Application of BnaA02g04730D gene in improvement of salt tolerance of rape

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an application of a BnaA02g04730D gene in improving the salt tolerance of oilseed rape. According to the invention, a high-salt-tolerance germplasm 'Xiangong saline-alkali oil No.1' is obtained by screening, and a key candidate gene BnaA02g04730D responding to salt stress is excavated by combining absolute quantitative transcriptomics analysis. Yeast stress phenotype identification and transgenic rape verification both prove that the gene positively regulates the salt tolerance of rape. Functional verification shows that under the stress of 0.24% NaCl, the SOD activity, the CAT activity and the POD activity of the overexpressed plant are remarkably improved compared with those of a wild type, and the MDA content is remarkably reduced. The invention preliminarily illustrates a molecular mechanism for endowing rape with salt tolerance by regulating and controlling an anti-oxidation defense system and a hormone signal channel. A new perspective is provided for analysis of a salt-tolerant molecular mechanism of the rape, and a gene resource with application value is also provided for molecular breeding of a new salt-tolerant variety.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically relates to a gene that improves the salt tolerance of rapeseed. Background Technology

[0002] Currently, China's edible oil self-sufficiency rate is only around 30%, and the supply-demand imbalance is becoming increasingly prominent. Rapeseed, as my country's largest domestically produced vegetable oil source, accounts for nearly 50% of the total oil production of domestic oilseed crops, occupying a core position in the domestic edible oil supply system. Therefore, increasing rapeseed oil production is of irreplaceable strategic significance for ensuring my country's edible oil supply security. Expanding the rapeseed planting area is the most direct and effective way to increase rapeseed oil production, but the existing arable land area is insufficient to meet the demand for expanded rapeseed cultivation. my country has approximately 100 million mu of saline-alkali land reserves. The efficient development and utilization of saline-alkali land has become an important breakthrough for tapping the potential for expanded rapeseed cultivation and overcoming the limitations of arable land resources. Salt stress, as the main environmental limiting factor in saline-alkali land, severely inhibits rapeseed seed germination, root growth, and photosynthetic metabolism through mechanisms such as osmotic stress, ion toxicity, and oxidative damage. This leads to hindered rapeseed growth and development, decreased stress resistance, and ultimately a significant reduction in yield, severely restricting the large-scale application of saline-alkali land in rapeseed cultivation. Currently, my country's comprehensive utilization of saline-alkali land has established a strategic direction of "shifting from controlling crops adapted to saline-alkali land to selecting and breeding salt-tolerant plants adapted to saline-alkali land." Therefore, screening superior salt-tolerant germplasm and cultivating new salt-tolerant rapeseed varieties have become key technological pathways to overcome the constraints of salt stress on rapeseed growth, achieve efficient agricultural utilization of saline-alkali land resources, increase total rapeseed oil production, and strengthen the national edible oil security defense line. These technologies have significant strategic value and practical implications.

[0003] Salt stress damages plants primarily through ion toxicity, osmotic stress, and oxidative damage. Under high salinity, reactive oxygen species (ROS) accumulate in large quantities within plant cells, triggering membrane lipid peroxidation and disrupting cell membrane structural integrity. To cope with oxidative stress, plants have evolved multiple stress-resistance regulatory mechanisms. On one hand, through an antioxidant enzyme defense system composed of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), plants can maintain high antioxidant enzyme activity under salt stress, effectively scavenging ROS and reducing the accumulation of malondialdehyde (MDA), thereby mitigating oxidative damage. On the other hand, they maintain cell turgor pressure and ensure metabolic stability by accumulating osmotic regulators such as proline and soluble sugars. Furthermore, endogenous hormones play a crucial role in plant salt-resistance signal transduction. Abscisic acid (ABA), as a core stress-response hormone, can be induced to accumulate under salt stress, thereby activating SnRK2 protein kinase and reducing water loss by regulating stomatal closure. Salicylic acid (SA) has also been shown to participate in stress resistance regulation; exogenous SA treatment can significantly reduce Na+ in plants. + Accumulate and increase endogenous SA levels to enhance salt tolerance.

[0004] In the study of salt tolerance gene function in rapeseed, several key genes have been confirmed to participate in salt tolerance regulation. For example, patent 202510832614.4 discloses the application of the BnaCP15A gene in improving the salt tolerance of Brassica napus, which enhances the salt tolerance of plants by increasing the expression of the BnaCP15A gene; patent 202411552758.6 discloses the application of the BnaC04.bZIP16 gene in improving the salt tolerance of plants, showing that transgenic Brassica napus overexpressing this gene can grow better under salt stress, verifying that this gene can act as a positive regulator to enhance the salt tolerance of rapeseed. However, a large-scale, systematic salt-tolerant germplasm screening system for Brassica napus is still lacking, and research focusing on the discovery of key salt tolerance genes and the analysis of regulatory mechanisms in Brassica napus itself is relatively scarce. Therefore, conducting systematic screening of salt-tolerant germplasm of Brassica napus and discovering specific key salt tolerance genes is of great significance for promoting the progress of molecular breeding for salt tolerance in rapeseed. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes the application of the BnaA02g04730D gene in improving the salt tolerance of rapeseed.

