Application of BcNCED3 gene in regulating heat tolerance of pakchoi

Abstract

The application belongs to the field of molecular biology and relates to BcNCED3 Application of a gene in regulating heat tolerance of Brassica chinensis L. BcNCED3 The application takes the gene as a research object and explores the biological function of the gene in high-temperature stress response, so as to provide a theoretical basis and gene resources for heat tolerance breeding of the Brassica chinensis L. BcNCED3 Overexpression of the gene can enhance the heat tolerance of the Brassica chinensis L., and knockout of the gene can seriously weaken the heat tolerance.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology and relates to the application of the BcNCED3 gene in regulating the heat tolerance of non-heading Chinese cabbage. Background Technology

[0002] Non-heading Chinese cabbage has a long history of cultivation in my country, especially in the southern regions. Due to its significant economic value, it has become an important vegetable crop globally. Non-heading Chinese cabbage prefers cool climates and thrives best in environments between 18 and 25°C. Sustained high temperatures inhibit its growth, resulting in stunted plants, yellowing leaves, reduced nutritional value, lower survival rates, and increased susceptibility to viral diseases, severely impacting yield and quality. In production practice, non-heading Chinese cabbage commonly suffers from insufficient heat tolerance, severely restricting its summer production capacity. Therefore, analyzing the response mechanism of non-heading Chinese cabbage to high-temperature stress and breeding heat-resistant new varieties has become an urgent need to ensure its summer production and year-round supply. Summary of the Invention

[0003] This invention provides the application of the BcNCED3 gene in regulating the heat tolerance of non-heading Chinese cabbage. This invention uses the BcNCED3 gene as the research object to explore its biological function in response to high-temperature stress, aiming to provide a theoretical basis and gene resources for heat-tolerant breeding of non-heading Chinese cabbage.

[0004] To achieve the above objectives, the present invention adopts the following technical solution:

[0005] The application of overexpression of the BcNCED3 gene in improving the heat resistance of non-heading Chinese cabbage, wherein improving the heat resistance of non-heading Chinese cabbage includes reducing the degree of chlorosis and wilting of leaves under high temperature stress treatment and reducing the MDA content under high temperature stress treatment, and the CDS sequence of the BcNCED3 gene is shown in SEQ ID NO.1.

[0006] Furthermore, improving the heat resistance of non-heading Chinese cabbage includes increasing the endogenous ABA content under high-temperature stress treatment.

[0007] The present invention also provides a method for improving the heat resistance of non-heading Chinese cabbage, the method comprising overexpressing the BcNCED3 gene in the target non-heading Chinese cabbage to obtain transgenic non-heading Chinese cabbage with higher heat resistance than the target plant, wherein the CDS sequence of the BcNCED3 gene is shown in SEQ ID NO.1.

[0008] Furthermore, RT-qPCR was used to detect transgenic non-heading Chinese cabbage.

[0009] Furthermore, the method for overexpressing the BcNCED3 gene in non-heading Chinese cabbage includes constructing an overexpression vector, wherein the primer sequences used to construct the overexpression vector are shown in SEQ ID NO.4 and SEQ ID NO.5.

[0010] Furthermore, the vector plasmid with the correct sequence alignment was transformed into Agrobacterium rhizogenes K599 competent cells, and positive clones were identified for subsequent infection experiments.

