Medicago sativa MsILL6 gene and application thereof

Abstract

The invention discloses a medicago sativa MsILL6 gene. A nucleotide sequence of a coding region of the medicago sativa MsILL6 gene is as shown in SEQ ID NO. 1; a nucleotide sequence of a coding region of the medicago sativa MsILL6 gene is as shown in SEQ ID NO. The amino acid sequence coded by the nucleotide sequence of the coding region of the alfalfa MsILL6 gene is as shown in SEQ ID NO. 2. The invention also constructs an MsILL6 overexpression vector and successfully transfers the MsILL6 overexpression vector into arabidopsis thaliana and medicago sativa to obtain a transgenic plant. Tests developed by using transgenic arabidopsis thaliana and medicago sativa show that overexpression of MsILL6 reduces the tolerance of transgenic plants to alkali stress.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to alfalfa MsILL6 Genes and their applications. Background Art

[0002] Alfalfa is known as the "king of forage grass". It is a perennial leguminous forage grass widely planted throughout the world and occupies an important position in the development of agriculture and animal husbandry. Alfalfa is an important feed source for the world's animal husbandry. Alfalfa is rich in protein, has high nutritional value, good palatability, and high yield. As a feed, it can significantly improve the economic benefits of green animals. Alfalfa is an excellent forage plant. Proper addition of alfalfa to feed can enhance the growth performance and rumen degradation capacity of ruminants, increase the lean meat rate and weight of pigs, improve the egg-laying performance of poultry, and reduce the cost of animal breeding. In addition, feeding meat rabbits with alfalfa can improve their production performance and meat taste. Even alfalfa grown in saline-alkali land does not affect the development of meat rabbits.

[0003] Plants adapt to saline-alkali stress by changing the morphological structure of their organs. Under different pH, salt or saline-alkali concentration treatments, the root weight and stem and leaf weight of alfalfa decreased as the stress factors increased, among which the changes in the roots were particularly obvious; the unit mass of the leaves decreased as the environmental stress intensified. Salt-alkali stress produces osmotic imbalance or osmotic stress at the root-soil interface, resulting in a decrease in the plant's water absorption capacity, which in turn causes physiological drought and restricts the growth and development of plants. During saline-alkali stress, high concentrations of Na in the soil + It will destroy the homeostasis of cells and cause plant damage, such as destroying the cell membrane structure and inducing ion toxicity. Salt-alkali stress will cause the generation and accumulation of reactive oxygen species (ROS) in plants, thus causing damage to cells. Excessive accumulation of ROS in plants will destroy the normal physiological functions of cells and cause metabolic disorders.

[0004] ILL belongs to the carboxypeptidase family. The members of this family regulate IAA activity by hydrolyzing the amide bond between IAA and amino acids. In Arabidopsis, the ILL family consists of ILR1 (IAA-LEU RESISTANT1), ILL1 (ILR1-like1), ILL2 (ILR1-like2), ILL3 (ILR1-like2), ILL5 (ILR1-like5), ILL6 (ILR1-like6) and IAR3 (IAA-ALARESISTANT3). At present, the research on ILL family members is mainly focused on auxin metabolism.

[0005] Three hydrolases, ILL2, ILR1 and ILL2, participate in the activation of auxin signaling by hydrolyzing IAA-Ala, IAA-Leu and IAA-Phe into biologically active IAA, while ILL6 has very low or even no hydrolysis activity in vitro.

[0006] ILL6 is a member of the Arabidopsis ILL family and is also associated with jasmonic acid (JA) metabolism, an important signaling molecule for plant defense and plant growth and development. ILL6 and IAR3 are involved in chlorophyll degradation and anthocyanin synthesis. AtILL6 The gene plays a role in regulating plant growth and enhancing plant defense. ILL6 and IAR3 play redundant roles in the metabolism of JA-Ile and 12OH-JA-Ile (12-hydroxy-JA-Ile) in vivo, while ILL6 plays a limited role in the overall JA metabolism; in addition, ILL6 cannot hydrolyze exogenous IAA conjugates, but the mechanism is still unclear.

[0007] at present, ILL6 Most of the research on genes focuses on auxin metabolism, and no research has shown whether it responds to salt-alkali stress. ILL6 Functional effects on salt-alkaline tolerance in alfalfa and Arabidopsis thaliana. Summary of the invention

[0008] The present invention aims to solve the above problems and provides a kind of alfalfa MsILL6 Genes and their applications. Specifically, the technical solutions provided by this application are as follows: In the first aspect, the present invention clones a kind of alfalfa for the first time MsILL6 Gene, the alfalfa MsILL6 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.1.

[0009] Furthermore, the alfalfa MsILL6 The amino acid sequence encoded by the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.2.

[0010] In the second aspect, based on the same invention, the present invention also provides the above-mentioned alfalfa MsILL6 The application of genes can make alfalfa MsILL6 The gene is prepared into a recombinant expression vector, an expression cassette, a transgenic cell line, a recombinant bacterium or a recombinant virus to regulate the salt-alkali tolerance of the plant.

[0011] Furthermore, the plant is alfalfa or Arabidopsis thaliana.

[0012] In a third aspect, the present invention also provides a pCAMBIA3301- MsILL6Overexpression vector, the overexpression vector is composed of alfalfa MsILL6 The nucleotide sequence of the coding region of the gene and the construction of the pCAMBIA3301 vector were obtained.

[0013] Furthermore, the primer sequence is: MsILL6 -F:5'-AAGACAGCGACGAACT-3'; MsILL6 -R:5'-GCTCTAACGGCAACAA-3'.