[0006] The technical solution of this invention is implemented as follows:

[0007] On the one hand, the present invention proposes the application of the BnaA02g04730D gene or biomaterials containing the BnaA02g04730D gene in improving the salt tolerance of rapeseed. The nucleotide sequence of the BnaA02g04730D gene is shown in SEQ ID No.1, and the amino acid sequence of the protein it encodes is shown in SEQ ID No.2.

[0008] Preferably, the above-mentioned biological material is an expression cassette, expression vector, or recombinant bacteria containing the BnaA02g04730D gene.

[0009] Preferably, the above-mentioned improvement is achieved by increasing the expression level of the BnaA02g04730D gene in rapeseed to enhance its salt tolerance. Increasing the expression level of the BnaA02g04730D gene in rapeseed can enhance the activity of antioxidant defense enzymes, reduce membrane lipid peroxidation damage, and positively regulate the salt tolerance of rapeseed.

[0010] Preferably, the rapeseed mentioned above is Brassica napus. The Brassica napus type is either Xiangnong Salt-Alkali Oil No. 1 or Zhongshuang 11.

[0011] Secondly, the present invention also proposes a method for improving the salt tolerance of rapeseed and prolonging its survival time, the steps of which are: overexpressing the BnaA02g04730D gene in rapeseed plants, thereby improving the salt tolerance of rapeseed and prolonging its survival time.

[0012] Preferably, the nucleotide sequence of the BnaA02g04730D gene is shown in SEQ ID No. 1, and the rapeseed is Brassica napus. The Brassica napus is either Xiangnong Salt-Alkali Oil No. 1 or Zhongshuang 11.

[0013] Thirdly, a method for cultivating salt-tolerant transgenic rapeseed includes the following steps: constructing an overexpression vector containing the BnaA02g04730D gene, transferring it into the rapeseed plant to be improved using Agrobacterium-mediated transformation, and obtaining salt-tolerant transgenic rapeseed plants through callus induction, differentiation culture, and rooting screening.

[0014] Preferably, the nucleotide sequence of the BnaA02g04730D gene is shown in SEQ ID No. 1.

[0015] Preferably, the rapeseed mentioned above is Brassica napus. The Brassica napus type is either Xiangnong Salt-Alkali Oil No. 1 or Zhongshuang 11.

[0016] The present invention has the following beneficial effects:

[0017] This invention screened and obtained a highly salt-tolerant germplasm, 'Xiangnong Salt-Alkali Oil No. 1'. A comprehensive screening was conducted based on plant phenotype, physiological indicators, and gene expression patterns, identifying a key candidate gene, BnaA02g04730D, in response to salt stress. The expression level of BnaA02g04730D showed highly significant correlations with SOD, CAT activities, and MDA content at multiple time points (|r|>0.70), indicating that it is located upstream in the antioxidant regulatory network. Yeast stress phenotype identification and transgenic rapeseed validation both confirmed that the gene BnaA02g04730D can significantly improve the salt tolerance survival rate of eukaryotic cells and positively regulate the salt tolerance of rapeseed. Functional validation showed that under 0.24% NaCl stress, the SOD, CAT, and POD activities of overexpressing plants were significantly increased by 1.8 times, 3.1 times, and 2.7 times, respectively, compared to the wild type, while the MDA content was significantly reduced by approximately 25%. This application not only screened and obtained valuable salt-tolerant germplasm resources, but also identified a key salt-tolerant gene, BnaA02g04730D, with significant application value. This provides a new perspective for elucidating the molecular mechanism of salt tolerance in rapeseed and also offers a gene resource with important application value for the molecular breeding of new salt-tolerant varieties. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art 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.

[0019] Figure 1 The field growth of the finally selected salt-tolerant material 'Xiangnong Salt-Alkali Oil No. 1' in saline-alkali land.

[0020] Figure 2 The growth phenotypes of 'Xiangnong Salt-Alkali Oil No. 1' under different concentrations of NaCl stress are shown in Figure A, which shows the growth of the aboveground parts of the plant at 12, 24, 36 and 48 days after treatment, and Figure B shows the growth of the root system of the plant at 12, 24, 36 and 48 days after treatment.

[0021] Figure 3 Figure A shows the changes in physiological indicators of roots and leaves under different salt concentration treatments, while Figure B shows the content of physiological indicators of roots under different salt concentration treatments.

[0022] Figure 4 The content of endogenous hormones in roots under different salt concentration treatments.

[0023] Figure 5GO enrichment analysis of differentially expressed genes under different treatments; Figure A shows the GO enrichment results of differentially expressed genes in the Y1 treatment group, Figure B shows the GO enrichment results of differentially expressed genes in the Y2 treatment group, and Figure C shows the GO enrichment results of differentially expressed genes in the Y3 treatment group.

