Alfalfa Msjaz1 gene, transcription factor, interacting protein, promoter and application thereof

Abstract

The application discloses alfalfa MsJAZ1 gene, transcription factor, interactive protein, promoter and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the MsJAZ1 gene is shown in sequence 1, the nucleotide sequence of the interactive protein MsCCCH16 is shown in sequence 2, the nucleotide sequence of the promoter is shown in sequence 3, and the nucleotide sequence of the transcription factor MsCPRF2 is shown in sequence 4. The application contributes to the cultivation of excellent cold-resistant plants.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to an alfalfa MsJAZ1 gene, its transcription factors, interacting proteins, promoters, and their applications. Background Technology

[0002] Alfalfa (Medicago sativa L.) is a high-quality perennial leguminous forage crop characterized by wide adaptability, strong resistance, high yield, good palatability, and high crude protein content. Widely cultivated worldwide, it is known as the "King of Forages." Alfalfa has a well-developed root system, with the taproot penetrating deep into the soil and symbiotic root nodules. Alfalfa harvested at the initial flowering stage typically has a crude protein content of around 20%, and the processed forage products are rich in vitamins, flavonoids, and polysaccharides. Due to its excellent economic efficiency, high yield potential, symbiotic nitrogen fixation with rhizobia (SNF), and outstanding nutritional value, alfalfa has long been a focus of global agricultural biological research. In my country, the overall planting area of ​​alfalfa is small, the hay yield is low, and the supply to the livestock industry is insufficient. The self-sufficiency rate of high-quality alfalfa is only 64%, requiring large-scale imports. Furthermore, my country's alfalfa breeding level is relatively backward, mostly focusing on high yields and lacking superior, stress-resistant varieties, failing to meet the needs of domestic livestock development. Therefore, developing high-quality, resistant varieties that can adapt to different regions of China is crucial for the development of the alfalfa industry.

[0003] In northern my country, cold winters and frequent late spring frosts cause widespread alfalfa death and failure to regrow due to freezing damage, indicating that low temperatures have severely restricted alfalfa development in the region. When plants are exposed to non-freezing temperatures for extended periods, they acquire greater frost resistance—a phenomenon known as cold adaptation—and there are significant differences in cold tolerance among different alfalfa varieties. Therefore, exploring the molecular mechanisms of alfalfa's low-temperature perception and response at the physiological and molecular levels is crucial for breeding cold-resistant, high-yielding alfalfa varieties, improving alfalfa's overwintering and regrowth capabilities, ensuring safe overwintering, and achieving stable and high alfalfa yields in northern China. Summary of the Invention

[0004] Therefore, the purpose of this invention is to provide an alfalfa MsJAZ1 gene and its transcription factors, interacting proteins, promoters and their applications, so as to cultivate superior alfalfa varieties.

[0005] The present invention solves the above problems through the following technical solutions:

[0006] A MsJAZ1 gene of alfalfa, the nucleotide sequence of which is shown in Sequence 1.

[0007] An interacting protein, MsCCCH16, is capable of interacting with the MsJAZ1 gene, the nucleotide sequence of which is shown in Sequence 1, and the amino acid sequence of the interacting protein, MsCCCH16, is shown in Sequence 2.

[0008] A promoter for the alfalfa MsJAZ1 gene, wherein the nucleotide sequence of the MsJAZ1 gene is shown in Sequence 1, and the promoter is:

[0009] A. A nucleotide sequence as shown in sequence 3;

[0010] B. Nucleotide sequences with equivalent or similar promoter functions obtained by substituting, deleting, or adding one or more nucleotide sequences as shown in Sequence 3;

[0011] C. A nucleotide sequence as shown in Sequence 3 that has at least 90% homology and is a derived nucleotide sequence with equivalent function.

[0012] A transcription factor MsCPRF2 that can bind to the promoter of claim 3, the nucleotide sequence of said transcription factor MsCPRF2 being shown in sequence 4.

[0013] Furthermore, the application of any one or more of the MsJAZ1 gene according to claim 1, the interacting protein MsCCCH16 according to claim 2, and the transcription factor MsCPRF2 according to claim 4 in regulating the plant's resistance to cold, cold tolerance, or low temperature resistance.

[0014] Furthermore, methods to improve the plant's resistance to cold, cold tolerance, or low temperature resistance include knocking out, silencing, or inhibiting one or more of the MsJAZ1 gene, transcription factor MsCPRF2, and interacting protein MsCCCH16.

[0015] A biomaterial, wherein the biomaterial is any one of the following:

[0016] a. A gene expression cassette containing one or more of the MsJAZ1 gene as described in claim 1, the promoter as described in claim 3, and the transcription factor MsCPRF2 as described in claim 4;

[0017] b. A recombinant vector containing one or more of the MsJAZ1 gene as described in claim 1, the promoter as described in claim 3, and the transcription factor MsCPRF2 as described in claim 4;

[0018] Furthermore, it includes host cells, engineered bacteria, or recipient plant cells containing the biomaterial as described in claim 7.

[0019] A method for improving plant cold resistance, the method comprising reducing or weakening the expression or activity of any one or more of the MsJAZ1 gene as described in claim 1, the interacting protein MsCCCH16 as described in claim 2, and the transcription factor MsCPRF2 as described in claim 4 in the plant.

[0020] Furthermore, the method can induce the expression of the low-temperature response genes MsCBF3 and MsCOR47.

[0021] Furthermore, the cold resistance of plants can be improved by inducing the expression of the low-temperature response genes MsCBF3 and MsCOR47.

[0022] Furthermore, the plant described in this invention is either alfalfa or tobacco.

[0023] Beneficial effects:

[0024] 1. Reducing, silencing, or inhibiting the expression of the MsJAZ1 gene can increase the survival rate of alfalfa under low temperature stress, reduce cell membrane damage, decrease the accumulation of reactive oxygen species, improve the ability to scavenge reactive oxygen species and the content of osmotic regulatory substances, and improve the cold resistance of alfalfa by inducing the expression of low temperature response genes MsCBF3 and MsCOR47.

[0025] 2. Yeast one-hybrid and dual luciferase experiments verified that MsCPRF2 can bind to the MsJAZ1 promoter and regulate the cold resistance and frost resistance of alfalfa.

[0026] 3. Yeast two-hybrid and bimolecular fluorescence complementation experiments verified that MsJAZ1 and the interacting protein MsCCCH16 interact in the cell nucleus.

[0027] 4. GUS staining experiments can prove that the MsJAZ1 promoter has high activity.

