Brassica plant drought resistance negative regulation factor BnaA9.TZF5 and application thereof

Abstract

The invention discloses a brassica plant drought resistance negative regulation factor BnaA9.TZF5 and application of the brassica plant drought resistance negative regulation factor BnaA9.TZF5. The BnaA9.TZF5 is a transcription factor gene for coding CCCH type zinc finger protein in brassica napus, and a knockout plant obtained by performing targeted editing on the BnaA9.TZF5 through CRISPR / Cas9 shows that the drought resistance is remarkably enhanced along with phenotypic changes such as increase of leaf epidermis wax deposition and reduction of water loss rate; and an overexpression plant of the gene shows that the wax is reduced, the water loss is accelerated and the drought resistance is weakened, so that the BnaA9.TZF5 is a negative regulation factor for the drought resistance of the brassica plant. The BnaA9.TZF5 gene expression is knocked out to improve the drought resistance of brassica plants, the BnaA9.TZF5 gene can be used for molecular breeding of crops such as brassica napus, Chinese cabbage, cabbage and mustard, and a novel drought-resistant improved target gene is provided.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology and crop genetics and breeding, specifically relating to the negative regulator of drought resistance in Brassica plants, BnaA9.TZF5, and its uses. Background Technology

[0002] Drought is a limiting factor for rapeseed (Brassica napus) Brassica napus Drought is a major abiotic stress factor affecting the yield and quality stability of rapeseed and other Brassica species. Under drought conditions, rapeseed often exhibits premature leaf senescence, decreased photosynthetic efficiency, inhibited silique development, and reduced grain plumpness, severely impacting stable and high yields. Currently, drought-resistant breeding of Brassica crops mainly relies on field phenotypic identification and conventional hybridization breeding methods, which suffer from problems such as long identification cycles, low screening efficiency, and a lack of molecular markers. Therefore, identifying and utilizing key drought-resistant regulatory genes is a crucial technical approach for achieving drought-resistant molecular breeding in Brassica crops.

[0003] The Tandem CCCH-type zinc finger protein (TZF) family is a class of transcription factors containing tandem CCCH zinc finger domains. They are primarily involved in RNA metabolism and gene expression regulation, playing a crucial role in plant growth, development, and stress responses. Previous studies have shown that Arabidopsis TZF5 participates in light, abscisic acid, and gibberellin-mediated seed germination; potato TZF5 promotes the accumulation of reducing sugars at low temperatures. However, there are no reports on whether TZF5 participates in drought resistance regulation. Overexpression of OsTZF5 in rice enhances the plant's drought resistance, but rice is a monocotyledonous crop, and its OsTZF5 shares only about 13.5% amino acid sequence homology with the Brassica napus gene BnaA9.TZF5, and they belong to different TZF subfamilies (BnaA9.TZF5 and Arabidopsis TZF5 both belong to the C3H61 subfamily, while OsTZF5 belongs to the C3H33 subfamily). Therefore, OsTZF5 and BnaA9.TZF5 are not direct homologous genes, and there are significant differences between them in phylogeny and functional regulation. This indicates that the biological function of BnaA9.TZF5 in Brassica plants has not been studied, and provides a new research direction for exploring the role of TZF family members in drought resistance regulation in cruciferous crops.

[0004] This invention, through a systematic analysis of the TZF family members in the whole genome of Brassica napus, identifies BnaA9.TZF5 for the first time as a negative regulator associated with drought resistance. Functional validation results show that knocking out BnaA9.TZF5 using CRISPR / Cas9 technology significantly enhances the plant's drought resistance, while overexpression of BnaA9.TZF5 leads to a decrease in drought resistance. Further research revealed that this gene can affect the accumulation of epidermal wax in leaves, thereby regulating the plant's water loss rate, providing molecular evidence for its mechanism of action in drought resistance development.

