Cpgpx16 gene for improving cold resistance of elephant grass and application thereof

Abstract

This invention relates to the field of genetic engineering for enhancing plant cold resistance, and specifically to a method for improving the cold resistance of elephant grass. CpG ODN 1018 Genes and their applications. This invention is the first to clone and functionally identify an elephant grass-derived gene. CpG ODN 1018 Genes, and through experimental verification, it was found that elephant grass CpG ODN 1018 Genes can increase a plant's cold resistance through heterologous overexpression. CpG ODN 1018 The gene significantly improved the growth status of yeast under cold stress at the single-cell level, and the silent elephant grass CpG ODN 1018 The gene significantly increased the accumulation of ROS levels in leaves, disrupting cell membrane integrity and thus significantly reducing cold resistance. This invention identifies... CpG ODN 1018 The genes provide the molecular basis for the promotion and cultivation of forage and energy crops in cold regions, and have significant agricultural production and ecological value.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering for enhancing plant cold resistance, and specifically to a method for improving the cold resistance of elephant grass. CpGPX16 Genes and their applications. Background Technology

[0002] Low temperature is a crucial environmental factor affecting plant growth and development, limiting the geographical distribution of many plant species and being a major limiting factor for plant productivity in arable land worldwide. Therefore, improving crop yield and stability under low-temperature conditions and enhancing their cold resistance have become key areas of research in crop genetic improvement and stress physiology. Plants activate a series of complex response mechanisms under low-temperature stress. Existing studies have shown that a large number of differentially expressed genes appear in plants after low-temperature stress treatment. These genes are typically enriched in pathways such as plant hormone signal transduction, redox processes, photosynthesis, membrane system protection, and transcription factor regulation. Key pathways and genes within these pathways are considered important candidate regulatory factors involved in plant cold resistance mechanisms.

[0003] genus *Pennisetum* ( Pennisetum *Pennisetum* is one of the most important genera in the Poaceae family, containing approximately 140 species, widely distributed in various ecological environments worldwide. Elephant grass, belonging to the genus *Pennisetum*, is a perennial, clump-forming, large herbaceous plant. It is one of the most important forage and potential energy grasses in tropical and subtropical regions of Asia, Africa, and the Americas. Its soft texture, good palatability, high crude protein content, and moderate crude fat and nitrogen-free extract content make it suitable for feeding various animals such as cattle, sheep, pigs, rabbits, and fish. *Pennisetum* introduced from Guangxi and Fujian in China is an excellent forage resource, exhibiting strong drought and disease resistance, and high sugar content. However, elephant grass is sensitive to low temperatures, generally exhibiting weak cold tolerance and lacking adaptability to cold environments, which is a major factor limiting its large-scale production and efficient utilization. Therefore, in-depth research on its molecular response mechanism to low-temperature stress is of great significance for expanding its ecological adaptability and provides a theoretical basis for the discovery and molecular breeding of cold-resistant germplasm. However, to date, research on the cold-resistant genes of *Pennisetum* is limited, and its cold-resistance mechanism remains unclear.

[0004] The glutathione peroxidase (GPX) gene family is a core component of the intracellular antioxidant defense system. Its main function is to catalyze the reduction of hydrogen peroxide and organic hydroperoxides by reduced glutathione, thereby protecting cells from oxidative damage. Therefore, GPX has been extensively studied in various life systems, including microorganisms, plants, animals, and humans. Genome-wide identification and characterization of GPX genes have been performed in model plants and crops such as Arabidopsis thaliana, sorghum, cotton, quinoa, maize, tobacco, and soybean. GPX also exhibits differential spatiotemporal expression patterns in responses to various stresses, regulation of growth and development, and signal transduction of reactive oxygen species (ROS) and immune responses. In Arabidopsis thaliana, AtGPX1 , AtGPX2 and AtGPX7 Responsible for clearing reactive oxygen species from the cytoplasm, among which AtGPX1 It is significantly upregulated under drought stress, and counteracts dehydration damage by reducing intracellular hydrogen peroxide (H2O2). In rice OsGPX3 Overexpression can enhance salt tolerance. AtGPX7 It also participates in regulating the abscisic acid signaling pathway, affecting stomatal closure and osmotic balance. In transgenic tobacco, overexpression of GPX enhances reactive oxygen species scavenging capacity, reduces membrane damage, and improves tolerance to salt and low-temperature stress. Among the six GPX gene members in chickpeas... CaGPX3 It is a key gene in the salt stress response. CaGPX3 Under salt stress, a synergistic expression regulatory pattern was observed, clarifying... CaGPX3 Genes play a central role in salt tolerance mechanisms. Aluminum toxicity in the roots of *Lobelia chinensis* reduces the expression of all GPX genes; conversely, cadmium toxicity causes [a decrease in GPX expression] after 1 hour. GPX3 , GPX4 and GPX5 The increase, and caused within 3-24 hours GPX1 , GPX2 , GPX4 and GPX6 Downregulation. Furthermore, GPXs regulate root structure through redox and hormone-mediated pathways. Similarly, knockdown... OsGPX1 or OsGPX3 It severely affected the growth and development of rice plants. All eight members of the cotton GPX family showed varying degrees of upregulation under abiotic stress. GPX2 , GPX3A and GPX4A-1 All three genes were significantly upregulated under low temperature, drought, and salt stress treatments. Under abiotic stress, the DREB1D-COX11-GPX regulatory module plays a key role in protecting cells from oxidative damage and enhancing plant stress resistance, jointly enhancing reactive oxygen species scavenging capacity and maintaining cellular redox homeostasis.