[0028] In summary, the key cold-resistance gene MsJAZ1 in alfalfa was successfully identified, and a cold-resistance molecular regulatory network of MsCPRF2-MsJAZ1-MsCCCH16 with MsJAZ1 as the node was constructed. The results show that MsJAZ1 is not only regulated by the MsCPRF2 transcription factor, but also interacts with MsCCCH16 to jointly regulate the expression of CBF-dependent low-temperature response genes, affecting membrane permeability, antioxidant enzyme activity, and the content of osmotic regulators, thus regulating the cold-resistance of alfalfa and contributing to the breeding of superior varieties. Attached Figure Description

[0029] Figure 1 Results of PCR amplification of MsJAZ1 gene, MsCPRF2 transcription factor, and MsCCCH16;

[0030] Figure 2 Subcellular localization of MsJAZ1 protein and MsCPRF2 protein;

[0031] Figure 3 : Analysis of MsJAZ1 expression patterns in tissues and under low temperature stress; (A) Analysis of MsJAZ1 expression patterns in tissues; (B) Analysis of MsJAZ1 expression patterns in aboveground tissues under 4℃ stress; (C) Analysis of MsJAZ1 expression patterns in underground tissues under 4℃ stress; (D) Analysis of MsJAZ1 expression patterns in aboveground tissues under -5℃ stress; (E) Analysis of MsJAZ1 expression patterns in underground tissues under -5℃ stress;

[0032] Figure 4 Genetic transformation process of alfalfa. (A) Alfalfa leaves; (B) Callus induction; (C) Adventitious bud differentiation; (D) Adventitious root induction;

[0033] Figure 5 Detection of transgenic positive plants. (A) Bar test strip detection in OE plants; (B) RT-PCR detection of Bar gene in OE plants; (C) Bar test strip detection in RNAi plants; (D) RT-PCR detection of Bar gene in RNAi plants;

[0034] Figure 6 Determination of relative expression levels of MsJAZ1 in transgenic plants. (A) OE; (B) RNAi;

[0035] Figure 7 Phenotypic and survival rate analysis of plants subjected to low temperature stress. (A) Phenotypic; (B) Survival rate;

[0036] Figure 8 Plant plasma membrane stability under low temperature stress. (A) Relative conductivity; (B) MDA content;

[0037] Figure 9 ROS detection in alfalfa under low temperature stress. (A) NBT staining; (B) O2- content; (C) DAB staining; (D) H2O2 content; Note: The scale bar in Figures A and C is 2 cm long;

[0038] Figure 10 Analysis of ROS scavenging capacity of alfalfa under low temperature stress. (A) SOD activity; (B) POD activity; (C) CAT activity; (D) Flavonoid content;

[0039] Figure 11 Content of osmotic regulators in alfalfa under low-temperature stress. (A) Soluble protein; (B) Soluble sugar; (C) Proline;

[0040] Figure 12Chlorophyll content of alfalfa under low temperature stress. (A) Chlorophyll a; (B) Chlorophyll b; (C) Total chlorophyll;

[0041] Figure 13 Relative expression levels of low-temperature response genes. (A) MsICE1; (B) MsCBF1; (C) MsCBF2; (D) MsCBF3; (E) MsCOR47;

[0042] Figure 14 GUS transient staining results of tobacco leaves; Note: The scale bar in the figure is 0.2 cm long;

[0043] Figure 15 MsCPRF2 expression pattern analysis. (A) Tissue expression pattern analysis of MsCPRF2; (B) Expression pattern analysis of MsCPRF2 in aboveground tissues under 4℃ stress; (C) Expression pattern analysis of MsCPRF2 in aboveground tissues under -5℃ stress;

[0044] Figure 16 Prediction of interaction sites between the MsCPRF2 and MsJAZ1 promoters;

[0045] Figure 17 Verification was performed using yeast mono- and heterozygous interactions between the MsCPRF2 and MsJAZ1 promoters;

[0046] Figure 18 Dual-LUC fluorescence images;

[0047] Figure 19 MsCCCH16 expression pattern analysis. (A) Tissue expression pattern analysis of MsCCCH16; (B) Expression pattern analysis of MsCCCH16 in aboveground tissues under 4℃ stress; (C) Expression pattern analysis of MsCCCH16 in aboveground tissues under -5℃ stress;

[0048] Figure 20 Prediction of interaction sites between MsJAZ1 and MsCCCH16;

[0049] Figure 21 The interaction between MsJAZ1 and MsCCCH16 yeast was used for verification.

[0050] Figure 22 Verification of bimolecular fluorescence complementarity between MsJAZ1 and MsCCCH16. Detailed Implementation

[0051] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings:

[0052] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods. Unless otherwise specified, the materials and reagents used in the embodiments of this invention are commercially available.

[0053] The alfalfa variety “Dongnong No.1” (Medicago sativa L. cv. Dongnong No.1) and tobacco (Nicotiana benthamiana) used in this invention were provided by the Key Laboratory of Forage Germplasm Resources and Breeding of Heilongjiang Province.

[0054] Table 1-1 Primer sequences

[0055]

[0056] Example 1: Cloning of MsJAZ1 gene, transcription factor MsCPRF2, promoter, and interacting protein MsCCCH16

[0057] Total RNA was extracted from alfalfa using the "Dongnong No. 1" RNA extraction kit and then reverse transcribed into cDNA using a reverse transcription kit. The reverse transcription system was as follows:

[0058] Step 1: Add 1 μg Total RNA + 4 μL 4×gDNA wiper Mix to 16 μL RNase-free Water, 42℃; 2 min;

[0059] Step 2: 5×HiScript. Ⅱ Select qRT Super Mix Ⅱ 4µL, 50℃, 15 min, 85℃, 5 s, Total volume 20μL.

[0060] I. Cloning of the MsJAZ1 gene

[0061] (1) Using alfalfa cDNA of "Dongnong No. 1" as a template, specific primers were designed using the MS.gene057422 sequence as a reference (Table 1-1). PCR amplification was then performed in a 20 µL system. The reaction system is shown in RT-PCR 20 µL reaction system (Table 1). The reaction program was: 95℃, 3 min; 95℃, 15 s, 58℃, 15 s, 72℃, 60 s for 35 cycles; 72℃, 5 min. The fragment length was verified by agarose gel electrophoresis. After obtaining fragments of the correct length, amplification was performed again in a 50 µL system. The system is shown in Table 1. The reaction program was: 95℃, 3 min; 95℃, 10 s, 58℃, 20 s, 72℃, 40 s for 35 cycles; 72℃, 5 min. The fragment length was verified by agarose gel electrophoresis. After verification, the target fragment was cut, and the DNA fragment was purified using a product purification kit. The product concentration was recorded.

[0062] The purified DNA fragments were ligated to the pCE2 TA / Blunt-Zero vector using the 5 min TA / Blunt-Zero Cloning Kit. The ligation system is shown in Table 2, and the experimental procedures were performed according to the manufacturer's instructions. The ligation product was transformed into DH5α E. coli competent cells and transferred to LB solid medium containing 50 µg / mL Kan. The cells were incubated overnight at 37°C. Single colonies were picked for colony PCR identification. Positive colonies were amplified and cultured in LB liquid medium containing 50 µg / mL Kan at 37°C and 200 rpm. The colonies were then sent to Jilin Kumei Biotechnology Co., Ltd. for sequencing. The MsJAZ1 CDS sequence was obtained by comparing the sequencing results. The bacterial culture was mixed with 30% glycerol at a 1:1 ratio and added to a 1.5 mL sterile centrifuge tube and stored at -80°C. The amplified 753 bp nucleotide sequence is shown below. Figure 1 As shown in the figure, the nucleotide sequence is shown in Sequence 1, and the 250 amino acids encoded are shown in Sequence 5. The resulting gene is named MsJAZ1.