[0011] Beneficial effects

[0012] Through Agrobacterium-mediated genetic transformation, we obtained BcNCED3 overexpressing plants and LbCas12a-mediated gene-edited plants. After high-temperature treatment, the phenotype, heat-response gene expression, and physiological index changes of the overexpressing plants were consistent with those of seedlings treated with exogenous ABA, while the BcNCED3-edited plants exhibited a sensitive phenotype, indicating that BcNCED3 has a positive regulatory effect on the heat tolerance of non-heading Chinese cabbage. Overexpression of BcNCED3 can enhance the heat tolerance of non-heading Chinese cabbage, while its knockout severely weakens heat tolerance. This invention demonstrates that BcNCED3 synergistically enhances the heat tolerance of non-heading Chinese cabbage through a triple mechanism of increasing endogenous ABA, activating the heat shock pathway, and enhancing antioxidant scavenging of reactive oxygen species, fully revealing the molecular, hormonal, and physiological regulatory network of leafy vegetables under high-temperature stress. The same NCED3 gene may have different functions and regulatory amplitudes on ABA levels in different species (and even different varieties of the same species). The existing NCED family gene effects in current technology cannot be 100% extrapolated to non-heading Chinese cabbage from the conclusions drawn from model plants or rice. Attached Figure Description

[0013] Figure 1 Phenotypic changes and BcNCED3 expression levels in non-heading Chinese cabbage varieties 'Suzhou Green' (left) and 'Dwarf Yellow' (right) under high-temperature treatment; Figure 1 In the middle, A represents a phenotypic comparison. Figure 1 B represents the change in the expression level of NHCC001; Figure 1 C represents the change in the expression level of NHCC002;

[0014] Figure 2 To investigate the phenotype and ABA content of non-heading Chinese cabbage under high temperature after exogenous ABA pretreatment; Figure 2 Type A represents the non-heading cabbage phenotype; Figure 2 In the middle section, B represents the expression level of BcNCED3 in the control group and the ABA-treated group; Figure 2 C represents the endogenous ABA content in the control group and the ABA-treated group; all data are expressed as mean ± SD for three replicates (Student's t-test, **p < 0.01, ***p < 0.001).

[0015] Figure 3 The expression levels of heat-related genes in non-heading Chinese cabbage under high temperature after exogenous ABA pretreatment; among them, Figure 3 A in the middle is BcHSP101, Figure 3 In the middle, B is BcHSFA1. Figure 3 C in the middle is BcHSFA2, Figure 3 D is BcDREB2A, Figure 3 E in the middle is BcGolS1 and Figure 3 F in the middle is BcMBF1c;

[0016] Figure 4 Identification of BcNCED3 overexpressing plants; Figure 4 Image A shows a GFP fluorescence image of regenerated seedling roots observed with LUYOR3415. Figure 4 In the figure, B represents the relative expression level of BcNCED3 in WT and BcNCED3 overexpressing plants; Figure 4 C represents the PCR detection of the BcNCED3 gene in overexpressing plants;

[0017] Figure 5 Phenotypes and ABA content of WT wild-type and OE overexpressing plants under high-temperature treatment; Figure 5 In Figure A, the phenotypes of WT and BcNCED3 overexpressing plants (OE-1, OE-2, OE-3) after treatment at room temperature (25℃) and high temperature (38℃) are shown. Figure 5 In the figure, B represents the endogenous ABA content of WT and BcNCED3 overexpressing plants (OE-1, OE-2, OE-3) before and after high-temperature treatment;

[0018] Figure 6 The expression levels of heat-related genes in WT wild-type and OE overexpression plants under high-temperature treatment; among them, Figure 6 A in the middle is BcHSP101, Figure 6 In the middle, B is BcHSFA1. Figure 6 C in the middle is BcHSFA2, Figure 6 D is BcDREB2A, Figure 6 E in the middle is BcGolS1 and Figure 6 F in the middle is BcMBF1c;

[0019] Figure 7 Physiological parameters of WT wild-type and OE-overexpressing plants under high-temperature treatment were determined.

[0020] Figure 8 PCR detection of the Cas12a fusion gene;

[0021] Figure 9 This is a CRISPR / Cas12a-induced BcNCED3 open reading frame mutation type; Figure 9Image A shows the third-generation sequencing peak diagrams and alignment results for WT and KO plants. Figure 9 In the middle B, the mutation type at target site 1 is found in KO plants; Figure 9 C represents the mutation type at target site 2 in KO plants;

[0022] Figure 10 Phenotypic and ABA content of WT wild-type and KO edited plants before and after high-temperature treatment; Figure 10 In section A, the phenotypes of plants (KO-1, KO-2, KO-3) before and after high-temperature treatment were edited for WT and BcNCED3. Figure 10 The content of endogenous ABA in plants (KO-1, KO-2, KO-3) before and after high-temperature treatment was edited by B for WT and BcNCED3.