[0005] In summary, the discovery and functional analysis of BnaA9.TZF5 reveals for the first time the role of TZF family members in regulating drought resistance in Brassica crops, providing a new molecular target for molecular breeding of drought resistance in rapeseed and its closely related crops. This invention establishes a complete technical system from gene discovery and functional verification to molecular breeding applications, providing a directly usable technical foundation for the efficient improvement of drought resistance in rapeseed and its closely related crops, and has significant scientific value and application prospects. Summary of the Invention

[0006] Drought is a significant abiotic stress limiting the stable yield and quality of Brassica crops. Current drought-resistant breeding mainly relies on field phenotypic screening and conventional hybridization, which suffers from problems such as long cycles, low efficiency, and a lack of molecular markers, making it difficult to meet the needs of modern, efficient drought-resistant breeding. The purpose of this invention is to provide a novel molecular target gene, BnaA9.TZF5, and its homologous genes. By regulating the expression level of this gene, the drought resistance of Brassica plants can be improved, providing a directly usable technical means and application basis for drought-resistant molecular design breeding.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: The use of the BnaA9.TZF5 gene in improving the drought resistance of Brassica plants: By editing the BnaA9.TZF5 gene to induce a functional deletion mutation, the drought resistance of Brassica plants is enhanced. The nucleotide sequence of the BnaA9.TZF5 gene is shown in SEQ ID NO:1. Preferably, the Brassica plant is Brassica napus, and the BnaA9.TZF5 gene is knocked out using CRISPR-Cas9 editing technology, with the target sequence shown in SEQ ID NO:2 or SEQ ID NO:3.

[0008] A breeding method for obtaining drought-resistant Brassica plants: The BnaA9.TZF5 gene in Brassica plants is edited to induce a functional deletion mutation, thereby obtaining plants with enhanced drought resistance. The nucleotide sequence of the BnaA9.TZF5 gene is shown in SEQ ID NO:1. Preferably, the Brassica plant is Brassica napus, and the BnaA9.TZF5 gene is knocked out using CRISPR-Cas9 editing technology, with the target sequence shown in SEQ ID NO:2 or SEQ ID NO:3.

[0009] The use of a gene-editing vector for knocking out the BnaA9.TZF5 gene in improving drought resistance in Brassica plants, said gene-editing vector comprising a guide sequence for targeting the knockout of the nucleotide sequence shown in SEQ ID NO:1. Preferably, the targeting sequence is as shown in SEQ ID NO:2 or SEQ ID NO:3.

[0010] Compared with the prior art, the present invention has the following significant advantages and technical effects: 1. This invention is the first to discover and verify the negative regulatory role of the BnaA9.TZF5 gene in drought resistance of Brassica napus. By knocking out the BnaA9.TZF5 gene, plants under normal growth conditions exhibited increased leaf wax accumulation and decreased water loss rate, providing a physiological basis for enhanced drought resistance under drought stress. Under drought stress, the knockout plants showed significantly increased leaf water content and survival rate, demonstrating enhanced drought resistance. Simultaneously, the knockout operation did not affect plant yield, showing good application value. Overexpression of the BnaA9.TZF5 gene, on the other hand, resulted in reduced wax accumulation, accelerated water loss, and decreased drought resistance.

[0011] 2. The BnaA9.TZF5 gene is an important molecular target for drought resistance, and regulating its expression can significantly enhance drought resistance. Experiments have confirmed the feasibility of this technique in Brassica napus and suggest its application in other Brassica species. Given the existence of conserved homologous genes in other Brassica species, those skilled in the art can reasonably expect that regulating the expression of these homologous genes may also improve drought resistance, providing potential applications for molecular design breeding of Brassica crops.

[0012] 3. This invention is the first to propose using molecular techniques such as gene editing to directionally regulate the expression levels of TZF family members—especially BnaA9.TZF5—to enhance crop drought resistance. This strategy provides a novel regulatory pathway for CCCH-type TZF transcription factors, enriching the theory of molecular regulation of drought resistance and offering new ideas and technical pathways for molecular design breeding of drought resistance in Brassica crops. Attached Figure Description

[0013] Figure 1 This diagram illustrates the targeted editing and mutation types of the BnaA9.TZF5 gene in knockout lines. The diagram shows different mutations generated by the CRISPR / Cas9 system targeting the exon region of BnaA9.TZF5.

