CN112626086B - Application of Medicago truncatula gene MtREVOLUTA in improving the salt tolerance of alfalfa, a forage relative of leguminous family - Google Patents

Abstract

The invention discloses a medicago truncatula gene MtREVOLUTA and application of a dicotyledonous plant expression vector pEARLEYGATE100 containing the medicago truncatula gene MtREVOLUTA in improving salt tolerance of leguminous kindred forage grass alfalfa. The invention also discloses application of the medicago truncatula gene MtREVOLUTA in cultivating salt-tolerant medicago sativa. Experiments prove that the gene MtREVOLUTA of the invention is a regulatory gene responding to abiotic stress, can be expressed in large quantity in the alfalfa of the leguminous forage grasses of the kindred species, and obviously enhances the salt tolerance of the transgenic alfalfa. The survival rate of the transgenic plant after high salt treatment is higher than that of wild alfalfa SY 4D. The application of the medicago truncatula gene MtREVOLUTA in improving the salt tolerance of the medicago truncatula in the kindred forage grass of leguminosae is shown to have important significance and economic value.

Description

Application of the Medicago truncatula gene MtREVOLUTA in improving the salt tolerance of alfalfa, a forage relative of the leguminous family

technical field

The invention relates to the application of the Medicago truncatula gene MtREVOLUTA, in particular to the application of a Medicago truncatula gene MtREVOLUTA in improving the salt tolerance of alfalfa, a closely related forage grass of the leguminous family, and belongs to the technical field of biological genetic engineering.

Background technique

Legumes belong to dicotyledonous plants, with about 650 genera and 18,000 species, with a wide distribution range. Alfalfa is a leguminous plant and is an important perennial high-quality pasture in the world. It has high protein content and good palatability. Whether it is used as pasture, silage or hay, it is the most economical source of feed. Therefore, alfalfa is called "King of Pastures".

The study on the salt tolerance mechanism of alfalfa can provide theoretical basis and technical support for the genetic improvement and breeding of new high-quality alfalfa lines. However, alfalfa is a cross-pollinated plant and is a highly heterozygous autotetraploid (2n=4x=32), which makes it very difficult to study its salt tolerance mechanism. Recent studies have reported that under different concentrations of salt treatment, alfalfa can adapt to medium and low concentrations of salt stress, and some varieties have strong salt tolerance and have the potential to be promoted in salty land. Although alfalfa has a certain tolerance to salt stress, the increasing soil salinity seriously affects the yield and quality of alfalfa.

After more than ten years of research by scientists from many countries, Medicago truncatula has become a model plant for legume biology and genomics research. Medicago truncatula is a diploid (2n=16) plant with self-pollination, small genome, short life cycle and high genetic transformation efficiency, which is convenient for research. Therefore, the study of Medicago truncatula will help to reveal the salt tolerance mechanism of alfalfa, and accelerate the research process of the regulation mechanism of alfalfa tolerance to salt stress.

Plants usually contain many miRNAs, of which miR166 is derived from a multigene family with independent genes loci. miR166 is highly conserved targeting HD-Zip III family genes, including the gene REVOLUTA (REV). The applicant's previous studies have confirmed that the MtREVOLUTA gene, a member of the Medicago truncatula HD-ZIP III gene family, is involved in regulating the number of leaflets and the ratio of leaf to stem in legumes. Knockout of this gene can obtain transgenic legumes with higher leaflet number and leaf-to-stem ratio than wild-type plants. Experiments show that the number of leaflets in the knockout mutant lines changes from three to four or five ratios. Significantly increased, reaching 81.5%, while the leaf-to-stem ratio increased by about 11%. And this achievement applied for the patent "application of REVOLUTA gene in regulating the number of small leaves and leaf-to-stem ratio of legumes (patent number CN201710672282.3)". The applicant's research also confirmed that in the transgenic Medicago truncatula plants overexpressing MtREVOLUTA, the expression level of MtREVOLUTA did not change significantly, and the expression level could only be obtained by constructing transgenic Medicago truncatula plants with MtREVOLUTA mutated at the target site of MtmiR166. In the transgenic Medicago truncatula plants with significantly increased mMtREVOLUTA, although the leaves of a single compound leaf increased, the whole plant was weak. Therefore, the expression level of transgenic MtREVOLUTA in Medicago truncatula is tightly regulated by MtmiR166, and the expression level of MtREVOLUTA without mutation cannot be changed by conventional transgene overexpression. After searching, it was found that MtREVOLUTA was different from the previous discovery that MtREVOLUTA was only responsible for regulating the development of leaves. It was confirmed that MtREVOLUTA is a Medicago truncatula regulated gene in response to salt stress, and the Medicago truncatula gene MtREVOLUTA could show different differences in its related species, alfalfa forage legumes. The literature or patent about the expression level and the large amount of expression has not yet been reported.