[0024] Figure 6 Figure A shows the gene expression levels under different salt concentrations; Figure B shows the expression levels after 12 days of treatment, Figure C shows the expression levels after 36 days of treatment, and Figure D shows the expression levels after 48 days of treatment.

[0025] Figure 7 For the TLC validation of three key genes; Figure A shows the TLC validation of BnaC07g08360D, Figure B shows the TLC validation of BnaA10g15320D, and Figure C shows the TLC validation of BnaA02g04730D; the dilutions from left to right are 100, 10, and 10, respectively. -1 10 -2 times.

[0026] Figure 8 Phenotypic comparison photos of BnaA02g04730D overexpression lines under different salt concentration stresses.

[0027] Figure 9 The relative expression levels of BnaA02g04730D in wild-type (WT) and overexpression (OE) lines.

[0028] Figure 10 The effects of different salt concentrations on physiological parameters of wild-type (WT) and BnaA02g04730D overexpression (OE) plants were investigated. Figure A shows the MDA content in leaves and roots, B shows the SOD activity in leaves and roots, C shows the CAT activity in leaves and roots, and D shows the POD activity in leaves and roots. Detailed Implementation

[0029] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0031] In the early stages of this invention, a large-scale salt tolerance evaluation was conducted using 1609 Brassica napus accessions, and high-salt-tolerant superior germplasm was screened out. Combined with root transcriptomics analysis and gene function verification, a key gene regulating salt tolerance in rapeseed was successfully discovered and identified, providing a reference for the breeding of new salt-tolerant rapeseed varieties and the study of molecular mechanisms.

[0032] (1) Test materials

[0033] All the rapeseed materials of this invention were provided by Hunan Agricultural University. Among them, the rapeseed material 'Xiangnong Salt-Alkali Oil No. 1' was cultivated by the applicant and obtained in May 2019 at Hunan Agricultural University in Changsha, Hunan Province, using 'Xiangyou No. 15' as the parent, through multiple generations of self-pollination and screening. It was selected from 2,000 rapeseed materials through indoor germination period salt tolerance identification and pot experiment screening.

[0034] (2) Data statistics and analysis:

[0035] All physiological and biochemical indicators and qRT-PCR data in this invention were performed in triplicate. Data were processed using Microsoft Excel 2019 and statistically analyzed using SPSS 27.0 software. Gene expression heatmaps were generated using TBtools.

[0036] Example 1: Screening of salt-tolerant germplasm

[0037] In the early stages of this application, the germination box method was used to evaluate the salt tolerance of 1609 materials during the germination period. Based on the overall performance of each material under salt stress, 50 materials with high, medium and low tolerance were initially selected (Table 1).

[0038] Based on its growth in saline-alkali soil (total soluble salt: 52.36 g / kg) in Sunfuji Township, Liangyuan District, Shangqiu City, Henan Province (34.5920°N, 115.4983°E), YLS2060 ('Xiangnong Salt-Alkali Oil No. 1') was selected from highly salt-tolerant materials. Subsequent testing showed that this variety is a high-oil, low-erucic acid (0%) material, with an oil content as high as 49.33%, 222 siliques per plant, and a thousand-seed weight of 4.60 g. Field growth is as follows... Figure 1 As shown.

[0039] Table 1. 50 high, medium and low quality materials selected in the preliminary screening

[0040]

[0041] Phenotypic and physiological differences in 'Xiangnong Salt-Alkali Oil No. 1' under different salt concentration stress:

[0042] Germinated 'Xiangnong Salt-Alkali Oil No. 1' seeds were planted in nutrient soil (5 seedlings per pot, 4 pots per treatment). Different concentrations of NaCl were applied at the 2-3 leaf stage (CK: water control; Y1: 0.12% NaCl; Y2: 0.24% NaCl; Y3: 0.36% NaCl). Samples were taken at 12, 24, 36, and 48 days after treatment, and root and leaf tissues were collected, flash-frozen in liquid nitrogen, and stored at -80℃.

[0043] The activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were determined using the nitroblue tetrazolium (NBT) photochemical reduction method, the guaiacol method, and the ultraviolet absorption method, respectively. The content of malondialdehyde (MDA) was determined using the thiobarbituric acid (TBA) method. The content of soluble protein was determined using the Coomassie Brilliant Blue G-250 staining method. The activities of APX and glutathione peroxidase (GPX), the content of proline (Pro), and the contents of endogenous hormones (ABA, SA, GA, IAA) were determined using the corresponding ELISA (enzyme-linked immunosorbent assay) kits.