[0014] Figure 2 A plot showing the mRNA levels in the BnaA9.TZF5 knockout line. The plot demonstrates a significant decrease in the transcriptional level of the BnaA9.TZF5 gene in the knockout line compared to the wild type.

[0015] Figure 3Comparative figures show the growth status of BnaA9.TZF5 knockout lines, overexpression lines, and wild-type materials at different stages of drought stress treatment. The figures sequentially show plant morphology before drought treatment, after 8 days of water cessation, and after 13 days of water cessation followed by 2 days of water resumption. After 8 days of water cessation, wild-type plants showed significant wilting, while the wilting of overexpression lines was further aggravated; in contrast, the BnaA9.TZF5 knockout lines maintained normal growth. After 13 days of water cessation followed by 2 days of water resumption, the survival rate of knockout lines was significantly higher than that of wild-type, while most overexpression lines failed to recover growth.

[0016] Figure 4 Scanning electron microscopy (SEM) images of the epidermal wax structure of leaves from the BnaA9.TZF5 knockout, overexpression, and wild-type materials. Compared to the wild type, the knockout lines showed denser epidermal wax crystal deposition, while the overexpression lines showed sparser epidermal wax crystal distribution. Scale bar in the image is 5 μm.

[0017] Figure 5 This figure shows the comparison of leaf wax content among BnaA9.TZF5 knockout lines, overexpression lines, and wild-type materials. Wax content was determined by gas chromatography. Compared with the wild type, the total wax content in the knockout lines was significantly increased, while the total wax content in the overexpression lines was significantly decreased. The data in the figure represent the average of four biological replicates, and the error bars represent the standard deviation. * , ** , *** The values ​​indicate significant differences at the P<0.05, P<0.01, and P<0.001 levels, respectively.

[0018] Figure 6 The figures show the leaf water loss rates of the BnaA9.TZF5 knockout, overexpression, and wild-type materials. The results show that the leaf water loss rate of the knockout line was significantly lower than that of the wild type, indicating enhanced cuticle barrier function; while the leaf water loss rate of the overexpression line was significantly higher than that of the wild type, indicating increased cuticle permeability and weakened barrier function.

[0019] Figure 7 The image shows GUS staining results of the BnaA9.TZF5 gene promoter in different tissues and organs of Arabidopsis thaliana. af represents seedlings, rosette leaves, cauline leaves, second internode of stem, leaf cross-section, and stem cross-section, respectively. GUS staining results show that BnaA9.TZF5 has significant expression activity in leaves and stems and is distributed in the epidermal cell layer. This tissue expression characteristic supports the regulatory role of BnaA9.TZF5 in epidermal-related physiological processes.

[0020] Figure 8This is a schematic diagram of the subcellular results of the BnaA9.TZF5 protein. After transient expression of the 35S::GFP-BnaA9.TZF5 fusion protein in tobacco leaves, the green fluorescence signal was mainly localized in the nucleus, overlapping with the red fluorescence signal of the nuclear localization marker mCherry; the fluorescence signal of the empty vector GFP could be detected in both the cytoplasm and the nucleus. These results indicate that the BnaA9.TZF5 protein is mainly localized in the nucleus, consistent with its regulatory function as a transcription factor. The scale bar in the figure is 20 μm. Detailed Implementation

[0021] The plant material used is Brassica napus 'Zhongshuang 11'. The technical solution of the present invention is further illustrated below through specific embodiments and accompanying drawings. Unless otherwise stated, the experimental methods used in the embodiments are conventional techniques in the art, and the relevant reagents and materials are commercially available. It should be understood that the following embodiments are only used to illustrate the technical concept and implementation of the present invention and are not intended to limit the scope of protection of the present invention.

[0022] The gene editing system, promoter type, expression vector backbone, screening marker, and Agrobacterium strain described in this specification are all specific examples used in the embodiments. Those skilled in the art can choose other technical means with the same or similar functions for equivalent substitution or modification according to actual needs, without departing from the technical concept and expected technical effect of this invention, and all such modifications should fall within the protection scope of this invention.