[0005] Although the GPX family plays an important role in plant growth, development, and stress response, there is still much to be done regarding elephant grass GPX ( CpGPX Research on the family (of a specific family) is still lacking. This invention systematically analyzes... CpGPX Family members, analyzed CpGPX The expression levels of the gene in roots, stems, and leaves were determined, and the expression was analyzed using qRT-PCR technology. CpGPX The expression patterns of gene families under cold stress were discovered through yeast cold stress experiments. CpGPX16 It has the ability to enhance cold resistance, and molecular docking technology was used to further predict... CpGPX16It can closely bind to molecules related to redox reactions during physiological metabolism, thus playing a role in eliminating reactive oxygen species. This invention provides new insights into the study of cold resistance in plants of the genus *Pennisetum*.

[0006] Furthermore, on the one hand, there are differences in understanding among those skilled in the art; on the other hand, the inventors studied a large number of documents and patents when making this invention, but due to space limitations, not all details and contents were listed in detail. However, this does not mean that the present invention does not possess the features of these prior art. On the contrary, the present invention already possesses all the features of the prior art, and the applicant reserves the right to add relevant prior art to the background art. Summary of the Invention

[0007] This invention relates to the field of genetic engineering for enhancing plant cold resistance, and specifically to a method for improving the cold resistance of elephant grass. CpGPX16 Genes and their applications.

[0008] To address the aforementioned technical problems, one of the objectives of this invention is to provide a method for improving the cold resistance of elephant grass. CpGPX16 Gene, CpGPX16 The amino acid sequence of the gene is shown in SEQ ID NO.6.

[0009] According to a preferred embodiment, the encoding CpGPX16 The nucleotide sequence of the gene's amino acid sequence is shown in SEQ ID NO. 5.

[0010] One of the objectives of this invention is to provide elephant grass CpGPX16 Application of genes and related materials in improving plant cold resistance, elephant grass CpGPX16 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.6, and the plant is Arabidopsis thaliana or elephant grass.

[0011] According to a preferred embodiment, improving the cold resistance of plants is achieved by improving the cold resistance of elephant grass. CpGPX16 This is achieved through gene expression levels.

[0012] According to a preferred embodiment, the relevant material includes materials that enhance the properties of elephant grass. CpGPX16 Recombinant vectors or recombinant strains for gene expression.

[0013] One of the objectives of this invention is to provide a recombinant expression vector comprising a vector and an elephant grass connected to the vector. CpGPX16 The gene and vector are pCaBS-γbLIC or pCaBS-γbLIC-CpGPX16.

[0014] One of the objectives of this invention is to provide a recombinant strain comprising the above-mentioned recombinant expression vector and a host cell, wherein the host cell is Agrobacterium GV3101.

[0015] One of the objectives of this invention is to provide elephant grass CpGPX16 A method for constructing tobacco transient overexpression plants using gene therapy includes the following steps: obtaining transient overexpression plants via Agrobacterium-mediated transformation and observing eGFP fluorescence using a Leica super-resolution confocal microscope.

[0016] One of the objectives of this invention is to provide a method for using elephant grass. CpGPX16 A method for constructing a yeast strain for heterologous gene expression, comprising the following steps: Elephant grass cold-resistant genes CpGPX16 Insert the vector to obtain the heterologous expression vector pYES2- CpGPX16 Elephant grass's cold-hardiness gene CpGPX16 For example, a CDS sequence.

[0017] One of the objectives of this invention is to provide a product containing elephant grass. CpGPX16 A subcellular localization vector for a gene, comprising the following steps: Elephant grass CpGPX16 The gene is inserted into the vector to obtain a subcellular localization vector. CpGPX16-EGFP .