[0044] 1. Growth phenotypic characteristics of 'Xiangnong Salt-Alkali Oil No. 1' under different salt concentration stress

[0045] The growth phenotypes of 'Xiangnong Salt-Alkali Oil No. 1' under different salt concentrations (Y1, Y2, Y3) and different treatment time points (12 d, 24 d, 36 d, 48 d) were systematically observed according to the above pot experiment method. Figure 2 ),Depend on Figure 2 It was observed that under low salt stress (Y1), the plants exhibited strong adaptability, with only slight growth inhibition at 12 days of treatment. Growth rate gradually recovered with prolonged adaptation, with only slight yellowing of leaves observed under long-term stress (48 days). However, under medium-to-high salt stress (Y2, Y3), plant growth was significantly inhibited, and the inhibition occurred earlier. The Y3 treatment group showed stunted growth as early as 12 days, and by 48 days, the entire plant was severely yellowed, with leaf edges scorched and even wilted. With increasing salt concentration, taproot development was significantly hindered; lateral root density showed an increasing trend in the early stages of stress (12-24 days), indicating that the plants attempted to enhance their absorption capacity to adapt to adversity by reshaping their root system architecture. However, lateral root growth was also severely inhibited in the later stages of long-term high salt stress (36-48 days), ultimately leading to irreversible damage. While plants may partially adapt to low salt concentrations under long-term salt stress, high salt concentrations can cause irreversible damage.

[0046] 2. Analysis of the differences in physiological indicators of 'Xiangnong Salt-Alkali Oil No. 1' under different salt concentration stress

[0047] (1) Measure the physiological indicators of roots and leaves under different salt concentrations (Y1, Y2, Y3) and treatment times.

[0048] Depend on Figure 3 As shown in Figure A, SOD activity in the root system exhibited a trend of first increasing and then decreasing. In the early stages of stress (12-24 days), the SOD activity of all treatment groups was significantly higher than that of the control (CK). At 24 days, the activity of the Y3 treatment group reached a peak of 499 U / g, significantly higher than that of the CK (380 U / g) by 31.32%. However, as the stress duration increased to 48 days, the SOD activity of the Y2 and Y3 treatment groups significantly decreased to 314-329 U / g, lower than the CK level. POD activity was higher than that of the CK throughout the entire stress process. At 48 days, the POD activity of the Y1 low-salt treatment group was the highest, reaching 128 U / g, significantly higher than that of the CK (83 U / g) and other treatment groups. CAT activity peaked at 28 U / g at 24 days under the Y3 treatment, 1.75 times that of the control (CK, 16 U / g). SOD, POD, and CAT activities in the roots increased slowly over time under the Y1 treatment, demonstrating strong adaptability. However, in the early stages of the Y2 and Y3 treatments (24 days), the antioxidant enzyme system was rapidly activated to scavenge reactive oxygen species. But as the stress duration increased to 48 days, enzyme activities decreased to varying degrees, indicating that long-term high salt stress led to damage to the enzyme system. At 36 days, MDA content in the Y2 and Y3 treatment groups increased sharply, reaching 1.48 and 1.98 µmol / g, respectively, approximately 1.8 times and 2.4 times that of the control (CK, 0.82 µmol / g). Soluble protein content showed the most significant response to salt stress at 24 days, reaching 0.68 mg / g in the Y1 treatment group, a 23.64% increase compared to the control (CK, 0.55 mg / g). APX and GPX activities and Pro content showed a continuous upward trend with stress duration. At 48 days, the Pro content in the Y3 treatment group reached approximately 2918 ng / L, significantly higher than that in the control group (2779 ng / L), indicating that under long-term stress, the root system still relies on proline accumulation to maintain osmotic balance.

[0049] Depend on Figure 3As shown in Figure B, SOD activity in leaves responded rapidly in the early stages of stress. At 24 days, the activity in the Y3 treatment group peaked at approximately 538 U / g, significantly higher than the control (CK) (426 U / g) by about 26.3%. However, at 48 days, the SOD activities in the Y2 and Y3 treatment groups decreased significantly, to 352 U / g and 328 U / g, respectively, lower than the CK level. POD activity responded most strongly to salt stress. At 24 days, all salt treatment groups reached highly significant levels, with the Y3 treatment group exhibiting the highest activity at 31 U / g, 3.52 times that of the CK (8.8 U / g). Subsequently, the activity gradually decreased, reaching only 7.78 U / g in the Y3 treatment group at 48 days. CAT activity peaked at 24 days under the Y2 and Y3 treatments, at 23.2 U / g and 33.2 U / g, respectively, significantly higher than the CK (7.5 U / g), representing increases of approximately 3.1 times and 4.4 times. The changes in antioxidant enzyme activity in leaves showed a similar trend to those in roots, but the decline was more dramatic in the later stage of the Y3 treatment (48 days). Changes in MDA content directly reflected the degree of membrane damage. At 24 days, the MDA content in the Y2 and Y3 treatment groups increased sharply, reaching 4.77 and 6.28 µmol / g, respectively, approximately 1.42 times and 1.86 times that of the control (CK) (3.37 µmol / g). Soluble protein content showed the most significant response to salt stress at 24 days, reaching 0.82 mg / g in the Y3 treatment group, a 43.86% increase compared to the control (CK) (0.57 mg / g). APX and GPX activities and Pro content showed a continuous upward trend with stress duration. At 48 days, the Pro content in the Y3 (R3) treatment group reached 3093 ng / L, significantly higher than that of the control (CK) (2982 ng / L). Furthermore, APX and GPX activities showed a continuous upward trend with increasing salt concentration and time, indicating their important role in long-term salt stress response.