[0063] The physicochemical properties, structural prediction, and subcellular localization of the MsJAZ1 protein are summarized below:

[0064] MsJAZ1 is a hydrophilic and unstable protein with a molecular weight of approximately 27.147 kDa and an isoelectric point of 8.83, making it slightly basic. Its structure is highly flexible, primarily consisting of random coils, and it contains multiple potential phosphorylation sites, located in the cell nucleus. Figure 2 ).

[0065] II. Promoter Cloning

[0066] (2) The upstream 2,000 bp sequence of the CDS region of the MsJAZ1 gene was extracted using TBtools software, and this sequence was used as a reference for cloning the promoter of the MsJAZ1 gene. DNA was extracted from alfalfa 'Dongnong No. 1' using a DNA extraction kit. Specific primers MsJAZ1pro-F and MsJAZ1pro-R were used to perform PCR on the alfalfa DNA. The primer sequences are shown in Table 1-1. The reaction system (50 µL) is shown in Table 1. The reaction program was: 95℃, 3 min; 95℃, 10 s, 58℃, 20 s, 72℃, 40 s, 35 cycles, 72℃, 5 min. The final MsJAZ1 promoter sequence was obtained, and its nucleotide sequence is shown in Sequence 3.

[0067] III. Cloning of the transcription factor MsCPRF2

[0068] (3) Following (1), using "Dongnong No. 1" alfalfa as a template, specific primers were designed based on the MS.gene24570 sequence to amplify it. The results are as follows: After sequencing and alignment, a full-length nucleotide sequence of 1,176 bp was obtained, as shown in Sequence 4. The nucleotide sequence was then translated into an amino acid sequence using Snapgene software, as shown in Sequence 7. The gene was named MsCPRF2. MsCPRF2 is a hydrophilic and unstable transcription factor with abundant potential phosphorylation modification sites.

[0069] The physicochemical properties, structural characteristics, and subcellular localization of the MsCPRF2 protein are summarized below: it is located in the cell nucleus and cell membrane. Figure 2 This suggests that it may regulate alfalfa's low-temperature stress response by receiving membrane-related signals and translocating to the cell nucleus, thereby binding to the promoters of downstream genes.

[0070] IV. Cloning of the interacting protein MsCCCH16

[0071] (4) Following (1), using "Dongnong No. 1" alfalfa as a template, specific primers were designed based on the MS.gene031723 sequence to amplify it. The results are as follows: Figure 1 As shown in Sequence 2, the full-length nucleotide sequence of 1,158 bp was obtained after sequencing alignment. The nucleotide sequence was then translated into an amino acid sequence using Snapgene software, as shown in Sequence 6. The gene was named MsCCCH16.

[0072] The physicochemical properties, structural characteristics, and subcellular localization of the MsCCCH16 protein are summarized below: MsCCCH16 is a highly flexible, unstable, hydrophilic protein with numerous potential phosphorylation sites, and it is located in the cell nucleus and cell membrane. Figure 22 Together with MsJAZ1, they may interact in the nucleus or membrane-associated signaling complex, thereby participating in the regulation of alfalfa's low-temperature stress response.

[0073] Table 1 RT-PCR reaction system

[0074]

[0075] Table 2 TA / Blunt-Zero Cloning Reaction System

[0076]

[0077] Example 2: Analysis of MsJAZ1 gene expression pattern and verification of gene function

[0078] I. Analysis of MsJAZ1 gene expression pattern

[0079] Select plump "Dongnong No. 1" alfalfa seeds, wash them with 75% ethanol for 1 min, then treat them with NaClO for 10 min, and finally rinse them three times with distilled water. After vernalization in distilled water at 4℃ for 2 days, the seeds were placed in petri dishes with moistened filter paper for germination. Once the cotyledons unfolded, select alfalfa seedlings of uniform growth and transplant them into soil (nutrient soil: vermiculite = 1:1) for cultivation. The cultivation conditions were 16 h light / 8 h darkness, 23℃ during the day / 20℃ at night, a light intensity of 10,000 Lux, and 50% humidity. The seeds were irrigated every 3 days with alfalfa-modified nutrient solution.

[0080] Five-leaf stage alfalfa plants with uniform growth were selected and placed in a low-temperature incubator to be subjected to 4℃ (cold) and -5℃ (freezing) stress. Sampling of the aboveground and underground parts of plants under 4℃ stress was performed at 0 h, 2 h, 4 h, 8 h, 12 h, 24 h, and 48 h. Sampling of the aboveground and underground parts of plants under -5℃ stress was performed at 0 h, 2 h, 4 h, 6 h, 8 h, and 12 h, with three biological replicates for each time point. To prevent stress response in the plants, the incubator temperature was decreased at a rate of 1℃ / h, and sampling began when the temperature reached the required stress level (0 h). After sampling, the tissues were flash-frozen in liquid nitrogen and stored at -80℃ for analysis of the expression pattern of alfalfa MsJAZ1.

[0081] Roots, stems, young leaves, old leaves, and flowers of alfalfa were collected. After sampling, the tissues were flash-frozen in liquid nitrogen and stored at -80°C for analysis of the tissue expression pattern of MsJAZ1. RNA was extracted from different tissues under different treatments and converted into cDNA, as described in Example 1. The cDNA concentration of each sample was measured using a micro-nucleic acid protein concentration analyzer. The cDNA concentration was diluted to 100-120 ng / µL using ddH2O. RT-qPCR was performed on the obtained cDNA using the internal reference genes MsActin and MsJAZ1, respectively. The reaction system is shown in Table 3, and the primers are shown in Table 1-1. The program was set as follows: 95°C, 30 s; 95°C, 10 s; 60°C, 30 s, 40 cycles. The target gene was compared with the internal reference gene using 2- ΔΔCt The relative expression level was calculated, and all responses were repeated three times.

[0082] Table 3 RT-qPCR reaction system

[0083]

[0084] Results Analysis: The expression patterns of MsJAZ1 in different tissues of alfalfa and in the aboveground and underground parts under low-temperature stress were analyzed. Figure 3 As shown in Figure A, MsJAZ1 expression was highest in stems and lowest in roots. Under 4℃ treatment, the relative expression level of MsJAZ1 in the aboveground parts showed a trend of first increasing and then decreasing, reaching its highest level at 24 h, which was 18.04 times that of the control (CK). Figure 3 B). The relative expression level in the underground part showed a trend of first increasing, then decreasing, and then increasing again, reaching its highest level at 8 h, which was 4.18 times that of CK. It decreased to the CK level at 12 h, and then increased to 2.36 times that of CK at 24 h. Figure 3 C). Under -5℃ treatment, the relative expression level of the aerial parts reached a peak at 6 h, partially declined at 8 h, and then rose again to the highest level at 12 h, reaching 7.21 times that of the control (CK). Figure 3 D). The relative expression level of the underground part did not change significantly in the first 6 hours, increased significantly to 2.72 times that of CK at 8 hours, then began to decrease, and dropped to 2.35 times that at the start of treatment at 12 hours. Figure 3 E).