[0018] One of the objectives of this invention is to provide a primer pair for detecting the cold resistance of elephant grass, comprising one or more of the following primer pairs: For example, the upstream primer q shown in SEQ ID NO.1 CpGPX16 -F and downstream primer q as shown in SEQ ID NO.2 CpGPX16 -R; The upstream primer as shown in SEQ ID NO.11 CpGPX16 -F and downstream primers as shown in SEQ ID NO. 12 CpGPX16 -R.

[0019] One of the objectives of this invention is to provide the application of the above-mentioned primer pairs in screening cold-resistant elephant grass varieties or identifying the low-temperature stress response of elephant grass.

[0020] One of the objectives of this invention is to provide a method for improving the cold resistance of plants, the method comprising the following steps: Elephant grass or Arabidopsis thaliana were transformed using a vector-mediated method to obtain elephant grass overexpressing the amino acid sequence of the protein encoded as shown in SEQ ID NO. 6. CpGPX16 Transgenic strains of genes.

[0021] According to a preferred embodiment, the carrier-mediated method is the Agrobacterium-mediated method.

[0022] Preferably, the Agrobacterium-mediated transformation method includes: obtaining transgenic plants through Agrobacterium-mediated transformation and screening, and verifying their cold resistance under low-temperature conditions. The low-temperature conditions are no higher than 10°C. More preferably, the low-temperature conditions are 4°C.

[0023] The beneficial effects of this technical solution are: This invention utilizes transcriptome sequencing technology to screen and identify a gene highly associated with low-temperature stress response. CpGPX16 This gene can significantly enhance the tolerance of elephant grass to low-temperature adversity by regulating the expression levels of genes related to cold stress response. The cold-resistance improvement technology provided by this invention has significant application value in ensuring stable agricultural production and reducing production losses caused by low-temperature disasters. CpGPX16 Overexpression of the gene can significantly enhance the adaptability of perennial elephant grass to low-temperature stress, thereby expanding its planting area to colder climate regions and enhancing its survival and growth ability in diverse ecological environments. Attached Figure Description

[0024] Figure 1 This invention presents a phylogenetic tree of GPX protein sequences from elephant grass, rice, wheat, maize, and Arabidopsis thaliana, wherein elephant grass... CpGPX Genes are highlighted in red; Figure 2 Elephant grass as shown in this invention CpGPX Tissue-specific gene expression; Figure 3 As shown in the present invention CpGPX16 Gene expression analysis under cold stress; Figure 4 As shown in the present invention CpGPX16 Gene clone PCR electrophoresis image; Figure 5 As shown in the present invention CpGPX16 Colony PCR electrophoresis image; Figure 6 As shown in the present invention CpGPX16 Subcellular localization results of genes; Figure 7 Expressions shown in this invention CpGPX16 Verification analysis of yeast's sensitivity to cold stress; Figure 8 As shown in the present invention CpGPX16 Analysis of cold stress sensitivity of gene-silenced elephant grass; Figure 9 As shown in the present invention CpGPX16 Analysis of cold stress sensitivity in Arabidopsis thaliana with gene overexpression, including, Figure 9 A shows the growth status of Arabidopsis thaliana at 23 / 4℃, and the overexpressing Arabidopsis thaliana showed stronger growth than the control group; Figure 9 B shows overexpression under 4°C low temperature conditions. CpGPX16 Identification of RNA levels in Arabidopsis thaliana; Figure 9 C shows the root length of Arabidopsis thaliana under a low-temperature treatment at 4℃. Detailed Implementation

[0025] In the description of this invention, terminology is used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly defined.

[0026] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials, reagents or instruments used, unless otherwise specified by the manufacturer, are all commercially available reagents and materials; the conditions not specified in the examples are all carried out according to conventional conditions or conditions recommended by the manufacturer. At the same time, the present invention does not limit the source of the raw materials used. Unless otherwise specified, the raw materials used in the present invention are all commercially available products in this technical field.