[0023] Figure 11 The expression levels of heat-related genes in WT wild-type and KO edited plants under high-temperature treatment;

[0024] Figure 12 Physiological parameters of WT wild-type and KO edited plants under high-temperature treatment were determined. Detailed Implementation

[0025] 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.

[0026] Example 1

[0027] BcNCED3 (BraC01g041920.1) is 2299 bp in length and has a CDS length of 1797 bp. It contains only one exon and no introns, encoding 598 amino acids. Physicochemical analysis shows that the BcNCED3 protein has a relative molecular mass of 65.76 kDa, a theoretical isoelectric point of 5.57, and an instability coefficient of 41.45. It is generally hydrophilic and lacks transmembrane structures, suggesting it is an acidic, unstable, soluble protein. Secondary structure prediction results indicate that the BcNCED3 protein is mainly composed of random coils (66.39%), extended chains (20.40%), and α-helices (13.21%). Tertiary structure prediction, using the Arabidopsis thaliana NCED3 protein as a template (91.47% similarity), showed a structure largely consistent with the secondary structure analysis results. Domain analysis showed that the BcNCED3 protein contains a typical RPE65 conserved domain in the 127-529 aa range. Subcellular localization predicted that the protein is mainly located in chloroplasts, indicating that it belongs to the NCED protein family and may have oxidoreductase activity and participate in the biosynthesis of ABA in chloroplasts.

[0028] Analysis of the BcNCED3 gene structure revealed that its coding region consists of a single exon. The absence of introns may mean that RNA splicing is not required during gene expression, thus enabling rapid transcription and translation while maintaining high precision in post-transcriptional processing, allowing for efficient ABA synthesis to cope with environmental changes. The BcNCED3 protein contains the highly conserved RPE65 homology domain of the NCED family. This domain possesses dioxygenase activity, catalyzing the cleavage of neoxanthin and participating in ABA biosynthesis. Phylogenetic analysis showed that BcNCED3 shares high amino acid homology with other NCED family members in Brassicaceae species, indicating a high degree of conservation within this family during evolution. Subcellular localization prediction and subsequent experimental validation results showed that BcNCED3 is located in chloroplasts, further confirming its functional location in the ABA synthesis pathway.

[0029] The CDS sequence of the BcNCED3 gene is shown in SEQ ID NO.1:

[0030]

[0031] The DNA sequence of the BcNCED3 gene is shown in SEQ ID NO.2:

[0032]

[0033] The amino acid sequence of the BcNCED3 protein is shown in SEQ ID NO.3:

[0034] *

[0035] Example 2

[0036] To investigate the response of BcNCED3 to heat stress in Chinese cabbage, RT-qPCR was used to detect the expression levels of BcNCED3 in four-week-old seedlings of the heat-resistant variety 'Suzhou Green' (NHCC001) and the heat-sensitive variety 'Aijiao Huang' (NHCCOO2) at 40℃ for 0 h, 3 h, 6 h, and 12 h. Phenotypic changes are shown below. Figure 1 As shown in Figure A, the heat-sensitive varieties exhibit more severe leaf wilting compared to the heat-resistant varieties. Quantitative analysis results are as follows... Figure 1 China B and Figure 1As shown in Figure C, the expression level of gene BcNCED3 was significantly higher in heat-resistant materials than in heat-sensitive materials. However, regardless of heat resistance, the expression level of this gene was significantly increased under heat stress, especially during the period of 3-6 h, reaching a peak and then decreasing at 9 h. These results indicate that BcNCED3 responds to heat stress, its expression level is strongly induced, and it is positively correlated with heat resistance in non-heading Chinese cabbage.

