BrSTM16 gene and use thereof

Abstract

The application belongs to the field of plant genetic engineering, and relates to a BrSTM16 gene and application. The nucleotide sequence of the BrSTM16 gene is shown in SEQ ID NO. 1. The application provides the BrSTM16 gene and application which can solve the problem of the molecular mechanism of the response of the white cabbage type winter rape to low-temperature stress and can cultivate new cold-resistant varieties (lines).

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, and relates to a cold-resistant gene and its application, particularly to a BrSTM16 gene and its application in enhancing the cold resistance of rapeseed. Background Technology

[0002] Rapeseed is a high-yield oilseed crop in my country, playing a crucial role in ensuring the security of edible oil supply. Northern China experiences cold winters and spring droughts, with extreme temperatures reaching -32℃. While the northward migration of winter rapeseed offers both economic and ecological benefits, the risk of frost damage is high from the approaching winter to the greening stage, and early spring frosts exacerbate yield reductions. Analyzing its low-temperature stress response pattern is crucial for cold-resistant breeding and frost damage control. Plants are often constrained by abiotic stresses such as drought, salinity, and low temperatures, leading to the evolution of multi-layered adaptation mechanisms. Drought reduces leaf area and enhances root growth; salinity causes denser epidermal cells and a thicker cuticle and waxy layer to retain water and prevent salt ingress. Stress also induces the accumulation of reactive oxygen species, triggering a cascade response that regulates stress resistance. Chinese cabbage-type rapeseed exhibits excellent cold resistance and is the main winter rapeseed type cultivated in northern China. Its above-ground growth is slow before winter, with a well-developed root system and a creeping growth cone. The growth cone, as the apical meristem, is the core of cell division and regulates subsequent plant growth and development. Therefore, improving the cold resistance of winter rapeseed and elucidating its cold resistance gene regulation mechanism are of great significance for ensuring the safe overwintering of plants, reducing low temperature stress damage, and improving production efficiency and yield.

[0003] The STM gene is a core member of the class I KNOX family. It plays a unique and crucial role in maintaining apical meristem (SAM) homeostasis by regulating the division and differentiation of meristems by inhibiting stem cell differentiation. This gene is stably expressed throughout the SAM region, but not in the early stages of organ primordium development; its loss of function directly hinders SAM formation. The characteristics of STM and its orthologs indicate that its intracellular transport processes are closely related to its function in regulating plant growth and development. Specifically, the nuclear localization of STM protein and its function in the SAM depend on the heterodimer it forms with BLH-type homologous domain proteins; simultaneously, as a core regulator of SAM, STM can also enhance plant drought tolerance.

[0004] To date, the characteristics of the STM gene in Chinese rapeseed and its functional mechanism in the low-temperature stress response process remain unclear, and there are still significant gaps in research in this field. Summary of the Invention

[0005] In order to solve the above-mentioned technical problems in the background art, the present invention provides a BrSTM16 gene and its application that can solve the problem of the molecular mechanism of the response of Chinese cabbage-type winter rapeseed to low temperature stress and can be used to cultivate cold-resistant new varieties (lines).

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A BrSTM16 gene, characterized in that: the nucleotide sequence of the BrSTM16 gene is shown in SEQ ID NO.1.

[0007] The amino acid sequence encoded by the BrSTM16 gene is shown in SEQ ID NO.2.

[0008] The BrSTM16 gene mentioned above regulates the low-temperature stress resistance of Chinese cabbage-type rapeseed.

[0009] The BrSTM16 gene positively regulates the low-temperature stress resistance of Chinese cabbage-type rapeseed.

[0010] The BrSTM16 gene has been previously described in its application in screening, identifying, distinguishing, or breeding plants with cold-resistant traits.

[0011] As previously described, the BrSTM16 gene is used in the screening, identification, differentiation, or breeding of rapeseed with cold-resistant traits.

[0012] As previously described, the BrSTM16 gene is used in the screening, identification, differentiation, or breeding of winter rapeseed of the Chinese cabbage type with cold-resistant traits.

[0013] Biological materials containing the BrSTM16 gene as described above are characterized in that: the biological material is an expression cassette, expression vector, host cell, cloning vector, or engineered bacteria.