[0027] Example 1 I. Elephant Grass CpGPX Gene family identification and phylogenetic tree analysis Using both BLASTP and HMM (Pfam ID: PF00255), 18 genes were identified in the elephant grass genome (https: / / www.ncbi.nlm.nih.gov). CpGPX Genes. Elephant grass was constructed using the maximum naturalness method. CpGPX Phylogenetic tree of protein sequences from rice (OsGPX), maize (TaGPX), wheat (AtGPX), and Arabidopsis thaliana (ZmGPX). The results showed that these genes can be divided into three main groups (Group A, Group B, and Group C). Figure 1 Specifically: Group A contains 26 GPX members (11 of whom are...). CpGPX Group B contains 11 GPX members (3 OsGPX, 2 OsGPX, 6 TaGPX, 4 ZmGPX, and 3 AtGPX), significantly more than other groups, suggesting that this subfamily may play an important biological role; Group B contains 11 GPX members (3 OsGPX, 6 TaGPX, 4 ZmGPX, and 3 AtGPX). CpGPX Group C contains 1 OsGPX, 3 TaGPX, 2 ZmGPX, and 2 AtGPX; Group C includes 14 GPX members (4... CpGPX (2 OsGPX, 5 TaGPX, and 3 AtGPX). Overall, GPX genes in the same subgroup may have similar functions.

[0028] two, CpGPX16 Tissue differential expression analysis RNA seq data was used to analyze the 18 RNA molecules identified in Example 1. CpGPX Analysis of gene expression patterns revealed that members of the same subfamily exhibited similar expression characteristics, suggesting potential functional redundancy. CpGPX16 The expression levels of this gene were higher in all tissues than in other tissues, indicating that this gene may play a central role in abiotic stress in elephant grass. Furthermore, the expression level of this gene in leaves was FPKM=118.41, significantly higher than in other tissues, suggesting that this gene may function in elephant grass leaves. Based on this analysis, we selected [the following tissues] for further analysis. CpGPX16 The expression of genes under cold stress was analyzed.

[0029] three, CpGPX16 Expressing response to cold stress Elephant grass (variety: Guimin Yin) was obtained from plants grown in our laboratory. Stem segments were separated and immersed in Hoagland's nutrient solution, then cultured for 10 days in a growth chamber (daytime temperature 26℃ / nighttime temperature 23℃, 12-hour light cycle), followed by 30 days of cultivation in nutrient soil. Plants with consistent growth were selected and transferred to a 4℃ incubator (12-hour light / 12-hour darkness) for cold stress treatment. Leaf samples were collected at 0, 6, 12, 24, and 48 hours, with three replicates collected at each time point.

[0030] Total RNA was extracted from samples collected at different time points under cold treatment conditions. RNA extraction was performed using the RNAprep PurePlant Plus Kit (catalog number: DP432; brand: Tiangen Biotech, China). 1 µg of cDNA was synthesized using the HiScript III first-strand cDNA synthesis kit (+gDNA wiper) (Novizan, China). qRT-PCR analysis was performed using the Bio-Rad CFX Connect real-time quantitative PCR system with a 2×Taq Pro Universal SYBR qPCR Master Mix (Novizan, China). ACTIN was used as an internal control gene, and 2... CT Method calculation CpGPX Gene expression levels. CpGPX16 The primer sequences for the quantitative analysis of the internal reference gene ACTIN are shown in Table 1. Three biological replicates and three technical replicates were set up for each sample. Statistical significance was assessed using ANOVA and Fisher's LSD post-hoc test. Error bars represent standard errors (SE), and the significance threshold was p < 0.05.

[0031] CpGPX16The expression analysis of genes in elephant grass leaves under different low-temperature stress treatment times is shown in [the figure]. Figure 3 At 1, 2, 4, 8, 12, 24, and 48 hours, CpGPX16 There was a significant difference in expression levels between the control group and the 4℃ treatment group (P<0.05), and the expression level in the treatment group was significantly upregulated at 48 h. P <0.05); CpGPX 16 At 8 h, the expression level in the treatment group was significantly upregulated ( P <0.05).

[0032] Table 1. Specific primers for RT-PCR

[0033] IV. Elephant Grass CpGPX16 Gene cloning Based on the above research results, it is shown that CpGPX16 Genes are involved in the cold stress response of elephant grass. Therefore, primers were designed to clone elephant grass. CpGPX16 The gene sequence was analyzed to conduct in-depth research on its biological functions.

[0034] The primer sequences are as follows: SEQ ID NO.11: CpGPX16 -F CTCTCCGCTCAATTCTTCGC; SEQ ID NO.12: CpGPX16 -R CTGCAGCCTCTGAAATACGG.

[0035] The PCR system is shown in Table 2.

[0036] Table 2 Elephant Grass CpGPX16 PCR system for gene cloning

[0037] The PCR procedure is shown in Table 3.

[0038] Table 3 Elephant Grass CpGPX16 PCR program for gene cloning

[0039] The PCR products were subjected to 1% agarose gel electrophoresis. The electrophoresis results are shown in the figure. Figure 4 Based on the marker (lane M), a specific band was observed at 650 bp, indicating that cloning was successful.