[0050] In summary, 'Xiangnong Salt-Alkali Oil No. 1' alleviates oxidation and osmotic pressure in the early stage of salt stress by rapidly increasing the activity of antioxidant enzymes such as SOD, POD, and CAT and accumulating osmotic regulators; however, under long-term high salt stress (48 days), the root system showed stronger tolerance than the leaves, while the leaves suffered more severe oxidative damage.

[0051] (2) Changes in the content of endogenous hormones in the roots of 'Xiangnong Salt-Alkali Oil No. 1' under different salt concentration stress

[0052] The contents of GA, IAA, SA and ABA in roots at different treatment times were determined using an ELISA kit. Figure 4 ),Depend on Figure 4It was found that the contents of the four hormones generally increased with increasing salt concentration and treatment time. Among them, abscisic acid (ABA), as a core signaling molecule for plants to respond to abiotic stress, showed a significant increase in content with increasing salt concentration, especially in the later stage of treatment (48 days). The ABA contents of the CK, Y1, Y2, and Y3 treatment groups were 356 ug / L, 375 ug / L, 362 ug / L, and 368 ug / L, respectively. The ABA contents of each salt treatment group remained at a high level, indicating that salt stress induces plants to produce more abscisic acid to cope with adverse environments, which is beneficial for inducing stomatal closure and the expression of stress resistance genes. The content of salicylic acid (SA) in the Y2 and Y3 treatment groups was significantly higher than that in the control, and continued to accumulate over time, indicating that high salt stress strongly stimulated SA synthesis to enhance stress resistance. Although the contents of auxin (IAA) and gibberellin (GA) increased overall, the growth slowed down or even decreased slightly in the later stage of the Y3 treatment. At 48 days, the GA content in the Y1 treatment group reached 628 pg / mL, which was significantly increased by 14.8% compared with the CK (547 pg / mL) at the same time. The content in the Y3 treatment group was 596 pg / mL, which was higher than the CK but significantly lower than the Y1 treatment group. At 48 days, the auxin (IAA) content in the Y1 treatment group reached a peak of 106 ug / L, which was significantly higher than the CK (99 ug / L) and the Y2 and Y3 treatment groups. This indicates that low salt stress may promote root growth to adapt to the adversity by maintaining higher levels of GA and IAA, while higher salt concentration may have a certain inhibitory effect on plants.

[0053] Example 2: Identification of key salt tolerance genes based on transcriptome analysis

[0054] 'Xiangnong Salt-Alkali Oil No. 1' was planted in soils with different salt concentrations (CK: water control; Y1: 0.12% NaCl; Y2: 0.24% NaCl; Y3: 0.36% NaCl). Root samples were collected after 12 days of treatment. Libraries were constructed using absolute quantitative transcriptome sequencing and high-throughput sequencing was performed. Differentially expressed genes showing significant responses to salt stress were screened as candidate salt-tolerance genes using bioinformatics analysis. Simultaneously, total RNA was extracted from samples of different treatments and reverse transcribed into cDNA. The expression levels of five candidate genes were detected using qRT-PCR (primers are shown in Table 2), with BnActin as an internal control. -ΔΔCt The relative expression level was calculated and its correlation with physiological indicators was analyzed.

[0055] Table 2 qRT-PCR primer information

[0056]

[0057] 1. Root transcriptome analysis and candidate gene screening under different salt concentration stresses

[0058] The root absolute quantitative transcriptome sequencing of 'Xiangnong Salt-Alkali Oil No. 1' under different salt concentrations (CK, Y1, Y2, Y3) was performed, and 2380, 2737 and 8715 DEGs were identified, respectively. There were 727 core differentially expressed genes between each treatment group and the control group, including 483 upregulated genes and 230 downregulated genes.

[0059] Functional enrichment analysis was performed on the differentially expressed genes. The results showed that the DEGs identified in this study were significantly enriched in categories such as "phenylpropanone biosynthesis," "plant hormone signal transduction," and "metabolic pathways." Particularly under high salt stress, the expression of a large number of genes involved in redox processes (oxidoreductase activity) and transmembrane transporter activity changed significantly. These results indicate that 'Xiangnong Salt-Alkali Oil No. 1' mainly adapts to salt stress through multiple mechanisms, including activating phenylpropanone metabolism, hormone signal transduction, and regulating ion transport. Therefore, the focus was on screening candidate genes responding to salt stress from these significantly enriched key pathways.