SUMMARY OF THE INVENTION

Aiming at the deficiencies of the prior art, the problem to be solved by the present invention is to provide the application of a Medicago truncatula gene MtREVOLUTA in improving the salt tolerance of alfalfa, a closely related forage of the leguminous family.

The application of the Medicago truncatula gene MtREVOLUTA of the present invention in improving the salt tolerance of alfalfa, a closely related forage grass of the leguminous family.

Wherein: the Medicago truncatula gene MtREVOLUTA is a Medicago truncatula regulated gene in response to salt stress, and the abundant expression of the Medicago truncatula gene MtREVOLUTA in its close relative legume forage alfalfa can improve the salt tolerance of alfalfa.

Wherein: the alfalfa is Zhongmu No. 1, Zhongmu No. 3, Gongnong, Nongjing or alfalfa SY4D, more preferably alfalfa SY4D.

The applicant subjected Medicago truncatula to abiotic stress treatment of salt, and it was confirmed that the expression of the gene MtREVOLUTA was significantly increased with the increase of treatment time compared with the control, indicating that the gene MtREVOLUTA participated in the salt stress response of plants, as shown in Figure 1.

The invention also provides the application of a dicotyledonous plant expression vector pEARLEYGATE100 containing the alfalfa gene MtREVOLUTA in improving the salt tolerance of alfalfa, a closely related forage grass of the leguminous family.

The application of the Medicago truncatula gene MtREVOLUTA in cultivating salt-tolerant alfalfa is characterized in that: the method for cultivating the salt-tolerant alfalfa is to construct a dicot expression vector pEARLEYGATE100/MtREVOLUTA by using the Medicago truncatula gene MtREVOLUTA and transform the alfalfa to obtain Transgenic alfalfa containing the Medicago truncatula gene MtREVOLUTA.

Through the experiment of expressing the Medicago truncatula gene MtREVOLUTA in alfalfa, it was confirmed that the dicot expression vector pEARLEYGATE100/MtREVOLUTA was constructed by using the Medicago truncatula gene MtREVOLUTA and transformed into alfalfa. See Figure 2.

The identified transgenic alfalfa containing the Medicago truncatula gene MtREVOLUTA and the wild-type control alfalfa SY4D were treated under high-salt conditions for 5 weeks to identify the role of the gene MtREVOLUTA in alfalfa stress resistance. With the prolongation of the treatment time, the leaves withered and withered until death, while the transgenic alfalfa still survived, as shown in Figure 3.

The survival rate of the treated MtREVOLUTA transgenic alfalfa and wild-type control alfalfa SY4D was counted, and it was found that MtREVOLUTA transgenic alfalfa was significantly improved compared with the wild-type control alfalfa SY4D, as shown in Figure 4.

In the present invention, the applicant discloses and confirms for the first time that the Medicago truncatula gene MtREVOLUTA is a Medicago truncatula responsive salt stress regulatory gene, which is different from the previously discovered MtREVOLUTA that is only responsible for regulating leaf development, and also confirms that the transgenic alfalfa through the Medicago truncatula gene MtREVOLUTA can Altering the expression level of the MtREVOLUTA gene to improve the salinity tolerance of alfalfa, a forage relative of the legume family, has outstanding substantive characteristics and significant progress. Further, the Medicago truncatula gene MtREVOLUTA disclosed in the present invention can be widely used for cultivating new varieties of crops that are closely related to the leguminous family with resistance to stress, and can provide a theoretical basis for in-depth clarification of the mechanism of plant stress resistance.