[0085] In summary, MsJAZ1 was expressed at the highest level in stems and the lowest level in roots, suggesting that its function may be mainly related to aboveground growth and development or stress response. The expression increase induced by cold stress (4℃) was much higher than that frost damage (-5℃), especially in the aboveground parts at 24 h, where the difference was significant. Alfalfa responded faster in the aboveground parts than in the underground parts under low temperature treatment.

[0086] II. Obtaining positive MsJAZ1 transgenic alfalfa plants

[0087] This experiment used Agrobacterium-mediated transformation to perform genetic transformation of alfalfa, as follows: Figure 4 As shown. The specific steps are as follows:

[0088] (1) Select plump "Dongnong No. 1" alfalfa seeds, grind them with sandpaper, disinfect them with 75% ethanol for 5 min, rinse them with sterile water for 1 min, disinfect them with 84 disinfectant for 3 min, and rinse them with sterile water 3 times. Sow the disinfected alfalfa seeds on germination medium (pH=5.8, 2.22g / L 1 / 2MS + sucrose 15g / L + agar 0.8%), 16 h light / 8 h dark, daytime 23℃ / nighttime 20℃ for 2-3 months. Cut the sterile alfalfa leaves into small pieces and place them on pre-culture medium (pH=5.8, 4.44g / L MS + sucrose 30g / L + 6-BA 1mg / L + agar 0.85%).

[0089] (2) Take the activated Agrobacterium overexpression and interference vector bacterial suspension and resuspend it in antibiotic-free YEB medium to OD600=0.2~0.3. Place the leaves that have been pre-cultured for 2~3 days in Agrobacterium resuspension for 10~15 min to inoculate them. After inoculation, the infected alfalfa leaves are inoculated onto filter paper and dried. After drying, they are inoculated into co-medium (pH=5.2, 4.44g / L MS + sucrose 30g / L + 6-BA 1mg / L + acetylsyleugenol 100µM + agar 0.85%) and incubated in the dark for 48~72 h.

[0090] (3) The co-cultured leaves were placed in a selection medium (pH=5.8, SH 14.21g / L + sucrose 20g / L + 6-BA 0.5mg / L + 2,4-D 4mg / L + thiazolyl 200mg / L + termethin 200mg / L + glufosinate 2mg / L + agar 0.8%) and cultured for 60 days until callus tissue grew.

[0091] (4) Select suitable callus tissue and transfer it to differentiation medium containing glufosinate (pH=5.8, MS 4.44g / L + sucrose 30g / L + 6-BA 0.5mg / L + kinetin 1mg / L + thiazomycin 200mg / L + termethin 200mg / L + glufosinate 1mg / L + agar 0.8%), 16 h light / 8 h dark, daytime 23℃ / nighttime 20℃ for about 60 days.

[0092] (5) After the callus tissue produces buds, it is transferred to elongation medium (pH=5.8, SH 14.21g / L + sucrose 10g / L + thiazomycin 200mg / L + termethin 200mg / L + glufosinate 1mg / L + agar 0.8%) for 16 h light / 8 h dark, daytime 23℃ / nighttime 20℃ for about 60 days.

[0093] (6) After the callus grows into seedlings, remove the excess callus, dip the bottom of the seedlings in 100 mg / L indolebutyric acid for 2 min and then place them in rooting medium (pH=5.8, 2.22 g / L MS + 8 g / L sucrose + 0.7% agar) for about 30 days until adventitious roots grow.

[0094] (7) After the roots have grown and new leaves have emerged above ground, loosely open the sterile bottle cap to harden the seedlings. After 2 days, open the bottle cap, take out the seedlings, wash off the culture medium and excess callus tissue with running water, remove the withered and yellow small leaves, and transplant them into a small box containing soil (nutrient soil: vermiculite = 1:1);

[0095] (8) After the new leaves grow, the seedlings were tested for Bar protein using a transgenic rapid detection kit; leaf RNA was extracted and reversed into cDNA, and RT-PCR was performed using Bar gene-specific primers; MsJAZ1 gene expression was detected by RT-qPCR using MsJAZ1 fluorescence quantitative specific primers. The primers are shown in Table 1-1. Finally, positive MsJAZ1 transgenic plants were identified.

[0096] Because the vector backbone carries a Bar resistance (glufosinate) selection gene, after detecting positive plants using Bar test strips, specific primers were used to amplify the Bar gene in the initially screened positive plants by PCR, resulting in 9 OE-positive and 9 RNAi-positive MsJAZ1 transgenic alfalfa plants. Figure 5 ).

[0097] RT-qPCR was performed on positive plants using specific primers to detect the expression level of the MsJAZ1 gene. The expression level in OE plants was significantly increased (p<0.05), while in RNAi plants, 2 plants showed no decrease in expression, and 7 plants showed a significant decrease (p<0.05). MsJAZ1-OE6 and MsJAZ1-OE8, with higher expression levels, and MsJAZ1-RNAi1 and MsJAZ1-RNAi5, with lower expression levels, were selected for subsequent experiments. Figure 6 ).

[0098] III. Cold Resistance Determination of MsJAZ1 Transgenic Alfalfa

[0099] Alfalfa was propagated by cuttings in plug trays, with two cuttings per hole. Alfalfa plants of uniform growth, 28 days old, were placed in a low-temperature incubator to simulate low-temperature stress. The growing conditions were: 16 h light / 8 h darkness, 23°C daytime / 20°C nighttime, 10000 Lux light intensity, and 50% humidity. To simulate natural cooling, the incubator was set to cool down at 1°C every 2 h, with stress beginning when the temperature reached 4°C. After 7 days of stress, the above-ground parts of the alfalfa were collected, quickly wrapped in aluminum foil, flash-frozen in liquid nitrogen, and stored at -80°C.

[0100] After 7 days of treatment at 4℃, the low-temperature incubator was programmed to cool at a rate of 1℃ / h. Once the temperature reached -5℃, this was maintained for 8 hours to continue simulating frost stress on the alfalfa plants. After stress, samples were taken from the aboveground tissue above 10 cm. The samples were quickly wrapped in aluminum foil and flash-frozen in liquid nitrogen, then stored at -80℃. The remaining plants were incubated at 4℃ overnight, and then moved to room temperature for 3 days to recover. The survival rate was calculated as follows: Survival rate = Surviving plants after stress / Total number of plants.

[0101] The SOD activity, POD activity, CAT activity, MDA content, H2O2 content, and O2 content of plant samples were measured using Suzhou Keming Physiological Reagent Kit. - Content, Pro content, SS content, SP content, and flavonoid content were determined according to the instructions. DAB and NBT staining methods were performed according to the kit instructions. Leaf chlorophyll was extracted using 95% ethanol, following the principles and techniques of plant physiological and biochemical experiments. Relative conductivity was determined using the water bath method.

