CN118006658A - TaMAT1 gene and application thereof - Google Patents

Abstract

The invention discloses TaMAT gene and application thereof, belonging to the technical field of genetic engineering. The CDS region nucleotide sequence of TaMAT gene is shown in SEQ ID NO. 1. According to the invention, through cloning and analyzing the wheat TaMAT gene and combining a BSMV-VIGS technology and an over-expression technology, the function of the gene is verified, taMAT gene expression is closely related to crop salt tolerance, taMAT gene silencing obviously reduces the salt tolerance of plants, taMAT gene over-expression obviously improves the salt tolerance of plants, and theoretical basis and related genes are provided for wheat salt tolerance breeding and production.

Description

TaMAT1 gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to TaMAT genes and application thereof in regulating and controlling salt stress tolerance of plants.

Background

Soil salinization is an environmental stress caused by human activities or natural factors, and is a major threat affecting agricultural sustainable development in arid and semiarid regions (Daliakopoulos et al, 2016). Soil salt damage is one of the major stresses affecting crop yield and quality. At present, the salinization level of global soil is on the rise, and more than 9.5 hundred million hectares of land are affected by salinization, which accounts for about 10% of the global land area, and more seriously, land salinization is developing at a rate of about 3 hectares/minute (Ghassemi et al, 1995; shabala et al, 2014). With the increase of global climate warming, the influence of soil salinization on agricultural production is further increased, and the world grain safety production is seriously affected.

Plant salt tolerance is a complex quantitative trait involving a series of physiological and biochemical reactions in which numerous genes are involved, and plants evolved a variety of mechanisms including active oxygen scavenging mechanisms in order to accommodate high salt environments. Salt stress can increase the production of Reactive Oxygen Species (ROS), which are highly toxic to cells, and in addition to disrupting cellular redox homeostasis, excess ROS in cells can promote protein and enzyme degradation and lipid peroxidation, further deteriorating electron transport systems, PSII systems, and various membrane structures (Li et al, 2017).

Many genes related to salt tolerance are found in different species by adopting molecular genetics and functional genomics technologies, such as KUP/HAK/KT families, and K+ absorption is increased by driving potassium ion transportation, and a higher K+/Na+ ratio is maintained, so that the salt tolerance of plants is improved (Zhao Chang and the like, 2007). Halophytes maintain the balance of active oxygen production and scavenging systems by antioxidant and non-antioxidant enzyme systems (Valentina et al, 2004; yoshimura et al, 2004; begara-Morales et al, 2014, wei et al, 2020).

Wheat is one of the most important food crops worldwide, and after thousands of years of crossing, the now-planted domesticated wheat is polyploid, containing more than two sets of genomes, which also gives the wheat an extremely complex genome that gives it many excellent traits, but also loses many other traits including salt tolerance (Munns, et al 2012). Compared with crops such as rice, fewer salt tolerance related genes have been identified in wheat. Therefore, the development of the wheat salt tolerance key genes has important significance for cultivating new salt tolerance varieties.

Disclosure of Invention

The invention aims to provide a gene which is cloned from wheat and participates in regulation of wheat salt tolerance, and is used for cultivating a wheat variety with strong salt tolerance.

In order to achieve the above purpose, the invention adopts the following technical scheme:

The invention identifies a gene involved in wheat salt tolerance based on the results of the analysis of the GWAS and transcriptome sequencing association, and the gene is named TaMAT < 1 >. The salt tolerance germplasm X118 identified in the earlier stage of the subject group is taken as a material, the full-length CDS sequence of the gene is cloned, and the corresponding nucleotide sequence is shown as SEQ ID NO. 1.

TaMAT1 gene CDS total length 1323bp, coding 440 amino acids, the amino acid sequence is shown as SEQ ID NO. 2. The molecular weight of the protein is 47.56kDa, and the isoelectric point is 5.88. Protein domain predictions through SMART and Protter websites show that TaMAT protein contains 1 important Pfam domain TRANSFERASE, which is an acylase of BAHD (Amino-polyamine-organocation) family, and is used for carrying out acylation modification on secondary metabolic substances in plants. The protein tertiary structure prediction shows that the protein tertiary structure prediction has the crystal structures of anthocyanin 5,3' -aromatic acyl transferase H174A and caffeoyl-CoA mutant, and is an anthocyanin 5-aromatic acyl transferase.

Analysis of the expression pattern of TaMAT gene showed that the relative expression level of TaMAT gene in the resistant genotype germplasm was significantly increased after 48h of salt treatment; taMAT1 has high expression level in aerial leaf and leaf sheath.