[0040] The cloned PCR product was ligated into a T-vector, and sequencing comparison confirmed the successful cloning of the gene's CDS. The nucleic acid and protein sequences are as follows: CDS sequence (SEQ ID NO.5) > CpGPX16 ATGGCAGCCGCGTCGTCCGCGACCTCCGTCCACGATTTCACCGTCAAGGATGCAAGCGGGAAGGATGTTGACCTCAGCACCTACAAGGGCAAGGTTCTCCTTATTGTCAACGTCGCATCCCAGTGTG GATTAACTAACTCCAACTACACCGAGCTGGCCCAGCTCTATGAGAAGTACAAGGACCAAGGTTTTGAGATCCTGGCTTTTCCTTGCAACCAGTTTGGCGGGCAGGAGCCTGGCACAAACGAGGAGAT TGTCCAGTTTGCTTGCACACGCTTCAAGGCTGAGTACCCCATCTTCGACAAGGTTGATGTCAACGGTAACGATGCTGCGCCCATCTACAAGTTCCTGAAGTCTGGCAAGGGTGGCCTCTTCGGTGAC AGCATCAAGTGGAACTTCTCCAAGTTCTTGGTTGACAAGGAGGGGCGTGTTGTGGATCGCTATGCCCCGACCACTTCCCCCCTGAGCATCGAGAAGGATATCAAGAAGCTGCTTGGGAGCTCTTAA; Amino acid sequence (SEQ ID NO.6) > CpGPX16 MAARSSATSVHDFTVKDAQAGKDVDLSTYKGKVLLIVNVASQCGLTNSNYTELAQLYEKYKDQGFEILAFPCNQFGQQEPGTNEEIVQFAYTRFKAEYPIFDKVDVNGNDAAPIYKFLKSGKGGLFGDSIKWNFSKFLVDKEGRVVDRYAPTTSPLSIEKDIKKLLGSL.

[0041] five, CpGPX16 Gene subcellular localization (a) Tobacco cultivation Sow tobacco seeds in pots filled with nutrient soil, water thoroughly, and allow them to grow to 5 leaves (about 4-5 weeks) under conditions of 14 hours of light / 10 hours of darkness, a temperature of 25℃, and a relative humidity of 60%-70%.

[0042] (two) CpGPX16 Construction of EGFP plant expression vector S1, Primer Design according to CpGPX16 The CDS sequence of the gene is used to design primers with vector homologous arms.

[0043] Table 4 CpGPX16 Subcellular localization vector construction primers

[0044] S2, PCR amplification products and recovery The PCR system is the same as in Table 2, except the primers are replaced with G- CpGPX16 -F and G- CpGPX16 -R; PCR program: 95℃ 5 min; 95℃ 30 s, 60℃ 30 s, 72℃ 1 min, 30 cycles; store at 4℃. PCR products were purified by 1% agarose gel electrophoresis.

[0045] S3 and pCAMBIA1381-EGFP vector were digested with enzymes, and the digestion system is shown in Table 5.

[0046] Table 5. Vector Enzyme Digestion System

[0047] Enzyme digestion procedure: 37℃ for 30 min; 65℃ for 20 min; store at -20℃.

[0048] S4. The amplified product with homologous arms CpGPX16 The product was connected to the linearized carrier, and the system is shown in Table 6.

[0049] Table 6 CpGPX16 Linkage system with linearized carrier

[0050] The PCR instrument was set at 50℃ for 30 minutes, and then stored at 4℃.

[0051] S5. Transform the ligation product into competent E. coli cells. Thaw 50 μL of competent E. coli cells on ice, add all the ligation product to the competent cells, and gently tap to mix. Incubate the competent cells in an ice box for 30 min. Then heat shock in a 42°C water bath for 30 s, followed by an ice bath for 2 min. Add 500 μL of LB liquid medium and incubate at 37°C with shaking for 1 h. Remove the competent cells, plate them on Kana (50 mg / L) solid LB agar plates, and incubate overnight at 37°C until single colonies grow.

[0052] S6. PCR identification of positive clones using bacterial culture. Single colonies from the agar plate were transferred to sterile centrifuge tubes, and LB liquid medium (containing 50 mg / L kan) was added. The centrifuge tubes were then incubated at 37°C and 200 rpm for 8 h in a shaker. The bacterial culture was amplified using 35S-f and GFP-r primers, and the amplification system is shown in Table 7.