[0102] The conclusions drawn are as follows:

[0103] (1) Analysis of plant phenotype and survival rate under low temperature stress

[0104] like Figure 7 As shown, the function of the MsJAZ1 gene was analyzed by subjecting transgenic and wild-type plants to -5℃ low-temperature stress. The results showed no significant phenotypic differences in alfalfa plants treated at 4℃ for 7 days. However, after further treatment at -5℃ for 8 hours, the leaves of WT and OE plants wilted and the plants collapsed. The RNAi plants showed less damage than the WT and OE plants, indicating that short-term freezing damage is far greater than cold damage. Phenotypic characteristics reflect the degree of damage to the aboveground parts of the plant, and survival rate directly reflects the differences in cold tolerance among plants. Therefore, we measured the survival rate of transgenic and wild-type plants after thawing and returning to normal conditions for 3 days following -5℃ stress. We found that the survival rates of RNAi1 and RNAi5 were significantly higher than those of WT and OE lines (p<0.05), indicating that interfering with MsJAZ1 expression improved the survival rate of alfalfa after low-temperature stress.

[0105] (2) Determination of the stability of the alfalfa plasma membrane under low temperature stress

[0106] The MDA content and relative conductivity of plants can reflect the degree of damage to the plant plasma membrane. Figure 8 The results showed that the relative conductivity and MDA content after low-temperature stress were consistent after treatment at 4℃ and -5℃, with OE plants showing higher values ​​than WT plants and RNAi plants showing the lowest values. Low temperature led to an increase in relative conductivity and MDA content in alfalfa, indicating that RNAi plants could mitigate the damage to the plasma membrane caused by low temperature and improve the cold resistance of alfalfa. Overexpression of MsJAZ1, however, reduced the plant's cold resistance. Considering the survival rate, although the relative conductivity of alfalfa leaves under -5℃ stress reached 61.46% for WT plants and 51.42% and 54.88% for RNAi plants, respectively, the actual survival rate was better than that under ion leakage stress. This suggests that low temperature primarily damages alfalfa leaves, while the root damage from low-temperature freezing in the soil may be less than the direct damage to the leaves. After relief at 4℃ and restoration to normal conditions, some plants could still regrow. The relative electrical conductivity of OE6 and OE8 plants was 71.25% and 69.55% respectively after low-temperature stress, but their actual survival rates were only 37.50% and 40.63%, indicating that both the aboveground and underground parts of the OE plants were damaged by low-temperature stress, and this damage could not be alleviated after returning to normal conditions. Furthermore, this suggests that overexpression of MsJAZ1 increases electrolyte extravasation in alfalfa, accelerating the damage to the alfalfa membrane system caused by low temperature, while interfering with MsJAZ1 expression reduces the damage to plasma membrane stability caused by low temperature.

[0107] (3) ROS detection in alfalfa under low temperature stress

[0108] The production of reactive oxygen species (ROS) affects cell membrane function and disrupts cell membrane structure. Histochemical staining with diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) can visually reflect the H2O2 and O2 content in alfalfa leaves under low-temperature stress. - The accumulation. For example... Figure 9 As shown in Figures A and B, alfalfa leaves treated at -5℃ exhibited more brownish-yellow and blue hues than those treated at 4℃ and CK. Furthermore, under the same treatment, OE plants showed more brownish-yellow and blue hues than WT and RNAi plants, while RNAi plants showed higher levels of H2O2 and O2. - The accumulation is minimal. For H2O2 and O2 - Content determination revealed that, consistent with staining results, the content of both was highest in OE plants and second highest in WT plants under both 4℃ and -5℃ low-temperature treatments, while the content was lowest in RNAi plants. Figure 9 -C, D). This also indicates that under low temperature stress, OE plants produce more ROS than WT plants, while RNAi plants produce the least amount of ROS, which can reduce the toxic effects of excessive peroxide accumulation in plants.

[0109] (4) Analysis of ROS scavenging capacity of alfalfa under low temperature stress

[0110] Antioxidant enzymes can promote the clearance of ROS and alleviate the damage caused by ROS accumulation. SOD activity was increased at 4℃, with only RNAi5 significantly higher than WT (p<0.05), reaching 466.81 U·g. -1 After being subjected to -5℃ stress, the SOD activity of RNAi plants continued to increase, significantly higher than that of WT (p<0.05); while the SOD activity of OE plants increased but remained the lowest. Figure 10 -A). Treatment at 4℃ and -5℃ both increased the POD activity of all plants, with RNAi plants showing the highest POD activity, followed by WT, and OE plants showing the lowest. Figure 10 -B). CAT activity increased at 4℃ and decreased at -5℃, with OE6 showing an activity of 60.81 U·g at -5℃. -1 The levels were significantly lower than WT (p<0.05), with RNAi1 and RNAi5 at 72.98 U·g, respectively. -1 and 73.45 U·g -1 Significantly higher than WT (p<0.05) Figure 10 -C). Flavonoids, as members of the non-enzymatic antioxidant system, play an important role in scavenging ROS. Results showed that under 4℃ stress, except for OE6, whose flavonoid content was significantly lower than WT (p<0.05), the flavonoid content of other lines was not significantly different from WT; under -5℃ stress, the flavonoid content of RNAi1 and RNAi5 increased by 1.11 times and 1.18 times respectively compared to 4℃, and their contents were significantly higher than those of WT and OE plants (p<0.05). Figure 10 -D). The results indicate that interfering with MsJAZ1 expression can increase the activity of antioxidant enzymes and flavonoid content in plants under low-temperature stress, increase ROS scavenging capacity, and reduce oxidative damage.

[0111] (5) Effects of low temperature stress on the content of osmotic regulators in alfalfa

[0112] Osmotic regulators within plants can maintain cell osmotic pressure and alleviate damage caused by low-temperature stress. We measured the contents of proline, soluble sugars, and soluble proteins to analyze the changes in the content of osmotic regulators in alfalfa under low-temperature stress. Figure 11As shown, under 4℃ treatment, the proline and soluble sugar contents of RNAi plants were significantly higher than those of WT and OE plants, while the soluble sugar content of OE plants was significantly lower than that of WT (p<0.05). Compared with 4℃, the soluble sugar content of WT and OE plants did not change significantly under -5℃ stress, but the soluble sugar contents of RNAi1 and RNAi5 increased by 1.18 times and were significantly higher than those of WT and OE (p<0.05). The proline level increased under -5℃ stress in all plants, but the content was highest in RNAi plants, followed by WT, and the content was lowest in OE plants, significantly lower than that of WT and RNAi (p<0.05). The results indicate that MsJAZ1 interference promotes the accumulation of osmotic regulatory substances under low temperature stress, while overexpression in plants reduces the accumulation of osmotic regulatory substances.