[0014] The application of biomaterials as described above in enhancing plant resistance to low-temperature stress.

[0015] The aforementioned plants are Arabidopsis thaliana or Chinese rapeseed.

[0016] The present invention has the following technical effects: This invention uses rapeseed (Brassica napus) subjected to low-temperature stress as the research object. Based on transcriptome data under drought stress, a rapeseed STM gene named BrSTM16 was identified. Molecular characterization analysis showed that the BrSTM16 protein possesses a highly conserved Homeobox_KN domain. Tissue expression results indicated that this gene is highly expressed mainly in the plant meristematic tissues, especially at the growth cone. Abiotic stress response assays confirmed that the BrSTM16 gene exhibits a significant response to low-temperature stress. Further research showed that stable expression of BrSTM16 in Arabidopsis thaliana enhanced the cold resistance of transgenic plants. Comparison of wild-type and overexpressing Arabidopsis thaliana at the same planting time and under the same growth conditions revealed that the leaves of the STM-overexpressing Arabidopsis thaliana seedlings were larger and wider than those of the wild-type. Furthermore, compared to wild-type plants, BrSTM16 transgenic Arabidopsis thaliana exhibited lower malondialdehyde (MDA) content, higher proline (Pro) accumulation, and stronger SOD, POD, and CAT activities. These findings indicate that the BrSTM16 gene plays a positive regulatory role in the response of Chinese rapeseed (Brassica napus) to low-temperature stress, and that overexpression of the BrSTM16 gene can improve plant cold resistance. In other words, through systematic functional analysis of this gene, this invention provides a solid theoretical basis for elucidating the regulatory mechanism of the STM gene in the low-temperature stress response of Chinese rapeseed (Brassica napus), and also provides key technical support for the breeding of new cold-resistant winter rapeseed varieties, possessing significant practical application value. Attached Figure Description

[0017] Figure 1 This is a graph showing the transcriptome analysis results of BrSTM family gene expression under cold stress; Figure 2 This is a schematic diagram of the prediction results of the control elements in the promoter region of BrSTM16; Figure 3 This is a diagram showing the tissue expression analysis results of the BrSTM16 gene; Figure 4 These are the results of qPRC analysis of BrSTM16 gene expression levels under low temperature stress. Figure 5 This is a diagram showing the identification results of genetically modified Arabidopsis thaliana; Figure 6 Arabidopsis thaliana phenotype transformed with the BrSTM16 gene; Figure 7 These are phenotypic observation diagrams of transgenic lines and wild-type plants under low-temperature treatment; Figure 8 This is a statistical chart of MDA content in transgenic lines and wild-type plants under low-temperature treatment; Figure 9 This is a statistical chart of Pro content in transgenic lines and wild-type plants under low-temperature treatment; Figure 10 This is a statistical graph showing the activity of antioxidant enzymes (SOD, POD, and CAT) in transgenic lines and wild-type plants under low-temperature treatment. Detailed Implementation

[0018] This invention provides a BrSTM16 gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the amino acid sequence encoded by which the BrSTM16 gene is shown in SEQ ID NO.2. The BrSTM16 gene provided by this invention regulates the low-temperature stress resistance of Chinese cabbage-type rapeseed, particularly positively regulating its low-temperature stress resistance.

[0019] The application of the BrSTM16 gene provided by this invention in screening, identifying, distinguishing or cultivating plants with cold-resistant traits, especially in rapeseed with cold-resistant traits, for example, the rapeseed can be Chinese cabbage type winter rapeseed.

[0020] Furthermore, this invention also provides a biomaterial containing the BrSTM16 gene, which is an expression cassette, expression vector, host cell, cloning vector, or engineered bacteria. Exemplary applications of this biomaterial include enhancing plant resistance to low-temperature stress, particularly in Arabidopsis thaliana or Chinese rapeseed.

[0021] For example, the present invention also provides a method for enhancing the cold resistance of plants, specifically including the following steps: including genetically transforming the above-mentioned BrSTM16 gene into a plant, causing it to be overexpressed in the plant, and cultivating a transgenic plant with cold resistance.