[0014] Further preferably, the MsILL6 -F sequence 5' end also includes a homology arm sequence acgggggactcttgacc; MsILL6 The 5' end of the -R sequence also includes the homology arm sequence ggggaaattcgagctggtcacc.

[0015] Preferably, transgenic technology is an important tool for gene function research and crop genetic improvement, and vector construction is the basis for obtaining transgenic plants. Homologous recombination and enzyme digestion are common methods for vector construction. Compared with enzyme digestion, homologous recombination is simpler in primer design and operation steps, and can construct vectors efficiently and quickly. In actual operation, it was found that the pCAMBIA3301 vector Nco Ⅰ The restriction site contains ATG bases, so when designing primers, it is necessary to cut off the ATG on the vector and design homology arms to avoid affecting the expression of the target gene. In addition, since the concentration of the restriction product of the pCAMBIA3301 vector is low, the reaction system can be appropriately expanded during restriction digestion to increase the product concentration.

[0016] In a fourth aspect, the present invention also provides alfalfa MsILL6 Use of genes in plant salt-alkali tolerance, the MsILL6 Gene overexpression can reduce the tolerance of plants in saline-alkali environments; MsILL6 Gene silencing expression can improve plant tolerance in saline-alkali environments.

[0017] The present invention has the following beneficial effects: Alfalfa Auxin ILL Amidohydrolase MsILL6 IAA-amino acid hydrolase ILR1-like6, ILL6 belongs to the auxin amidohydrolase (IAA-Leucine Resistant1-like Hydrolase, ILR1-likehydrolase, ILL) family. MsILL6 The gene coding region sequence is 1431 bp in length and encodes 476 amino acids. MsILL6It is highly conservative and is a stable acidic protein. MsILL6 With Medicago truncatula ( Medicago truncatula ) The sequence similarity of MtILL6 was as high as 98.53%. MsILL6 Gene overexpression vector (pCAMBIA3301- MsILL6 ).

[0018] Response of transgenic Arabidopsis and mutants to alkaline stress. Arabidopsis was transformed using the Agrobacterium-mediated floral dip method, and 21 positive strains were obtained after screening and identification. Phenotypic observations of wild-type and transgenic Arabidopsis under alkaline stress revealed that overexpression MsILL6 The gene reduces the tolerance of Arabidopsis to alkaline stress. There is no significant difference between transgenic Arabidopsis and wild type under salt stress and drought stress. atill6-1 Homozygous mutant lines; the germination rate of atil16-1 mutant seeds under alkaline stress was significantly higher than that of wild type. There was no significant difference in the germination rate of mutant and wild type Arabidopsis seeds under salt and salt-alkali mixed treatments.

[0019] Genetic transformation of alfalfa and screening of Medicago truncatula mutants. 22 strains of alfalfa were obtained by Agrobacterium-mediated transformation of alfalfa. MsILL6 Positive plants. Under alkaline stress, overexpression MsILL6 The tolerance of transgenic alfalfa to alkaline stress was reduced. MtILL6 1 mutant strain.

[0020] This application builds MsILL6 The overexpression vector was successfully transferred into Arabidopsis and alfalfa to obtain transgenic plants. Experiments using transgenic Arabidopsis and alfalfa showed that overexpression MsILL6 Reduced the tolerance of transgenic plants to alkaline stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0022] Figure 1 : Gel electrophoresis of the target gene clone of the present invention; wherein M: 2000 DNA Marker; 1-8: MsILL6 .

[0023] Figure 2: Construction of the vector of the present invention; wherein A: gel electrophoresis of linear vector; B: pCAMBIA3301- MsILL6 Overexpression vector map.

[0024] Figure 3 : The present invention MsILL6 Gene structure and protein hydrophilicity analysis diagram; A: MsILL6 Gene structure diagram; B: affinity analysis of MsILL6 protein.

[0025] Figure 4 : The prediction results of the signal peptide, protein transmembrane domain and conserved domain of the MsILL6 protein of the present invention; wherein A: protein signal peptide distribution diagram; B: protein transmembrane domain prediction results; C: protein conserved domain prediction results.

[0026] Figure 5 : Prediction of the secondary and tertiary structures of the protein of the present invention; wherein A: prediction result of the secondary structure of the protein, in which blue represents α-helix, red represents extended chain, green represents β-turn, and purple represents random coil; B: protein tertiary structure model.

[0027] Figure 6 : Identification of transgenic positive seedlings of the present invention; wherein A: screening results of transgenic Arabidopsis T1 generation. B: gel electrophoresis diagram of transgenic Arabidopsis identification; WT is wild-type Arabidopsis, and numbers represent transgenic Arabidopsis strains.

[0028] Figure 7 : The present invention MsILL6 Results of differential expression analysis of transgenic Arabidopsis thaliana by qRT-PCR.

[0029] Figure 8 : Morphological changes of Arabidopsis thaliana treated with saline-alkali in the present invention; A: control group; B: alkali treatment group (200 mM NaHCO3); C: salt treatment group (300 mM NaCl); D: salt-alkali mixed treatment group (200 mM NaCl+100 mM NaHCO3). The first row of pictures shows the growth of Arabidopsis thaliana before saline-alkali stress; the second row of pictures shows the growth of Arabidopsis thaliana after 25 days of saline-alkali treatment.

[0030] Fig. 9 : Survival rate of wild type and transgenic Arabidopsis thaliana after treatment with 200 mM NaHCO3 of the present invention.

[0031] Fig.10 : Chlorophyll fluorescence parameters of wild-type and transgenic Arabidopsis thaliana under alkali treatment of the present invention; A, B and C represent the initial fluorescence, maximum optical efficiency and relative chlorophyll content of wild-type and transgenic Arabidopsis thaliana under 200 mM NaHCO3 treatment, respectively, and different lowercase letters represent significant differences at the 0.05 level.