[0037] qPCR was used to detect the expression dynamics of BcNCED3 in heat-resistant and heat-sensitive materials under 40℃ high-temperature stress. The results showed that BcNCED3 responded rapidly to high-temperature induction, and its expression level differed significantly between the two materials. The change in BcNCED3 expression was positively correlated with the heat resistance of the materials. This result suggests that BcNCED3 may play a positive regulatory role in the resistance of non-heading Chinese cabbage to high-temperature stress by regulating ABA synthesis.

[0038] Example 3

[0039] To verify the effect of ABA on the heat tolerance of non-heading Chinese cabbage, 'Dwarf Yellow' seedlings pretreated with 10 μM exogenous ABA and a water control group were treated for 3 days under high temperature conditions (38℃ / 25℃, 16h light / 8h darkness). Phenotypic analysis showed that... Figure 2 In the ABA-treated group (CK), under heat stress, the lower two true leaves of the water-treated group showed yellowing and necrosis, while the upper true leaves suffered more severe water loss and wilting. In contrast, the plants in the ABA-treated group (CK) showed less damage and demonstrated stronger tolerance to heat stress. Samples were collected before and after heat stress to measure the endogenous ABA content and BcNCED3 gene expression levels in the control and treated groups. Figure 2 In studies involving B and C, exogenous ABA spraying induced upregulation of the NCED3 gene expression and increased endogenous ABA levels. Similarly, after high-temperature treatment, the expression level of the BcNCED3 gene and the endogenous ABA content in the treatment group were significantly higher than those in the control group, indicating that exogenous ABA spraying can promote the accumulation of endogenous ABA and alleviate the damage of high temperature to seedlings.

[0040] Example 4

[0041] From the perspective of relative gene expression levels, the expression levels of heat-related genes BcHSP101, HcHSFA1, BcHSFA2, BcDREB2A, BcGolS1, and BcMBF1c in seedlings pretreated with exogenous ABA under high temperature stress were significantly increased compared to the water control group. Figure 3 This result indicates that BcNCED3 can enhance the heat resistance of NHCC002 by regulating the expression of the HSR gene.

[0042] Example 5

[0043] Identification of BcNCED3 overexpressing plants

[0044] Based on the NruI restriction site of the pER8 vector, the BcNCED3 stop codon was removed. Specific homologous primers pER8-BcNCED3-F / R and universal primers pER8-F / R for vector detection were designed. Amplification was performed using the CDS fragment as a template. Agarose gel electrophoresis was used to recover the correct gene bands. Homologous recombination was performed using the OK Clon DNA Ligation Kit III Ver.2 (Aikerui, China), inserting the open ORF of BcNCED3 into the PER8-WPR-superGFP overexpression vector. This vector was transformed into *E. coli* DH5α and the bacterial culture was identified. After sequencing alignment, the plasmid was extracted. Subsequently, the correctly aligned vector plasmid was transformed into *Agrobacterium rhizogenes* K599 competent cells (TOLOBIO, China), and positive clones were identified. The plasmid was stored in 50% glycerol (glycerol:water = 1:1) at -80°C for subsequent infection experiments.

[0045] The 35S:LbCas12a-RRV (Addgene ID: 223423) expression cassette was cloned into the WUS2-IPT-PLT5-Ruby backbone to construct the LbCas12a-RRV vector. The ORF sequences of BcPDS and BcNCED3 were submitted to the CRISPR-P v2.0 design tool (http: / / crispr.hzau.edu.cn / CRISPR2 / ) at Huazhong Agricultural University to design crRNAs capable of editing the BcPDS and BcNCED3 ORF sequences. These crRNAs were then sent to GenScript Biotech Co., Ltd. to complete the construction of the editing vector. After the company returned the vector plasmid, it was transformed into *Agrobacterium rhizogenes* K599 using a freeze-thaw method. After bacterial testing, it was stored at -80℃ for later use.