[0022] Specifically, the technical solution provided by this invention will be described in detail through the following experiments: 1. Materials and Methods 1.1 Plant materials and growing conditions Plant materials: Chinese cabbage-type rapeseed varieties “Longyou 7” (hereinafter referred to as L7) and “Longyou 99” (hereinafter referred to as L99) and Arabidopsis thaliana were provided by the rapeseed research group of the State Key Laboratory of Arid Habitat Crops, Gansu Agricultural University.

[0023] Longyou 7 and Longyou 99 were selected, the former being a strongly cold-resistant variety and the latter a weakly cold-resistant variety. Healthy seeds were selected, surface-sterilized with sodium hypochlorite, and thoroughly rinsed with distilled water. The seeds were then germinated in petri dishes. After transplanting the seedlings into pots, they were cultured until the 7-leaf stage. A control group (CK) without low-temperature treatment was used. Seedlings were then treated at 0℃, 4℃, and -4℃ for 72 hours, with three replicates for each treatment. Leaves, roots, and cones of rapeseed were collected, frozen in liquid nitrogen, and then stored at -80℃ for total RNA extraction and reverse transcription.

[0024] Vectors: The plant expression vector pEarlyGate101 was provided by the State Key Laboratory of Arid Habitat Crops, Gansu Agricultural University.

[0025] Agrobacterium: Agrobacterium tumefaciens GV3101, provided by the State Key Laboratory of Arid Habitat Crops, Gansu Agricultural University.

[0026] Primer sequences are shown in Table 1: Table 1 Primer sequences Table 2 Real-time quantitative PCR reaction system Table 3 Real-time quantitative PCR reaction procedure Table 4 Cloning PCR Reaction System Table 5. Cloning PCR reaction procedure 1.2 Screening and characterization of the BrSTM16 gene Total RNA was extracted from samples of Chinese rapeseed (L7 and L99) using the RNAout kit (Tiandz, Beijing, China), and then reverse transcribed into cDNA using the RevertAid first-strand cDNA synthesis kit (MBI, USA). A novel STM gene named BrSTM16 was identified using specific primers in a database of low-temperature treated Chinese rapeseed seedlings. The gene sequence was analyzed using the NCBI database. Details of the primers used are shown in Table 1, SEQ ID NO. 7 and SEQ ID NO. 8.

[0027] 1.3 Expression analysis of the BrSTM16 gene This invention investigated the tissue-specific expression of the BrSTM16 gene in Chinese rapeseed (Brassica napus) and its response to various abiotic stresses. For abiotic stress, normally grown Chinese rapeseed (Brassica napus) at the seven-leaf stage was exposed to low-temperature environments of 4℃, 0℃, and -4℃. Samples were collected at different time intervals (0, 3, 24, and 72 h) for gene expression analysis. For precision, each stress treatment was repeated three times. It should be noted that qPCR was used for the expression analysis of the BrSTM16 gene. The reaction system and procedure are detailed in Tables 2 and 3. The primers (F and R) used in the reaction system shown in Table 2 are SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6 from Table 1, respectively.

[0028] 1.4 Starter Cistern Component Analysis The 2000 bp promoter sequence upstream of the start codon of the BrCUC2 gene was extracted using TBtools. Subsequently, cis-regulatory elements in the sequence were analyzed using the PlantCARE website (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ).

[0029] 1.5 Cloning and overexpression vector construction of the cold resistance gene BrSTM16 in Chinese cabbage-type winter rapeseed. (1) Primers containing adapters were designed using Premier 5.0 software. The primers BrSTM16-F and BrSTM16-R for constructing transgenic vectors (see SEQ ID NO.7 and SEQ ID NO.8 in Table 1), the universal primers attBF and attBR (see SEQ ID NO.9 and SEQ ID NO.10 in Table 1), and the primers BrSTM16-PF and BrSTM16-PR for constructing tissue localization vectors (see SEQ ID NO.11 and SEQ ID NO.12 in Table 1) were synthesized by Shanghai Sangon Biotech Co., Ltd.

[0030] (2) Cloning of BrSTM16: Amplification of the target fragment: Amplification was performed using Gateway technology. The total PCR reaction volume was 25 μL (see Table 4 for details). The PCR reaction procedure used a two-step method (see Table 5 for details).