[0060] A preliminary screening of expression levels was conducted on 727 core genes, focusing on genes that responded most strongly to salt stress, based on an absolute value of the logFC (log value of expression difference) > 1.5 and a p-value < 0.05. Secondly, functional annotation was screened, with a focus on genes closely related to plant salt tolerance mechanisms based on GO and KEGG enrichment analyses. Figure 5 The specific screening criteria included: (1) genes annotated as transcription factors (such as AP2 / ERF, MYB, WRKY, etc.), which, as upstream regulators, may initiate the entire salt tolerance response; (2) genes involved in the antioxidant system (such as peroxidase, glutathione S-transferase); (3) genes involved in ion transport and osmotic regulation (such as SPX domain proteins); and (4) genes involved in hormone signal transduction (such as auxin-responsive proteins). Based on gene annotations and the NCBI database, 20 differentially expressed genes were identified (Table 3).

[0061] Table 3. 20 differentially expressed genes obtained through screening

[0062]

[0063] Under salt stress, excessive absorption of Na +It competitively inhibits the absorption of other nutrient ions, leading to abnormal plant growth. This study investigated the effects of salt stress on ion osmotic regulation and antioxidant enzyme regulation, as well as the influence of salt stress on phosphate transport proteins. Genes with functional annotations related to salt tolerance were screened from differentially expressed genes. Further screening of differentially expressed genes using gene function data from the NCBI database yielded five key differentially expressed genes: BnaA07g33310D, BnaA10g15320D, BnaA02g04730D, BnaC03g23980D, and BnaC07g08360D.

[0064] 2. Analysis of expression patterns of candidate key genes under different salt concentration stresses

[0065] Given that roots are a key site for salt stress response, qRT-PCR was used to detect the expression of five candidate genes (BnaA07g33310D, BnaA10g15320D, BnaA02g04730D, BnaC03g23980D, and BnaC07g08360D) in roots under different salt concentrations and treatment time points. Figure 6 ).Depend on Figure 6 It was found that all five genes were significantly induced by salt stress, but the response patterns differed. Specifically, BnaA02g04730D expression was suppressed in the early stages of low-salt (Y1) treatment, but under medium-high salt (Y2, Y3) stress, although initially downregulated, it showed an adaptive recovery trend over time. Meanwhile, BnaA10g15320D expression increased sharply in the later stages of treatment (48 days). Similarly, BnaC03g23980D expression reached 19.67 times that of the control at 48 days of high-salt (Y3) treatment. These results confirm that the selected candidate genes are indeed involved in the molecular response of rapeseed to salt stress. BnaC07g08360D was significantly upregulated only in the early stages of low salt (Y1), while fluctuating significantly under other conditions, suggesting that this gene may be related to a specific response. The expression levels of the BnaA10g15320D gene showed extreme changes in the Y1 and Y3 treatment groups, suggesting that this gene may play a regulatory role in certain acute stress responses. Based on the above expression characteristics, it is speculated that genes such as BnaA02g04730D occupy a core position in the rapeseed salt tolerance regulatory network.

[0066] 3. Correlation analysis between candidate gene expression levels and physiological indicators and hormone levels

[0067] To explore the intrinsic relationship between candidate genes and salt tolerance physiological mechanisms, a correlation analysis was conducted on the expression levels of five candidate genes and root physiological indicators and endogenous hormone content.

[0068] Table 4. Correlation between expression levels of candidate key genes and content of physiological indicators

[0069]

[0070] Note: *At the 0.05 level (two-tailed), the correlation is significant. **At the 0.01 level (two-tailed), the correlation is significant.

[0071] Table 5. Correlation between expression levels of candidate key genes and hormone levels

[0072]

[0073] Note: *At the 0.05 level (two-tailed), the correlation is significant. **At the 0.01 level (two-tailed), the correlation is significant.

[0074] Tables 4-5 show that all five genes were significantly induced by salt stress. Among them, the expression level of gene BnaA02g04730D showed a highly significant negative correlation with SOD activity, CAT activity, and MDA content in the early stages of treatment (12 days and 24 days) (r < -0.7, P < 0.01). Given that SOD and CAT are key enzymes for ROS scavenging, and that this gene also showed a highly significant negative correlation with ABA and GA content at 36 days, it is speculated that BnaA02g04730D may act as an upstream regulator, negatively regulating oxidative damage or participating in feedback regulation of the antioxidant enzyme system. Furthermore, the highly significant negative correlation between this gene and ABA and GA content at 36 days suggests that it may be involved in hormone signaling interactions. The expression level of gene BnaA10g15320D showed a significant negative correlation with multiple antioxidant indicators at 24 days of treatment, but became highly significantly positively correlated with APX activity and Pro content at 48 days (r>0.9), suggesting that it may participate in osmotic regulation and assist in antioxidant processes under long-term stress. The expression level of gene BnaC07g08360D showed a highly significantly positive correlation with GPX, APX activity, and Pro content at 12 days of treatment, but became highly significantly negatively correlated with these activities at 24 days. It also showed a highly significantly positive correlation with hormone (IAA, ABA, etc.) content in the early stages, but the correlation turned negative over time, exhibiting a complex regulatory pattern. Therefore, the significant changes in the expression levels of BnaA02g04730D, BnaA10g15320D, and BnaC07g08360D genes and their strong correlation with physiological and biochemical indicators may indicate that they are key genes affecting salt tolerance in rapeseed.