[0046] Agrobacterium-mediated genetic transformation

[0047] (1) Seed disinfection: Take an appropriate amount of 'Dwarf Yellow' seeds and disinfect them in a clean bench after UV sterilization for 30 min. Prepare a 75% alcohol solution with anhydrous ethanol and sterile water at a ratio of 3:1 for 2 min, and rinse with sterile water 2-3 times. Then disinfect with 20% sodium hypochlorite solution for 10-12 min, during which time the seeds can be placed on a vortex mixer to ensure full contact with the sodium hypochlorite solution. Finally, rinse with sterile water 3-5 times until the seeds are completely clean. After flaming and cooling with tweezers, sow the seeds evenly in 1 / 2 MS medium (pH 5.8) and culture in a tissue culture room (24℃, 16 h light / 8 h dark) for 5-6 days until the seeds germinate and grow to a suitable size.

[0048] (2) Preparation of Agrobacterium infection solution: K599 bacterial culture stored at -80℃ was activated by streaking on LB solid medium and then inoculated into LB liquid medium containing the corresponding antibiotic. It was cultured overnight at 28℃ on a shaker for 12-16 h. After centrifugation at 6000 rpm for 5 min at room temperature, the supernatant was discarded, and the bottom bacterial block was resuspended in MS liquid medium (pH 5.2) and the OD was adjusted to 0.6-0.8. Then, 150 μmol·L⁻¹ was added. -1 Acetyleugenol can improve the conversion efficiency of K599. The prepared inoculation solution should be left to stand at room temperature in the dark for 3-4 hours before use.

[0049] (3) Explant infection and co-culture: In a clean bench, the seedlings were cut below the growing point using a tissue culture scalpel, and the part with two cotyledons and a 1 cm hypocotyl was used as the explant for infection. The explants were soaked in the prepared Agrobacterium infection solution for 10 min and then placed on sterile filter paper to allow the surface liquid to be absorbed. They were then inserted into the co-culture plate with tweezers and placed in the dark at 24°C for 2 days.

[0050] (4) Differentiation culture: Wash the explants 3-4 times with sterile water containing antibiotics to remove residual Agrobacterium on the surface, then transfer them to differentiation culture medium and culture them in the tissue culture room for 20-25 days.

[0051] (5) Plant tissue regeneration and subculture: After roots, callus and adventitious buds differentiate from the hypocotyl cut of the explant, the callus is transferred to a new MS medium to differentiate into buds, and the buds with growth points are also cultured until they grow into seedlings.

[0052] (6) Rooting culture and hardening: Transgenic seedlings are placed in rooting medium to induce rooting. Adventitious roots are observed with a LUYOR-3415RG handheld fluorescent lamp. Seedlings with green fluorescent roots are selected and hardened after the roots are strong and transplanted into the substrate for further culture.

[0053] The culture medium formula used is as follows:

[0054] Co-culture: MS (4.74 g / L) + sucrose (30 g / L) + agar (7.5 g / L) + estradiol (50 mg / L), pH 5.2.

[0055] Differentiation / subculture: MS (4.74 g / L) + sucrose (30 g / L) + agar (7.5 g / L) + estradiol (50 mg / L) + Carb (250 mg / L) + TMT (250 mg / L), pH 5.8.

[0056] Rooting culture: MS (4.74 g / L) + sucrose (30 g / L) + agar (7.5 g / L) + NAA (0.2 mg / L) + Carb (250 mg / L) + TMT (250 mg / L), pH 5.8.

[0057] After seedlings selected by GFP green fluorescent visual labeling were hardened and transplanted, newly grown leaves were taken, flash-frozen in liquid nitrogen, and ground into powder in a pre-cooled grinder. Genomic DNA was extracted, and positive transgenic plants were identified by PCR using the specific gene clone BcNCED3-F and the vector detection primer pER8-R as upstream and downstream primers.