[0031] 1.6 Construction of BrSTM16 expression vector (1) BP reaction system: 0.5 μL of carrier (pDONR), 1.5 μL of gel recovery product, 0.5 μL of BPIClonase, total system 2.5 μL. After ligation, incubate overnight in a metal bath at 25°C. The reaction time should not exceed 18 h, and the product should be transformed and plated after at least 4 h. The BP reaction product was transformed into Escherichia coli (DH5α) according to the E. coli instructions. The antibiotic used in the plate coating process was bleomycin (ZEO).

[0032] (2) LR reaction system: 1.5 μL of plant overexpression vector (pEarlyGate101), 0.5 μL of BP plasmid, 0.5 μL of LRIIClonase, total 2.5 μL. After ligation, incubate overnight at 25°C in a metal bath. The reaction should not exceed 18 h, and transformation and plating should be performed at least 4 h later. The LR reaction product was transformed into Escherichia coli (DH5α) according to the E. coli instructions. The antibiotic used in the plating process was kanamycin (Kan).

[0033] (3) Preparation of Agrobacterium competent cells: Follow the instructions for competent cell operation. Add 3-4 μL LLR plasmid to 50 μL Agrobacterium competent cells. Add Kan antibiotic when plating. Incubate at 28℃ for 48-72 h. Pick a single colony and place it in LB liquid medium containing Kan and RIF. Shake at 28℃ and 220 rpm for 60 h. Finally, perform bacterial PCR. The positive bacterial solution is the required fusion vector.

[0034] 1.7 Genetic transformation in Arabidopsis thaliana (1) Preparation of infecting bacterial suspension: First, activate the Agrobacterium suspension containing the overexpression vector in a 2 mL collection tube, expand the bacterial suspension in LB medium to 50 mL, centrifuge at 6000 rpm for 5 min, and then resuspend it evenly with 5% sucrose solution. 600 After adjusting the value to 0.8, add 3 / 10000 of surfactant (Silwet L-77) for later use.

[0035] (2) Infection of Arabidopsis thaliana by flower immersion: Before infection, cut off the fully opened flowers and mature siliques, immerse the entire inflorescence in a petri dish containing bacterial solution for 5-10 seconds, wipe off the excess bacterial solution, incubate in the dark at 25°C for 24 h, and then continue to incubate under light. Infect again after one week, for a total of 3 infections. Harvest seeds after Arabidopsis thaliana matures.

[0036] Screening of homozygous Arabidopsis plants with the 1.8BrSTM16 gene The seeds of Arabidopsis thaliana harvested from individual plants were vernalized for 3 days in a 4°C incubator. After sterilization with 75% anhydrous ethanol and 15% sodium hypochlorite, they were sown in 1 / 2 MS medium containing the herbicide Basta (1 / 10000) to screen for resistant seedlings (surviving plants were considered positive). These seedlings were then transplanted and cultured. After the seedlings matured, leaves were harvested from the Arabidopsis thaliana plants, and DNA was extracted from the leaves using a DNA extraction kit for PCR detection. The seeds from mature Arabidopsis thaliana plants were then harvested. The seeds from the harvested individual plants were vernalized again and then sown in 1 / 2 MS medium containing Basta to screen for homozygous transgenic Arabidopsis thaliana plants, resulting in homozygous transgenic Arabidopsis thaliana plants carrying the BrSTM16 gene.

[0037] 1.9 Identification of BrSTM16 transgenic resistant plants DNA PCR identification: Genomic DNA was extracted from transgenic Arabidopsis thaliana (i.e., homozygous Arabidopsis thaliana plants transgenic with the BrSTM16 gene). Primers are shown in SEQ ID NO.13 and SEQ ID NO.14 in Table 1. The amplification program and system were the same as those for the gene clone (see Tables 4 and 5 for details). RT-PCR identification: Primer sequences and reaction conditions are shown in Tables 1, 2, and 3 for details.

[0038] 1.10 Measurement of physiological indicators Transgenic and wild-type Arabidopsis thaliana plants, approximately 30 days old, were treated in a -4℃ incubator for 3 h, 6 h, 12 h, and 24 h, respectively, before resuming normal growth. Plant survival rates were observed and tallied one week later. The contents of cold-resistance-related antioxidant enzymes (superoxide dismutase SOD, peroxidase POD, and catalase CAT) in the transgenic and wild-type Arabidopsis thaliana plants after low-temperature treatment were determined. Specifically, SOD activity was measured using the nitroblue tetrazolium (NBT) photoreduction method, POD activity using the guaiacol method, and CAT activity using the ultraviolet absorption method.