The application of the Medicago truncatula gene MtREVOLUTA disclosed in the present invention in improving the salt tolerance of the leguminous relative forage grass alfalfa and the method for improving the salt tolerance of the leguminous relative forage grass alfalfa have the following beneficial effects: using existing The present invention transgenic expression of the Medicago truncatula gene MtREVOLUTA in alfalfa is the first time that the salt stress response gene MtREVOLUTA is transgenic in alfalfa. Through comparative analysis, it is proved that the Medicago truncatula gene MtREVOLUTA can be expressed in a large amount in its related species, the legume forage grass, alfalfa. And the salt tolerance of transgenic alfalfa of MtREVOLUTA was obviously enhanced. Biological replicate statistical analysis showed that the survival rate of transgenic plants was higher than that of wild-type alfalfa SY4D after high salt treatment. It is predicted that the application of the Medicago truncatula gene MtREVOLUTA in the present invention in improving the salt tolerance of alfalfa, a closely related forage of the leguminous family, has important significance and economic value.

Description of drawings

Figure 1: Up-regulation of MtREVOLUTA in Medicago truncatula transgenics in response to salt stress detected by qRT-PCR.

The bar graph at 0 h in the figure represents the expression level of MtREVOLUTA in leaves of wild-type Medicago truncatula R108 one month old before 100 mM NaCl treatment. The bar graph at 12h in the figure represents the expression level of MtREVOLUTA in leaves of one-month-old wild-type Medicago truncatula R108 at 12h after 100 mM NaCl treatment. The bar graph at 24 h in the figure represents the expression level of MtREVOLUTA in the leaves of one-month-old wild-type Medicago truncatula R108 at 24 h after 100 mM NaCl treatment. Three biological replicates; * indicates P<0.05, 25 seedlings per batch of plants.

Figure 2: RT-PCR detection of MtREVOLUTA expression in leaves of 35S:MtREV alfalfa transgenic plants of different lines has obvious differences, the high expression is named 35S:MtREV#1, and the low expression is named 35S:MtREV #2.

The upper row of gels indicated the expression levels of MtREVOLUTA in leaves of wild-type alfalfa SY4D and 35S:MtREV alfalfa transgenic plants of different lines. The bottom row of gums indicated the expression levels of housekeeping gene MsActin in leaves of wild-type alfalfa SY4D and 35S:MtREV alfalfa transgenic plants of different lines.

Figure 3: Salt-treated one-month-old 35S:MtREV#1 transgenic cuttings of alfalfa improved salt tolerance compared to control wild-type alfalfa SY4D cuttings.

A, B, C, D, I, J, K, L, Q, R, S, T shown in the figure show wild-type alfalfa SY4D and 35S:MtREV#1 flowers of the control group without saline, respectively Growth of alfalfa transgenic plants before treatment and 1, 2, 3, 4, and 5 weeks after treatment. E, F, G, H, M, N, O, P, U, V, W, X shown in the figure show the wild-type alfalfa SY4D and 35S:MtREV of the treatment group applied with 1.5% NaCl saline, respectively. 1 Growth of alfalfa transgenic plants before treatment and 1, 2, 3, 4, and 5 weeks after treatment.

Figure 4: t-test statistical analysis shows that the survival rate of 35S:MtREV alfalfa transgenic plants 35S:MtREV#1 was significantly improved compared to wild-type SY4D after salt treatment.

Three biological replicates; * indicates P<0.05, 25 seedlings per batch of plants.

Detailed ways

The content of the present invention will be described in detail below with reference to the specific drawings and embodiments. The following examples are only preferred embodiments of the present invention. It should be noted that the following descriptions are only for explaining the present invention, and do not limit the present invention in any form. Any simple modifications, equivalent changes and modifications made all fall within the scope of the technical solutions of the present invention.

In the following examples, the involved alfalfa R108 was purchased from Noble Research Institute Mutant collections; alfalfa SY4D was purchased from Noble Research Institute Mutant collections. Escherichia coli DH5α and Agrobacterium EHA105 were purchased from Shanghai Weidi Biotechnology; vector pENTR-TOPO and expression vector pEARLYGATE100 were purchased from Invitrogen. The nucleotide sequence of the Medicago truncatula gene MtREVOLUTA has been disclosed in patent CN201710672282.3, and its nucleotide sequence is shown in SEQ ID No.1.

Other materials, reagents, etc. used, unless otherwise specified, are obtained from open commercial sources.