[0058] Two to three weeks after red callus formation from the hypocotyls of Chinese cabbage explants, approximately 30 callus tissues were collected, mixed, and ground. DNA was extracted using a kit, and specific primers were designed for PCR amplification. The amplified products were then sent for high-throughput sequencing. Genomic DNA was extracted from the leaves of BcNCED3-edited regenerated plants. PCR amplification was performed using Cas12a vector-specific primers and gene CDS-specific primers, followed by gel electrophoresis. The target band fragment was excised and sent for third-generation sequencing to identify positive transgenic plants and detect the editing results.

[0059] 'Dwarf Yellow' regenerated plants were obtained through Agrobacterium infection and plant tissue regeneration culture. Irradiation of the plant roots with a handheld fluorescent lamp (LUYOR-3415RG) revealed obvious GFP-green fluorescence in the transgenic plants under a yellow filter. Genomic DNA was extracted from the regenerated plants and amplified by PCR using vector-specific primers pER8-BcNCED3-F and pER8-R, followed by electrophoresis. Wild-type was used as a control. The results showed that a specific band of approximately 2000 bp was amplified in three plants. Figure 4 The expression levels of BcNCED3 in three plants (OE-1, OE-2, and OE-3) and WT were detected by RT-qPCR. The results showed that the expression levels of BcNCED3 gene in the overexpressing plants were 30-fold, 12-fold, and 13-fold higher than those in the wild type, respectively, all of which were significantly higher than those in WT.

[0060] Table 1 Primer Sequences

[0061]

[0062] Table 2 CRISPR / Cas12a gene editing target site sequences

[0063]

[0064] Example 6

[0065] Phenotypic and ABA content of WT and overexpressing plants before and after high-temperature treatment

[0066] After stressing WT wild-type and OE overexpressing plants at 38℃ for 3 days, it was observed that the leaves of WT plants showed obvious chlorosis and wilting, while the overexpressing plants showed relatively mild stress, except for the yellowing of the lower older leaves and yellowing and wilting of the new leaves. Figure 5 The study measured the endogenous ABA content of each plant before and after high-temperature treatment and found that high temperature induced an increase in endogenous ABA content. At the same time, the endogenous ABA content of overexpressing plants was significantly higher than that of WT under both normal temperature and heat treatment. This indicates that overexpression of the BcNCED3 gene can improve the endogenous ABA content of plants in response to high temperature, thus adapting to adversity, alleviating the damage caused by high temperature, and enhancing their own stress resistance.

[0067] Example 7

[0068] BcNCED3 overexpression enhances thermostable gene response and antioxidant capacity.

[0069] To elucidate the potential molecular mechanisms underlying the heat tolerance of BcNCED3, RT-qPCR was used to detect the transcriptional levels of six heat-related genes in transgenic and wild-type plants under control and heat stress conditions. For example... Figure 6 As shown, under high-temperature stress, the expression levels of these genes in the overexpression lines were significantly higher than those in the WT line. These results indicate that BcNCED3 activates heat-response genes under heat stress, thereby enhancing its resistance to high-temperature stress.

[0070] Example 8

[0071] BcNCED3 overexpression enhances antioxidant capacity

[0072] To reveal the potential physiological mechanism by which BcNCED3 enhances the heat tolerance of non-heading Chinese cabbage, the MDA content, SOD activity, POD activity, and CAT activity of BcNCED3 transgenic and wild-type seedlings under heat stress were measured. Malondialdehyde (MDA) can reflect the rate and intensity of plant membrane lipid peroxidation and can also indirectly reflect the potential antioxidant capacity of plant cells. Under control conditions, there was no significant difference in MDA content between transgenic and wild-type seedlings, but the increase in MDA content in overexpression lines was significantly lower than that in WT. The activities of antioxidant enzymes in transgenic and wild-type seedlings under heat stress were measured. Figure 7 As shown, after 3 days of heat stress, the activities of all three enzymes in both WT and transgenic lines were higher than before the high-temperature treatment, and the increase in antioxidant enzyme activity in transgenic plants was significantly greater than that in WT. This suggests that the BcNCED3 overexpression lines may maintain cell structure and function under heat stress by regulating the activity of their own enzyme systems to scavenge free radicals and other toxic substances.