[0023] Example 1: Targeted editing of the BnaA9.TZF5 gene and obtaining mutants Based on the nucleotide sequence of the BnaA9.TZF5 gene (SEQ ID NO:1), two specific targeting sequences, sgRNA1 and sgRNA2 (as shown in SEQ ID NO:2 and SEQ ID NO:3, respectively), were designed in its exon region. The sgRNA sequences were then cloned into the Cas9 expression vector pP1C.4 to construct the BnaA9.TZF5 gene editing vector.

[0024] The gene editing vector was transformed into Escherichia coli DH5α competent cells using the conventional CaCl2 method. After PCR identification and confirmation, the recombinant plasmid was extracted. Subsequently, the recombinant plasmid was transformed into Agrobacterium GV3101 competent cells using the freeze-thaw method. Positive strains carrying hygromycin resistance markers were screened for subsequent plant genetic transformation.

[0025] Genetic transformation of Brassica napus was performed using Agrobacterium-mediated hypocotyl transformation. Specifically, after germination of seeds of the Brassica napus variety 'Zhongshuang 11', hypocotyls of 0.8-1.0 cm in length were harvested as explants and placed in Agrobacterium-mediated transformation solution containing acetylsyringone for approximately 10 min. Following co-culture, callus induction, shoot differentiation, and rooting induction, regenerated plants were obtained. The relevant procedures were performed according to the conventional rapeseed genetic transformation system.

[0026] Positive transformed plants were obtained through hygromycin resistance screening. Using their genomic DNA as templates, PCR amplification and sequencing analysis were performed using specific primers targeting the BnaA9.TZF5 gene to confirm the mutation type at the target site. Several independent gene-edited lines with different mutation types at the two target sites were identified. Representative lines were selected and named KO#8 and KO#16 (see...). Figure 1 ) is used for subsequent phenotypic identification and functional analysis, and its mRNA level is as follows Figure 2 As shown, the transcription level of the BnaA9.TZF5 gene was significantly reduced in the knockout strain compared to the wild type.

[0027] Example 2: Phenotypic and drought resistance analysis of BnaA9.TZF5 knockout lines (1) Drought stress treatment and phenotypic observation Drought stress experiments were conducted using BnaA9.TZF5 gene-edited lines (KO#8, KO#16) verified by sequencing and wild-type 'Zhongshuang 11' as controls. Plants that had grown for approximately 4-5 weeks were dewatered for 13 days, followed by rehydration. Results showed that after 8 days of dewatering, wild-type leaves exhibited significant wilting and yellowing, while the knockout plants remained upright with good leaf water content, demonstrating significantly enhanced drought tolerance. After 13 days of continuous drought followed by 2 days of rehydration, the knockout lines rapidly recovered growth, with a significantly higher survival rate than the wild-type. Figure 3 ).

[0028] (2) Detection of epidermal wax and physiological indicators related to leaf water loss Scanning electron microscopy was used to observe the epidermal wax structure of leaves from different materials. The results showed that the deposition of wax crystals on the leaf surface of the knockout plants was significantly increased. Figure 4 The total wax content was determined by gas chromatography. The specific method was as follows: leaf wax was extracted with chloroform, derivatized with bis(trimethylsilyl)trifluoroacetamide (BSTFA), and then analyzed by gas chromatography (GC) to determine its wax components and total amount. The results showed that, compared with the wild type, the total wax content of the knockout lines KO#8 and KO#16 was significantly increased. Figure 5 The results of leaf water loss rate measurements showed that the water loss rate of the knockout plants was significantly lower than that of the wild type. Figure 6 ).

[0029] The above results indicate that knocking out the BnaA9.TZF5 gene can enhance the accumulation of waxy cuticle in the leaves of Brassica napus, reduce water transpiration loss, and thus improve the plant's drought resistance.

[0030] (3) Analysis of production and application prospects The yield of individual plants of knockout and wild type was investigated, and the results showed that no significant differences were observed in the main yield traits between the two.