[0039] 2. Results 2.1 Screening and Molecular Characterization Analysis of BrSTM16 STM (strained micrometers) play a crucial role in plant growth, development, and responses to abiotic stress. Therefore, a thorough analysis of the STM gene family in early stages was conducted, identifying 31 STM gene sequences. Subsequently, transcriptomic data were used to analyze the expression profiles of all STM genes in two rapeseed varieties with different levels of cold resistance. This analysis aimed to identify key STM genes playing a role in the response of rapeseed to low-temperature stress. Ultimately, the gene Brapa09T005046.1, which showed a significant response to low-temperature stress, was identified and named BrSTM16. Figure 1 As shown.

[0040] Nucleotide sequence of the BrSTM16 gene (SEQ ID NO.1) The amino acid sequence of the BrSTM16 gene (SEQ ID NO.2) MESGSNSTSCPMAFAGDNSDGPMCPMMMMMMPVITSHQQHHGHDQQHQHQQQHDGYAYQSHHQQSSSLFLQSLTPPSQEAKNKVTSSCSPSSGAPAYSFMEINHQNELLAGGLNPCSSASVKAKIMGHPHYHRLLLAYVNCQKVGAPPEVQARLEETCSSAAAAAASMMGPTGSLGEDPGLDQFMEAYCEML VKYEQELSKPFKEAMVFLQHVECQFKSLSLSSPSSFSGYGEAAIERNNNGSSEEEVDMNNEFVDPQAEDRELKGQLLRKYSGYLGSLKQEFMKKRKKGKLPKEARQQLLDWWSRHYKWPYPSEQQKLALAESTGLDQKQINNWFINQRKRHWKPSEDMQFVVMDATHPHHYFMDNVMGNPFPIDHISSTML 2.2 Low temperature stress enhances BrSTM16 promoter activity To further elucidate the mechanism by which the BrSTM16 gene responds to stress, a 2000bp promoter fragment upstream of the BrSTM16 start codon was obtained using TBtools. Plant CARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ) is used to predict regulatory elements (such as...) within the promoter region. Figure 2 (As shown). The results showed that, in addition to the CAAT-box and CAT-box core promoter elements, this promoter sequence also contained multiple photoresponsive elements such as 3-AF1 binding site, G-box, Box4, GA-motif, GT1-motif, and TCT-motif, as well as hormone-related functional elements ABRE and AuxRR-core. ABRE responds to abiotic stress and participates in the abscisic acid response, while AuxRR-core responds to auxin. Furthermore, it contained the meristematic tissue expression-related element CAT-box, the anaerobic essential element ARE, and the TC-rich repeats element involved in stress responses.

[0041] 2.3 Expression analysis of the BrSTM16 gene The BrSTM16 promoter region contains both a low-temperature response element and a meristematic expression regulatory element. Transcriptome analysis showed that this gene was consistently expressed at high levels in all tissues and remained stable under different stress conditions. To further clarify the tissue expression characteristics of BrSTM16, RT-qPCR was used to detect its expression levels in leaves, stems, growth cones, and roots. The results showed significant differences in BrSTM16 expression among different tissues in the two Chinese rapeseed varieties (Longyou 99 and Longyou 7), with the highest expression level in the growth cone. The expression levels in the growth cones of Longyou 99 and Longyou 7 were 61 times and 33 times higher than those in leaves, respectively, indicating that this gene mainly functions in the growth cone. (See details below.) Figure 3 .