[0053] Table 7 PCR amplification system

[0054] Table 8 PCR Procedure

[0055] Bacterial cultures with positive bands were selected and sent to Sangon Biotech for sequencing. Comparative analysis of the sequencing results confirmed successful ligation and the absence of mutations. CpGPX16 -EGFP vector. Results are shown below. Figure 5 Using the marker as a reference, the electrophoresis results show that their respective target fragments were successfully detected.

[0056] S7, Instantaneous Tobacco Conversion extract CpGPX16 -EGFP vector was used to transform Agrobacterium GV3101 competent cells. Single clones of Agrobacterium transformed with the expression plasmid were picked and cultured overnight at 28°C and 200 rpm in 2 mL LB broth containing the appropriate antibiotic. 1 mL of the overnight Agrobacterium culture was transferred to 20 mL LB broth containing the appropriate antibiotic and cultured at 28°C and 200 rpm until the logarithmic growth phase (OD50) of Agrobacterium. 600 =0.6-0.8). The cells were collected by centrifugation at 6000 rpm for 10 min at room temperature. The Agrobacterium cells were then resuspended in a staining buffer (containing 10 mM MgCl2, 10 mM MES, 150 μM acetylsylgenone, pH=5.6) to OD0.05. 600 =1.0. After standing at room temperature for 2 h, tobacco leaves were injected. Injected plants were placed in darkness for 12 h, then cultured normally in a greenhouse for 48 h. The green fluorescence signal of the Agrobacterium-injected area on the tobacco leaves was then captured using a laser confocal microscope. CpGPX16 Gene subcellular localization results as follows Figure 9 As shown in the figure. The results indicate that this gene is expressed in both the cell nucleus and the cell membrane.

[0057] six, CpGPX16 Analysis of heterologous expression in yeast under cold stress Constructed CpGPX16 Overexpression of yeast strain. Under normal culture conditions and a 4°C treatment for 48 h, INVSC1(pYES2- CpGPX16 The growth status of the engineered bacteria was basically the same as that of the control INVSC1 (pYES2). Figure 7 No significant growth difference was observed. However, under treatment at -20℃ for 48 h, overexpression... CpGPX16 The yeast strain enhanced its low-temperature resistance. Experimental results show that heterologous overexpression... CpGPX16 It can significantly enhance the cold resistance of yeast cells.

[0058] seven, CpGPX Phenotype of Elephant Grass under Cold Stress After Gene Silencing This embodiment studies transiently silent elephant grass. CpGPX16 The effects of low-temperature stress on the growth of the target herbaceous plant were investigated, and the specific process is as follows: (I) Materials and Reagents The seed nodes of *Eriocaulon buergerianum* during the seedling stage, the three plasmid vectors of the BSMV-VIGS system (pCaBS-α, pCaBS-β, pCaBS-γbLIC), and *Agrobacterium* strain GV3101 (pSoup).

[0059] (II) Silent Elephant Grass CpGPX16 Gene S1. Construction of recombinant silencing vector 1) Gene fragment design According to sequencing CpGPX16 The nucleic acid sequence of the gene was used for multiple sequence alignment of homologous genes in the genome. A specific region of approximately 300 bp was selected as the target sequence. CpGPX16 To determine the specific silencing fragment of a gene, primers were designed as follows: pCaBS- CpGPX -F: AAGGAAGTTTAAATCACCCAAGGACCAAGGTTTTG (SEQ ID NO. 9); pCaBS- CpGPX -R: AACCACCACCACCGTAGTAGTTCTCGATGCTCAGGGG (SEQ ID NO. 10).

[0060] 2) pCaBS-γbLIC- CpGPX16 Silent carrier construction Using primer pCaBS- CpGPX -F and pCaBS- CpGPX -R, with T- CpGPX16 Using plasmids as templates, high-fidelity enzyme PCR was used for amplification. CpGPX16 Specific fragments. PCR products were purified by gel extraction. The purified fragments were inserted into the pCaBS-γbLIC linearized vector according to the LIC cloning method to construct the recombinant plasmid pCaBS-γbLIC- CpGPX16The recombinant plasmid was transformed into E. coli DH5α, and positive clones were screened and sequenced for verification.

[0061] S2 Infection Solution Preparation 1) Verify the correct recombinant plasmid pCaBS-γbLIC- CpGPX16 The cells, along with helper plasmids pCaBS-α and pCaBS-β, were transformed into Agrobacterium GV3101 competent cells. The cells were then plated on LB agar plates containing kanamycin (50 mg / L) and rifampin (25 mg / L), and incubated upside down at 28°C for 2-3 days. Single colonies were then picked.