Example 1: Expression analysis of MtREVOLUTA

(1) Cultivation of materials

The hard outer skin of Medicago truncatula R108 seeds was moderately sanded with sandpaper to break the dormancy of the seeds. The seeds were cultured on moist filter paper at 4°C for about a week, and then the germinated seeds were transferred to a seedling tray and grown in an incubator. The environmental conditions of the incubator were as follows: long sunshine (16h/8h), temperature of 23°C, humidity of 65%, and light intensity of 150mmol m ^-2 s ^-1 . It was grown in the seedling tray for about two weeks (the first compound leaf developed), transferred into a large pot, and placed in the greenhouse for growth. The normal growth conditions in the greenhouse are: long sunshine (16h/8h), temperature 22℃～25℃, 60%-70% humidity, light intensity 150mmol m ^-2 s ^-1 . Salt treatment with 100 mM NaCl was initiated when cultured to one month old. After treatment for different time, the leaf RNA of seedlings was extracted by Trizol method.

(2) RNA extraction

Preparations: RNase-free gun tip, RNase-free EP tube (2.0mL, with steel balls; 1.5mL), liquid nitrogen, 75% ethanol; 70% alcohol for sterilizing the tabletop and centrifuge instruments;

DEPC water preparation: prepare 1000 mL of DEPC treated water, add 1 mL of DEPC, make up to 1000 mL with ultrapure water, and stir with a magnetic stirrer overnight. Then high pressure steam sterilization at 123 ℃, 40min (DEPC is toxic, but decomposes into carbon dioxide and alcohol after sterilization), try to avoid pollution, refrigerate at 4 ℃ or room temperature for use.

The total RNA of leaves was extracted by TrizoLRT. The main steps are as follows:

1) Take the sample (≤100mg) and put it into a 2mL EP tube with steel balls, put it into liquid nitrogen immediately, and crush it with a grinder. The grinder parameters: 25-30HZ for 60 seconds;

2) Add 1 mL of TrizoLRT, vortex the constant temperature mixer, 2000rpm, 3min;

3) Add 400 μL of DEPC water, vortex with a constant temperature mixer, 2000 rpm, 3 min; let stand for 5 min, centrifuge at 12000 rpm at room temperature, 15 min;

4) Transfer the supernatant into a 1.5mL EP tube (in duplicate, 600uL/tube), add an equal volume of isopropanol, mix gently, stand at room temperature for 15 minutes, centrifuge at 12000 rpm for 10 minutes at room temperature, and discard the supernatant;

5) Add 800 μL of 75% ethanol to wash the precipitate, gently bounce the precipitate, and centrifuge at 4000 rpm for 3 min at room temperature; repeat the washing with 75% ethanol, discard the supernatant, and centrifuge briefly. Clean the workbench for 1 to 2 minutes, until the edge is translucent and taken out.

6) Add 50 μL of DEPC water, flick, 60 ℃ metal bath for 10 minutes;

7) Place at room temperature for 2 min, and measure the concentration on ice.

(3) Reverse transcription to obtain cDNA

Reverse transcription kit: 5×All-In-One RT MasterMix (with AccuRT Genomic DNARemoval Kit)

1) Put the extracted RNA and the freezing reagents in the reverse transcription kit on ice, centrifuge briefly after dissolving, and collect the samples at the bottom of the tube. All the following steps are performed on ice;

2) Put the RNase-free 200μL PCR tube on a 96-well ice plate, generally do three biological replicates, and the total volume of one reaction is 20μL;

3) Add reaction system 1 to the tube first:

4) Put the above reaction system 1 into the PCR machine, incubate at 42°C for 2 min, and place the tube on ice to cool after the reaction is over;

5) Add reaction system 2 to the tube:

Denature the template and primer mixture to stop the reaction;

6) Finally, add reaction system 3 to the tube:

7) Carefully mix the above total system, centrifuge at the bottom of the tube briefly, and put it into the PCR machine. The reaction procedure is as follows:

After the reaction, the product was stored in a -20°C refrigerator.

(4) Real-time fluorescence quantitative PCR

BIO-RAD real-time PCR instrument; SYBR green Mix;

The primer sequences are as follows:

MtREV-qF: GGAAAATGAGAGGCTGCAGAAG; MtREV-qR: TGCTGGCGCATAAATCCAT; MtUBQ-qF: CTGACAGCCCACTGAATTGTGA; MtUBQ-qR: TTTTGGCATTGCTGCAAGC.

cDNA was diluted to 15ng/μL as template; forward primer and reverse primer were mixed in advance, 2.5μM

ReaL-time PCR system (10μL):

SYBR 5μL

primers 2μL

cDNA template 3μL

Real-time PCR procedure:

The relative expression level was calculated according to the 2 ^-ΔΔCT method. Statistical analysis using t-test; three biological replicates; * indicates P<0.05, the results are shown in Figure 1.