[0032] Fig.11 : The effect diagram of drought treatment of Arabidopsis thaliana of the present invention; Fig.12 : Screening of homozygous mutants of Arabidopsis thaliana of the present invention; wherein A: T0 generation mutant atill6-1 Identification gel electrophoresis, strains 1-6 are homozygous mutants. B: T0 generation mutants atill6-2 Identification gel electrophoresis Figure 1-2 All 5 strains are wild type. Numbers represent mutant plants. G bands verify gene integrity, and T bands verify whether T-DNA is inserted.

[0033] Fig.13 : The wild type and atill6 Germination of mutant Arabidopsis thaliana under different concentrations of salt and alkali treatments; A / B: wild type and mutant Arabidopsis thaliana under different concentrations of NaCl treatments atill6-1 Germination and germination rate of mutant Arabidopsis thaliana. C / D: Wild type and mutant Arabidopsis thaliana under different concentrations of NaHCO3 treatment atill6-1 Germination status and germination rate of mutant Arabidopsis thaliana. Differential expression analysis was performed using the t-test method. “*” represents P <0.05, “**” represents P <0.01.

[0034] Fig.14 : The wild type and atill6 Germination of mutant Arabidopsis thaliana under different concentrations of salt-alkali mixture treatment; A / B: germination of wild type and mutant Arabidopsis thaliana under different concentrations of NaCl and NaHCO3 treatment atill6 Germination status and germination rate of mutant Arabidopsis thaliana. Differential expression analysis was performed using t Inspection method, "*" represents P <0.05, “**” represents P <0.01.

[0035] Fig.15 : A diagram of the genetic transformation process of alfalfa of the present invention; wherein A: co-cultivation of explants in SH3a medium; B / C: callus development in SH3a selection medium; D: MSBK medium induces callus to turn green and differentiate into mature embryonic buds; E / F: SH9a medium induces regeneration of plants.

[0036] Fig.16 : Identification of positive alfalfa plants of the present invention; wherein M: DNA Marker; 1-26: MsILL6 Genetically modified alfalfa.

[0037] Fig.17 : The relative expression difference results of the transgenic alfalfa of the present invention.

[0038] Fig.18 : Salt-treated alfalfa of the present invention; A: 300 mM NaCl treated for 0 d; B: 300 mM NaCl treated for 8 d; C: 300 mM NaCl treated for 15 d; D: 300 mM NaCl treated for 20 d.

[0039] Fig.19 : Alfalfa treated with alkali according to the present invention; wherein A: 0 mM NaHCO3 treated for 0 d; B: 200 mM NaHCO3 treated for 7 d.

[0040] Fig. 20 :The screening of homozygous mutants of Medicago truncatula of the present invention; wherein the T0 generation mutant mtill6 is identified by gel electrophoresis, strains 1-4 and 8 are wild type; strains 6-9 are heterozygous mutants; the numbers represent mutant plants. G band verifies gene integrity, T band verifies Tnt1 Whether to insert.

[0041] Fig.21 :The results of gene cloning and sequencing of the present invention are consistent with MsILL6 Transcriptome DNA sequence alignment.

[0042] Fig. 22 :The results of gene cloning and sequencing of the present invention are consistent with MsILL6 Transcriptome protein sequence alignment; Fig.23 : The present invention MsILL6 Comparison of overexpression vector sequencing results. DETAILED DESCRIPTION

[0043] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. The following embodiments are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way. Based on the embodiments in the present invention, all other embodiments obtained by those skilled in the art without making creative work are within the scope of protection of the present invention. The experimental methods used in the present invention are conventional methods unless otherwise specified. The materials, reagents, etc. used in the present invention, unless otherwise specified, can be obtained from commercial channels. In addition, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0044] Embodiment 1: MsILL6 Gene cloning and overexpression vector construction 1.1 Materials and methods 1.1.1 Test materials The plant material was alfalfa SY4D, and Hoagland nutrient solution was used during plant growth. The pCAMBIA3301 vector was used in this experiment. The experimental materials and reagents are shown in Table 1. Primer synthesis and sequencing were completed by Sangon Biotech (Shanghai) Co., Ltd. The culture medium preparation is shown in Table 2.

[0045] Table 1 Summary of experimental materials and reagents

[0046] Table 2 LB medium configuration

[0047] 1.1.2 MsILL6 Cloning of gene coding regions RNA was extracted from leaves of alfalfa SY4D according to the instructions (Vazyme, Nanjing, China). The extracted RNA was reverse transcribed (Vazyme, Nanjing, China) to obtain cDNA products. Primers were designed using Primer3.0 (Table 3).

[0048] Table 3 Gene cloning primers

[0049] Through specific primer pairs MsILL6 Amplify and sequence, and obtain the target band by comparing and analyzing the sequencing results. The total reaction system is 10 µL (Table 4).

[0050] Table 4 Gene cloning PCR system

[0051] 1.1.3 Construction of overexpression vector Use restriction enzymes Bst EⅡ and Nco Ⅰ Double enzyme digestion was performed on the pCAMBIA3301 vector. The enzyme digestion reaction system is shown in Table 5.

[0052] Table 5 Enzyme digestion reaction system

[0053] Mix the prepared solution by pipetting, centrifuge briefly, and react at 37°C for 1 h. Use homologous recombination enzyme to homologously recombine the digested plasmid with the target gene. The reaction system is shown in Table 6.