[0073] Example 9

[0074] Knocking out the BcNCED3 gene reduces the heat resistance of non-heading Chinese cabbage.

[0075] Genomic DNA was extracted from vigorous regenerated plants after hardening. Using the DNA as a template and the sequence-specific Cas12a-F and Cas12a-R primers from the Cas12a vector as upstream and downstream primers, PCR amplification and 1.2% agarose gel electrophoresis were performed. Wild-type (WT) and positive plasmid (P) were used as controls. The results showed that all 5 seedlings amplified specific bands of about 800 bp, and were preliminarily identified as transgenic positive plants. Figure 8 ).

[0076] Based on the locations of the two target sequences, upstream of CrRNA1 and downstream of CrRNA2 respectively, a pair of specific detection primers was designed using the NCBI website. PCR amplification and gel electrophoresis were performed using DNA from transgenic positive plants as templates. The target gene band was then excised and sent to Qingke Biotechnology Co., Ltd. for third-generation sequencing analysis. Based on the peak diagram results (… Figure 9 According to the mutation statistics table (A) and the mutation types of the gene, three mutation types were found in the five positive transgenic plants, all of which were small deletion mutations with different numbers of bases at the two designed target sites. Figure 9 China B and Figure 9 (C)

[0077] After subjecting WT wild-type and OE overexpression plants to stress at 38℃ for 3 days, we observed significant wilting and necrosis in three gene-edited positive plants. Furthermore, we found that under the 3-day high-temperature environment, the growth rate of the edited plants was significantly faster than that of the WT plants. Figure 10 (A). Measurements of endogenous ABA content in plants before and after high-temperature treatment revealed that high temperature induced an increase in endogenous ABA content. However, the ABA content in the edited plants was significantly lower than that at WT both before and after treatment, indicating that the loss of function of the BcNCED3 gene inhibited the ABA synthesis rate under abiotic stress. The plants exhibited low sensitivity to high-temperature stress and were unable to respond promptly to environmental changes, failing to transition from vegetative growth to a state of resistance to abiotic stress. Figure 10 (B)

[0078] BcNCED3 editing reduced the expression of heat-related genes in non-heading Chinese cabbage under high temperatures:

[0079] RT-qPCR was used to detect the transcriptional levels of six heat-related genes in edited and wild-type plants under control and heat stress conditions, such as... Figure 11 As shown, the expression levels of all six genes in the edited plants under high-temperature treatment were significantly lower than those under whole temperature (WT). This indicates that after the loss of function of BcNCED3, heat-related genes in the plant cannot respond to high temperatures in a timely manner, thus weakening its heat tolerance.

[0080] BcNCED3 reduces the antioxidant capacity of non-heading Chinese cabbage at high temperatures:

[0081] like Figure 12 As shown, from a physiological perspective, under normal conditions, there was no significant difference in antioxidant enzyme activity and MDA content between gene knockout plants and WT plants. Enzyme activity increased after high-temperature treatment, but the increase in knockout plants was significantly lower than that in WT plants. Conversely, MDA content increased more than that in WT plants, reflecting their lower antioxidant capacity and more severe damage to the cell membrane system.

[0082] High temperature stress often leads to damage to plant membrane systems and metabolic disorders. To cope with this adversity, plants have evolved a series of protective mechanisms, including reactive oxygen species scavenging and heat shock protein accumulation. Hormonal signaling plays a key regulatory role in these mechanisms, especially ABA, which is a recognized stress hormone whose biosynthesis is tightly controlled by the NCED gene family.