[0113] (6) Effects of low temperature stress on chlorophyll content in alfalfa leaves

[0114] The determination of chlorophyll a, chlorophyll b, and total chlorophyll content in alfalfa leaves revealed that low-temperature stress led to a decrease in chlorophyll content in all plants. The chlorophyll a, chlorophyll b, and total chlorophyll contents of RNAi plants treated at -5℃ were significantly higher than those of WT and RNAi plants (p<0.05), while there was no significant difference between WT and RNAi plants. After treatment at 4℃, although there was no significant difference in chlorophyll levels among the plants, the trend was the same as after treatment at -5℃, i.e., RNAi plants had higher contents, and OE plants had lower contents. Figure 12 This indicates that low temperatures can impair a plant's photosynthetic capacity, leading to a decrease in chlorophyll content. However, RNAi plants can reduce the damage to photosynthetic capacity caused by low temperatures and alleviate the decrease in chlorophyll content.

[0115] In summary, MsJAZ1 acts as a negative regulator in the low-temperature response of alfalfa. Its overexpression exacerbates membrane damage, ROS accumulation, antioxidant inhibition, weakened osmotic regulation, and photosynthetic damage. Interference with its expression improves cold resistance through multiple pathways, such as stabilizing the plasma membrane, enhancing ROS scavenging, accumulating osmotic substances, and protecting chlorophyll, ultimately resulting in a higher survival rate after freezing.

[0116] Example 3: Expression of MsCBF3 and MsCOR47, low-temperature response genes, in MsJAZ1 transgenic alfalfa

[0117] Based on Example 2, RNA was extracted from plant samples in the control and treatment groups and converted into cDNA, as described in Example 1. The cDNA concentration of each sample was measured using a micro-nucleic acid protein concentration analyzer. The cDNA concentration was diluted to 100-120 ng / µL using ddH2O. RT-qPCR was performed on the obtained cDNA using the internal control gene MsActin and the stress response gene fluorescent quantitative primers, respectively. The reaction system is shown in Table 3, and the primers are shown in Table 1-1. The program was set as follows: 95℃, 30 s; 95℃, 10 s; 60℃, 30 s, 40 cycles. Using 96-well samples, the target gene was compared with the internal control. 2- ΔΔCt The relative expression level was calculated, and all responses were repeated three times.

[0118] Results analysis:

[0119] The results obtained are as follows Figure 13 As shown, the relative expression levels of all genes increased after cold stress, and the expression levels of RNAi plants under 4℃ and -5℃ stress were significantly higher than those of WT and OE plants. Under CK conditions, the relative expression levels of MsCBF2 and MsCOR47 genes in RNAi plants were significantly higher than those in WT (p<0.05), while the relative expression levels of other genes were not significantly different from those in WT. However, the expression levels of all low-temperature response genes in overexpressing plants were not significantly different from those in WT. Under 4℃ stress, the expression levels of MsCBF2 and MsCBF3 in OE plants were not significantly different from those in WT, while the expression levels of other genes were significantly lower than those in WT (p<0.05). Under -5℃ stress, the relative expression levels of all low-temperature response genes in OE plants were significantly lower than those in WT (p<0.05). These results indicate that RNAi plants can induce high expression of low-temperature response genes under low-temperature stress, while the expression levels of low-temperature response genes in OE plants are reduced. Interference with MsJAZ1 expression improved the survival rate of alfalfa under low-temperature stress and reduced membrane permeability. Furthermore, the enhanced ability to scavenge reactive oxygen species (ROS) indicated that RNAi plants could maintain high antioxidant enzyme activity to scavenge ROS under both chilling and freezing stress, thus improving the cold tolerance of alfalfa.

[0120] In summary, the increased content of osmotic regulators in RNAi plants indicates stronger resistance to cold stress and slows down the degradation of chlorophyll content after low-temperature stress, thus improving cold resistance. Furthermore, the upregulation of low-temperature response gene expression in plants further demonstrates that interfering with MsJAZ1 gene expression can improve the cold resistance of alfalfa by increasing the relative expression levels of MsCBF2 and MsCOR47.

[0121] Example 4: Promoter Activity Analysis

[0122] Primers MsJAZ1pro-GUS-F and MsJAZ1pro-GUS-R were designed based on the MsJAZ1pro sequence for the pCAMBIA3301-GUS expression vector amplification. Primer sequences are shown in Table 1-1. PCR amplification of the MsJAZ1pro-T plasmid was performed using a PCR system (50 µL) as shown in Table 1. The agarose gel electrophoresis was performed, and the DNA fragments were obtained using a product purification kit. After measuring the concentration, the fragments were stored at -20°C.

[0123] The pCAMBIA3301-GUS vector was double-digested with HindIII and NcoI, as shown in Table 4. Homologous recombination of the linearized vector and insert fragment was performed using the one-step cloning kit in Table 4. The recombinant product was transformed into DH5α *E. coli* competent cells to obtain the GUS recombinant vector. Plasmids were extracted from correctly sequenced bacterial cultures using a plasmid extraction kit. After measuring the concentration, the plasmid DNA was transformed into EHA105 *Agrobacterium* competent cells using chemical transformation.

[0124] Table 4. Double enzyme digestion system and one-step cloning reaction system

[0125]

[0126] Agrobacterium tumefaciens suspension (pCAMBIA3301-MsJAZ1pro-GUS) was added to liquid YEB medium containing the corresponding antibiotic at a ratio of 1:100 and activated at 28°C and 180 rpm on a shaker until OD was reached. 600 =0.6, the activated bacterial solution was reactivated under the same conditions at a ratio of 1:10 until the OD value reached 0.6. 600 =0.6. Take 50 mL of bacterial culture and centrifuge at 3000 rpm for 15 min at 4℃. Discard the supernatant and resuspend the bacterial pellet in working solution (100 mL of 0.5 M MES + 100 mL of 20 mM Na3PO4 + 5 g of D-glucose + 100 µL of 1 M acetylsylcholine + 1 L of ddH2O). Resuspend to OD of the Agrobacterium culture. 600 =0.4.

[0127] The resuspended Agrobacterium bacterial suspension was injected into tobacco leaves at the five-leaf stage for expression, and then incubated in the dark for 2 days. After dark incubation, the control group received no treatment, while the treatment groups were treated with 100 µM ABA, 100 µM MeJA, 100 µM IAA, 100 µM GA3, at 4℃, and with 20% PEG6000. After 2 days, infected portions of tobacco leaves were collected using a perforator for GUS staining, following the instructions of the GUS staining kit. After destaining, images were taken using a stereomicroscope.

[0128] Results analysis:

[0129] The results obtained are as follows Figure 14 As shown, GUS staining was darker under MeJA, IAA, 4℃, and drought treatments, indicating increased MsJAZ1 promoter activity and a response to MeJA, IAA, 4℃, and drought stress. Staining was not significant under ABA and GA3 treatments, indicating a lower response of MsJAZ1 promoter activity to ABA and GA3.

[0130] Example 5: Expression pattern analysis and gene function verification of transcription factor MsCPRF2

[0131] The expression pattern of transcription factor MsCPRF2 and gene function were analyzed and verified based on the experiment in Example 3.