Figure 1 shows: qRT-PCR detection of MtREVOLUTA up-regulated expression in Medicago truncatula transgenic in response to salt stress. The bar graph at 0 h in the figure represents the expression level of MtREVOLUTA in leaves of wild-type Medicago truncatula R108 one month old before 100 mM NaCl treatment. The bar graph at 12h in the figure represents the expression level of MtREVOLUTA in leaves of one-month-old wild-type Medicago truncatula R108 at 12h after 100 mM NaCl treatment. The bar graph at 24 h in the figure represents the expression level of MtREVOLUTA in the leaves of one-month-old wild-type Medicago truncatula R108 at 24 h after 100 mM NaCl treatment. Three biological replicates; * indicates P<0.05, 25 seedlings per batch of plants.

Example 2: Construction of Dicotyledonous Plant Expression Vector

Amplify MtREVOLUTA-specific primer sequences as follows:

MtREV-F: CACCATGGCTATGGCTGTTGCAC;

MtREV-qR:GGGGGTTTAAATCCAGCAATAGGTC.

1) PCR system:

2) PCR program:

95℃5min; 25～30cycles 95℃30s, 60℃30s, 72℃30s; 72℃5min.

3) Recycling connection conversion

After the PCR product was subjected to agarose gel electrophoresis, the gel block at the size of the target fragment was cut into a 1.5mL EP tube. The PCR product was recovered according to the instructions of the ordinary agarose gel DNA recovery kit (TIANGEN, DP209).

The recovered product was ligated into the pEntry-T vector (Thermo Fisher Scientific, product number 12536017), and the plasmid was cloned in E. coli DH5α by the method of transformation. LR reaction was performed to connect the gene MtREVOLUTA to the expression vector pEARLYGATE100, and then the plasmid was extracted, and the plasmid was transformed into Agrobacterium strain EHA105.

Example 3: Transformation of alfalfa and screening of transgenic plants

The medium required for genetic transformation of alfalfa was prepared according to the following formula.

1) Prepare medium for sterilization in advance, and sterilize secondary water, petri dish, filter paper, triangular flask, and 50mL centrifuge tube.

2) Agrobacterium preparation. Two days before the genetic transformation experiment, pick the successfully transformed Agrobacterium monoclonal or Agrobacterium stock described in Example 2, use 2 mL of LB liquid medium to load the corresponding antibiotics, and place it on a shaker at 200 rpm and 28°C overnight.

3) Proliferate 100 μL of the shaken Agrobacterium liquid, place it in 30-50 mL liquid medium (containing the corresponding antibiotics), and cultivate overnight at 200 rpm at 28° C. The OD ₆₀₀ nm of the Agrobacterium liquid is about 0.8-1.0.

4) Surface sterilization of alfalfa SY4D leaves. Young and fully expanded healthy leaves were selected for genetic transformation experiments. The removed leaves were placed on ice and brought back to the laboratory. If using the ultrasonic method, take a whole compound leaf; if the leaf disc is to be cut, take a single leaf.

5) Disinfection. Place the retrieved leaves in a 50mL centrifuge tube with a lid (do not have too many leaves, too much will lead to incomplete sterilization). Add 30% sodium hypochlorite solution and 1‰ Tween-20, and invert the centrifuge tube several times to ensure that after each leaf is infiltrated, place it on a horizontal shaker for 15 minutes at low speed (change a new sodium hypochlorite solution every 7 minutes). Pour out the sodium hypochlorite solution and wash with clean water 3-4 times. After washing, the leaves were placed on sterilized filter paper to absorb excess water.

6) The explants were inoculated with bacterial liquid and co-cultured. The bacterial solution was centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the bacterial solution was gently resuspended in SM4 liquid medium and diluted to an OD ₆₀₀ nm of 0.2.

7) Leaf cutting disc: Place the sterile leaves in a sterilized petri dish, cut off the leaf edge with a sharp sterile blade, and cut into a square leaf disc. When cutting, be careful not to over-dry the leaves.

8) Agrobacterium infection: The cut leaf discs were placed in Agrobacterium SM4 liquid medium. Gentle shaking keeps the blisks from overlapping each other.