[0042] To investigate the effect of low temperature on BrSTM16 expression, two varieties were treated at 0℃, 4℃, and -4℃ for 72 h, respectively. The results are as follows: Figure 4 As shown. The results indicate that the expression of this gene under low-temperature stress is tissue-specific, with particularly significant changes in expression in stems and growth cones. In Longyou 99, which has weaker cold resistance and a convex growth cone, the expression of BrSTM16 was generally higher than that in Longyou 7, which has stronger cold resistance and a concave growth cone. Specifically, in leaves, except for an upregulation at -4℃ (1.57-fold and 2.09-fold upregulation in Longyou 7 and Longyou 99, respectively), the expression was downregulated at other temperatures; in roots, only Longyou 7 showed a significant upregulation of 6.09-fold at -4℃, while Longyou 99 only showed an upregulation of 1.57-fold. In stems and growth cones, the expression level increased with decreasing temperature, reaching its highest level at -4℃. The expression levels in stems were 5.9 times (Longyou 7) and 18.3 times (Longyou 99) of the control, respectively, while in growth cones they were 20.1 times and 33.3 times (as shown in the figure). Figure 9 (As shown). In summary, the expression of BrSTM16 in different tissues was identified through low-temperature treatment. It was found that the BrSTM16 gene is mainly expressed in the growth cone of Chinese rapeseed and is strongly induced by low temperature, especially in the stem and growth cone. It is speculated that the plant regulates gene expression to enhance its cold resistance in order to cope with cold, and it plays an important role in the low-temperature response and development regulation of the growth cone.

[0043] 2.4 Screening and Identification of Arabidopsis thaliana Transgenic with BrSTM16 Gene Transgenic Arabidopsis thaliana seeds of generation T0 (BrSTM16) after vernalization were sown in sterilized nutrient soil. Two weeks later, the seedlings were sprayed with 10% Basta for screening, with each treatment occurring one week apart, for a total of three treatments. After three screenings, most seedlings showed yellowing and death, while some plants survived normally (e.g., ...). Figure 5 As shown in A, the T1 generation of Basta was screened. Seeds from surviving individual plants were harvested to obtain the T1 generation. The T1 generation seeds were sown in a Basta-containing medium, and the resistance segregation ratio met the requirement of 3:1 (e.g., ...). Figure 5As shown in B, T2 generation screening initially indicated that the exogenous gene had been integrated. After transplanting the resistant seedlings, T2 generation seeds were harvested and further screened in a Basta-containing medium. All T2 generation plants survived, indicating that homozygous transgenic lines (such as...) had been obtained. Figure 5 As shown in C, T3 generation screening). Genomic DNA was extracted from T2 generation transgenic Arabidopsis thaliana for PCR detection. The results showed that wild-type plants had no amplified bands, while all four transgenic plants amplified the target band of approximately 1500 bp (e.g., as shown in Figure C). Figure 5 As shown in Figure D, PCR detection of transgenic Arabidopsis thaliana (M: 2000 Marker; S1-S4: transgenic BrSTM16 gene; WT: wild type) preliminarily confirmed the successful transduction of the BrSTM16 gene. Further RT-qPCR analysis of RNA extracted from these four lines revealed a significant increase in BrSTM16 expression in all four lines compared to the wild type, with the highest expression level observed in line S2, which was 5.64 times that of the wild type (e.g., ...). Figure 5 As shown in E, the transgenic Arabidopsis thaliana was detected by RT-PCR, indicating that all tested lines were BrSTM16 overexpressing plants, and the S2 line had a higher transformation efficiency.

[0044] 2.5 Phenotypic observation of Arabidopsis thaliana overexpression of BrSTM16 gene Phenotypic observations of wild-type and overexpressing Arabidopsis thaliana S2 at the seedling, flowering, and maturity stages under the same planting period and growth conditions revealed that, during the seedling stage, the overexpressing Arabidopsis thaliana had larger and wider leaves than the wild-type. Figure 6 As shown in Figure A (seedling stage). Although there were differences in phenotype between Arabidopsis thaliana overexpressing the gene and wild-type Arabidopsis thaliana at the flowering and maturity stages, the differences were not significant. Figure 6 B (flowering period) and Figure 6 As shown in C (maturity stage).

[0045] 2.6 Phenotypic analysis of Arabidopsis plants overexpressing BrSTM16 under low-temperature treatment To investigate whether the BrSTM16 gene responds to low-temperature stress, four-week-old WT and overexpressing Arabidopsis plants were treated at −4°C for 3 h, 6 h, 12 h, and 24 h, respectively, followed by a 7-day recovery period at room temperature. Under this low-temperature treatment, all WT plants died after 12 h, while most overexpressing plants survived (e.g., ...). Figure 7 (As shown in the image). This observation indicates that overexpression of the BrSTM16 gene enhances the cold resistance of Arabidopsis thaliana.

