Plant cold tolerance regulation genes CnMPK1 and CnbHLH130 and application

Abstract

The application provides a plant cold tolerance regulation gene CnMPK1 and CnbHLH130 and application, and belongs to the technical field of genetic breeding. The application clones and verifies the function of a key cold tolerance gene CnMPK1 and CnbHLH130 from a wild Elymus nutans in Tibet, breaks the one-way regulation mode of a kinase to a transcription factor in the prior research, and first discovers and proves that CnMPK1 and CnbHLH130 constitute a two-way positive feedback regulation module. CnMPK1 stabilizes CnbHLH130 protein through phosphorylation, CnbHLH130 can transcribe and activate the expression of CnMPK1 gene, forms a self-strengthening signal amplification loop, and significantly enhances the low-temperature response efficiency and resistance of the plant. The application also provides a new theoretical basis for clarifying the molecular basis of the adaptation of native grass species in the Qinghai-Tibet Plateau to low temperature, and provides important gene resources for cultivating new cold-tolerant grass varieties by using excellent cold-tolerance genes in alpine regions.

Description

Technical Field

[0001] This invention relates to the field of genetic breeding technology, and more particularly to a gene regulating plant cold resistance. CnMPK1 and CnbHLH130 and its applications. Background Technology

[0002] The Qinghai-Tibet Plateau region holds a vital ecological position but suffers from a harsh environment. Prolonged periods of low temperatures and intense radiation severely constrain the stability of local grassland ecosystems and forage production. Breeding and utilizing highly cold-resistant forage varieties is crucial for consolidating the region's ecological security barrier and developing distinctive grass-based livestock farming. However, current forage breeding faces core challenges, including insufficient exploration of superior stress-resistant gene resources, in-depth understanding of key regulatory mechanisms, and a weak foundation in molecular design breeding. Therefore, identifying key cold-resistant genes with independent intellectual property rights from native grass species adapted to extreme high-altitude environments and elucidating their mechanisms of action is of significant theoretical and applied value for cultivating new cold-resistant forage varieties and enhancing the cold resistance of closely related grasses.

[0003] Plants have developed complex molecular networks for cold resistance through long-term evolution. Among these, the mitogen-activated protein kinase (MAPK) cascade signaling pathway and basic helical-loop-helical (bHLH) transcription factors are recognized as two core regulatory elements. In model plants such as Arabidopsis thaliana and rice, MPK kinases can phosphorylate bHLH transcription factors such as ICE1, affecting their stability and activity, thereby positively regulating downstream cold resistance pathways such as CBF-COR, ultimately enhancing the plant's low-temperature tolerance. This indicates that the "MPK-bHLH" synergistic regulatory module plays a conserved and important role in plant cold resistance. However, existing research has mostly focused on a few model plants and major crops. For important native grass species that have long adapted to the extreme environment of the Qinghai-Tibet Plateau, whether they possess a unique and highly efficient "MPK-bHLH" regulatory module, the specific molecular interaction mechanism of this module, and its function in the adaptive evolution of plateau plants remains a lack of systematic and in-depth research. This limits our ability to discover key genes with significant breeding value from local genetic resources.

[0004] Tibetan wild *Leymus chinensis* is a superior native grass species unique to the Qinghai-Tibet Plateau, possessing extremely strong cold resistance potential and making it an ideal material for discovering excellent stress-resistance genes. Previous studies have shown that its cold resistance is closely related to complex signal transduction, transcriptional reprogramming, and metabolic regulatory networks. In-depth analysis of the unique cold-resistance molecular modules of this species will not only enrich the theory of plant cold resistance but also provide effective genetic resources for molecular breeding, addressing the shortage of excellent cold-resistance genes and promoting the development of forage breeding and ecological restoration in high-altitude cold regions. Summary of the Invention

[0005] The purpose of this invention is to provide a gene for regulating plant cold resistance. CnMPK1 and CnbHLH130 The application of this technology has solved the problem of the shortage of excellent cold-resistant gene resources and provided important gene resources for the breeding of new cold-resistant forage varieties using excellent cold-resistant genes from high-altitude and cold regions.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a plant cold tolerance regulatory gene, wherein the plant cold tolerance regulatory gene is... CnMPK1 and CnbHLH130 (GenBank: OR567265.1); The CnMPK1 The gene sequence is shown in SEQ ID NO.1.

[0007] Preferably, CnMPK1 can improve the protein stability of CnbHLH130 through phosphorylation modification and inhibit its degradation via the 26S proteasome pathway.

[0008] Preferably, the CnbHLH130 is reverse-binded and activated. CnMPK1 Transcriptional expression.

[0009] The present invention also provides an application of the above-mentioned plant cold resistance regulatory gene in improving plant cold resistance.

[0010] This invention also provides an application of the above-mentioned plant cold resistance regulatory gene in cold-resistant plant breeding.

[0011] This invention also provides a method for breeding cold-resistant plants, comprising the following steps: Build CnMPK1 Gene overexpression vector, and CnMPK1 Cold-resistant plants were obtained by transferring gene overexpression vectors into plants.

