CN117660479A - Gene for improving aluminum tolerance of forage grass plants and application thereof - Google Patents
Gene for improving aluminum tolerance of forage grass plants and application thereof Download PDFInfo
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- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 title claims abstract description 58
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 52
- 239000004459 forage Substances 0.000 title claims abstract description 14
- 244000025254 Cannabis sativa Species 0.000 title abstract description 14
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- 235000017587 Medicago sativa ssp. sativa Nutrition 0.000 claims abstract description 37
- 241000196324 Embryophyta Species 0.000 claims description 8
- 239000004411 aluminium Substances 0.000 claims description 5
- 230000006872 improvement Effects 0.000 claims description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 abstract description 51
- 230000009261 transgenic effect Effects 0.000 abstract description 44
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- 238000010230 functional analysis Methods 0.000 description 7
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- COXVTLYNGOIATD-HVMBLDELSA-N CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O Chemical compound CC1=C(C=CC(=C1)C1=CC(C)=C(C=C1)\N=N\C1=C(O)C2=C(N)C(=CC(=C2C=C1)S(O)(=O)=O)S(O)(=O)=O)\N=N\C1=CC=C2C(=CC(=C(N)C2=C1O)S(O)(=O)=O)S(O)(=O)=O COXVTLYNGOIATD-HVMBLDELSA-N 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a gene for improving aluminum resistance of forage grass plants and application thereof, and belongs to the technical field of genetic engineering. The gene comprises at least one gene shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3. The aluminum tolerance and the citric acid secretion of the double-gene and triple-gene combined transgenic alfalfa constructed by the invention are obviously enhanced, and the double-gene and triple-gene combined alfalfa created by utilizing the molecular design of the citric acid synthesis and secretion metabolic pathway has feasibility and great significance, is hopeful to improve the yield of the alfalfa under the acid condition on the basis of not changing the quality of pasture, and promotes the development of the south livestock industry in China.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a gene for improving aluminum tolerance of forage grass plants and application thereof.
Background
Alfalfa is a quality leguminous grass, known as the "grass king", and is widely planted worldwide (Liu et al 2016). The south grass eating animal husbandry in China develops rapidly, the demand for high-quality leguminous grass increases rapidly, and the industry prospect of developing south alfalfa is wide (Cao Hong, etc., 2011). Aluminum is considered to be a major factor in acid soil limiting alfalfa planting and harvest. In addition, most of the southern soil in China is acidic, and the free aluminum ions (Al < 3+ > in the soil is the main) become key factors (Wu Daoming and the like, 2013) for limiting the southern cultivation of high-quality leguminous forage. The traditional solution method is to apply lime and organic fertilizer to soil, but the traditional lime soil improvement measure is difficult to eliminate the inhibition effect of deep acid soil on alfalfa growth, and has the problems of high cost, short time, lack of feasibility and the like (Sade et al, 2016). In addition, other species of alfalfa, including alfalfa species, lack aluminum-resistant germplasm, and it is difficult to obtain aluminum-resistant varieties using conventional breeding (Wei Yunmin et al, 2020). Therefore, it is particularly important to improve the aluminum resistance of alfalfa.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a gene for improving the aluminum resistance of forage grass plants.
In order to achieve the above purpose, the technical scheme adopted by the invention for solving the technical problems is as follows:
a gene for improving aluminium tolerance of forage grass plants comprises at least one gene shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
Further, the genes consist of three genes of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
Further, the gene consists of two genes of SEQ ID NO.1 and SEQ ID NO. 2.
The application of the gene in screening aluminum-resistant forage grass plants or forage grass plant germplasm resource improvement.
Further, the forage grass plant is alfalfa.
The invention has the beneficial effects that:
the aluminum tolerance and the citric acid secretion of the double-gene and triple-gene combined transgenic alfalfa constructed by the invention are obviously enhanced, and the double-gene and triple-gene combined alfalfa created by utilizing the molecular design of the citric acid synthesis and secretion metabolic pathway has feasibility and great significance, is hopeful to improve the yield of the alfalfa under the acid condition on the basis of not changing the quality of pasture, and promotes the development of the south livestock industry in China.