[0054] Table 6 Homologous recombination reaction system

[0055] The homologous recombination product was transformed into DH5a chemical competent cells (Weidi, Shanghai, China), and the steps were referred to the instructions. The transformed product was evenly spread on LB solid medium containing Kan (50 mg / mL), and placed upside down in a 37℃ incubator for 12-16 h; monoclonal colonies were selected for bacterial liquid PCR identification, and the primers are shown in Table 7. The amplification program of 2×Phanta MaxMaster Mix (Dye Plus) (Vazyme, Nanjing, China) was as follows: pre-denaturation at 98℃, 30 sec; denaturation at 98℃, 10 sec, annealing at 58℃, 10 sec, extension at 72℃, 10 sec, 34 cycles; complete extension at 72℃, 1 min. The bacterial liquid with the expected band was sequenced and the corresponding bacterial liquid was saved (bacterial liquid: 50% glycerol = 1:1).

[0056] Table 7 Vector identification primers

[0057] The bacterial solution with expected sequencing results was activated, the plasmid was extracted, and it was transferred into EHA105 Agrobacterium competent cells (Weidi, Shanghai, China). The experimental steps were referred to the instructions. The transformation product was evenly spread on LB solid medium containing Kan (50 mg / mL) and Rif (50 mg / mL), and placed upside down in a 28℃ incubator for 60 h. 8-24 single colonies were selected, activated with LB (Kan 50 mg / mL) liquid medium, and then identified by PCR and gel electrophoresis.

[0058] 1.1.4 MsILL6 Gene bioinformatics analysis MsILL6 The genetic structure of genes was analyzed by GSDS. MsILL6 The physicochemical properties of proteins and the hydrophilicity / hydrophobicity of amino acid sequences were analyzed using the ExPasy proteomics analysis database, ProtParam, and Protcale. MsILL6 The signal peptide of the protein was predicted by SignalP-4.1 serve online software, and the transmembrane structure prediction was analyzed using DeepTMHMM. MsILL6 Subcellular localization prediction was performed using the WoLF PSORT server.

[0059] Protein secondary structure prediction was analyzed by SOPMA. MsILL6 Protein conserved domain structure prediction was performed using NCBI's CD-search, and protein tertiary structure prediction was performed using SWISS-MODLE.

[0060] 1.2 Results and Analysis 1.2.1 MsILL6Cloning of gene coding regions After RT-PCR amplification and gel electrophoresis, fragments of the expected size were obtained ( Figure 1 The fragment was separated, purified and sequenced. After the sequencing result was compared with the transcriptome of "Zhongmu No. 1", its sequence was basically consistent with the predicted protein sequence, such as Fig.21 and Fig. 22 shown.

[0061] 1.2.2 MsILL6 Construction of overexpression vector Use restriction enzymes Nco Ⅰ and Bst EⅡ double-digested the pCAMBIA3301 vector, and connected the linearized vector with the target gene. The gel electrophoresis diagram is shown in Figure 2 As shown in A, after transformation into E. coli, the single clone was sequenced, and the sequencing results were compared with the transcriptome sequence. The results show that ( Fig.23 ), successfully constructed pCAMBIA3301- MsILL6 Overexpression vector ( Figure 2 B).

[0062] 1.2.3 MsILL6 Gene Biology Information Analysis 1.2.3.1 MsILL6 Analysis of gene structure and basic physical and chemical properties MsILL6 Gene( Figure 3 A) The total length is 8331 bp, the coding region is 1431 bp, with 5 exons and 4 introns. Analysis by ExPasy database MsILL6 The protein has a relative molecular weight of 52.62 kDa and 476 amino groups, of which alanine (Ala) has the highest proportion, accounting for 9.9%. MsILL6 The protein is a stable acidic protein with an instability coefficient of 31.57 and a theoretical isoelectric point (pI) of 5.9. MsILL6 Medicago truncatula MtILL6 The sequence similarity is as high as 98.53% (Appendix 4). In addition, MsILL6 The extinction coefficient of the protein is 43340.

[0063] MsILL6 The hydrophilicity of protein amino acids is shown in Figure 3 B, the total average hydrophilic index is -0.49, the fat coefficient is 68.9, and it is a hydrophilic protein. Mature proteins must be in specific subcellular organelles to play a stable biological function. Subcellular localization prediction by WoLF PSORT found MsILL6 The encoded protein is mainly localized in the tonoplast and chloroplasts.

[0064] 1.2.3.2 Analysis of protein signal peptides, transmembrane domains and conserved domains MsILL6 The mean S (the average S-score of each amino acid from the N-terminal to the cleavage site) of the protein is 0.717, and the D-score (the weighted average of mean S and Y-max) is 0.623, indicating that the protein has a signal peptide, and the cleavage site of the signal peptide is between amino acids 29 and 30 ( Figure 4 A). TMHMM2.0 server prediction MsILL6 The protein has only one transmembrane structure ( Figure 4 B), contains 20.9 amino acids, and the probability that the N-terminus is on the outside of the cell membrane is 0.98, suggesting that it may be a secretory protein. MsILL6 The protein has a total of 476 amino acids, of which 472 constitute conserved domains. MsILL6 The conserved domain of the gene is between the 5th and 476th amino acids, and the protein is highly conserved ( Figure 4 C).

[0065] 1.2.3.3 Protein secondary and tertiary structure prediction MsILL6 The proportions of random coil (Cc), alpha helix (Hh), extended strand (Ee) and beta turn (Tt) in the protein are 42%, 38.87%, 14.5% and 4.83% respectively. The specific distribution is as follows: Figure 5 As shown in A. Overall, MsILL6 The secondary structure of protein is a random coil. The tertiary structure model of protein is as follows Figure 5 As shown in B, the model consistency is 89.08 and the reliability is 0.87.