[0075] Example 3: Validation of candidate salt tolerance gene BnaA02g04730D and construction of transgenic materials

[0076] 1. Construction of yeast expression vector and identification of salt tolerance

[0077] Total RNA was extracted from the roots of 'Xiangnong Salt-Alkali Oil No. 1' and reverse transcribed into cDNA. The CDS sequence of the candidate gene was amplified and ligated into the pYES2-NTB vector to construct a recombinant yeast expression vector. The recombinant plasmid was transformed into Saccharomyces cerevisiae INVSC1 strain using the lithium acetate method. The growth of the transformed strain on SG-Ura medium containing 1.0 mol / L NaCl was compared by plate testing to identify the salt tolerance function of the gene.

[0078] Identification of yeast stress phenotypes of key genes

[0079] To identify the salt tolerance function of candidate genes, yeast expression vectors pYES2-NTB containing BnaA10g15320D, BnaA02g04730D, and BnaC07g08360D were constructed and transformed into *Saccharomyces cerevisiae* INVSC1 strain. The results of the plate test are as follows: Figure 7 As shown, all strains exhibited consistent growth on normal medium (SC-U). However, on stress medium containing 1.0 mol / L NaCl, significant differences emerged among the strains: the strain expressing BnaA02g04730D showed the strongest growth, significantly superior to the empty vector control (pYES2), indicating that this gene can significantly enhance the salt tolerance of yeast cells; the growth of the strain expressing BnaA10g15320D was inhibited, with no significant difference from the control; and the growth of the strain expressing BnaC07g08360D was even weaker than the control, suggesting that it may play a negative regulatory role under salt stress. Based on these results, we identified BnaA02g04730D as the most critical positive salt tolerance regulatory gene.

[0080] 2. Construction and genetic transformation of BnaA02g04730D overexpression vector

[0081] The overexpression vector PC1300s-SPX was constructed, transformed into Agrobacterium GV3101, and then transformed into the hypocotyl of Brassica napus 'Zhongshuang 11' using Agrobacterium-mediated transformation to obtain regenerated plants.

[0082] Identification and Expression Analysis of Transgenic Plants: Hypocotyls of Brassica napus 'Zhongshuang 11' were transformed using Agrobacterium-mediated transformation. After callus induction, differentiation, rooting, and resistance screening, regenerated plants were successfully obtained. DNA was extracted from the leaves of the regenerated plants, and PCR detection was performed using primers specific to the hygromycin resistance gene (HYG). The results showed that the expected 557 bp target band was amplified using the recombinant plasmid as a positive control, while no amplification was observed using wild-type (WT) plant DNA or water as negative controls. Among the 16 plants tested, 14 amplified the target band of the same size as the positive control, with a positive rate of 87.5%, confirming that the hygromycin resistance gene (HYG) had been successfully integrated into the genome of these plants.

[0083] Salt stress was applied to BnaA02g04730D gene overexpression (OE) lines and wild-type (WT) lines, and their phenotypic differences were compared. Figure 8 ).Depend on Figure 8 As shown, under normal control conditions (CK), the OE line was superior to the WT line in terms of plant height and leaf size, indicating that overexpression of BnaA02g04730D itself does not negatively affect the normal growth and development of rapeseed. Twelve days after Y2 treatment, WT plants exhibited growth inhibition, stunted growth, yellowing and wilting of lower functional leaves, and chlorosis in newly emerging upper leaves. In contrast, under the same stress conditions, OE plants showed significantly enhanced salt tolerance, with a significantly greater overall biomass than WT plants. Their leaves remained mostly green, with only a few older leaves showing yellowing edges, and their growth was significantly better than the wild type. Furthermore, even when the OE line died in the later stages of Y3 treatment, the overexpressing plants survived longer than the wild type. These phenotypes demonstrate that overexpression of BnaA02g04730D can effectively alleviate the damage of salt stress to rapeseed and significantly improve its salt tolerance.

[0084] 3. Identification and expression level analysis of transgenic positive plants

[0085] Leaf DNA was extracted and amplified by PCR using hygromycin resistance gene (HYG) specific primers (forward primer: 5'-ACACTACATGGCGTGATTTCAT-3', reverse primer: 5'-TCCACTATCGGCGAGTACTTCT-3') to screen for positive transformed plants. Total RNA was extracted from positive plants and reverse transcribed into cDNA. The expression level of the target gene was detected by qRT-PCR. Two... -ΔΔCt The relative gene expression level was calculated to verify the overexpression level of BnaA02g04730D in transgenic plants.