Drawings
FIG. 1 is pSuper1300-GmM4-ΔGmHA4-schematic representation of the structure of the T-DNA region of the double gene combination vector of eGFP;
FIG. 2 is a diagram of pSuper1300-GmM4-ΔGmHA4-MsCSX1-schematic representation of T-DNA region structure of eGFP three-gene combination vector;
FIG. 3 shows the expression levels of GmM 4.DELTA.GmHA 4 in transgenic tobacco of (GmM 4.DELTA.GmHA 4) and 35S for the double gene combination;
FIG. 4 is a phenotype and functional analysis of 35S: (GmM 4-. DELTA.GmHA 4) transgenic tobacco; wherein A: root relative elongation; b: root tip citrate secretion; c: 0. 50 mu mol/L AlCl 3 The aluminum content of the root tip of the tobacco is treated for 24 hours; WT: wild type; OE: an over-expression type; the different letters on the bar graph represent P<Significance of 0.05;
FIG. 5 is a functional analysis of GmM, mM7, gmMATE13 transgenic tobacco; wherein A: root Relative Elongation (RRE); b: root tip citrate secretion; c: root tip aluminum content; WT: wild type; OE: an over-expression type; different letters on the bar graph represent significance of P < 0.05;
FIG. 6 is a functional analysis of ΔGmHA4 transgenic tobacco; wherein A: root Relative Elongation (RRE); b: root tip citrate secretion; c: root tip aluminum content; different letters on the bar graph represent significance of P < 0.05;
FIG. 7 is a graph showing the gene expression level of (GmM- ΔGmHA4-MsCSX 1) transgenic tobacco for a three gene combination 35S, wherein WT: wild type; OE: an over-expression type; different letters on the bar graph represent significance of P < 0.05;
FIG. 8 is a functional analysis of 35S: (GmM 4-. DELTA.GmHA 4-MsCSX 1) transgenic tobacco; wherein A: root Relative Elongation (RRE); b: root tip citrate secretion; c: root tip aluminum content; WT: wild type; OE: an over-expression type; different letters on the bar graph represent significance of P < 0.05;
FIG. 9 is a functional analysis of GmFER84 transgenic tobacco, A: citric acid secretion amount; b: root tip aluminum content; c: malondialdehyde content; d: root Relative Elongation (RRE); WT: wild type; OE: an over-expression type; 0. 25, 50 mu mol/L AlCl 3 Treating for 24 hours; the different letters on the bar graph represent P<Significance of 0.05;
FIG. 10 is a graph of M811, (GmM 4-. DELTA.GmHA 4) identification of aluminum tolerance function of transgenic alfalfa roots; wherein A: root tip citrate secretion; b: root tip soluble protein content; c: root tip aluminum content; d: root tip H 2 O 2 The content is as follows; e: evan blue staining of root tip. F: root Relative Elongation (RRE). WT: wild type; OE: an over-expression type; 0. 25 mu mol/L AlCl 3 Treating for 24 hours; the different letters on the bar graph represent P<Significance of 0.05;
FIG. 11 is an analysis of the aluminum tolerance function of (GmM- ΔGmHA4-MsCSX 1) transgenic alfalfa; wherein A: the citric acid secretion amount of the root tip of the alfalfa; b: the soluble protein content of the root tip of the alfalfa; c: the aluminum content of the root tip of the alfalfa; d: alfalfa root tip H 2 O 2 Is contained in the composition; e: the alfalfa root tip is dyed with Evan blue; f: root Relative Elongation (RRE); WT: wild type; OE: an over-expression type; the different letters on the bar graph represent P<Significance of 0.05.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and all the inventions which make use of the inventive concept are protected by the spirit and scope of the present invention as defined and defined in the appended claims to those skilled in the art.