[0132] 1. Analysis of the expression pattern of transcription factor MsCPRF2

[0133] The expression pattern of MsCPRF2 in different tissues and aboveground parts of alfalfa under low temperature stress was analyzed. As shown in the figure, the expression level of MsCPRF2 was the highest in new leaves and the lowest in old leaves. Figure 15 A). Under 4℃ stress, the expression level of MsCPRF2 showed a trend of first decreasing and then increasing, with the lowest expression level at 8 h, which was 0.17 times that of CK. After 8 h, the expression level gradually increased, and the highest expression level at 48 h was 3.83 times that of CK. Figure 15 B). The expression pattern under -5℃ stress also showed a trend of first decreasing and then increasing, reaching a minimum of 0.52 times that of CK at 8 h, and returning to a level with no significant difference compared to CK at 12 h. Figure 15 C).

[0134] 2. Predictive analysis of interaction sites between transcription factor MsCPRF2 and promoter

[0135] Predictive analysis was performed to determine whether the MsCPRF2 transcription factor interacts with the MsJAZ1 promoter. The results showed that MsCPRF2 and MsJAZ1 interact with each other, with a confidence level of 0.9086. Hydrogen bonds were the primary interaction force, and 14 hydrogen bond interaction sites were detected. Figure 16 Table 5 shows the hydrogen bond interaction sites.

[0136]

[0137] 3. Verification of MsCPRF2 and MsJAZ1 promoter yeast one-hybrid (Y1H)

[0138] Single colonies successfully verified in the experimental group (pHIS2-MsJAZ1pro+pGADT7-MsCPRF2), the control group (pHIS2-MsJAZ1pro+pGADT7), and the positive control (pGAD53m+pHIS2-p53) were resuspended in 2 mL of ddH2O and adjusted to OD200. 600 =0.002, concentration set to three gradients of 10 0 10 -1 10 -2 Take 10 μL for plate testing, and spot it onto SD-TL, SD-TLH, and SD-TLH+75 mM 3AT medium, respectively. Spot three spots on each plate and incubate at 30℃ for 3~5 days.

[0139] Results analysis:

[0140] In the one-to-one validation of yeast one-hybrid assays, the positive control pGAD53m+pHIS2-p53 grew normally on all plates. The control group pHIS2-MsJAZ1pro+pGADT7 could not grow on SD-TL or SD-TLH plates, nor on SD-TLH with 75 mM 3AT. The experimental group pHIS2-MsJAZ1pro+pGADT7-MsCPRF2 grew in the same manner as the positive control. Figure 17 The results indicate that the MsJAZ1 promoter interacts with MsCPRF2.

[0141] 4. Dual-LUC assay

[0142] Agrobacterium bifidum culture containing empty vectors pGreenⅡ-62SK and pGreenⅡ-0800-LUC was added to liquid YEB medium containing the corresponding antibiotics and activated at 28°C and 180 rpm on a shaker until OD was reached. 600 =0.6, the activated bacterial solution was reactivated under the same conditions at a ratio of 1:10 until the OD value reached 0.6. 600 =0.6. Take 50 mL of bacterial culture and centrifuge at 3000 rpm for 15 min at 4℃. Discard the supernatant and resuspend the bacterial pellet in working solution (MES 10 mM + MgCl2 10 mM + acetylsyleugenol 10 µM). Resuspend to OD of Agrobacterium tumefaciens. 600 =0.5, 28℃, protected from light for 2~3 hours.

[0143] The resuspended Agrobacterium bacterial suspension was mixed at a ratio of 9:1 for each of the following formulations: pGreenⅡ-62SK-MsCPRF2:pGreenⅡ-0800-MsJAZ1pro-LUC, pGreenⅡ-62SK-MsCPRF2:pGreenⅡ-0800-LUC, pGreenⅡ-62SK:pGreenⅡ-0800-MsJAZ1pro-LUC, and pGreenⅡ-62SK:pGreenⅡ-0800-LUC. The mixture was then injected into the underside of 3-5 leaves below the top of 4-6 week old tobacco plants. After injection, the plants were incubated in the dark for 1 day, followed by light incubation for 1-2 days. The undersides of the infected tobacco leaves were then coated with potassium fluorescein and incubated in the dark for 10 minutes before being imaged using a fully automated chemiluminescence imaging system.

[0144] Results analysis:

[0145] Recombinant vectors MsCPRF2-62-SK and MsJAZ1pro-LUC were constructed, and their binding activity was verified by transient transformation of tobacco. Figure 18 As shown, the fluorescence intensity of LUC in tobacco leaves co-transformed with the MsCPRF2 and MsJAZ1 promoters was higher than that in tobacco leaves transformed with the 62-SK and MsJAZ1 promoters. This indicates that MsCPRF2 can regulate the expression of the MsJAZ1 promoter.

[0146] Example 6: Expression pattern analysis and gene function verification of the interacting protein MsCCCH16

[0147] Analysis of the expression pattern and verification of gene function of the interacting protein MsCCCH16 based on the experiment in Example 3.

[0148] 1. Analysis of MsCCCH16 expression pattern

[0149] Results analysis:

[0150] The expression pattern of MsCCCH16 in different tissues and aboveground parts of alfalfa under low temperature stress was analyzed. As shown in the figure, the expression level of MsCCCH16 was highest in new leaves, and relatively high in stems and flowers, with no significant difference compared to new leaves; the expression level was lower in old leaves and roots, but lowest in old leaves, with no significant difference between the two. Figure 19 A). Under 4℃ stress, the expression level gradually decreased before 8 hours, but there was no significant difference compared with CK. After 8 hours, the expression level gradually increased, reaching its highest level at 48 hours, which was 9.45 times that of CK. Figure 19 B). Expression levels under -5℃ stress decreased at 2 h, but showed no significant difference compared to the control (CK). Expression levels then gradually increased, reaching a peak at 12 h, which was 2.19 times that of the CK. Figure 19 C).

[0151] 2. Predictive analysis of interaction sites between MsJAZ1 and MsCCCH16

[0152] Predictive analysis was performed to determine whether MsJAZ1 and MsCCCH16 interact and the possible interaction sites. The results showed that MsJAZ1 and MsCCCH16 do interact, with a confidence level of 0.9808. A total of 12 hydrogen bond groups and 1 salt bridge interaction site were detected. Figure 20 As shown in Tables 6 and 7:

[0153] Table 6 Hydrogen bond interaction sites

[0154]

[0155] Table 7 Salt Bridge Interaction Sites

[0156]

[0157] 3. Verification of MsJAZ1 and MsCCCH16 yeast two-hybrid (Y2H)

[0158] The experimental group pGBKT7-MsJAZ1+pGADT7-MsCCCH16, the control group pGBKT7+pGADT7-MsCCCH16, pGBKT7-MsJAZ1+pGADT7, the positive control pGBKT7-p53+pGADT7-largeT, and the negative control pGBKT7-laminC+pGADT7-largeT, all of which had been validated by PCR, were resuspended in 2 mL of sterile ddH2O at three concentration gradients of 100, 10⁻¹, and 10⁻². 10 µL of each was then used for plate testing on defective plates containing SD-TL, SD-TLH+0.25 mM 3AT, SD-TLH+0.25 mM 3AT+X-α-gal, SD-TLHA+0.25 mM 3AT, and SD-TLHA+0.25 mM 3AT+X-α-gal.