9) Vacuuming: vacuumize the Agrobacterium SM4 solution 0.5 Mbar put into the leaf disc for 10 min. To avoid damage to the cells, the vacuum switch should be performed slowly. Shake slowly for another 5-10min.

10) In the ultra-clean workbench, suck up the bacterial liquid, and transfer the infected leaves to sterilized filter paper to absorb the excess bacterial liquid around the leaves, but be careful to avoid the wind in the workbench from blowing the leaves too dry. . The leaves from the above steps were transferred to SM4 solid medium (containing 1 mL of cefo). Try to keep the lower surface of the leaf disc in contact with the medium.

11) After 5-7 days, transfer the leaf discs to SM4 solid medium containing carrier-corresponding resistant antibiotics (add cefo 500mg/L, and add screening antibiotics such as Kan 50mg/L, PPT 3mg/L, hyg 10mg/L), Incubate in the dark at 24°C for 4-6 weeks in a plant incubator.

12) When the callus is large enough (generally 4-5 weeks in SM4 screening medium), transfer to regeneration medium MSBK and cultivate for 2 weeks.

13) Transfer to elongation medium MSS until plants grow, typically 6-8 weeks.

14) Transfer the plants to culture flasks, and the medium is MSR. After the seedlings have taken root, move the robust plants to the soil.

15) Medicago truncatula DNA extraction. Prepare 2×CTAB (pH8.0) extract (1L) in advance:

Note: First make the volume to 1L, add CTAB powder, stir and dissolve, and then place it in a 65°C oven to accelerate the dissolution, and store at room temperature.

16) For the genomic DNA extracted from alfalfa, use the vector primer GCACAATCCCACTATCCTTC and the gene reverse primer to perform PCR amplification detection. The RNA reverse cDNAs of the above-mentioned positive transgenic plants and wild plants were extracted for RT-PCR detection using the gene forward primer TACACTGCTGAACAGATTGAAG and the gene reverse primer ACCAGGCTTCATCCCAGGCATC. The results are shown in Figure 2.

Internal reference used MsACTIN-F: TTTGAGACTTTCAATGTGCCCGCC and MsACTIN-R: TAGCATGTGGGAGTGCATAACCCT.

The results are shown in Figure 2.

Figure 2 shows: RT-PCR detection of MtREVOLUTA expression in leaves of 35S:MtREV alfalfa transgenic plants of different lines has obvious differences, the high expression is named 35S:MtREV#1, and the low expression is named 35S: MtREV#2. The upper row of gels indicated the expression levels of MtREVOLUTA in leaves of wild-type alfalfa SY4D and 35S:MtREV alfalfa transgenic plants of different lines. The bottom row of gums indicated the expression levels of housekeeping gene MsActin in leaves of wild-type alfalfa SY4D and 35S:MtREV alfalfa transgenic plants of different lines.

Example 4: Salt tolerance analysis of 35S:MtREV purple flower transgenic cutting seedlings

The wild plants and transgenic plants 35S:MtREV purple flower cuttings were selected, and 25 seedlings with the same cutting survival status were selected, cultivated for one month, and the seedlings in the small pots were treated with high-salt 1.5% NaCl for 5 weeks.

The results are shown in Figures 3 and 4.

Figure 3 shows that the salt-treated one-month-old 35S:MtREV#1 transgenic cutting plants of alfalfa have improved salt tolerance compared to the control wild-type alfalfa SY4D cutting plants. A, B, C, D, I, J, K, L, Q, R, S, T shown in the figure show wild-type alfalfa SY4D and 35S:MtREV#1 flowers of the control group without saline, respectively Growth of alfalfa transgenic plants before treatment and 1, 2, 3, 4, and 5 weeks after treatment. E, F, G, H, M, N, O, P, U, V, W, X shown in the figure show the wild-type alfalfa SY4D and 35S:MtREV of the treatment group applied with 1.5% NaCl saline, respectively. 1 Growth of alfalfa transgenic plants before treatment and 1, 2, 3, 4, and 5 weeks after treatment.