EXAMPLE 1 cloning of the Gene of interest and vector construction
1. Cloning
At 2017, month 9, at 0.5mmol/L CaCl 2 After pretreatment for 12 hours at pH 4.5, 50. Mu. Mol/L was addedAlCl 3 And (3) processing for 24 hours, extracting RNA, taking cDNA obtained by reverse transcription as a template, designing specific primers by comparing a Phytozome gene library (https:// Phytozome. Jgi. Doe. Gov/pz/portal. Html#) with NCBI comparison sequences (https:// blast. NCBI. Lm. Nih. Gov/blast. Cgi), and carrying out PCR amplification by using the full-length coding sequences of soybean genome GmM (SEQ ID NO. 1), gmHA4 (SEQ ID NO. 2) and MsCSX1 (SEQ ID NO. 3) to obtain the full-length coding sequence. The primer sequences were as follows:
GmM4-35S-F：5’-GAGAACACGGGGGACGAGCTCATGGACGAGAATAGAAGTTCC-3’；
GmM4-35S-R：5’-AATGTTTGAACGATCGTCGACTCATTTGCAGTGTCCTTGTTG-3’；
GmHA4-35S-F：5’-GAGAACACGGGGGACGAGCTCATGGGCGGTATCAGCCTTGA-3’；
GmHA4-35S-R：5’-AATGTTTGAACGATCGTCGACTCAACTCAGGACGTAGCGGAT-3’；
MsCSX1-35S-F：5’-GAGAACACGGGGGACGAGCTCATGTCAACCGATTCACAACC-3’；
MsCSX1-35S-R：5’-AATGTTTGAACGATCGTCGACCTACATCCTAGATCCAGCAAG-3’；
the PCR product is subjected to 1% agarose gel electrophoresis to detect a GmFER84 target fragment (other genes refer to the method), the target fragment is recovered by using an OMEGA gel recovery kit after the band size is correct, and the target fragment is connected with a pMD19-T vector (16 ℃ for 8 hours), and the GmFER84 is used for transforming E.coli DH5 alpha competent cells.
Picking up single colony of GmFER84 with a sterile gun head, detecting PCR of the colony of GmFER84 with a detection primer M13F/R, picking up positive colony, culturing at 37 ℃ and extracting GmFER84 plasmid (OMEGA plasmid extraction kit), after positive detection by electrophoresis, sequencing by a Optimaceae gene company, and comparing the obtained DNA sequence of the DANBAOHEI soybean aluminum-resistant related gene with the sequence in a soybean genome to obtain the target gene. Meanwhile, the GmHA4 gene is modified to express the gene without a C-terminal self-repressing region, and the modified gene is named delta GmHA4.
2. Vector construction
(1) Construction of double-Gene Combined vector
Inserted Gene Expression Cassette (GEC): the vector pSuper1300-eGFP is taken as a framework, poly (A) -NOS-35SPPDK-35S is synthesized by genes, and the primers XbalI-poly (A) and 35S-SalI are utilized to clone and insert the primers between MAS and eGFP of the vector in one step, so that 3 GECs are formed between MAS at the RB end and eGFP at the LB end: MAS-poly (A), NOS-35SPPDK, 35S-eGFP-NOS.
F:5’-CTCGATACACCAAATCGACTCTAGAGATCGTTCAAACATTTGGCA-3’；
R:5’-TATTTAAATGTCGACCCCGGGGGAGCAAGGGGAGAGGGAG-3’。
Construction of a double-gene combination 35S (GmM-delta GmHA 4) vector: the modified vector is taken as a framework, and 2 GECs are utilized: MAS-poly (A), NOS-35SPPDK, and the primer XbalI was designedGmM4-XbalI、SmaI-ΔGmHA4-SmaI, one-step cloning, construction of an over-expression vector comprising MAS, gmM4, poly (A), NOS, delta GmHA4, 35SPPDK, 35S, eGFP, NOS, 35S, hygR, poly (A) and other elements, and the constructed vector is named pSuper1300-GmM4-ΔGmHA4eGFP (FIG. 1).
XbalI-GmM4-XbalI-F:5’-ATACACCAAATCGACTCTAGAATGGACGAGAATAGAAGTTCC-3’；
XbalI-GmM4-XbalI-R:5’-AAATGTTTGAACGATCTCTAGACTAAGGCAGTGAGCGACCTCT-3’；
SmaI-ΔGmHA4-SmaI-F：5’-CTCTCCCCTTGCTCCCCCGGGATGGGCGGTATCAGCCTTGA-3’；
SmaI-ΔGmHA4-SmaI-R:5’-GTATTTAAATGTCGACCCCGGGTCAACTCAGGACGTAGCGGATT-3’；
(2) Construction of three-Gene combination vector
With the double gene combination vector pSuper1300-GmM4-ΔGmHA4eGFP as skeleton, designing the primer SalI-MsCSX1SalI, one-step cloning, construction of a plant expression vector comprising the three-gene combination 35S of MAS, gmM4, poly (A), NOS, ΔGmHA4, 35SPPDK, 35S, msCSX1, eGFP, NOS, 35S, hygR, poly (A), etc. (GmM. DELTA.GmHA 4-MsCSX 1), and designated pSuper1300-GmM4-ΔGmHA4-MsCSX1eGFP (FIG. 2).