[0062] 2) Pick samples containing pCaBS-α, pCaBS-β, and pCaBS-γbLIC- respectively. CpGPX16 Single colonies of Agrobacterium were cultured with shaking in LB broth containing the corresponding antibiotic until OD200. 600 ≈1.5. Collect bacterial cells by centrifugation at 6000 rpm for 10 minutes, resuspend the cells in the infection solution, and set the OD value accordingly. 600 Adjust to 1.0. After standing at room temperature for 2 hours, it can be used for infection.

[0063] S3 Infects Elephant Grass Sprouts (1) Three Agrobacterium-based bacterial solutions (GV3101 / pCaBS-α, GV3101 / pCaBS-β, GV3101 / pCaBS-γbLIC-) were prepared. CpGPX16 Mix them at a volume ratio of 1:1:1 to obtain the mixed bacterial solution of the treatment group.

[0064] (2) Negative control: GV3101 / pCaBS-γbLIC- CpGPX16 Replace with GV3101 / pCaBS-γbLIC (empty vector), and mix at the same ratio of 1:1:1.

[0065] (3) Completely immerse the elephant grass seedlings in the mixed bacterial solution and treat them for 60 minutes under a vacuum pressure of 1.0 bar using a vacuum pump. Transplant the infected plants into nutrient soil and cultivate them for 25 days under the conditions of day / night temperature of 24 / 22℃ and light / dark cycle of 16 / 8 h.

[0066] (4) Samples were taken 30 days after infection, using the elephant grass Actin gene as an internal control. CpGPX16 qRT-PCR detection using specific primers CpGPX16 Gene expression levels. Silent lines and control groups were simultaneously treated with 8°C for 20 days. Results are as follows: Figure 8 As shown.

[0067] Figure 8 A shows CpGPX16 Comparison of growth status of Silent Elephant Grass under cold stress. CpGPX16 Gene-silenced plants exhibit a phenotype that is more sensitive to cold stress, manifested by wilting of leaves.

[0068] Figure 8 B shows elephant grass CpGPX16 RT-qPCR identification of gene expression. Results showed that, compared to the control group, silent plants showed significantly higher levels of gene expression in their leaves. CpGPX16 The significantly reduced mRNA expression level of the gene demonstrates the successful implementation and silencing of the gene using the BSMV-VIGS system. CpGPX16 Gene.

[0069] Figure 8 C and Figure 8 D shows CpGPX16 The deeper ROS staining in the leaves of gene-silenced elephant grass plants indicates a significant increase in ROS levels.

[0070] Figure 8 E illustrates the condition under cold stress. CpGPX16 The relative electrical conductivity of leaves of gene-silenced elephant grass plants was significantly higher than that of the control group, indicating that cell membrane damage was more severe in the silent strains.

[0071] Figure 8 F indicates the condition under cold stress. CpGPX16 The hydrogen peroxide content in the leaves of gene-silenced elephant grass plants was significantly higher than that in the control group. In conclusion, CpGPX16 The gene enhances the cold resistance of elephant grass by clearing low-temperature-induced ROS levels.

[0072] 8. Overexpression CpGPX16 Phenotype of Arabidopsis thaliana after cold stress This embodiment uses Arabidopsis thaliana as an example. CpGPX16 The cold stress resistance of genes was studied, and the specific process was as follows: S1, Agrobacterium culture Two days before the conversion, the vaccine containing 35S::CpGPX16 Agrobacterium vector was transferred to 2 mL of LB broth containing antibiotics (rifampin 50 mg / L, kanamycin 50 mg / L) and cultured overnight (approximately 12 h) at 28°C with shaking. 2 mL of the cultured Agrobacterium was then transferred to 100 mL of LB broth containing antibiotics and cultured at 28°C with shaking for approximately 8 h. The Agrobacterium was then transferred to a 50 mL centrifuge tube and centrifuged at 6000 rpm for 10 minutes at room temperature. The supernatant was then discarded, and the precipitate was resuspended in staining buffer (5% sucrose solution with 0.01% Silwet L-77) (OD). 600 =0.8), and the Agrobacterium suspension was transferred to an open sterile petri dish.

[0073] S2, Infection Select robust Arabidopsis thaliana plants, remove open flowers and pods, and immerse the inflorescences in a container containing Agrobacterium suspension for 3 minutes. After infection, place the Arabidopsis thaliana in a dark chamber, sprinkle water to maintain humidity, and incubate in the dark for 12 hours. Transfer the Arabidopsis thaliana plants to a light condition of 22-25℃ and culture normally until flowering, fruiting, and harvesting of mature seeds.