[0031] Based on the phenotypic, physiological indicators, and wax accumulation results under drought stress, it is evident that knocking out the BnaA9.TZF5 gene can improve the drought resistance of rapeseed without significantly affecting yield traits. Therefore, this mutant material can serve as an intermediate material or superior parent for drought-resistant breeding, possessing good application potential.

[0032] Example 3: Construction and validation of BnaA9.TZF5 overexpression strain (1) Construction and transformation of overexpression vectors To verify the function of the BnaA9.TZF5 gene, the coding sequence of BnaA9.TZF5 shown in SEQ ID NO:1 was cloned into the plant expression vector pCAMBIA1300 driven by a strong promoter (such as the CaMV35S promoter), thus constructing a BnaA9.TZF5 overexpression recombinant plasmid. This recombinant plasmid was introduced into Agrobacterium GV3101 competent cells and transformed into the Brassica napus variety 'Zhongshuang 11' via Agrobacterium-mediated hypocotyl transformation. After hygromycin resistance screening and molecular detection, several positive transgenic plants were obtained. Real-time quantitative PCR analysis showed that the expression level of BnaA9.TZF5 in these transgenic plants was significantly higher than that in the wild-type control. Two representative overexpression lines with high expression levels were selected and named OE#10 and OE#14 for subsequent phenotypic and functional analysis.

[0033] (2) Phenotypic analysis of drought stress The drought stress experiment used the same treatment conditions as in Example 2, namely, stopping watering for 13 days and then restoring water supply for 2 days. The results showed that after 8 days of watering cessation, the overexpressing plants exhibited obvious wilting, curling, and water loss symptoms in their leaves, with significantly lower relative leaf water content than the wild type, demonstrating clear water deficit characteristics. After 13 days of continuous drought followed by 2 days of rehydration, most wild-type plants recovered growth, while the recovery ability of the overexpressing lines was extremely weak, with most plants failing to return to normal growth; their recovery ability was significantly weaker than that of the wild type (see Example 2). Figure 3 ).

[0034] (3) Detection of epidermal wax and physiological indicators related to leaf water loss Scanning electron microscopy revealed that the epidermal wax crystals of the overexpressing strains were sparse and unevenly arranged. Figure 4 The total wax content was determined using the same method as in Example 2. The results showed that the total wax content in the leaves of the overexpression lines was significantly lower than that of the wild type. Figure 5 The results of water loss rate detection showed that the overexpression lines had a significantly faster leaf water loss rate. Figure 6 This indicates that the stratum corneum barrier function is impaired.

[0035] In summary, overexpression of BnaA9.TZF5 significantly weakened the growth adaptability and survival ability of rapeseed plants under drought stress. This result is the opposite of the performance of the gene knockout strain in Example 2, further confirming that BnaA9.TZF5 plays a negative regulatory role in the drought resistance response of Brassica napus.

[0036] Example 4: Analysis of BnaA9.TZF5 promoter activity and tissue-specific expression To analyze the tissue-specific and spatial expression characteristics of BnaA9.TZF5 in plants, and thus provide molecular expression evidence for its function in wax synthesis and drought resistance regulation, a promoter sequence (SEQ ID NO:4) approximately 2.0 kb upstream of the start codon of the BnaA9.TZF5 gene was cloned from the genomic DNA of the Brassica napus cultivar ZS11. This promoter fragment was cloned upstream of the GUS gene in the reporter gene vector pBI121 to construct the promoter reporter expression vector BnaA9.TZF5Pro::GUS.

[0037] The recombinant vector was transformed into Arabidopsis thaliana using the Agrobacterium-mediated flower-dipping method to obtain T1 generation transgenic plants. GUS histochemical staining was performed on seedlings, stems, leaves, and other tissues of the transgenic plants to detect the tissue expression pattern of the promoter. All experimental conditions and staining methods employed were conventional techniques in the field.

[0038] GUS staining results show (see) Figure 7 The BnaA9.TZF5 promoter exhibits significant expression activity in leaves and a strong GUS signal was also detected in stem tissue. Further observation revealed that the GUS signal is mainly distributed in the epidermis of leaves and stems, showing a significant blue precipitate in epidermal cells, while the signal is relatively weak in internal tissues.