SalI-MsCSX1-SalI-F:5’-TGTTACTAGATCCCCGGGGTCGACTGAGACTTTTCAACAAAGGG-3’；
SalI-MsCSX1-SalI-R:5’-CCACTAGTATTTAAATGTCGACCATCCTAGATCCAGCAAGTCTC-3’。
Example 2 transformation of tobacco and functional identification
1. Screening and functional identification of double-gene combination 35S (GmM 4-delta GmHA 4) transgenic tobacco
(1) 35S (GmM 4-delta GmHA 4) screening of transgenic tobacco
pCAMBIA1300-35S was prepared by the Agrobacterium (A. Tumefaciens) Gv 3101-mediated leaf disc method (Wei et al 2020)GmM4-ΔGmHA4Tobacco transformed by mCherry, screening positive buds by LUYOR-3415RG, continuing culturing until rooting, and performing fluorescence rechecking, wherein 4 strains with stronger fluorescence are selected from each gene, namely OE-1, OE-2, OE-3 and OE-4. The results of RT-qPCR showed that the relative expression levels of GmM, Δgmha4 genes in transgenic tobacco were significantly up-regulated compared to WT (P<0.05, fig. 3).
(2) Phenotypic analysis of 35S: (GmM 4-. DELTA.GmHA 4) transgenic tobacco under aluminum stress
Root tip cell walls are the primary target of aluminum toxicity (Lavhale et al, 2018). At 50 mu mol/L AlCl 3 At (pH 4.5), 35S: (GmM 4-. DELTA.GmHA 4) the Root Relative Elongation (RRE) of the transgenic tobacco was 1.39-1.48 times (P) as high as that of WT<0.05, fig. 4A), demonstrating that the growth inhibition of the transgenic tobacco root system is significantly reduced compared to WT.
(3) Functional analysis of (GmM 4-delta GmHA 4) tobacco over-expressed double-gene combination 35S
At 50 mu mol/L AlCl 3 Under the condition of (pH 4.5), 35S is that the root tip citric acid secretion amount of (GmM 4-delta GmHA 4) transgenic tobacco is 4.94-5.97 times (P) of WT<0.05, fig. 4B); root tip aluminum content was 0.706 times that of wild type (P<0.05, fig. 4C). The above shows that the over-expression of 35S under the aluminum stress (GmM 4-delta GmHA 4) can obviously improve the secretion of root tip citric acid, thereby improving the aluminum tolerance of transgenic plants.
(4) Comparison of overexpressed double Gene combinations with monogenic GmM4, deltaGmHA 4 transgenic tobacco
50μmol/L AlCl 3 Under the condition of (pH 4.5), the comparison result of the double-gene combination 35S: (GmM-delta GmHA 4) and the single gene 35S:: gmM4, 35S::: delta GmHA transgenic tobacco shows that the transgenic tobaccoRRE of grass was 1.39-1.48, 1.23-1.37, 1.15-1.33 times that of WT (P<0.05, FIG. 4A, FIG. 5A, FIG. 6A), the amount of citric acid secreted is 4.94-5.94, 3.39-4.53, 1.98-3.10 times (P) that of WT, respectively<0.05, FIG. 4B, FIG. 5B, FIG. 6B) and root tip aluminum content was 0.706, 0.735, 0.884 times (P) as high as that of WT, respectively<0.05, fig. 4C, fig. 5C, fig. 6C).
Comparison of aluminum resistance of the three: the transgenic tobacco of (GmM-delta GmHA 4) > single citrate porin gene GmM or single hydrogen pump gene delta GmHA4 is probably related to the fact that the citrate porin GmM4 provides a channel for citric acid secretion, and the modified hydrogen pump delta GmHA4 provides continuous power for citric acid secretion (Briskin et al, 1990; zhou et al, 2013) and verifies the feasibility of molecular design of a citric acid-proton transport system.
