE1 and pCXSN-Myc-CdSTAR1;
s5, escherichia coli transformation: respectively adding recombinant plasmids pCXSN-Myc-CdMATE1 and pCXSN-Myc-CdSTAR1 into competent cells of escherichia coli Trans T1, adding an LB culture medium for culturing for 50-70 min, coating bacterial liquid on an LB solid culture medium containing kanamycin, and culturing for 12-16h in an inverted mode;
s6, colony verification by PCR: and (3) detecting colony products cultured by the solid culture medium in the step (S5), selecting positive clone colonies, placing the positive clone colonies into an LB liquid culture medium containing kanamycin for amplification culture, and selecting positive clone bacterial liquid for sequencing analysis to obtain CdMATE1 and CdSTAR1 full-length cDNA sequences.
4. The method for cloning the aluminum response gene of bermuda grass according to claim 3, wherein the specific operations of the hydroponic rooting in the step S1 are as follows: cutting creeping stem segments of aluminum-resistant bermuda grass germplasm A22, immersing stem segments in a nutrient solution for water culture rooting, wherein the nutrient solution is 1/2Hoagland nutrient solution, and the pH value is 5.8.
5. A method of cloning a bermudagrass aluminum responsive gene according to claim 3, wherein the PCR amplification system in step S2 is: 5 XPS GXL Buffer:10 μL,2.5mM dNTP mix:1.6 μL, forward primer:0.5 μl, reverse primer: 0.5. Mu.L, 20-fold dilution of cDNA:1 mu L, GXL Enzyme：0.4μL，ddH 2 O：12μL，Total：20μL；
The PCR reaction parameters are as follows:
6. the method for cloning the aluminum response gene of bermuda grass according to claim 3, wherein the reaction system for adding the A tail in the step S3 is as follows: 10 XA-Tailing Buffer:5 μL,2.5mM dNTP mix: 4. Mu.L, A-tagging Enzyme: 0.5. Mu.L, 0.5-5. Mu.g of end smooth DNA fragment: 10 mu L ddH 2 O:30.5 μL, total: 50. Mu.L; reaction conditions: 72 ℃ for 20min; placing on ice for 2min; preserving at 4 ℃.
7. The method for cloning the aluminum response gene of bermuda grass according to claim 3, wherein the reaction system of the cleavage reaction in the step S4 is as follows: plasmid:3 μg (xμL), 10 XNEB Buffer:3 μL, xcmI:1 mu L ddH 2 O: (26-x) μL, total: 30. Mu.L; reaction conditions: the reaction is carried out for more than 3 hours at 37 ℃.
8. A method of cloning an aluminum response gene from bermuda grass according to claim 3, wherein the linkage system of the linearized plant expression vector pCXSN-Myc in step S4 is: 50ng Vector:1 μL,50ng Insert DNA:2 mu L,10 xT 4 ligase Buffer：1μL，5U/L T4 ligase：1μL，ddH 2 O:6 μL, total: 10. Mu.L; reaction conditions: the reaction was carried out at 4℃overnight.
9. The method for cloning the aluminum response gene of bermuda grass according to claim 3, wherein in the step S6, colony PCR products are detected by adopting 1% agarose gel electrophoresis, and the reaction system of the colony PCR is as follows: 2×Green r Taq mix:10 μL, forward primer:0.5 μl: reverse primer:0.5 μl: and (3) a template: 2 mu L, ddH 2 O:7 μL, total: 20. Mu.L; colony PCR reaction parameters were as follows:
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007119381A1 (en) * 2006-03-16 2007-10-25 National University Corporation Okayama University Gene participating in aluminum tolerance and utilization of the same
JP2008054532A (en) * 2006-08-29 2008-03-13 Okayama Univ Gene participating in aluminum tolerance and use thereof
CN101883572A (en) * 2007-05-17 2010-11-10 美国政府农业部 Sorghum aluminum tolerance gene, sbmate
CN102154318A (en) * 2011-01-30 2011-08-17 浙江大学 Snailflower citric acid transporter gene VuMATE and use thereof
CN104561026A (en) * 2013-10-29 2015-04-29 中国农业大学 Application of peanut AhFRDL1 gene in improving aluminum toxicity stress resistance of plants

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007119381A1 (en) * 2006-03-16 2007-10-25 National University Corporation Okayama University Gene participating in aluminum tolerance and utilization of the same
JP2008054532A (en) * 2006-08-29 2008-03-13 Okayama Univ Gene participating in aluminum tolerance and use thereof
CN101883572A (en) * 2007-05-17 2010-11-10 美国政府农业部 Sorghum aluminum tolerance gene, sbmate
CN102154318A (en) * 2011-01-30 2011-08-17 浙江大学 Snailflower citric acid transporter gene VuMATE and use thereof
CN104561026A (en) * 2013-10-29 2015-04-29 中国农业大学 Application of peanut AhFRDL1 gene in improving aluminum toxicity stress resistance of plants

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