[0074] S3, Screening of transgenic Arabidopsis thaliana (1) Disinfection of Arabidopsis thaliana seeds Autoclave centrifuge tubes, pipette tips, petri dishes, and other consumables. Place an appropriate amount of seeds into a 1.5 mL centrifuge tube, add 1 mL of sterile water, and vernalize at 4°C for 3 days (to promote synchronous germination). Discard the sterile water, add 1 mL of 75% ethanol, shake for 1 minute, and immediately discard the ethanol (to avoid alcohol residue damaging the seeds). Add 1 mL of 10% sodium hypochlorite (containing 0.1% Triton X-100 to enhance permeability), shake to sterilize for 10 minutes. Discard the sodium hypochlorite, rinse 8 times with sterile water, and absorb all water after each rinse.

[0075] (2) Sowing Prepare and sterilize the culture medium (0.8% agar + 1 / 2 MS medium, pH 5.7); add 30 mg / L hygromycin to the melted culture medium and pour it into a petri dish (about 0.5 cm thick), and let it cool and solidify; use a sterile pipette tip to draw up the seed suspension and sow it evenly on the surface of the culture medium; seal the petri dish with sealing film.

[0076] S4, Overexpression CpGPX16 Evaluation of cold resistance in Arabidopsis thaliana (1) Arabidopsis thaliana control group and overexpression vector CpGPX16 The disinfection and sowing methods for Arabidopsis thaliana seeds were the same as the previous step. After germination at room temperature (3 days), they were transferred to a 4℃ cold stress condition for cultivation, and compared with Arabidopsis thaliana. CpGPX16 Gene expression and phenotypic changes, results as follows Figure 9 As shown.

[0077] The results showed that the virus was detected in the genomes of three independent Arabidopsis thaliana lines. CpGPX16 Gene transfer. Semi-quantitative results showed that gene transfer was detected in all three overexpressing Arabidopsis lines under cold treatment conditions. CpGPX16 Gene transcripts were expressed, while no expression was observed in the empty vector control group. Figure 9 A shows the growth status of Arabidopsis thaliana at 23 / 4℃, with the overexpressing Arabidopsis thaliana showing stronger growth than the control group. Figure 9 B shows overexpression under 4°C low temperature conditions. CpGPX16 Identification of RNA levels in Arabidopsis thaliana; Figure 9 C shows the root length of Arabidopsis thaliana under 4℃ low-temperature treatment. The results indicate that under cold stress, overexpression of [the virus / organism]... CpGPX16The root length of Arabidopsis thaliana with the gene was significantly increased compared to the control group.

[0078] It should be noted that the specific embodiments described above are exemplary, and those skilled in the art can devise various solutions inspired by the disclosure of this invention. These solutions all fall within the scope of this invention and its protection. Those skilled in the art should understand that this specification and its accompanying drawings are illustrative and not intended to limit the scope of the claims. The scope of protection of this invention is defined by the claims and their equivalents.

Claims

1. A gene for improving cold tolerance of Pennisetum purpureum, characterized by, CpGPX16 The CpGPX16 The amino acid sequence of the gene is shown as SEQ ID NO.

6. ​ 2. The method of claim 1, wherein the cold tolerance of the elephant grass is improved CpGPX16 gene, characterized by The nucleotide sequence encoding the amino acid sequence of the gene is shown as SEQ ID NO.

5. CpGPX16 The nucleotide sequence encoding the amino acid sequence of the gene is shown as SEQ ID NO.

5.

3. Stipa CpGPX16 application of the gene in regulating cold tolerance of plants, characterized in that, Elephant grass CpGPX16 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

6. The plant is Arabidopsis thaliana or elephant grass, and the gene is overexpressed in elephant grass. CpGPX16 Genes designed to improve the cold hardiness of Arabidopsis thaliana, silent elephant grass CpGPX16 Genes were modified to reduce the cold hardiness of elephant grass.

4. A recombinant expression vector, characterized in that, comprising a vector and a gene as claimed in claim 1 or 2 linked to the vector CpGPX16 gene.

5. A recombinant bacterial strain, characterized in that, It comprises the recombinant expression vector as described in claim 4 and a host cell, wherein the host cell is Agrobacterium GV3101.

6. A method for improving cold tolerance in plants, characterized by, The method includes the following steps: The vector-mediated method is used to transform Arabidopsis, and a transgenic strain overexpressing the amino acid sequence of the protein as shown in SEQ ID NO. 6 is obtained CpGPX16 The transgenic strain of the gene.

7. The method for improving plant cold resistance according to claim 6, characterized in that, The vector-mediated method is the Agrobacterium-mediated method.

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