The function of TaMAT gene is verified by using BSMV-VIGS method, and the result shows that under the condition of salt stress, the biomass and the relative water content of the overground parts of wheat plants inoculated with BSMV: taMAT1 are obviously lower than those of plants inoculated with BSMV: gamma.

Culturing and screening by using agrobacterium-mediated wheat young embryo genetic transformation technology to obtain TaMAT gene over-expression plants. Through salt stress test, phenotype identification is carried out on genetically transformed plants, and the result shows that the survival rate of the over-expressed strain is obviously higher than that of the wild type under the salt stress condition.

The results show that TaMAT gene expression is closely related to crop salt tolerance.

Thus, the present invention provides the use of TaMAT gene in regulating tolerance of plants to salt stress.

Specifically, taMAT gene silencing reduces salt tolerance of plants; the TaMAT gene over-expression improves the salt tolerance of the plant.

Further, the plant is wheat.

Further, taMAT gene overexpression promotes active oxygen scavenging. Under salt stress conditions, the content of superoxide anions and hydrogen peroxide in TaMAT gene over-expression lines is significantly lower than that of wild type.

Further, the application includes: inserting TaMAT genes into an over-expression vector to construct a recombinant plasmid, then introducing a target gene fragment into a receptor plant by using an agrobacterium-mediated technology, and screening to obtain a transgenic plant obtained functionally.

The recipient plant may be, but is not limited to, wheat.

Preferably, the over-expression vector is pLGY-OE3 vector.

Preferably, the agrobacterium-mediated technique employs agrobacterium EHA105 as a host.

The invention has the beneficial effects that:

According to the invention, through cloning and analyzing the wheat TaMAT gene and combining a BSMV-VIGS technology and an over-expression technology, the function of the gene is verified, taMAT gene expression is closely related to crop salt tolerance, taMAT gene silencing obviously reduces the salt tolerance of plants, taMAT gene over-expression obviously improves the salt tolerance of plants, and theoretical basis and related genes are provided for wheat salt tolerance breeding and production.

Drawings

FIG. 1 is a TaMAT protein structure prediction, where (a) is the SMART predicted protein domain; (b) a predicted protein domain for Protter; (c) protein tertiary structure prediction for TaMAT a 1.

FIG. 2 is a TaMAT gene expression pattern, where (a) is the spatiotemporal expression pattern of the TaMAT1 gene in X118 and X276; (b) Is TaMAT expression pattern of gene in different tissues of field.

FIG. 3 is a diagram showing the verification of TaMAT gene function using the BSMV-VIGS method, wherein (a) is the plant phenotype of TaMAT1 wheat silenced under salt stress; (b) The change of gene expression quantity of the overground part and the underground part TaMAT of the wheat plant; (c) - (e) are the effects of salt stress on aerial biomass, relative water content and aerial Na ⁺ content, respectively.

FIG. 4 is a schematic representation of the function of the TaMAT gene verified by overexpression TaMAT1, wherein (a) is the plant phenotype of the TaMAT wheat overexpressed under salt stress; (b) is the survival rate of wheat under salt stress; (c) And (d) the effect of salt stress on superoxide anion and hydrogen peroxide accumulation, respectively.

Detailed Description

The invention will be further illustrated with reference to specific examples. The following examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Wheat salt tolerance germplasm X118 and sensitive genotype germplasm X276 (published in Zhang,et al.Genotypic variation in grain cadmium concentration in wheat:Insights into soil pollution,agronomic characteristics,and rhizosphere microbial communities.Environmental Pollution,2024,340(1):122792) as main materials to verify TaMAT gene function) previously selected by the subject group in the following examples.

Example 1: taMAT1 Gene CDS region cloning and analysis

1. TaMAT1 Gene CDS region cloning

Based on the results of the analysis of the GWAS and transcriptome sequencing association, a gene involved in wheat salt tolerance was identified, and the full-length CDS sequence of the gene was cloned using salt tolerance germplasm X118 identified earlier in the subject group as a material and designated TaMAT.

Total RNA from X118 leaves was extracted using the total RNA extraction kit, DNaseI was used to remove genomic DNA contamination, and was subsequently reverse transcribed into single stranded cDNA using PRIMESCRIPFFMII.sup.1: 1st Strand cDNASynthesis Kit. Designing a primer according to the Blast sequence, and adopting KOD ONE high-fidelity enzyme amplification, wherein the primer sequence is as follows:

TaMAT1-CDS-F：5’-ATGTCTCCGGTGAGAGTAATCC-3’；

TaMAT1-CDS-R：5’-TTAACCGACTAACTCGAGCAAC-3’；

And (3) connecting the amplified product with a pMD18-T vector, transforming escherichia coli DH5 alpha, and picking up monoclonal sequencing. The corresponding nucleotide sequence is shown as SEQ ID NO. 1.