[0039] The above results indicate that the expression region driven by the BnaA9.TZF5 promoter is highly consistent with the main accumulation sites of wax in the rapeseed epidermis, suggesting that BnaA9.TZF5 mainly functions in the epidermis. This tissue-specific expression characteristic is consistent with its physiological role in wax synthesis and cuticle structure regulation, providing molecular expression-level support for its function in drought resistance formation.

[0040] Example 5: Subcellular localization analysis of BnaA9.TZF5 To determine the intracellular localization characteristics of the BnaA9.TZF5 protein and provide evidence at the cellular level for its role as a transcriptional regulator in drought resistance and wax metabolism regulation, this embodiment analyzed the subcellular localization of BnaA9.TZF5. Using cDNA from the rapeseed variety 'Zhongshuang 11' as a template, the coding sequence of BnaA9.TZF5 without a stop codon was amplified and cloned into the subcellular localization vector pCAMBIA1302-GFP to construct a 35S::GFP-BnaA9.TZF5 fusion expression vector. After verification by E. coli amplification and sequencing, the recombinant vector was transformed into Agrobacterium GV3101 competent cells.

[0041] Agrobacterium strain carrying 35S::GFP-BnaA9.TZF5 was transformed into tobacco leaves using a conventional transient expression system. Empty vector 35S::GFP and the nuclear localization marker mCherry were used as controls. After cultivation under suitable conditions, the distribution of GFP fluorescence signal was observed using laser confocal microscopy. The results showed (see...) Figure 8 The green fluorescence signal of the 35S::GFP-BnaA9.TZF5 fusion protein was highly concentrated in the nuclear region and showed a clear overlap with the red fluorescence signal of the nuclear localization marker mCherry; in contrast, the fluorescence signal of the empty vector 35S::GFP could be detected in both the cytoplasm and the nucleus, showing a diffuse distribution.

[0042] The above results indicate that the BnaA9.TZF5 protein is located in the cell nucleus, consistent with its functional characteristics as a transcription factor involved in gene expression regulation. This subcellular localization further supports the transcriptional regulatory role of BnaA9.TZF5 in regulating drought resistance and epidermal wax metabolism in rapeseed.

Claims

1. Use of BnaA9.TZF5 gene in improving drought resistance of Brassica plants, characterized in that, The BnaA9.TZF5 gene is edited by gene editing to cause functional deletion mutation, so as to enhance the drought resistance of the Brassica plant, and the nucleotide sequence of the BnaA9.TZF5 gene is shown as SEQ ID NO:

1.

2. Use according to claim 1, characterized in that, The Brassica plant is Brassica napus.

3. Use according to claim 2, characterized in that, The BnaA9.TZF5 gene of Brassica napus is knocked out by using CRISPR-Cas9 editing technology, and the target sequence is shown as SEQ ID NO:2 or SEQ ID NO:

3.

4. A breeding method for obtaining a Brassica plant with enhanced drought resistance, characterized in that, The BnaA9.TZF5 gene in the Brassica plant is edited by gene editing to cause functional deletion mutation, so as to obtain a plant with enhanced drought resistance, and the nucleotide sequence of the BnaA9.TZF5 gene is shown as SEQ ID NO:

1.

5. The breeding method according to claim 4, characterized in that, The Brassica plant is Brassica napus.

6. The breeding method according to claim 5, characterized in that, The BnaA9.TZF5 gene is edited by using CRISPR-Cas9 editing technology, and the target sequence is shown as SEQ ID NO:2 or SEQ ID NO:

3.

7. Use of a gene editing vector for knocking out the BnaA9.TZF5 gene in improving drought resistance of Brassica plants, characterized in that, The gene editing vector comprises a guide sequence for targeting knockout of the nucleotide sequence shown as SEQ ID NO:

1.

8. Use according to claim 7, characterized in that, The BnaA9.TZF5 gene of Brassica napus is knocked out by using CRISPR-Cas9 editing technology, and the target sequence is shown as SEQ ID NO:2 or SEQ ID NO:3.

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