Figure 4 shows: t-test statistical analysis shows that the survival rate of 35S:MtREV alfalfa transgenic plants 35S:MtREV#1 was significantly improved compared to wild type SY4D after salt treatment. Three biological replicates; * indicates P<0.05, 25 seedlings per batch of plants.

sequence listing

<110> Shandong University

Application of <120> Medicago truncatula gene MtREVOLUTA in improving the salinity tolerance of alfalfa, a closely related leguminous forage grass

<141> 2022-03-11

<160>1

<210> 1

<211> 4380

<212> DNA

<213> Artificial sequences

<221> Nucleotide sequence of Medicago truncatula gene MtREVOLUTA

<222>(1)…(4380)

<400>1

atggctatgg ctgttgcaca acaacaaaga gataacagca ttgagagaca ccttgattcg 60

tctggcaaat atgtgaggta cactgctgaa cagattgaag ctttggaaaa ggtttatgtg 120

gaatgcccta agcctagttc attgagaagg caacagctga ttcgggagtg cccggttctg 180

gccaacattg agcctaagca gatcaaggtt tggtttcaga ataggaggta atggagattc 240

tgattcacct ttttttgttt gttttgaatt tgtgtcgtgt ggaagggttt ggctcttttt 300

ggttgtgtga tttgatttgt gtctttcttg ttttgcaggt gtagggagaa gcagagaaaa 360

gaggcttctc agcttcagag tgtgaacagg aaactttctg cgatgaataa gctgttgatg 420

gaggaaaatg agaggctgca gaagcaggtt tcacagctgg tgaatgagaa tggatttatg 480

cgccagcaac tacaccctgt aagccctatc attgttcatt ttcattcact actatacatt 540

ttttttttgt gaaatggtaa ttatcattgt ttggttgagg catgtagttg ttgtatgata 600

tgatatgatt tgggttattt tgaatgtaaa tttgtattgt tacttactgt catgtcattg 660

cagaccccag cagctccaaa tgctgacggt agtggcgttg attccgcggc tgctgctcct 720

atgaactcat tgagagatgc taatagccct gctgggtaat tttgaagttc ttgatttgag 780

tgctttttat ttattttaa ttatttcgct tgttgagctt tctttgattg atctttgcag 840

attcctatca attgcggagg agacattgac agagttcctt tcaaaggcta caggaactgc 900

tgtcgattgg gtccagatgc ctgggatgaa ggtagagaat catgtttcta gtggacaatt 960

ggttttttgct tttacaattt tgatactgtg atgattatga catggaggtt aatttccttc 1020

cactaatatc tcatactata tttactttca accattgttt gttagcctgg tccggattcg 1080

gttgggatat ttgccatttc tcaaggtggc aacggagtgg cagctcgagc ctgtggtctt 1140

gttagtttag aacctactaa ggtaattaaa aaggacatgt ggatggatat tttcagtaat 1200

ttcttttgat gttatttatg tagtttgtaa gctatctgaa ttttgaattg ctgaaaaatt 1260

tcaatagatt gtggagatat taaaagatcg cccaacttgg taccgtgatt gtcggagttc 1320

agaagttttc acaatgttcc cagctggaaa tggaggaaca attgaacttg tttacacaca 1380

ggtgaagaat caaatgtgaa taggatgctt tatttattat ttaatgagca ttttgcataa 1440

acgtttagtt tgctgcagac atatgctcca atgacactgg cttctgctcg cgacttctgg 1500

actctaagat acactacaaa tttggaaaac ggaagtgttg tggtgagtat atttgctgtc 1560

aaaagcgtta ctattgttgc atgggattat atatctagct gcatcgattt aatataattg 1620

cactttggaa ggtttgtgaa aggtcactgt ctggtactgg tgctggccct aatgctgcag 1680

ccgcctcaca gtttgagagg gctgaaatgc tccctagtgg ctatttgatt cgaccatgtg 1740

aaggtggagg atcgatcatc cacattgtag accacctaaa cctgcaggtt tgagttcttg 1800

accattagct agaataccta ctatattctt attttctttt catatacatt ttttttttttg 1860

accaaaatcc agaattttct tttcatatac attaattaac atgattaaca ttttcttcta 1920

ggcatggagt gtgccagaag tgctgcggcc gatctatgaa tcgtcgcaaa tggtagctca 1980

gagactgaca attgcggtaa gcatagtatt attaattcac gatgtaggat tctattgcaa 2040