[0012] The beneficial effects of this invention compared to the prior art are as follows: (1) This invention cloned and functionally verified the key cold-resistant gene from wild Leymus chinensis in Tibet. CnMPK1 and CnbHLH130 This discovery breaks through the existing unidirectional regulatory model of "kinase → transcription factor" and is the first to discover and confirm its function. CnMPK1 and CnbHLH130 This constitutes a bidirectional positive feedback regulatory module. CnMPK1 stabilizes the CnbHLH130 protein through phosphorylation, while CnbHLH130 can in turn activate transcription. CnMPK1 The expression of these signals forms a self-reinforcing signal amplification loop, significantly enhancing the plant's low-temperature tolerance.

[0013] (2) This invention elucidates that CnMPK1 inhibits the degradation of CnbHLH130 via the 26S proteasome pathway through specific phosphorylation modification, thereby enhancing its protein stability. This module works synergistically to ultimately improve the overall cold resistance of plants by reducing physiological effects such as membrane lipid peroxidation and inhibiting the accumulation of reactive oxygen species.

[0014] (3) This invention provides a new theoretical basis for elucidating the molecular basis of the adaptation of native grass species in the Qinghai-Tibet Plateau to low temperature, and also provides important gene resources for the cultivation of new cold-resistant forage varieties using excellent cold-resistant genes in high-altitude and cold regions. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a phylogenetic tree of CnMPK1 and MPK1 homologous proteins from different plants constructed using the neighbor-joining method in Example 1 of this invention; Figure 2 This is an amino acid multiple sequence alignment of CnMPK1 and MPK1s in Example 1 of the present invention; Figure 3 Analysis of conserved motifs and domains of CnMPK1 and MPK1s homologous proteins in Example 1 of this invention; Figure 4 In Embodiment 1 of the present invention CnMPK1 The cloning and expression pattern analysis results of CDS are shown in the figure, where a is... CnMPK1 PCR amplification results of CDS; b is CnMPK1 Relative expression levels in roots, stem bases, and leaves; c represents... CnMPK1 Relative expression levels at different treatment times under room temperature and low temperature; different lowercase letters indicate that the differences reached a significant level. P <0.05 indicates the subcellular localization of CnMPK1, with a scale bar of 10 μm; Figure 5 Cn in Embodiment 2 of the present invention MPK1 Heterologous expression in rice improved plant cold tolerance, where 'a' represents the WT and Cn values ​​after 7 days of treatment at 4℃ under normal and cold stress conditions. MPK1 Overexpression of rice plant phenotype; Bar = 5 cm; b represents different strains MPK1Gene expression analysis; c shows the Western blot results of target protein accumulation in different strains; d shows the results of relevant physiological indicators of each strain after low-temperature treatment, from left to right: fresh weight, relative conductivity, MDA content, and superoxide anion content; different lowercase letters indicate that the differences reached a significant level. P <0.05; Figure 6 The silence Cn in Embodiment 3 of the present invention MPK1 Figure 1 shows the results of reducing the cold tolerance of wild *Leymus chinensis* in Tibet, where a represents the uncontrolled BSMV. γ And the silent material BSMV: CnMPK1 Phenotypic changes under ambient and low temperature treatments; Bar = 5cm; b is CnMPK1 Relative expression levels in the unloaded control and silenced materials; c represents the physiological index measurements of the unloaded control and silenced materials under room temperature and low temperature treatments, from left to right: superoxide anion content, electrolyte leakage rate, and MDA content; different lowercase letters indicate significant differences. This indicates a highly significant difference compared to the control. P <0.001); Figure 7 This is a phylogenetic tree of CnbHLH130 and bHLH130 homologous proteins from different plants constructed using the neighbor-joining method in Example 4 of the present invention; Figure 8 This is for the amino acid multiple sequence alignment of CnbHLH130 and bHLH130s in Example 4 of the present invention; Figure 9 This is for the analysis of conserved motifs and domains of CnbHLH130 and bHLH130s homologous proteins in Example 4 of the present invention; Figure 10 In Embodiment 4 of the present invention CnMPK1 The results of the interaction between CnMPK1 and CnbHLH130 in vivo and in vitro are shown in the figure. In the figure, a is the yeast two-hybrid detection of the interaction between CnMPK1 and CnbHLH130; b is the detection result of luciferase complementation imaging (LCI); c is the detection result of GST pull-down; d is the detection result of Co-IP. Figure 11 This is a graph showing the results of CnMPK1 phosphorylating CnbHLH130 in vivo and in vitro in Example 5 of the present invention. Figure a shows the phosphorylation effect of CnMPK1 on CnbHLH130 detected by in vitro kinase reaction combined with Phos-tag gel electrophoresis; figure b shows the in vivo phosphorylation level of CnbHLH130 in single-overexpression and double-overexpression plants detected using an Anti-Ser / Thr phosphorylation antibody; the left figure is... CnbHLH130Changes in phosphorylation levels in GFP-OE plants treated at 4 ℃ for different durations; the right figure shows... CnbHLH130 -GFP-OE and CnMPK1 × CnbHLH130 Comparison of phosphorylation levels in GFP-OE plants treated at 4 ℃; the numbers in the figure represent relative phosphorylation signal intensity. Figure 12 This is a diagram showing the results of CnMPK1's main phosphorylation of Ser4 and Ser49 sites of CnbHLH130 in Example 5 of this invention. In diagram a, CnMPK1 phosphorylation of wild-type CnbHLH130 and its point mutants was detected by in vitro kinase reaction combined with Phos-tag gel electrophoresis. Based on the predicted phosphorylation sites from NetPhos-3.1, S... 4A S 49A Single-point mutants and S 4A / S 49A Two-point mutant 2M; numbers in the figure represent relative phosphorylation signal intensity; b represents co-expression in plant cells. CnMPK1 -GFP and CnbHLH130 -Myc or CnbHLH130 2M -Myc, its phosphorylation level in vivo was detected using Phos-tag gel; the numbers in the figure represent the relative phosphorylation signal intensity; Figure 13 This is the result of CnMPK1 phosphorylation and enhancement of CnbHLH130 protein stability in Example 5 of the present invention, where a is the result of in vitro cell-free degradation experiment analysis of CnMPK1 on wild-type CnbHLH130-His and double-point mutant CnbHLH130. 2M - Effect of His protein stability; protein abundance changes were detected at different time points under conditions with and without MG132; the numbers in the figure represent relative protein signal intensity; b is the effect of CnMPK1 on the stability of CnbHLH130 protein analyzed by in vivo CHX tracking experiments; comparison CnbHLH130 -GFP-OE and CnMPK1×CnbHLH130 Protein degradation dynamics in GFP-OE plants with or without MG132; the numbers in the figure represent relative protein signal intensity. Detailed Implementation