[0159] Results analysis:

[0160] The positive control pGBKT7-p53+pGADT7-LargeT grew normally on all defective plates and showed a blue color on SD-TLH and SD-TLHA plates supplemented with X-α-gal. The negative control pGBKT7-laminC+pGADT7-LargeT grew normally only on SD-TLH plates and could not grow on other defective plates. The control groups pGBKT7+pGADT7-MsCCCH16 and pGBKT7-MsJAZ1+pGADT7 showed the same growth as the negative control. The experimental groups showed the same growth as the positive control. Figure 21The results indicate that MsJAZ1 and MsCCCH16 interact.

[0161] 4. Verification of bimolecular fluorescence complementarity (BiFC) between MsJAZ1 and MsCCCH16

[0162] Centrifuge 50 mL of bacterial culture at 3000 rpm for 15 min at 4℃, discard the supernatant, and resuspend the bacterial cells using working solution (100 mL of 0.5 M MES + 100 mL of 20 mM Na3PO4 + 5 g of D-glucose + 100 µL of 1 M acetylsalicylic acid + 1 L of ddH2O). The OD of the resuspended bacterial culture is... 600 =0.1~0.2, and the OD values ​​are equal. Take equal volumes of bacterial suspension and mix them evenly. The bacterial suspension combination includes positive controls pCAMBIA1300-35S-N-YFPN-OsHAL3 and pCAMBIA1300-35S-C-YFPC-OsHAL3; and experimental groups pCAMBIA1300-35S-N-YFPN-MsCCCH16 and pCAMBIA1300-35S-C-YFPC-MsJAZ1.

[0163] The mixed Agrobacterium bacterial suspension was injected into tobacco leaves at the five-leaf stage and cultured in the dark for 36-60 h for transient expression. Tobacco leaves near the pinholes were taken to make sections, and the presence and location of fluorescence signals were observed using a laser confocal microscope.

[0164] Results analysis:

[0165] Expression vectors for C-YFP-MsJAZ1 and N-YFP-MsCCCH16 were constructed and transiently expressed in tobacco leaf epidermal cells via Agrobacterium-mediated transformation. Results showed that the positive controls C-YFP-OsHAL3 and N-YFP-OsHAL3 interacted in tobacco leaf epidermal cells, while C-YFP-MsJAZ1 and N-YFP-MsCCCH16 exhibited fluorescent signals in the nuclei of tobacco leaf epidermal cells. This indicates that the two fluorescent protein fragments of N-YFP and C-YFP interact with each other in the nucleus to produce fluorescent signals, demonstrating that MsJAZ1 and MsCCCH16 proteins can interact in the plant cell nucleus. Figure 22 ).

[0166] In summary:

[0167] This invention constructs a low-temperature response regulatory model centered on the negative regulatory factor gene MsJAZ1, integrating its upstream promoter, transcription factor MsCPRF2, and interacting protein MsCCCH16. Systematic experiments verified the crucial role of MsJAZ1 in the cold tolerance of alfalfa. Expression pattern analysis showed that MsJAZ1 exhibits tissue specificity and low-temperature inducibility, with its expression level in the aboveground parts increasing 18.04 times that of the control under 4℃ stress. Transgenic functional validation demonstrated that interfering with MsJAZ1 expression significantly improved cold tolerance, specifically manifested in a substantial increase in survival rate after -5℃ stress (RNAi plants significantly outperformed WT and OE), enhanced cell membrane stability (MDA content and relative conductivity lower than WT and OE), improved reactive oxygen species scavenging capacity (increased SOD and POD activity and flavonoid content), increased accumulation of osmotic regulators (proline and soluble sugars), and slowed chlorophyll degradation. At the molecular level, yeast one-hybrid (Y1H) and dual-luciferase (Dual-LUC) experiments confirmed that the transcription factor MsCPRF2 directly binds to and activates the MsJAZ1 promoter, while yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments demonstrated that MsJAZ1 and MsCCCH16 specifically interact within the cell nucleus, thus refining the low-temperature signal transduction network from transcriptional regulation to protein interaction. This model provides a new target for crop stress resistance breeding and has significant theoretical and applied value.

[0168] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. An alfalfa MsJAZ1 gene, characterized in that, The nucleotide sequence of the MsJAZ1 gene is shown in Sequence 1.

2. An interacting protein MsCCCH16, characterized in that, The interacting protein MsCCCH16 can interact with the MsJAZ1 gene, the nucleotide sequence of which is shown in Sequence 1, and the nucleotide sequence of the interacting protein MsCCCH16 is shown in Sequence 2.

3. A promoter for the MsJAZ1 gene of alfalfa, characterized in that, The nucleotide sequence of the MsJAZ1 gene is shown in Sequence 1, and the promoter is: A. A nucleotide sequence as shown in sequence 3; B. Nucleotide sequences with equivalent or similar promoter functions obtained by substituting, deleting, or adding one or more nucleotide sequences as shown in Sequence 3; C. A nucleotide sequence as shown in Sequence 3 that has at least 90% homology and is a derived nucleotide sequence with equivalent function.

4. A transcription factor MsCPRF2, characterized in that, It can bind to the promoter of claim 3, and the nucleotide sequence of the transcription factor MsCPRF2 is shown in sequence 4.

5. The application of any one or more of the MsJAZ1 gene according to claim 1, the interacting protein MsCCCH16 according to claim 2, and the transcription factor MsCPRF2 according to claim 4 in regulating the plant's resistance to cold, cold tolerance, or low temperature resistance.

6. The application according to claim 5, characterized in that, Methods to improve the plant's resistance to cold, cold tolerance, or low temperature include knocking out, silencing, or inhibiting one or more of the MsJAZ1 gene, transcription factor MsCPRF2, and interacting protein MsCCCH16.

7. A biomaterial, characterized in that, The biomaterial is any one of the following: a. A gene expression cassette containing one or more of the MsJAZ1 gene as described in claim 1, the promoter as described in claim 3, and the transcription factor MsCPRF2 as described in claim 4; b. A recombinant vector containing one or more of the MsJAZ1 gene as described in claim 1, the promoter as described in claim 3, and the transcription factor MsCPRF2 as described in claim 4.

8. Host cells, engineered bacteria, or recipient plant cells containing the biological material as described in claim 7.

9. A method for improving the cold resistance of plants, characterized in that, The method for improving plant cold resistance includes reducing or weakening the expression or activity of any one or more of the MsJAZ1 gene as described in claim 1, the interacting protein MsCCCH16 as described in claim 2, and the transcription factor MsCPRF2 as described in claim 4 in the plant.

10. The method according to claim 9, characterized in that, The method can induce the expression of the cryo-response genes MsCBF3 and MsCOR47.

11. The method according to claim 10, characterized in that, The cold resistance of plants can be improved by inducing the expression of the low-temperature response genes MsCBF3 and MsCOR47.

12. The plant in the application according to any one of claims 5-6 or the method according to any one of claims 9-11 is one of alfalfa and tobacco.

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