[0066] In this study, RT-PCR was used to clone MsILL6 The results showed that the full length of the gene was 1431 bp, encoding 476 amino acids, and the protein was highly conserved. MsILL6 The instability coefficient of the protein is 31.57 and the theoretical isoelectric point is 5.9, which makes it a stable acidic protein. MsILL6 The probability that the N-terminus of the protein is outside the cell membrane is 0.98, which indicates that it is a secreted protein. The results of subcellular localization prediction show that MsILL6 The protein may function in the vacuole membrane and chloroplast. Using homologous recombination, we constructed MsILL6 Overexpression vector (pCAMBIA3301- MsILL6 ), laying the foundation for the transformation of Arabidopsis and alfalfa.

[0067] Embodiment 2: MsILL6 Functional identification in Arabidopsis 2.1 Materials and methods 2.1.1 Test materials The plant material was Arabidopsis thaliana Columbia ecotype Col-0. The experimental materials and reagents are shown in Table 8.

[0068] Table 8 Summary of test materials and reagents

[0069] LB medium is the same as Table 1. The configuration of 1 / 2 MS medium is shown in Table 9.

[0070] Table 9 1 / 2MS medium configuration

[0071] 2.1.2 MsILL6 Screening and identification of transgenic Arabidopsis thaliana 2.1.2.1 Arabidopsis thaliana cultivation Take an appropriate amount of Arabidopsis seeds, wash and soak in dd H2O for 5 min, wash in 75% ethanol for 30 sec, and wash again in dd H2O; add 10% sodium hypochlorite solution and shake in a 28℃ shaking incubator for 5-10 min; wash with dd H2O 5-9 times, and then add dd H2O to swell for 30 min. Spread the washed Arabidopsis seeds evenly on 1 / 2 MS culture medium; vernalize at 4℃ for 2-4 d, grow in a 24℃ light incubator for 7-10 d, and then move into soil (vermiculite: nutrient soil = 3:1).

[0072] 2.1.2.2 Infection of Arabidopsis thaliana by floral dipping Kan (50 mg / mL) and Rif (50 mg / mL) were added to LB liquid medium at a ratio of 1:1000, and activated in a 28°C shaking incubator (200 rpm) for 2-3 days, and repeated once. 600 When the OD value is 1.2-2.0, centrifuge at 4500 rpm for 20 min at 4℃, discard the supernatant, keep the bacterial mass, and add resuspension solution (dd H2O: sucrose = 20:1) to adjust the OD value to 1.2-2.0. 600 Adjust to 0.8-1.2, activate in a 28℃ shaker (100 rpm) for 30 min, add surfactant, immerse Arabidopsis inflorescence in the bacterial solution for 10-15 sec, shade for 16-24 h, and carry out the second infection after 5-7 d.

[0073] 2.1.2.3 Screening and identification of Arabidopsis positive seedlings The mature F0 seeds of Arabidopsis thaliana infection were harvested and dried in a 37°C incubator for about 7 days. After cleaning and disinfection, the seeds were evenly spread on 1 / 2 MS selection medium containing PPT (7.5 mg / L). After vernalization for 2-4 days, they were cultured at 24°C for 8-15 days. The seedlings with 4 true leaves were transferred to soil (vermiculite: nutrient soil = 3:1). Arabidopsis leaf DNA was extracted for PCR verification, and the primers are shown in Table 10. RNA was extracted from positive seedlings and reverse transcribed for qRT-PCR analysis. The primer information is shown in Table 10. Each sample was repeated 3 times, and 2 −∆∆CT Methods Data were analyzed, and the error was SD. The three strains with the highest expression levels were selected, and the seeds of the T2 generation were harvested for subsequent experiments.

[0074] Table 10 Positive seedling identification primers and qRT-PCR primers

[0075] 2.1.3 Arabidopsis stress treatment methods and phenotypic analysis 2.1.3.1 Evaluation method of the effect of saline-alkali treatment on Arabidopsis seed germination Seeds of wild-type and homozygous mutant Arabidopsis lines were treated with salt (100 mM, 150 mM and 200 mM NaCl), alkali (10 mM, 15 mM and 20 mM NaHCO3), salt-alkali mixed treatment (90 mM NaCl +10 mM NaHCO3 and 80 mM NaCl +20 mM NaHCO3) and blank control group, with 3 biological replicates in each group. The culture dishes were placed in a 24℃ light incubator, and the germination rate of seeds was counted for 10 days. The phenotypic differences were observed and recorded. The differential expression analysis was performed using t Test, error analysis is SD.

[0076] 2.1.3.2 Evaluation method of saline-alkali stress and drought treatment on Arabidopsis seedling growth The salt-alkali treatment method is as follows: transgenic Arabidopsis and wild-type Arabidopsis were cultured in 1 / 2 MS medium for 8 days, moved into soil (vermiculite: nutrient soil = 6:1) and grown for 4 weeks. The plants were treated with salt (300 mM NaCl), alkali (200 mM NaHCO3) and salt-alkali mixed treatment (200 mM NaCl + 100 mM NaHCO3). Each treatment had 3 biological replicates. 1 L of the corresponding solution was added to the tray every 48 hours. A control group was set up at the same time to observe the morphological changes of the treated plants.

[0077] The drought treatment method is as follows: stop watering the plants until phenotypic differences appear or the leaves wilt and turn yellow.

[0078] 2.1.3.3 Initial fluorescence Fo, maximum optical efficiency Fv / Fm and relative chlorophyll content SPAD value The chlorophyll fluorescence parameters (initial fluorescence Fo and maximum optical efficiency Fv / Fm) of transgenic and wild-type Arabidopsis treated with 200 mM NaHCO3 solution were measured at different times (0 d, 2 d, 4 d, and 10 d) using a portable fluorescence meter (PAR-FluorPen FP 110) from 9:30 to 11:00 in the morning. Before each measurement, the leaves were kept in darkness for 15 min.