2. Screening and functional identification of transgenic tobacco (GmM-delta GmHA4-MsCSX 1) with three genes combined 35S
(1) Construction of three-gene combined expression vector and screening of tobacco positive plants
The three-gene combined vector 35S (GmM-delta GmHA4-MsCSX 1) is transformed into tobacco by an agrobacterium Gv3101 mediated leaf disc method (Wei et al, 2020), positive buds are screened by fluorescence, the tobacco is continuously cultured until rooting, the fluorescence is rechecked, and positive plants with stronger fluorescence are selected and named OE-1, OE-2, OE-3 and OE-4 for aluminum resistance function verification. The qRT-PCR results showed that the relative expression levels of transgenic tobacco root tips GmM4, Δgmha4, msCSX1 were significantly up-regulated compared to WT (P <0.05, fig. 7).
(2) Over-expression 35S under aluminum stress (GmM-delta GmHA4-MsCSX 1) tobacco root growth assay
At 50 mu mol/L AlCl 3 At (pH 4.5), the Root Relative Elongation (RRE) of transgenic tobacco (GmM-DeltaGmHA 4-MsCSX 1) is 1.42-1.55 times that of WT (P)<0.05, FIG. 8A), it is demonstrated that 35S: (GmM-DeltaGmHA 4-MsCSX 1) transgenic tobacco relieves aluminum toxicity by root growth under aluminum stress.
(3) 35S functional analysis of (GmM 4-. DELTA.GmHA 4-MsCSX 1) transgenic tobacco
At 50 mu mol/L AlCl 3 Under the condition of (pH 4.5), three genes are combined35S (GmM 4-delta GmHA4-MsCSX 1) the transgenic tobacco has 10.81-12.58 times (P) of WT<0.05, FIG. 8B), root tip aluminum content was 0.586 times (P)<0.05, fig. 8C). The above shows that (GmM-delta GmHA4-MsCSX 1) can obviously reduce the root tip aluminum content of transgenic tobacco and improve the root tip citric acid secretion amount under aluminum stress, thereby improving the aluminum resistance of the transgenic tobacco.
(4) Comparison of the aluminium tolerance of transgenic tobacco with the transcription factor GmFER84 and the double-Gene and triple-Gene combination
Under the stress of aluminum, the double gene combination 35S (GmM-delta GmHA 4) and the triple gene combination 35S (GmM-delta GmHA4-MsCSX 1) are compared with the aluminum-resistant function of the transcription factor (35S:: gmFER 84) transgenic tobacco, and the result shows that 50 mu mol/L AlCl is obtained 3 Under the condition of (pH 4.5), 35S: (GmM-delta GmHA 4), 35S: (GmM-delta GmHA4-MsCSX 1) and 35S:: (GmFER 84) the RRE of the transgenic tobacco is 1.39-1.48, 1.42-1.55 and 1.27-1.41 times (P) of that of the WT respectively<0.05, FIG. 4A, FIG. 8A, FIG. 9D), the amount of citric acid secreted was 4.94-5.94, 10.42-12.58, 1.70-1.79 times (P) that of WT, respectively<0.05, FIG. 4B, FIG. 8B, FIG. 9A) and root tip aluminum content was 0.706, 0.586, 0.74 times (P) as high as that of WT, respectively<0.05, fig. 4C, fig. 8C, fig. 9B).
Example 3
1. Double gene combination M811 (GmM-delta GmHA 4) alfalfa rooting aluminium tolerance identification
Under normal conditions (0. Mu. Mol/L AlCl) 3 pH 4.5), compared with the wild type, M811 is (GmM 4-delta GmHA 4) root tip aluminum content, soluble protein and H of transgenic alfalfa hairy root 2 O 2 The content is not significantly different (P>0.05, fig. 10, B, C, D).