2. TaMAT1 Gene sequence analysis

The CDS total length of the gene 1323bp codes 440 amino acids, the corresponding amino acid sequence is shown as SEQ ID NO.2, the molecular weight of the protein is 47.56kDa, and the isoelectric point is 5.88. Protein domain predictions via SMART and Protter websites showed that the TaMAT protein contained 1 important Pfam domain TRANSFERASE, an acylase of the BAHD (Amino-polyamine-organocation) family, to acylate secondary metabolites in plants (fig. 1 a). The protein tertiary structure prediction shows that the crystal structure of anthocyanin 5,3' -aromatic acyl transferase H174A and caffeoyl-CoA mutant (figure 1 c) is an anthocyanin 5-aromatic acyl transferase.

3. TaMAT1 expression Pattern analysis

Seeds of X118 (resistant genotype) and X276 (sensitive genotype) were soaked in 2% H ₂O₂ for 20min and thoroughly rinsed with distilled water, and whole healthy seeds were selected and sown in germination boxes at a culture temperature of 22 ℃/18 ℃. And selecting wheat seedlings with consistent healthy growth vigor in the two-leaf period, and transplanting the wheat seedlings into 1/5Hogland nutrient solution. After 5 days of preculture, salt treatment was carried out, and 2 treatments were set up for the test: control (minimal medium) and salt treatment (150 mM NaCl), samples were taken at 3h, 6h, 12h, 24h, 48h, 72h, respectively, for spatial-temporal expression analysis; samples of different tissue parts of the field in the grouting period of the soil culture test are taken, and the expression quantity is measured.

After extracting total RNA of different samples and reversely transcribing the total RNA into cDNA, analyzing TaMAT1 expression quantity change (qRT-PCR) by using SYBR green fluorescent enzyme complex and LIGHT CYCLER PCR instrument, wherein the primer sequences are as follows:

TaMAT1-qRT-PCR-F：5’-CTGGCACCGGCGTTCACTGT-3’；

TaMAT1-qRT-PCR-R：5’-TTCTCCGTCCGCATCGGCTTG-3’；

TaGADPH-F：5’-AGTTCACGGCCATGTTCA-3’；

TaGADPH-R：5’-ACGAGGTCGTTCATGTTGCT-3’；

The results of the spatial-temporal expression analysis showed that in the resistance genotype X118, the gene expression level was decreased within 24 hours, and significantly increased after 48 hours of salt treatment. While the expression level of sensitive genotype X268 was not significantly changed in 24h of salt treatment, but was significantly decreased after 48h (FIG. 2 a).

The results of tissue expression analysis showed that TaMAT1 was expressed in the aerial leaf and leaf sheath in high amounts (FIG. 2 b).

Example 2: BSMV-VIGS method for verifying TaMAT gene function

1. BSMV TaMAT vector construction

The specific primer is designed by taking the pMD 18-T-TaMAT-cDNA plasmid stored in a laboratory as a template, and the TaMAT gene fragment is amplified.

Primer sequences are (underlined as cleavage sites):

TaMAT1-γ-F:5’-GTACGCTAGCATCGTGCGTCTCGGTGAATC-3’；

TaMAT1-γ-R:5’-GTACGCTAGCCATCCTGGTGTTCTCCGTCC-3’；

and (3) after sequencing and comparison, the amplified TaMAT gene fragment is connected to a pMD18-T vector to transform the DH5 alpha competence of the escherichia coli, after overnight culture, the monoclonal is selected and sent to a company for sequencing, and positive monoclonal shaking bacteria with correct sequencing are extracted to obtain plasmids, and the plasmids are stored in a refrigerator at the temperature of minus 20 ℃.

The target gene fragment is subjected to enzyme digestion by NheI enzyme from a cloning plasmid vector, and then the target fragment is recovered; the gamma vector was digested with NheI and dephosphorylated with CIP to prevent self-ligation, and purified to ligate the gamma vector to the target gene fragment.

E.coli DH5 alpha is transformed by the connection products of TaMAT gene fragments, and the primers gamma-stand-F and TaMAT 1-gamma-F on the gamma vector are used for sequencing, and the monoclonal extraction plasmid of the gene fragments inserted into the gamma vector in the reverse direction is selected and named as gamma TaMAT1 and stored.