[0079] The SPAD values ​​of the relative chlorophyll content of transgenic Arabidopsis and wild-type Arabidopsis treated with 200 mM NaHCO3 solution were measured using SPAD-502Plus between 9:00 and 9:30 in the morning, and the data were recorded every 48 h. The measured data were subjected to analysis of variance and Duncan's multiple analysis using Excel and SPSS data processing software, and the error analysis was SD.

[0080] 2.1.4 Screening and identification of Arabidopsis mutants use MsILL6 The coding region sequence is in Tair ( https: / / www. Arabidopsis .org / index.jsp ) to obtain homologous genes ILL6 , gene number is AT1G44350. Two mutant strains were found on the Arashare website (https: / / www.arashare.cn / index / ) atill6-1 (N660351) and atill6-2 (N660665). The DNA of Arabidopsis mutants was identified by PCR using a double primer identification method. The primers are shown in Table 11. The identification method is as follows: atill6-1 -LP and atill6-1 -RP, LBb1.3 and atill6-1 -RP identification of mutant strains atill6-1 If the former has a band and the latter has no band, the plant is wild type; if both have bands, it is a heterozygous mutant; if the former has no band and the latter has a band, it is a homozygous mutant. atill6-2 The identification method is the same as above.

[0081] Table 11 Primers for mutant identification

[0082] 2.2 Results and Analysis 2.2.1 MsILL6 Obtaining transgenic Arabidopsis like Figure 6As shown in A, Arabidopsis T0 seeds infected by the floral dip method were spread on 1 / 2MS screening medium containing PPT (7.5 mg / L). Green and healthy seedlings that can grow normally are T1 positive seedlings. The selected seedlings were transplanted into the soil, and Arabidopsis leaf DNA was extracted for PCR identification. The amplified band was consistent with the size of the plasmid ( Figure 6 B) There is no band in wild-type Arabidopsis DNA and dd H2O. After harvesting and PCR identification, 21 positive plants were obtained.

[0083] The harvested T1 generation positive seedlings were screened again with 1 / 2 MS medium containing 7.5 mg / L PPT, and the positive seedlings with normal growth were selected and transplanted into the soil to harvest the T2 generation seeds. Figure 7 As shown, the T2 generation plants (OE3, OE8, OE9, OE12, OE14, OE21 and OE27) after propagation were subjected to qRT-PCR expression difference analysis, and the three strains with the highest expression levels (OE8, OE12 and OE21) were screened out.

[0084] 2.2.2 Effects of saline-alkali stress on transgenic Arabidopsis To verify MsILL6 Response of transgenic Arabidopsis to saline-alkali stress. Wild-type and overexpressing transgenic Arabidopsis lines grown for 4 weeks were treated with saline-alkali. Figure 8 As shown in the figure, there was no significant difference between transgenic Arabidopsis and wild type under salt treatment and salt-alkali mixed treatment, but the degree of leaf yellowing of transgenic Arabidopsis under alkali treatment was higher than that of wild type. Since the growth condition of transgenic Arabidopsis was worse than that of wild type, the survival rate of Arabidopsis after alkali treatment was statistically analyzed ( Fig. 9 ), and found that the survival rates of OE8 and OE12 were significantly lower than that of wild-type Arabidopsis.

[0085] 2.2.3 Changes in photosynthesis in Arabidopsis thaliana under alkaline stress Under alkaline stress, plant photosynthesis is affected, so the chlorophyll fluorescence parameters Fo and Fv / Fm, as well as the SPAD value are used to determine the strength of the transgenic Arabidopsis thaliana's ability to respond to alkaline stress.

[0086] Fo and Fv / Fm were measured in plants under 200 mM NaHCO3 stress at different times. Fig.10 As shown in A, there was no significant difference in Fo values ​​between wild-type and transgenic Arabidopsis under alkaline stress, except that OE12 and OE21 were higher than wild-type Arabidopsis on the 4th day of alkaline treatment ( P< 0.05). Fig.10 As shown in B, there is no significant difference in the maximum optical efficiency Fv / Fm between wild-type and transgenic Arabidopsis under alkali stress.

[0087] Soil and Plant Analyzer Development (SPAD) is a relative reading of chlorophyll (Chlorophyl) content, which reflects the greenness of plants, such as Fig.10 As shown in C, under alkaline stress, the SPAD values ​​of wild-type and transgenic Arabidopsis increased over time but there was no significant difference.

[0088] 2.2.4 Effects of drought stress on transgenic Arabidopsis like Fig.11 As shown, under drought stress, MsILL6 The degree of yellowing of transgenic Arabidopsis leaves was higher than that of the wild type, but the difference was not significant.

[0089] 2.2.5 Obtaining homozygous atill6 mutants For the sake of MsILL6 Genes were bidirectionally verified, and homologous genes in Arabidopsis thaliana were ILL6 Mutant strains atill6-1 and atill6-2 , for screening and identification. Fig.12 As shown, a homozygous mutant strain was obtained ( Fig.12 A), T0 generation atill6-2 All 25 strains were wild type ( Fig.12 B), so the subsequent experiments used atill6-1 Homozygous mutants completed.

[0090] 2.2.6 Effects of saline-alkali stress on seed germination of Arabidopsis mutants like Fig.13 As shown, on the 4th day after 150 mM NaCl treatment and the 5th day after 200 mM NaCl treatment, atill6-1 The seed germination rate of Arabidopsis mutants was significantly higher than that of wild-type Arabidopsis. Fig.13 As shown, from day 1 to day 10 of treatment with 10 mM NaHCO3, atill6-1 The germination rates of Arabidopsis mutants and wild-type Arabidopsis were significantly different, and the germination rate of mutant seeds was significantly higher than that of wild-type Arabidopsis. On the 4th day after treatment with 20 mM NaHCO3, atill6-1 The seed germination rate of Arabidopsis mutants was significantly higher than that of wild-type Arabidopsis.