[0017] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0018] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] CnMPK1 (SEQ ID NO.1):

[0023] Example 1 Embodiment 1 of the present invention was carried out CnMPK1 The cloning, bioinformatics analysis, and low-temperature response identification are performed using the following steps: 1.1 Plant materials and treatment Tibetan wild drooping elytra ( Campeiostachys nutans Seeds of *Griseb.* were collected from Damxung County, Tibet (30°28.535′ N, 91°06.246′ E, altitude 4678 m). Seeds were sterilized with 75% ethanol for 30–60 s, treated with 2.5% sodium hypochlorite for 5 min, rinsed with sterile water, and sown in sterilized vermiculite. They were then cultured in an artificial climate chamber with 16 h light / 8 h darkness and 22 / 18℃.

[0024] Four-week-old seedlings were subjected to low-temperature treatment: -10℃ stress for 12 h, followed by recovery at 4℃ for 7 days. Leaves were collected at different time points during the 4℃ treatment period and flash-frozen in liquid nitrogen for gene expression analysis. Materials for cold tolerance assessment were restored to room temperature for 3 days after the low-temperature treatment, and phenotypic and physiological indicators were statistically analyzed. Each treatment was performed in triplicate (30 seedlings per pot).

[0025] 1.2 Test Methods 1.2.1 Gene Cloning and Bioinformatics Analysis Candidate sequences for CnMPK1 were obtained based on transcriptome data, and their CDS (SEQ ID NO.1) were obtained through homologous cloning. Multiple sequence alignment, conserved motif and domain analysis, and phylogenetic analysis were performed using software such as NCBI, DNAMAN, MEME, and MEGA11. Visualization was performed using TBtools-II and iTOL. PlantCare was used to predict promoter cis-acting elements, with a focus on G-box elements. The results are as follows: Figure 1 As shown.

[0026] Figures 1-3 The results show that CnMPK1 encodes a typical MAPK family protein. Phylogenetic analysis indicates that CnMPK1 clusters with MPK1 homologs from grasses (Poaceae). Figure 1 It is most closely related to wheat TtMPK1 (VAI82478.1), rice OsMPK1 (NP_001389672.1), and maize ZmMPK1 (NP_001152745.2). Multiple sequence alignment showed that CnMPK1 shared 90.44% amino acid sequence identity with OsMPK1 and 88.24% with ZmMPK1. The protein kinase core region was highly conserved, with some differences observed in the N-terminal region. Figure 2Conserved motif and domain analysis revealed that CnMPK1 contains a typical STKc_TEY_MAPK kinase domain, belongs to the serine / threonine protein kinase family, and its motif composition and arrangement pattern are highly consistent with the homologous protein MPK1 of the Poaceae family. Figure 3 The above results indicate that CnMPK1 is an evolutionarily conserved member of the MAPK family.

[0027] 1.2.2 Expression pattern analysis and subcellular localization (1) RNA extraction and cDNA synthesis: RNA was extracted from roots, stem bases and leaves using the KK Ultrafast Plant Total RNA Extraction Kit (ZP405K), and cDNA was obtained by reverse transcription using the Zhuangmeng EX RT kit (gDNA remover, ZR108).