[0086] Three PCR-positive lines (OE#12, #4, #5) and the wild-type (WT) were selected for qRT-PCR analysis, such as... Figure 9As shown, under normal growth conditions (CK), the relative expression levels of BnaA02g04730D in all three lines were significantly higher than those in the wild type (WT), with the highest level observed in line OE#12, approximately 8.7 times that of WT, confirming effective constitutive high expression of this gene in transgenic plants. Furthermore, in WT, Y2 treatment upregulated the expression level of this gene by approximately 2.9-fold, indicating that BnaA02g04730D is a salt stress-induced gene. In different overexpression lines, the expression level of BnaA02g04730D also showed an increasing trend with increasing salt concentration. Under Y2 treatment, the expression levels of lines OE#4 and OE#5 were higher than their levels under CK conditions and significantly higher than those under salt stress in WT. Combining the PCR and qRT-PCR results, transgenic materials with high BnaA02g04730D expression were successfully obtained for subsequent salt tolerance functional analysis.

[0087] 4. Analysis of salt tolerance phenotype and physiological indicators of genetically modified rapeseed

[0088] T0 generation overexpression-positive plants with consistent growth and wild-type (WT) plants were selected. During the seedling stage, both transgenic lines and wild-type plants were treated with 0.24% NaCl salt stress. After 12 days of treatment, the plant growth phenotype was observed and recorded, and relevant physiological and biochemical indicators were measured. The measured indicators included SOD, POD, CAT activities, and MDA content. All indicators were measured using the methods described in Example 1. By comparing the phenotypic and physiological differences between overexpression plants and wild-type plants under salt stress, the effect of the BnaA02g04730D gene on the salt tolerance of rapeseed was comprehensively evaluated.

[0089] The antioxidant indices of roots and leaves in wild-type (WT) and overexpression (OE) plants under salt stress (0.24% NaCl) were determined. Figure 10 As shown in Figure A, the MDA content determination results indicated that, under normal growth conditions, there was no significant difference in MDA levels between wild-type (WT) and overexpression plants (OE). After Y2 treatment, the MDA content in the roots and leaves of WT plants increased sharply, indicating severe oxidative damage; in contrast, MDA accumulation in OE plants was significantly inhibited. Particularly in the roots, the MDA content of OE plants was significantly lower than that of WT (P < 0.05), indicating that overexpression of BnaA02g04730D effectively maintained cell membrane integrity.

[0090] Further analysis of the antioxidant enzyme system revealed that BnaA02g04730D has a significant positive regulatory effect on the activities of SOD, CAT, and POD. Figure 10(BD). Under salt stress, OE plants exhibited a more rapid and stronger antioxidant response than WT plants. Under Y2 treatment, the activities of SOD, CAT, and POD in the roots of OE plants reached 1.8 times, 3.1 times, and 2.7 times that of WT plants, respectively. Even under the higher salt concentration Y3 treatment, OE plants maintained extremely high enzyme activity levels. Therefore, BnaA02g04730D can significantly reduce the oxidative damage to the cell membrane system caused by salt stress by synergistically enhancing the activities of SOD, CAT, and POD, thus constructing an efficient reactive oxygen species scavenging network and ultimately conferring stronger salt tolerance to Brassica napus.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of the BnaA02g04730D gene or biomaterials containing the BnaA02g04730D gene in improving the salt tolerance of rapeseed.

2. The application according to claim 1, characterized in that: The nucleotide sequence of the BnaA02g04730D gene is shown in SEQ ID No.

1.

3. The application according to claim 2, characterized in that: The biological material is an expression cassette, expression vector, or recombinant bacteria containing the BnaA02g04730D gene.

4. The application according to claim 3, characterized in that: The improvement is achieved by increasing the expression level of the BnaA02g04730D gene in rapeseed, thereby enhancing the salt tolerance of rapeseed.

5. The application according to claim 4, characterized in that: The rapeseed in question is Brassica napus.

6. A method for improving the salt tolerance of rapeseed and prolonging its survival time, characterized in that, The steps are as follows: overexpress the BnaA02g04730D gene in rapeseed plants to improve their salt tolerance and prolong their survival time.

7. The method according to claim 6, characterized in that: The nucleotide sequence of the BnaA02g04730D gene is shown in SEQ ID No. 1, and the rapeseed is Brassica napus.

8. A method for cultivating salt-tolerant transgenic rapeseed, characterized in that, The steps are as follows: construct an overexpression vector containing the BnaA02g04730D gene, transform it into rapeseed plants to be improved using Agrobacterium-mediated transformation, and obtain salt-tolerant transgenic rapeseed plants through callus induction, differentiation culture and rooting screening.

9. The method according to claim 8, characterized in that: The nucleotide sequence of the BnaA02g04730D gene is shown in SEQ ID No.

1.

10. The method according to claim 9, characterized in that: The rapeseed in question is Brassica napus.

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