[0091] like Fig.14 As shown, under the salt-alkali mixed treatment, only the 80 mM NaCl and 20 mM NaHCO3 treatments on the 9th and 10th days, atill6-1 The seed germination rate of Arabidopsis mutants was significantly higher than that of wild-type Arabidopsis.

[0092] This experiment was successfully constructed MsILL6 The overexpression vector was introduced into Arabidopsis thaliana by Agrobacterium-mediated method, and 21 positive strains were obtained after screening and identification. Under alkali treatment, the growth of wild-type Arabidopsis thaliana was better than that of transgenic Arabidopsis thaliana, indicating that MsILL6 Gene overexpression reduces the tolerance of Arabidopsis to alkaline stress. atill6-1 Arabidopsis homozygous mutant strains. Under 10 mM NaHCO3 treatment, atill6-1 The germination rate of mutant strains was significantly higher than that of wild type. Under salt treatment and salt-alkali mixed treatment, there was no significant difference in phenotype between transgenic Arabidopsis and wild type Arabidopsis. Under drought treatment, the degree of yellowing of transgenic Arabidopsis leaves was greater than that of wild type Arabidopsis, but the difference was not significant.

[0093] Embodiment 3: MsILL6 Functional identification in alfalfa 3.1 Materials and methods 3.1.1 Test materials The plant material is alfalfa SY4D. The configurations of SH3a medium and SH9a medium are shown in Table 12.

[0094] Table 12 Culture medium configuration

[0095] 3.1.2 Genetic transformation process of alfalfa and identification of positive plants 3.1.2.1 Agrobacterium infection of alfalfa Bacterial liquid activation: Add Kan (50 mg / mL) and Rif (50 mg / mL) to LB liquid medium at a ratio of 1:1000, and activate at 28℃ shaker (200 rpm) for 2-3 days. Repeat once. OD of bacterial liquid 600 The value is 0.6-0.8. Prepare the resuspension: 4℃, 4000 rpm, 15 min. Discard the supernatant and add SH3a liquid culture medium to adjust the OD of the resuspension to 600 The value was adjusted to 0.2-0.4. Leaf disinfection: Take 3-4 leaves from the top of the stem of alfalfa SY4D, wash twice with 1 L of dd H2O; wash for 2 min with 0.1% Tween 20 (Tween: dd H2O=1:1000); put the leaves into a sterile tissue culture bottle, rinse with 75% ethanol for 15 sec, wash twice with dd H2O; 15% NaClO solution 100 rpm, shake incubator at 28℃ for 8-10 min, wash three times with dd H2O. Infection: Transfer the leaves to a new sterile culture bottle, add the prepared resuspension, vacuum for 10 min, ultrasonicate in an ice-water mixture for 3-5 sec, and vacuum again for 10 min.

[0096] 3.1.2.2 Regeneration of overexpression plants The leaves were air-dried in a clean bench, spread flat on SH3a co-culture medium and kept in the dark for 12-24 h, and then transferred to SH3a selection medium for dark culture until swollen callus tissue was produced; under the conditions of 24℃, 16 h light / 8 h dark, the callus was placed on MSBK selection medium and cultured until green buds were produced, and then transferred to SH9a selection medium for rooting; the rooted plants were transferred to soil for culture.

[0097] 3.1.2.3 Identification of overexpression positive plants DNA was extracted from leaves of regenerated plants (SDS method) for positive seedling identification, and the identified transgenic positive seedlings were verified by qRT-PCR, using the same method as in Example 2. Based on the qRT-PCR verification results, the three strains with the highest expression levels were selected for culture and cuttings for subsequent experiments.

[0098] 3.1.3 MsILL6 Functional analysis of transgenic alfalfa under saline-alkali stress Wild-type alfalfa SY4D and transgenic alfalfa were propagated by cuttings for 1-2 months, and alfalfa with consistent growth after cuttings was selected and transferred to new soil for salt-alkali stress.

[0099] Salt stress treatment method: Wild-type alfalfa and transgenic alfalfa were treated with salt (300 mM NaCl), with 3 replicates for each strain. Photos were taken and phenotypes were observed starting from the first day of treatment.

[0100] Alkali stress treatment method: Wild-type alfalfa and transgenic alfalfa were treated with alkali (200 mM NaHCO3), with 3 replicates for each strain. Photos were taken and phenotypes were observed starting from the first day of treatment.

[0101] 3.1.4 Screening and identification of Medicago truncatula mutants use MsILL6 The coding region sequences were compared at NCBI (https: / / www.ncbi.nlm.nih.gov / ) to obtain homologous genes MtILL6 , gene number is Medtr3g088590. A mutant strain was found in the Medicago truncatula mutant database (https: / / medicago-mutant.dasnr.okstate.edu / mutant / index.php) mtill6 (NF2836). The DNA of the Medicago truncatula mutant was identified by PCR using a double primer identification method. The primers are shown in Table 13. The identification method is as follows: mtill6 -F and mtill6 -R, LTR6-R and mtill6 -R identification of mutant strains mtill6 If the former has a band and the latter has no band, the plant is wild type; if both have bands, it is a heterozygous mutant; if the former has no band and the latter has a band, it is a homozygous mutant.