[0028] (2) Real-time quantitative PCR The ZF501 2×HQ SYBR qPCR Mix kit was used to perform the detection on a real-time quantitative PCR instrument, and the melting curve and Cp value were analyzed.

[0029] (3) Construction and transient expression of subcellular localization vectors Using a one-step cloning method (Novizan C112) to... CnMPK1 CDS was inserted into the pCambia1300:ubi:eGFP vector and transformed into *E. coli* DH5α, which was then verified by sequencing. The recombinant plasmid was transformed into *Agrobacterium* GV3101 (pSoup), and after resuscitation at 28°C, it was plated on LB agar plates containing antibiotics and rifampin. Positive clones were picked and shaken until OD=1.0-1.6, centrifuged to collect the cells, resuspended in the permeate, and then incubated with acetylsyleugenone in the dark for 2-3 hours. The cells were then injected into leaves of *Nicotiana benthamiana*, cultured at 25°C for 2-3 days, and observed using laser confocal microscopy.

[0030] Figure 4 The tissue expression analysis showed that CnMPK1 It is expressed in roots, stem base, and leaves, with the highest expression level in leaves, followed by stem base, and the lowest in roots. Figure 4 (b) Under low-temperature treatment (0, 3, 6, 12, 24 h), CnMPK1 The expression level showed a trend of first increasing and then decreasing: it began to rise at 3 h, reached its peak at 12 h, and decreased slightly at 24 h, but was still significantly higher than the control. Figure 4 c), indicating CnMPK1 This is a low-temperature response gene. Subcellular localization results showed that CnMPK1::eGFP fluorescence was mainly localized in the nucleus, co-localized with the nuclear marker NLS-mCherry, and also distributed in the cytoplasm and plasma membrane. Figure 4 (d).

[0031] In summary, CnMPK1 encodes a typical MAPK family protein, exhibits tissue-specific expression, is significantly induced by low temperature, and is located in the cell nucleus and cytoplasm, suggesting that it plays an important role in the plant's low-temperature response.

[0032] Example 2 CnMPK1 Construction and Cold Resistance Identification of Overexpression Rice Embodiment 2 of the present invention is for verification CnMPK1 Its function in regulating plant cold resistance, constructed in the ZH11 rice background. CnMPK1 The overexpression lines were then subjected to low-temperature treatment and cold resistance assessment. The specific steps are as follows: 2.1 Acquisition of genetically modified rice 2.1.1 Carrier Construction Using a one-step cloning method, CnMPK1 The full-length coding sequence (CDS) was cloned into the EcoRI site of the pCambia1300:ubi:eGFP vector. The successfully constructed plasmid was transformed into Agrobacterium tumefaciens EHA105, using the same method as in Example 1.2.2.

[0033] 2.1.2 Preparation of Rice Callus After removing the husks, mature rice seeds were sequentially soaked in 75% ethanol for 1-2 min, then sterilized with 1.25% sodium hypochlorite solution by shaking for 90 min, and finally soaked in 0.1% mercuric chloride solution for 30 min, followed by washing with sterile water 3-4 times. The sterilized seeds were then placed on callus induction medium and incubated in the dark at 26°C for approximately two weeks to induce callus formation. Subculture: every two weeks, pale yellow, dense callus tissue was selected for subsequent experiments.

[0034] 2.1.3 Co-cultivation Immerse the callus tissue in Agrobacterium suspension, gently shake at 28°C for 20 min, absorb excess bacterial solution with sterile filter paper, transfer to co-culture medium, and incubate in the dark at 26°C for 2-3 days.

[0035] 2.1.4 Screening and Differentiation of Resistant Callus After co-culture, the callus tissue was transferred to a selection medium containing kanamycin and cultured in the dark at 26°C for 14 days. Then, the medium was replaced with fresh selection medium for further culture. Light yellow resistant callus was selected and transferred to a differentiation medium containing kanamycin and Basta. After culturing in the dark at 26°C for 3 days, the culture was transferred to normal light. Green spots appeared after about 20 days, and seedlings differentiated after about one month.

[0036] 2.1.5 Rooting, Seedling Strengthening and Transplanting The differentiated shoots were transferred to a rooting medium containing Basta. After two weeks of culture, robust seedlings with well-developed root systems were selected, washed, and transplanted into greenhouse soil. The resulting plants were designated as the T1 generation. Seeds from the T2 generation were harvested through self-pollination for subsequent analysis.

[0037] 2.1.6 Identification of transgenic plants Two overexpression lines, OE#6 and OE#10, were obtained. qRT-PCR analysis showed that in both lines... CnMPK1 The expression levels of both were significantly higher than those of wild type ( Figure 5 (b); Western blot further verified the accumulation of the target protein ( Figure 5 (c)

[0038] 2.2 Low-temperature treatment and cold resistance assessment Select rice osmpk1 mutant lines osmpk1 #9 and osmpk1 #17 was used as a control material. Four-week-old plants were subjected to low-temperature stress treatment, followed by 3 days of recovery to room temperature. Survival rate and recovery growth were recorded. Dry and fresh weight, relative conductivity (REL), malondialdehyde (MDA) content, and superoxide anion content were also measured. Figure 5 ).