The sequence of primer gamma-stand-F is as follows:

5’-CAACTGCCAATCGTGAGTAGG-3’。

2. BSMV vector linearization and in vitro transcription

Single enzyme cutting RNA alpha, RNA gamma, gamma TaPDS and gamma TaMAT1 with restriction enzyme MluI; RNA β was single digested with the restriction enzyme SpeI-HF, and the digested products were linearized and transcribed in vitro using RiboMAXTM LargeScale RNAProduction System-T7 kit (Promega, USA) and the Ribo m7G Cap Analog kit (Promega, USA) according to the instructions. 1 μl of the posttranscriptional product was subjected to 1% (w/v) agarose gel electrophoresis to confirm that the bands were clear and non-diffuse.

In vitro transcribed RNA alpha, RNA beta, RNA gamma: GFP/gamma TaPDS/gamma TaMAT1 was mixed in a volume ratio of 1:1:1, diluted with a diploid volume of RNase-free water, then added with an equal volume of 2 XGKP buffer (1% bentonite, 1% diatomaceous earth 545, 50mM glycine, 30mM dipotassium hydrogen phosphate, pH adjusted to 9.2), thoroughly mixed, and the mixed product named BSMV: gamma, BSMV: taPDS, BSMV: taMAT1 for subsequent inoculation (Manmann, 2019).

3. BSMV inoculation verifies TaMAT gene function

X118 seeds are soaked in 2% H ₂O₂ for sterilization for 20 minutes, distilled water is used for thoroughly washing, and the whole healthy seeds are selected and sown in a germination box, wherein the culture temperature is 22 ℃/18 ℃. And selecting wheat seedlings with consistent healthy growth vigor in the two-leaf period, and transplanting the wheat seedlings into 1/5Hogland nutrient solution. After wheat seedlings grow to two leaves and one period, BSMV friction inoculation is carried out on the second leaves in RNase-free environment, a small amount of DEPC water is sprayed on the inoculated plants immediately, the plants are covered by a transparent plastic cover for moisturizing for 3 days, then the glass cover is taken off, continuous culture is carried out in a wheat growth culture room (22 ℃/18 ℃), and the phenotype of the plants is observed at regular time.

The test consisted of 4 treatments: (1) Inoculating BSMV: gamma and growing in normal culture solution (BNS); (2) inoculating BSMV: gamma and treating with 200mM NaCl; (3) inoculating BSMV TaMAT1 and growing in normal culture solution; (4) BSMV TaMAT 1:1 was inoculated and treated with 200mM NaCl.

When the infected seedlings in the BSMV-TaPDS system show the photo-bleaching phenomenon, the leaves of the wheat are selected to measure the PDS expression quantity, and whether the BSMV-VIGS system is successful or not is verified. After 5d of salt stress treatment, wheat plant leaves infected by the disease spots under control treatment and salt treatment in a BSMV: gamma and BSMV: taMAT1 system are respectively selected for sampling, and each treatment is repeated for three times, so that the determination of gene expression is carried out.

TaPDS primer sequences are shown below:

TaPDS-F:5’-ACCCTGACGAGTTATCCATGC-3’；

TaPDS-R:5’-CCTCACCACCCAAAGACTGA-3’；

The results show that compared with inoculated BSMV: gamma plants, the leaf of the BSMV-TaPDS plants has obvious albino phenotype, and the TaPDS expression quantity is reduced by 90 percent. The above results indicate that the BSMV-VIGS system was successfully inoculated in X118 and can be used to verify TaMAT functions.

Compared with wheat plants inoculated with BSMV: gamma, the expression level of the overground part TaMAT1 of plants inoculated with BSMV: taMAT1 was significantly reduced under both control and salt stress treatment (FIG. 3 b). Under control conditions, there was no significant change in the biomass in the aerial parts of inoculated BSMV TaMAT1 plants, but both the aerial parts biomass and the relative water content were significantly lower than in BSMV: gamma plants after salt treatment (FIG. 3 c). The Na ⁺ content of the overground part has no obvious difference.

Example 3: wheat over-expression verification TaMAT gene function

Specific primers were designed based on the sequences of the pre-clones, the primer sequences were as follows,

TaMAT1-OE-F：

5’-tacttctgcagccctaggcctATGTCTCCGGTGAGAGTAATCC-3’；

TaMAT1-OE-R：

5’-acgaacgaaagctctgagctcTTAACCGACTAACTCGAGCAAC-3’；

Amplifying TaMAT1 DNA sequences by KOD One DNA polymerase, connecting the obtained two haplotype DNA sequences into pLGY-OE3 vector by referring to a nonizane homologous recombination method, adopting StuI and SacI as restriction endonucleases for enzyme digestion, converting the constructed over-expression recombinant plasmid into agrobacterium EHA105 by a heat shock method, coating the over-expression recombinant plasmid onto LB solid medium containing rifampicin and kanamycin resistance, culturing overnight at 28 ℃, and picking up monoclonal bacterial colony for amplification culture. Then 200uL of bacterial liquid is sucked into 15mL of liquid culture medium (Kan+Rif), the bacterial liquid is placed on a shaking table at 28 ℃ for culture until the OD value is about 0.2, the supernatant is removed after centrifugation, and the prepared dye-flooding liquid is added to adjust the OD value to be 0.6-0.8.