[0046] 2.7 Survival analysis of plants overexpressing the BrSTM16 gene after low-temperature treatment The survival rate of Arabidopsis thaliana seedlings after low-temperature treatment was statistically analyzed (e.g., Figure 8As shown in the figure, after treatment at -4℃ for 3h and 6h, all plants grew normally; after 12h and 24h, there was a significant difference in the survival rate between wild-type and transgenic plants. All wild-type seedlings died, while most transgenic seedlings grew normally, with survival rates of 89% and 67%, respectively.

[0047] 2.8 Determination of physiological and biochemical indicators in plants overexpressing the BrSTM16 gene after low-temperature treatment To further elucidate the physiological mechanisms, physiological indicators related to cold resistance were measured. The results showed that under −4℃ treatment, the activities of superoxide dismutase (SOD) and peroxidase (POD) in both WT and overexpression plants generally increased, while catalase (CAT) activity and free proline (PR) content showed a trend of first increasing and then decreasing. In the early stages of treatment, the SOD, POD activities, and PR content of WT plants were all higher than those of the overexpression lines; as the treatment time prolonged, these three indicators gradually surpassed those of WT in the overexpression lines, while CAT activity remained consistently higher than that of WT. T and WT reached their peak values ​​at 3 h, while the overexpression lines reached their peak values ​​at 6 h. After 24 h of treatment, the SOD, POD activities, and PRO content of the overexpression lines were 9.6%, 28.5%, and 34.7% higher than those of WT, respectively. As a key enzyme for scavenging superoxide anions, the increased activity of SOD reflects the plant's strong reactive oxygen species scavenging ability under stress; the significant increase in POD activity indicates that the overexpression lines respond more rapidly and actively to stress; CAT is responsible for the efficient decomposition of hydrogen peroxide, and its sustained high activity helps alleviate oxidative damage (e.g., ...). Figure 9 As shown); PRO, as an important osmotic regulator, has a positive correlation between its accumulation level and the plant's stress resistance (e.g. Figure 10 (As shown).

[0048] In summary, overexpression of the BrSTM16 gene synergistically enhances the antioxidant enzyme activities of SOD, POD, and CAT, and promotes the accumulation of the osmotic regulator proline, thereby significantly improving Arabidopsis' ability to scavenge reactive oxygen species under low-temperature stress and maintaining cell membrane structure and osmotic pressure homeostasis, thus significantly enhancing its cold resistance. This study elucidates the molecular mechanism by which the BrSTM16 gene regulates plant cold resistance at the physiological level, providing important theoretical basis and gene resources for the genetic improvement of crop cold resistance.

Claims

1. A BrSTM16 gene, characterized in that: The nucleotide sequence of the BrSTM16 gene is shown in SEQ ID NO.

1.

2. The BrSTM16 gene according to claim 1, characterized in that: The amino acid sequence encoded by the BrSTM16 gene is shown in SEQ ID NO.

2.

3. The BrSTM16 gene according to claim 1 or 2, characterized in that: The BrSTM16 gene regulates the low-temperature stress resistance of Chinese cabbage-type rapeseed.

4. The BrSTM16 gene according to claim 3, characterized in that: The BrSTM16 gene positively regulates the low-temperature stress resistance of Chinese cabbage-type rapeseed.

5. The application of the BrSTM16 gene as described in claim 1, 2, 3, or 4 in the screening, identification, differentiation, or cultivation of plants with cold-resistant traits.

6. The application of the BrSTM16 gene as described in claim 1, 2, 3, or 4 in screening, identifying, distinguishing, or cultivating rapeseed with cold-resistant traits.

7. The application of the BrSTM16 gene as described in claim 1, 2, 3, or 4 in the screening, identification, differentiation, or cultivation of winter rapeseed of the Chinese cabbage type with cold-resistant traits.

8. A biomaterial containing the BrSTM16 gene as described in claim 1, 2, 3, or 4, characterized in that: The biomaterial is an expression cassette, expression vector, host cell, cloning vector, or engineered bacteria.

9. The application of the biomaterial as described in claim 8 in enhancing plant resistance to low temperature stress.

10. The application according to claim 9, characterized in that: The plant in question is Arabidopsis thaliana or Brassica napus.

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