[0039] 2.2.1 Determination of Fresh and Dry Weight Plants from the control group and the low-temperature treatment group were washed, dried, and weighed (5 seedlings per sample, 3 biological replicates). After drying at 65℃ to constant weight, the dry weight was weighed, and the dry-to-fresh weight ratio was calculated.

[0040] 2.2.2 Relative conductivity measurement Take 0.3 g of fresh leaves, cut them into 1 cm pieces, add 10 mL of ddH2O, shake at 200 rpm for 2 h at room temperature, and measure the initial conductivity (C1). After boiling for 20 min, measure the total conductivity (C2), using the conductivity of deionized water (C0) as a blank. Conductivity (%) = (C1-C0) / (C2-C0)×100%.

[0041] 2.2.3 MDA content determination Malondialdehyde (MDA) content determination kit (Protech Biotechnology). OD532 and OD600 were measured, ΔOD = OD532 - OD600. MDA content (nmol / g fresh weight) = 51.6 × ΔA ÷ W (W is the sample fresh weight).

[0042] 2.2.4 Determination of Superoxide Anion Content The absorbance at a wavelength of 530 nm was measured using spectrophotometry.

[0043] Figure 5 The results show: After treatment at 4℃ for 7 days, the phenotypic differences among the materials were significant. Figure 5 (a) CnMPK1The overexpression lines OE#6 and OE#10 showed less damage, with leaves remaining green and extended; while osmpk1#9 and osmpk1#17 The mutant leaves wilted, curled, and lost water severely, exhibiting greater sensitivity to cold. This indicates heterologous expression. CnMPK1 It can enhance the cold resistance of rice. OsMPK1 Functional deficiencies reduce cold resistance.

[0044] The results of physiological index measurements showed that ( Figure 5 (d) The fresh weight of the overexpression lines was significantly higher than that of the wild type and mutants, while the relative conductivity was significantly lower than that of the wild type, whereas the mutants showed the opposite. The MDA and superoxide anion contents in the overexpression lines were significantly lower than those in the wild type, while they were significantly higher in the mutants. This indicates... CnMPK1 Overexpression helps maintain plant growth at low temperatures, reduce increased membrane permeability, and inhibit membrane lipid peroxidation and excessive accumulation of reactive oxygen species.

[0045] In conclusion, CnMPK1 Overexpression significantly improves the cold resistance of rice seedlings, with its positive regulatory effects manifested in mitigating low-temperature phenotypic damage, maintaining higher fresh weight, reducing relative conductivity and MDA content, and decreasing superoxide anion accumulation. These results demonstrate that... CnMPK1 It is a key gene that positively regulates the cold resistance of plants.

[0046] Example 3 CnMPK1 Effects of gene silencing on the cold tolerance of Leymus chinensis Example 3 of this invention is for verification. CnMPK1 To investigate the function of wild *Leymus chinensis* in Tibet, a specific fragment (209 bp) was selected to construct the BSMV-VIGS silencing vector for gene silencing analysis. The specific steps are as follows: 3.1 Vector Construction and In Vitro Transcription: The BSMV-VIGS silencing vector was constructed using a one-step cloning method. The linearized plasmid was transcribed in vitro using RiboMAX™ Large Scale RNA Production Systems-T7. The reaction mixture (20 µL) consisted of: 4 µL 5×T7 Transcription Buffer, 1 µL each of γATP / UTP / CTP / GTP, 9 µL linearized plasmid, 1.5 µL Ribom7G CapAnalog, 2 µL Enzyme, and ddH2O to make up the volume. The mixture was incubated at 37°C for 2 h and then inactivated at 65°C for 10 min.

[0047] 3.2 Virus inoculation BSMV: α BSMV: β BSMV: γ (or BSMV:) CnMPK1 The in vitro transcription products were mixed at a 1:1:1 ratio, diluted 2-fold with ddH2O, and 5 µL of the mixture was added to 90 µL of FES buffer. The mixture was inoculated onto the second leaf of seedlings and cultured in the dark for 24 h, then transferred to normal culture at 25°C. Using BSMV: PDS The appearance of a photobleached phenotype is a sign of successful virus inoculation.

[0048] 3.3 Gene Silencing Validation Real-time quantitative PCR detection CnMPK1 The expression level and method are the same as in Example 2.2.3.

[0049] 3.4 Cold resistance test Silent plants were subjected to low-temperature stress (-10℃ for 12 h, followed by recovery at 4℃ for 7 d), and their phenotypes were observed and their electrical conductivity, MDA content, and superoxide anion radical content were measured.