Inoculating infection liquid into wheat (field) young embryo callus after induction differentiation by using an agrobacterium transformation mode, co-culturing, transferring into a selection medium containing a screening agent for screening, transferring the identified callus onto a differentiation medium and a rooting medium for culture respectively, and transplanting into a greenhouse after transgenic seedlings develop well to finish creation of over-expression materials.

According to the vector sequence and the DNA sequence TaMAT, positive seedling verification primers are designed to carry out PCR detection on the extracted tissue culture seedling DNA and transgenic plants are screened through sequence comparison. 2 transgenic lines L1, L2 and L3 were selected therefrom for functional identification of salt tolerance.

Wild Type (WT), over-expressed strains L1, L2 and L3 seeds were soaked in 2% h ₂O₂ for 20 min and rinsed thoroughly with distilled water. And selecting complete healthy seeds and sowing the seeds in the nutrient soil. Two leaves were incubated at one heart, and 150mM earth culture salt treatment was performed. After 15 days of salt treatment, the phenotype of the plants was observed; detecting the content of superoxide anions by using an NBT dyeing method; the DAB staining method detects the hydrogen peroxide content.

NBT dyeing method

Preparing NBT solution: 0.5mg/ml NBT reaction, pH=7.8, 50mM PBS buffer; preparation of 10×pbs buffer: 80g NaCl, 2g KCl, 14.4g Na ₂HPO₄ and 2.4g KH ₂PO₄ were added to 1L purified water, stirred until completely dissolved, pH adjusted to 7.4 with NaOH or HCl, and then water was added to make the total volume 1L.

Taking leaf parts of wild type subjected to salt treatment and TaMAT1 over-expressed plants, avoiding contacting with leaf veins, adding NBT in dark environment, vacuumizing for 30 minutes in dark, reacting at normal temperature in dark for 6 hours, pouring NBT, washing, treating with 90% ethanol and 90 ℃ water bath until chlorophyll is removed, and taking pictures.

DAB dyeing method

Preparing DAB solution: 1mg/ml DAB reaction, pH=5.5, 50mM Tris-HCl; i.e., 1.97g Tris-HCl to 250ml, adjusted to pH=5.5 with HCl, 0.25g DAB was added

Taking leaf parts of wild type subjected to salt treatment and TaMAT1 over-expressed plants, avoiding contacting with leaf veins, adding DAB in dark environment, vacuumizing for 30min in dark environment, reacting at normal temperature in dark environment for 1 hr, pouring DAB, washing, treating with 90% ethanol and 90 ℃ water bath until chlorophyll is removed, and photographing.

As shown in FIG. 4, after 15 days of 150mM NaCl treatment, the salt tolerance of the overexpressed lines L1, L2 and L3 was significantly higher than that of the wild-type. The survival rate of the over-expressed strain is significantly increased compared to WT; the superoxide anion and hydrogen peroxide content was significantly reduced (fig. 4c, fig. 4 d).

Claims (9)

1. The application of the TaMAT1 gene in regulating and controlling salt stress tolerance of plants is characterized in that the nucleotide sequence of a CDS region of the gene is shown as SEQ ID NO. 1.
2. 2. The use according to claim 1, wherein the TaMAT gene encodes a protein having the amino acid sequence shown in SEQ ID No. 2.
3. 3. The use of claim 1, wherein TaMAT a gene silencing reduces salt tolerance in the plant.
4. 4. The use of claim 1, wherein the TaMAT gene is overexpressed to increase salt tolerance in the plant.
5. 5. The use of claim 4, wherein TaMAT gene overexpression promotes active oxygen scavenging.
6. 6. The application of claim 4, wherein the application comprises: inserting TaMAT genes into an over-expression vector to construct a recombinant plasmid, then introducing a target gene fragment into a receptor plant by using an agrobacterium-mediated technology, and screening to obtain a transgenic plant obtained functionally.
7. 7. The use according to claim 6 wherein the over-expression vector is a pLGY-OE3 vector.
8. 8. The use according to claim 6, wherein the agrobacterium-mediated technique employs a host bacterium that is agrobacterium EHA105.
9. 9. The use according to claim 1, wherein the plant is wheat.