gctttggtgg actaatgtga tcaatattat ggcttttgtg caggcacttc gctatatcag 2100

gcaagtagct caagaaacaa gtggtgacgt ggtgtatagc atgggtcggc aacctgcagt 2160

tcttagaact tttagccaac ggttgagcag gtacgtcacg tgaaataaat ttatgcctca 2220

aaacctattt cagccttgct ttttacagaa cgatctgttg tgtttggtaa aaataaattt 2280

aaacatcatt cttgcagagg tttcaatgac gctgtcaatg gattcaatga taatggttgg 2340

tctgttctga actgtgatgg tgctgagggt gttactattt cagtaaattc aatcaagaat 2400

ttgagtggca cttctaatcc agcaagttcc ctttcactcc ttggaggaat tgtctgtgca 2460

aaagcttcta tgttactcca agtaagtgcg taaatccatt ggcatggcgc agtaatcggt 2520

ccttctataa tttaacattt agttcttcta atcgttacac ggaaagtttt cactttgttt 2580

tacttgcaga acaccactcc tgctgtttta gttcgctttc tgagggagca tcgctcggag 2640

tgggctgatt ttagtgttga tgccttttct gctgcatcac ttaaagctgg ctcctatggc 2700

tatcctggaa tgaggtctac aaagttcacc ggcaatcaag caatcatgcc tcttggacat 2760

acaattgaac atgaagaggt atgagagatt ttttgcttgc ttgaccttga ccatgtcttt 2820

ttaagatggt agattcatat ttgcatttgg ctaagatttg gtttatatac tttctatgat 2880

ttggtttata ctgatttctg ttgaaattcc agatgctaga aattatccgc cttgaaggtc 2940

ttgctcaaga tgattctttt gtttctaggg atgttcatct cttacaggtg cttcctctga 3000

cccttgttat ggtttgttta cctgcatgtt tatcggtttt ttgttgttgc ttaattctta 3060

tgatcaccat catcatcgtt tgaacagtta tgtactggaa ttgatgagaa tgctgtgggg 3120

gcttgttccg agctcatatt tgctccaatt gatgacatgt tcccagaaga tgctccctta 3180

gtgccttctg gtttccgcat tgtcctgttg aattctcaac cagttgagtc ccgtttcttg 3240

tatttgattt ttctctaatc ggtactgttc atatatgaag catagtaatc tagttgacat 3300

catattgtgt ttcagggtga tacaaagaac acaacaacag caaatcgaac cttggatttg 3360

acatctggtc ttgaagtaag cccggcaaca gctcatgcta acggagacgc atcgtgtcct 3420

aacaatcgat gtgtgttgac tgttgccttt cagtttcctt ttgagagcgg tctgcaggat 3480

aatgttgcag ccatggcacg tcaatatgtc cggcgtgtag tttctgccgt gcaggcggtt 3540

gcaacggcta tatctccatc cagtgttaac acttctggtg gagcaaagct ctcccctggc 3600

actccagaag cacttacact agctcaatgg atctgccaga gttataggta aagtctgcat 3660

gaatctctga tgttctcttt caaagtttct aacatgattc tctatttacc tctctttcta 3720

tatctcttgt ccgaaagcag tcatcatctg ggcgcgcaac tgctgagatc tgattctctt 3780

attggtgata tgctactgaa acatttgtgg catcatccag atgctatttt atgctgctct 3840

ttgaaggtat gtccatgatc tcattttatg gtacaattga atctggaagt gtataaataa 3900

tgcagtactt cgaagatttt aatgtaactt ctctatcctt gtttggtttt gcagcaagtg 3960

cccgtattca tctttgctaa ccaggctggc cttgacatgt tggaaacaac tctagtggct 4020

ctacaagata tcacactgga caaaatattt gatgagtctg cacgcaagaa tttgattgca 4080

tattttgcga agttaatgca gcaggtaatt tcctggagtt tggcatcatt agcttagctt 4140

ttttttatcg ttacaacctc aaatttgttt taaacaacac accatgctct taagtaaatg 4200

tctgaattgt tgcacggatt ttttttcgtt caggggtttg cttgtatgcc agctgggatc 4260

tgcatgtcaa caatggggcg acatgcttca tatgatcaag ccgtcgcgtg gaaagtgcat 4320

gctgaagaca acagtgttca ttgcttggct ttctcattca ttaattggtc atttatatga 4380

Claims (1)

1. Application of the Medicago truncatula gene MtREVOLUTA in improving the survival rate of alfalfa SY4D tolerant to 1.5% NaCl treatment, wherein the nucleotide sequence of the Medicago truncatula gene MtREVOLUTA is shown in SEQ ID No.1.

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