Wherein, the sequence of the M811 promoter is as follows:
CAAACTCATGACAATAGGAAGAACAATCTACAATATAGGAACAATATGACCTTATATAAATATTTTGTTTTGGTGTAACATTTTTCTTCCAATATGACCATTCACTTAACTACTTACTACAGTTTTATCGTTTCTTGCCAAAAAGTGAAAAGATGGCCATGTTAATGTCTATCTTGACTCTAGTATTCGAATAAAATTTATCTAAAAGTGAGTTGAACATAAAATTTGAGATTATATAGAATCAAAA。(SEQ ID NO.4)
25μmol/L AlCl 3 under the condition of (pH 4.5), M811: gmM 4-delta GmHA4 transgenic alfalfa hairy rootIs significantly increased compared to wild type (P<0.05, FIG. 10A), soluble protein had an upward trend (FIG. 10B); root tip aluminum content and H 2 O 2 The content is obviously reduced (P)<0.05, FIG. 10C, D), the root tip Evan blue staining is obviously shallower (FIG. 10E), showing that M811: (GmM-delta GmHA 4) the adsorption of aluminum ions on the root tip of transgenic alfalfa is obviously reduced compared with the wild type, and the overexpression of the M811: (GmM-delta GmHA 4) gene under the stress of aluminum can obviously reduce the aluminum content of the root tip and improve the citric acid secretion amount of the root tip, so that the aluminum tolerance of the alfalfa is improved.
Compared with WT, 25 mu mol/L AlCl 3 Under the condition of (pH 4.5), the growth inhibition effect of the transgenic alfalfa root system (GmM-delta GmHA 4) is obviously relieved, and the Root Relative Elongation (RRE) is 1.28-1.33 times that of WT (P<0.05, fig. 10F).
2. Three-gene combination M811 (GmM-delta GmHA4-MsCSX 1) alfalfa rooting aluminium tolerance identification
Under normal conditions (0. Mu. Mol/L AlCl) 3 ) Compared with wild type, M811 is (GmM-delta GmHA4-MsCSX 1) root tip aluminum content, soluble protein and H of alfalfa hairy root 2 O 2 The content is not significantly different (P>0.05, fig. 11, B, C, D).
25μmol/L AlCl 3 Under the condition of (pH 4.5), M811: gmM 4-delta GmHA4-MsCSX1, the secretion amount of root tip citric acid and soluble protein of the transgenic alfalfa hairy root are obviously increased (P) compared with the wild type<0.05, fig. 11, A, B), while root tip aluminum content and H 2 O 2 The content is obviously reduced (P)<0.05, FIG. 11C, D), the root tip Evan blue staining is obviously shallower (FIG. 11E), which shows that the aluminum ions adsorbed by the root tip are obviously reduced, and the M811: gmM 4-delta GmHA4-MsCSX1 gene can obviously reduce the root tip aluminum content of transgenic alfalfa and improve the root tip citric acid secretion amount, thereby improving the aluminum tolerance of the alfalfa.
25. Mu. Mol/L AlCl compared to wild type 3 (GmM-DeltaGmHA 4-MsCSX 1) the RRE of the rooting of transgenic alfalfa is 1.31-1.41 times (P) as high as that of WT at (pH 4.5)<0.05, FIG. 11F), indicating that the aluminum toxicity of the transgenic alfalfa root system was alleviated.
The results show that the aluminum resistance and the citric acid secretion of the three-gene combined transgenic alfalfa are obviously enhanced, and the three-gene combined transgenic alfalfa becomes a novel aluminum-resistant alfalfa germplasm created by the research.
Finally, it should be noted that the above-mentioned embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same, and although the present invention has been described in detail with reference to examples, it should be understood by those skilled in the art that modifications and equivalents may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and equivalents are intended to be encompassed in the scope of the claims of the present invention.
Claims (5)
1. A gene for improving the aluminum tolerance of forage plants, which is characterized in that the gene comprises at least one gene shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.
2. The gene according to claim 1, characterized in that it consists of three genes of SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3.
3. The gene according to claim 1, characterized in that it consists of two genes of SEQ ID No.1 and SEQ ID No. 2.
4. Use of a gene as claimed in any one of claims 1 to in the selection of aluminium tolerant forage plants or forage plant germplasm improvement.
5. The use as claimed in claim 4 wherein the forage plant is alfalfa.
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| CN117660451A (en) * | 2023-11-17 | 2024-03-08 | 广安市饲料工业管理站 | Alfalfa root tip specific promoter and application thereof |
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| CN117660451A (en) * | 2023-11-17 | 2024-03-08 | 广安市饲料工业管理站 | Alfalfa root tip specific promoter and application thereof |
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