[0050] Figure 6 The results show that under normal conditions, BSMV: γ No-load comparison with BSMV: CnMPK1 Silent plants showed no significant difference in phenotype; after low-temperature treatment, silent plants exhibited a significantly enhanced cold sensitivity phenotype, with leaves wilting, curling, and losing chlorosis, while control plants showed relatively good growth. Figure 6 (a) qPCR detection showed that in silent plants CnMPK1 Expression levels decreased significantly ( Figure 6 (b) Physiological index measurements showed that the electrical conductivity, MDA, and superoxide anion content of the silent plants after low-temperature treatment increased significantly more than those of the control. Figure 6 (c)

[0051] In summary, BSMV-VIGS-mediated CnMPK1 Silencing significantly reduced the cold tolerance of wild *Leymus chinensis* in Tibet. Silenced plants suffered more severe damage at low temperatures, with exacerbated oxidative damage and membrane injury, indicating... CnMPK1 It plays a positive role in cold resistance regulation.

[0052] Example 4 Example 4 of this invention involved the discovery and verification of CnMPK1 interacting proteins. The specific steps are as follows: To further explore the interacting proteins of CnMPK1, the downstream transcription factor CnbHLH130 was obtained through yeast two-hybrid library screening. This gene was cloned from wild *Leymus chinensis* from Tibet. Figure 7 The encoded protein has a molecular weight of 41.19 kDa and shares 72.3% sequence similarity with rice OsbHLH130 (XP_015650699.1). Phylogenetic analysis shows that CnbHLH130 clusters with homologous proteins of bHLH130 from various grasses in a clade ( Figure 7 Furthermore, it exhibits a close genetic relationship with homologous proteins from closely related species, indicating its high conservation within the Poaceae family. Multiple sequence alignment (…) Figure 8 ) and prediction of conservative motifs and domains ( Figure 9 The results show that CnbHLH130 has a typical bHLH conserved domain and its motif composition and arrangement pattern are consistent with most homologous proteins. It belongs to the bHLH transcription factor family and may be involved in plant stress response.

[0053] 4.1 Yeast two-hybrid (Y2H) A one-step cloning method was used to insert the CDS of CnMPK1 into the pGBKT7 vector (EcoRI / BamHI), and the CDS of CnbHLH130 into the pGADT7 vector (with the same restriction site). 5 µg of each plasmid, along with 10 µL of treated carrier DNA and 500 µL of PEG / LiAc, were co-transformed into competent yeast cells. The cells were incubated at 30°C for 30 min, heat-shocked at 42°C for 15 min, and then on ice for 5 min. After centrifugation and discarding the supernatant, the cells were washed with sterile water and plated onto SD / -Trp-Leu solid medium, and cultured at 30°C for 96 h. Single clones were then transferred to SD / -Trp / -His / -Ade-Leu quadruple-deficient medium and cultured at 30°C for 12 h. Results showed that the BD-CnMPK1+AD-CnbHLH130 combination and the positive control grew normally on the quadruple-deficient medium, while the negative control did not grow. Figure 10 (a) indicates that the two interact in yeast.

[0054] 4.2 Luciferase Complementation Imaging (LCI) Will CnbHLH130 and CnMPK1 The CDS were cloned into the pCAMBIA-nLUC and pCAMBIA-cLUC vectors, respectively. After transformation with Agrobacterium, the mixture was combined with P19 (OD600=0.3) and injected into leaves of Nicotiana benthamiana. The leaves were cultured at 25℃ for 48 h, and the luminescence signal was detected using 1 mM D-Luciferin. The results showed that leaves co-expressing CnMPK1-cLUC and CnbHLH130-nLUC showed strong fluorescence signals, while the negative control (empty vector combination or truncated mutant) showed extremely weak signals. Figure 10 (b) proves that the two interact within plant cells.

[0055] 4.3 GST pull-down Will CnMPK1 and CnbHLH130The CDS were cloned into the pGEX-4T-1 and pET28α vectors, respectively, and induced to express in E. coli BL21(DE3) (IPTG concentration: 0.5 mM for GST-CnMPK1, 0.25 mM for CnbHLH130-His; 100 rpm, 20 h). The target proteins were purified using the GST-tagged protein purification kit (Beyotime P2262) and the His-tagged protein purification kit (Beyotime P2226), respectively. Pull-down was performed according to the BeyoGold™ GST-tag Purification Resin (BeyoGold P2250) instructions: 50 µL of 50% gel suspension was taken, equilibrated with 20 times the volume of binding buffer, centrifuged, and the supernatant was discarded. The gel was resuspended in an equal volume of binding buffer to obtain a 50% equilibrated gel. Binding buffer, equilibrated gel, CnbHLH130-His, and GST-CnMPK1 (GST empty protein was added as a negative control) were added sequentially, and the mixture was incubated overnight at 4°C. The gel was centrifuged, and the gel was washed twice with 100 µL of binding buffer. 50 µL of elution buffer was added, and the mixture was incubated at room temperature for 10 min. Then, 25 µL of 2×SDS loading buffer was added, and the mixture was boiled for 5 min. The supernatant was centrifuged and used for Western blot analysis. Results showed that GST-CnMPK1 could pull down His-CnbHLH130, while the GST empty vector control could not. Figure 10 (c) indicates that the two bind directly in vitro.