[0102] Table 13 Primers for identification of Medicago truncatula mutants

[0103] 3.2 Results and Analysis 3.2.1 Genetic transformation process of alfalfa and acquisition of positive plants 3.2.1.1 Overexpression genetic transformation Using pCAMBIA3301- MsILL6 After the alfalfa leaves were infected with the overexpression vector Agrobacterium, regenerated plants were obtained through tissue culture. The alfalfa leaves were placed as explants on the SH3a co-culture medium and cultured for 12 h ( Fig.15 A), after switching to SH3a selective medium, the leaves showed varying degrees of curling after 5-7 days of culture ( Fig.15 B). After the callus is formed, it is placed on MSBK selection medium and cultured for 15-30 days. The leaves begin to swell and grow, and produce green dot-like particles ( Fig.15 C / D). SH9a selection medium was used for further culture for 2-5 months, and the callus took root and produced true leaves ( Fig.15 E). The adventitious buds with good growth conditions were transferred to culture bottles and cultured until complete regenerated plants were produced ( Fig.15 F). When the seedlings in the culture bottle grow to about 10 cm, they are transferred to soil.

[0104] 3.2.1.2 Identification of overexpression positive seedlings A total of 48 regenerated plants were obtained in this experiment. MsILL6 Overexpression of alfalfa regenerated plants. PCR identification was performed using universal primers on the pCAMBIA3301 vector, and the templates were WT, H2O, MsILL6 Plasmids and MsILL6 Transgenic alfalfa DNA, 23 of which overexpressed MsILL6 Alfalfa strips and MsILL6 The plasmid bands are consistent ( Fig.16 ), while no bands were amplified in wild-type alfalfa and H2O. This indicates that 23 of the 26 regenerated plants identified were positive seedlings.

[0105] 3.2.1.3 Analysis of overexpression positive expression The expression level of positive plants was analyzed by qRT-PCR ( Fig.17), OE9 had the highest expression, followed by OE4 and OE3, and 19 of the 23 positive seedlings MsILL6 The gene expression level was higher than that of wild-type alfalfa. MsILL6 The gene was successfully transferred into the genome of alfalfa through Agrobacterium-mediated method, and it was explained MsILL6 The gene was successfully expressed in alfalfa.

[0106] 3.2.2 Effects of salt stress on transgenic alfalfa like Fig.18 As shown in the figure, under 300 mM NaCl treatment, there was no significant difference in leaf phenotype between transgenic alfalfa and wild-type alfalfa. However, on the 20th day of salt treatment, the plant height of transgenic alfalfa was significantly lower than that of wild-type alfalfa.

[0107] 3.2.3 Effects of alkaline stress on transgenic alfalfa like Fig.19 As shown in the figure, on the 7th day after the treatment with 200 mM NaHCO3, the leaves of the transgenic alfalfa plants turned yellow and withered, and the growth of the plants was poor, while only the edges of the bottom leaves of the wild-type alfalfa turned yellow. The above morphological characteristics indicate that alkaline stress has a significant effect on the growth of transgenic alfalfa.

[0108] 3.2.4 Screening and identification of Medicago truncatula mutants MsILL6 The protein sequence similarity with MtILL6 of Medicago truncatula is as high as 98.53% (Appendix 4). MtILL6 Mutant strains mtill6 Screening and identification. Fig. 20 As shown, heterozygous mutant lines were obtained.

[0109] In this study, the Agrobacterium-mediated MsILL6 After the overexpression vector was successfully transferred into alfalfa, 23 positive strains were identified and the expression levels of positive plants were analyzed by qRT-PCR. The strains with the highest expression levels (OE3, OE4 and OE9) and wild-type alfalfa were selected for alkali treatment. The results showed that MsILL6 The gene reduced the tolerance of transgenic alfalfa to alkaline stress. Under salt treatment, the phenotypic differences between wild type and transgenic alfalfa were not obvious.

[0110] It is understandable that those skilled in the art can make equivalent substitutions or changes based on the technical solution and concept of the present invention, and all these changes or substitutions should fall within the protection scope of the claims attached to the present invention.

Claims

1. Alfalfa MsILL6 A gene characterized by Alfalfa MsILL6 The nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.

1.

2. Alfalfa according to claim 1 MsILL6 A gene characterized by Alfalfa MsILL6 The amino acid sequence encoded by the nucleotide sequence of the coding region of the gene is shown in SEQ ID NO.

2.

3. Alfalfa according to any one of claims 1 to 2 MsILL6 The application of genes is characterized in that Alfalfa MsILL6 The gene is prepared into a recombinant expression vector, an expression cassette, a transgenic cell line, a recombinant bacterium or a recombinant virus to regulate the salt-alkali tolerance of the plant.

4. The use according to claim 3, characterized in that: The plant is alfalfa or Arabidopsis thaliana.

5. A pCAMBIA3301- MsILL6 The overexpression vector is characterized in that The overexpression vector is the alfalfa described in claim 1 MsILL6 The nucleotide sequence of the coding region of the gene and the construction of the pCAMBIA3301 vector were obtained.

6. The overexpression vector according to claim 1, characterized in that The primer sequences are: MsILL6 -F:5’-AAGACAGCGACGAACT-3’; MsILL6 -R: 5'-GCTCTAACGGCAACAA-3'.

7. The overexpression vector according to claim 6, characterized in that Said MsILL6 -F sequence 5' end also includes a homology arm sequence acgggggactcttgacc; MsILL6 The 5' end of the -R sequence also includes the homology arm sequence ggggaaattcgagctggtcacc.

8. Alfalfa MsILL6 The use of the gene in plant salt-alkali tolerance is characterized in that: Said MsILL6 Gene overexpression can reduce the tolerance of plants in saline-alkali environments; MsILL6 Gene silencing expression can improve plant tolerance in saline-alkali environments.

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