[0083] Studies have shown that the application of exogenous plant hormones can effectively reduce heat stress-induced damage in plants and enhance their heat resistance. For example, spraying spinach leaves with low concentrations of exogenous ABA can alleviate the damage to photosynthetic organs caused by high-temperature stress and maintain leaf thermal stability. In rice, root irrigation pretreatment with 10 µM ABA can significantly reduce seedling leaf wilting rate and chlorophyll loss, and upregulate the expression of antioxidant enzyme genes and heat shock-related genes. This invention found that exogenous ABA treatment significantly upregulated the expression of the BcNCED3 gene in non-heading Chinese cabbage under high temperature, increased endogenous ABA content, and induced the expression of heat shock response genes (such as BcHSP101, BcHSFA1, and BcHSFA2), enhanced antioxidant enzyme activity, and reduced MDA accumulation. This indicates that exogenous ABA, by promoting the accumulation of endogenous ABA, synergistically activates the heat response transcription network and antioxidant defense system, thus mitigating membrane lipid peroxidation damage caused by high-temperature stress.

[0084] It is noteworthy that when transgenic positive plants were subjected to high-temperature treatment, the non-heading Chinese cabbage plants overexpressing BcNCED3 showed the same trend of change in phenotype, gene expression and physiological indicators as those treated with exogenous ABA, while the BcNCED3 knockout lines showed the opposite phenotype. This "positive and negative verification" proved that the BcNCED3 gene plays an important role in ABA-mediated heat tolerance.

[0085] The combination of stable genetic transformation systems and gene editing technology provides an efficient and precise verification method for plant gene function research. This invention utilizes Agrobacterium rhizogenes-mediated stable genetic transformation to construct BcNCED3 overexpressing plants in non-heading Chinese cabbage, and obtains BcNCED3 knockout mutants using the CRISPR / Cas12a system. Phenotypic comparison between overexpression and knockout materials not only clarifies the positive regulatory function of BcNCED3 in the heat tolerance of non-heading Chinese cabbage, but also avoids background interference that may arise from transient or heterologous expression systems. Furthermore, this invention verifies that the Cas12a system can achieve efficient and stable gene editing in non-heading Chinese cabbage, providing a feasible technical approach for further in-depth research on gene function in non-heading Chinese cabbage.

Claims

1. The application of overexpression of the BcNCED3 gene in improving the heat resistance of non-heading Chinese cabbage, characterized in that, The improvement of heat resistance in non-heading Chinese cabbage includes reducing the degree of chlorosis and wilting of leaves under high temperature stress treatment and reducing the MDA content under high temperature stress treatment. The CDS sequence of the BcNCED3 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, Improving the heat resistance of non-heading Chinese cabbage includes increasing the content of endogenous ABA under high-temperature stress treatment.

3. A method for improving the heat resistance of non-heading Chinese cabbage, characterized in that, The method involves overexpressing the BcNCED3 gene in the target non-heading Chinese cabbage to obtain transgenic non-heading Chinese cabbage with higher heat resistance than the target plant. The CDS sequence of the BcNCED3 gene is shown in SEQ ID NO.

1.

4. The method for improving the heat resistance of non-heading Chinese cabbage according to claim 3, characterized in that, RT-qPCR was used to detect transgenic non-heading Chinese cabbage.

5. The method for improving the heat resistance of non-heading Chinese cabbage according to claim 3, characterized in that, The method for overexpressing the BcNCED3 gene in non-heading Chinese cabbage includes constructing an overexpression vector. The primer sequences used to construct the overexpression vector are shown in SEQ ID NO.4 and SEQ ID NO.

5.

6. The method for improving the heat resistance of non-heading Chinese cabbage according to claim 5, characterized in that, The vector plasmid with the correct sequence alignment was transformed into Agrobacterium rhizogenes K599 competent cells, and positive clones were identified for subsequent infection experiments.

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