[0056] 4.4 Co-immunoprecipitation (Co-IP) Will CnMPK1 and CnbHLH130 CDS were cloned into pCambia1300-GFP and pCAMBIA1300-6myc vectors, respectively. After transformation with Agrobacterium, the CDS were mixed with P19 and injected into leaves of Nicotiana benthamiana. After culturing for 3-5 days, the leaves were collected, ground in liquid nitrogen, and total protein was extracted by adding NP40 lysis buffer. 500 µL of protein sample was taken, 10 µL of Anti-GFP magnetic beads (Beyotime P2132) was added, and the mixture was incubated overnight at 4℃; the mixture was separated by a magnetic rack, washed three times with 500 µL of 1×TBS; 100 µL of 1×SDS loading buffer was added, and the mixture was heated at 95℃ for 5 min. The supernatant was centrifuged and Western spectroscopy was performed. The results showed that in samples co-expressing GFP-CnMPK1 and CnbHLH130-MYC, the CnbHLH130-MYC signal could be detected after precipitation with Anti-GFP magnetic beads, while no signal was detected in the negative control. Figure 10 (d) proves that the two form a complex in the plant.

[0057] 4.5 Western blot SDS-PAGE was performed using a 10% gel of ABmart at 90 V for 10 min, followed by 150 V for approximately 30 min. The PVDF membrane was activated with methanol and transferred at a constant current of 400 mA for 30 min without ice bath. Blocking was performed with rapid blocking buffer at room temperature for 10 min, followed by overnight incubation with primary antibody (ABmart, 1:10000) at 4°C. The membrane was washed three times with PBST, incubated with secondary antibody (ABmart, 1:10000) at room temperature for 1 h, and then developed with ECL after washing with PBST.

[0058] In summary, the results of yeast two-hybrid imaging, luciferase complementation imaging, GST pull-down, and immunoprecipitation consistently indicate that CnMPK1 and CnbHLH130 can interact both in vitro and in vivo in plants.

[0059] Example 5: Phosphorylation of CnbHLH130 by CnMPK1 and Regulation of Protein Stability 5.1 In vitro phosphorylation detection GST-CnMPK1 and CnbHLH130-His proteins were purified according to the methods described in 4.3.3 and 4.3.4 of Example 4. 1.5 μg of GST-CnMPK1 and 10 μg of CnbHLH130-His were added to 3 μL of 10× kinase buffer (250 mM Tris-HCl pH 7.5, 100 mM MgCl2, 10 mM DTT, 500 μM ATP), along with protease inhibitors and phosphatase inhibitors. The mixture was reacted at 30°C for 60 min. An SDS-PAGE separating gel containing 50 μM MnCl2 and 100 μM Phos-tag™ was prepared, and the stacking gel was prepared as usual. After the reaction, the sample was treated with TCA precipitation: 1% DOC (final concentration) was added and incubated at 37℃ for 10 min, then an equal volume of 20% TCA was added, incubated on ice for 5 min, and centrifuged at 15000 rpm for 10 min at 4℃. The precipitate was washed three times with pre-cooled acetone, dried, and then added to SDS-PAGE loading buffer, and heated at 100℃ for 5-10 min. Electrophoresis and transfer were performed as described in Example 4.5, with 1-10 mM EDTA added to the transfer buffer to remove Mn. 2+ The transfer time was extended to 45 min (400 mA), and detection was performed using an antiphosphorylation antibody.

[0060] The results showed that no phosphorylation migration band was observed in the GST empty protein control; after the addition of GST-CnMPK1, a significant migration lag band appeared in CnbHLH130-His, which was attenuated by alkaline phosphatase CIAP, indicating that CnMPK1 directly phosphorylates CnbHLH130 in vitro. Figure 11 ).

[0061] 5.2 In vivo phosphorylation detection Take seedlings that are 4 weeks old CnbHLH130 -GFP-OE single overexpression rice and CnMPK1 × CnbHLH130 Rice plants double-overexpressing GFP-OE were treated at 4℃ for 0, 12, 24, and 36 h, respectively, and total protein was extracted. Western blot analysis was performed using an Anti-Ser / Thr phosphorylation antibody (method as in Example 4.5).

[0062] The results showed that in single-overexpression plants, the phosphorylation level of CnbHLH130 increased with prolonged low-temperature treatment, reaching a peak at 24 h and then slightly decreasing; in double-overexpression plants, the phosphorylation level was higher at 0 h than that of single overexpression, and the signal was further enhanced after 24 h of treatment at 4℃, demonstrating that CnMPK1 can increase the phosphorylation level of CnbHLH130 in vivo, and the promoting effect is more significant under low-temperature stress. Figure 11 ).

[0063] 5.3 Phosphorylation site prediction and point mutant construction The phosphorylation sites of CnbHLH130 were predicted using NetPhos-3.1 (https: / / services.healthtech.dtu.dk / services / NetPhos-3.1 / ), yielding two conserved MAPK phosphorylation sites, Ser4 and Ser49 (sequence characteristics: MYGSPASK / AASPDAAADNVFSR). Single / double point mutation vectors for S4A and S49A, and double point mutation vectors for S4A / S49A, were constructed using a one-step single / double point mutation kit (Novizan, C214). The primer sequences are as follows: S4A-F (SEQ ID NO.2): GATCCATGTACGGCgCGCCGGCGTCCAAGGATC S4A-R (SEQ ID NO.3): CGcGCCGTACATGAATTCGAAGCTTGAGCTCG S49A-F (SEQ ID NO.4): CGCCGCCgccCCCGACGCCGCCGCCGACAACGTCTT S49A-R (SEQ ID NO.5): CGTCGGGggcGGCGGCGCGGGGGCCGGCTGGA Using pET28α-CnbHLH130-His as a template, reverse amplification was performed. After digestion with Dpn I, recombination was catalyzed by Exnase II (37℃, 30 min), and the mixture was transformed into E. coli BL21. Sequencing was then used for verification.

[0064] In vitro kinase reactions (same as 5.1) showed that wild-type CnbHLH130-His exhibited a significant hysteresis band after co-incubation with GST-CnMPK1; phosphorylation signals were significantly weakened in the S4A and S49A single-point mutants; and the signal in the 2M double mutant was further reduced, indicating that Ser4 and Ser49 are the main phosphorylation sites of CnMPK1 on CnbHLH130. CnbHLH130 2M -Myc and CnMPK1 -GFP was co-expressed in *Elymus spp.* herbaceous plants. In vivo Phos-tag assays showed that co-expression of wild-type and CnMPK1 significantly increased phosphorylation levels, while the phosphorylation signal in the 2M mutant was significantly lower than that in the wild-type, confirming that Ser4 and Ser49 are important sites for CnMPK1 to promote phosphorylation in vivo. Figure 12 ).

[0065] 5.4 In vitro cell-free degradation experiment CnbHLH130-His and CnbHLH130 2M His was induced in *E. coli* BL21 with 0.25 mM IPTG at 20°C for 20 h, and recombinant protein was obtained using a His-tagged purification kit. Wild-type (WT) and... CnMPK1 Total protein was extracted from OE transgenic rice seedlings. The degradation reaction system (20 mM Tris-HCl pH 7.2, 2.5 mM MgCl2, 0.5 mM CaCl2, 1 mM MnCl2, 4 mM PMSF, 1 mM ATP, 2 mM DTT) contained 100 μg recombinant protein and 500 μg total rice protein. Some samples were treated with 20 μM MG132 (a 26S proteasome inhibitor). Samples were taken at 0, 0.5, 1, and 1.5 h, and the reaction was terminated by adding SDS loading buffer. The samples were boiled at 100℃ for 10 min and then analyzed by Western blot.

[0066] The results showed that without MG132, CnbHLH130-His in total WT protein degraded over time; degradation was significantly slowed in the system containing CnMPK1; and CnbHLH130... 2M -His continued to degrade rapidly in the CnMPK1-containing system, with a greater decrease in abundance than the wild type; the degradation of all groups was inhibited after the addition of MG132, indicating that the degradation was dependent on the 26S proteasome pathway.

[0067] 5.5 In vivo CHX tracking experiment Pick CnbHLH130 -GFP-OE overexpression and CnMPK1 × CnbHLH130-GFP-OE double overexpression seedlings were treated with 100 μM CHX (actinomycin) for 0, 2, 4, and 6 h, with some samples also treated with 50 μM MG132. Total protein was extracted, and CnbHLH130 protein abundance was detected by Western blot using H3 as an internal control.

[0068] The results showed that without MG132, the CnbHLH130 protein level in single-overexpression plants continuously decreased with CHX treatment, leaving only a low level after 6 h; the protein decline rate in double-overexpression plants was significantly slowed down, and they still maintained a high abundance after 6 h; both remained stable after the addition of MG132, further demonstrating that in vivo degradation depends on the proteasome pathway. Figure 13 ).

[0069] In summary, CnMPK1 significantly improves the stability of CnbHLH130 by phosphorylating the Ser4 and Ser49 sites, and this stabilizing effect depends on the two main phosphorylation sites mentioned above.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A gene regulating plant cold resistance, characterized in that, The plant cold resistance regulatory gene is: CnMPK1 and CnbHLH130 ; The CnMPK1 The gene sequence is shown in SEQ ID NO.

1.

2. The plant cold resistance regulating gene according to claim 1, characterized in that, The CnMPK1 can enhance the protein stability of CnbHLH130 through phosphorylation modification and inhibit its degradation via the 26S proteasome pathway.

3. The plant cold resistance regulating gene according to claim 1, characterized in that, The CnbHLH130 reverse-binds and activates CnMPK1 Transcriptional expression.

4. The application of the plant cold resistance regulating gene according to any one of claims 1 to 3 in improving plant cold resistance.

5. The application of the plant cold resistance regulating gene according to any one of claims 1 to 3 in the breeding of cold-resistant plants.

6. A method for breeding cold-resistant plants, characterized in that, Includes the following steps: Build CnMPK1 Gene overexpression vector, and CnMPK1 Cold-resistant plants were obtained by transferring gene overexpression vectors into plants.

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