Protein bnabzip12 and its application in improving salt tolerance of plants

Abstract

The application belongs to the technical field of plant genetic engineering, and particularly relates to a protein BnabZIP12 and application thereof in improving plant salt tolerance. The application discloses a Brassica napus stress resistance related protein BnabZIP12 and application of a gene thereof in plant salt tolerance regulation, and an amino acid sequence of the protein is shown as SEQ ID No. 1. In the embodiment, a CRISPR / Cas9 is used to obtain a BnabZIP12 homozygous knockout strain, a gene overexpression vector is constructed and transformed into a plant to obtain overexpression material. The salt stress experiment results show that, compared with a wild type, salt tolerance of the BnabZIP12 overexpression plant is significantly improved, and salt tolerance of the knockout strain is obviously reduced. It is confirmed that the BnabZIP12 can positively regulate the plant salt tolerance, and the gene can provide excellent gene resources for crop salt tolerance germplasm improvement and new variety breeding. The plant salt tolerance gene mined by the application has important research significance and practical application value for cultivating new salt tolerance varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to the protein BnabZIP12 and its application in improving the salt tolerance of plants. Background Technology

[0002] Plants continuously face various abiotic stresses during their growth and development, among which high soil salinity stress is one of the major abiotic hazards that restricts crop growth and development, limits arable land use, and affects crop yield and quality. Soil salinization is a widespread problem in agricultural areas globally. With climate change and the prevalence of irrational farming and irrigation practices, the degree of arable land salinization continues to worsen, severely encroaching on high-quality arable land resources and greatly restricting the large-scale, high-quality production of oil crops. High-salt environments disrupt the osmotic pressure balance of plant cells, damage cell membrane structure, inhibit photosynthetic metabolism and normal growth and development, resulting in reduced crop emergence rates, weakened growth, and a significant drop in yield, causing serious negative impacts on sustainable agricultural development.

[0003] Rapeseed (Brassica napus L.) is an important oilseed crop in my country, serving as a crucial source of edible vegetable oil and protein, and playing a vital role in the national economy and food security system. Salt stress significantly inhibits rapeseed seed germination, seedling growth, and reproductive development, leading to decreased stress resistance, reduced yield, and deteriorated quality. This is a key bottleneck restricting high and stable rapeseed yields and the development of rapeseed cultivation in saline-alkali land. As an important oilseed crop, rapeseed also possesses significant potential for improving saline-alkali soils. Therefore, identifying key endogenous salt-tolerant genes in rapeseed, elucidating its salt-tolerant regulatory mechanisms, and cultivating new salt-tolerant rapeseed varieties are core approaches to overcoming the limitations of rapeseed cultivation in saline-alkali lands, enhancing rapeseed stress resistance, and expanding rapeseed planting areas. These approaches have significant practical value and are essential for production.

[0004] Over long periods of evolution, plants have developed complex abiotic stress response mechanisms. Through a series of molecular regulatory pathways, including stress signal perception, signal transduction, and specific expression of stress-resistance-related genes, they regulate their physiological metabolism and growth status to adapt to adverse environments such as high salinity. Under salt stress, plants initiate a series of physiological and biochemical responses, regulating the expression of stress-resistance genes, modulating cellular osmotic balance, antioxidant metabolism, and stress signal transduction, reducing ion damage to cells, and thus enhancing the plant's salt tolerance.

[0005] Compared to the shortcomings of traditional hybridization breeding, such as long cycles, low breeding efficiency, and poor targeting of stress resistance improvement, genetic engineering breeding can precisely target and improve crop stress resistance traits, making it the mainstream and efficient method for current crop stress resistance breeding. Identifying key functional genes for stress resistance in rapeseed and clarifying their molecular functions in the salt stress response process can provide high-quality gene resources and theoretical support for the innovation of salt-tolerant rapeseed germplasm and the breeding of new varieties. Currently, the functions of many key stress resistance genes in rapeseed have not yet been fully explored and identified. Therefore, developing proteins and key genes related to salt tolerance has significant theoretical importance and broad prospects for the cultivation of highly salt-tolerant plant varieties and the efficient utilization of saline-alkali land. Summary of the Invention

[0006] The purpose of this invention is to provide a protein BnabZIP12 and its application in improving the salt tolerance of plants. The protein BnabZIP12 and its encoding gene described in this invention can achieve precise regulation of the salt tolerance of plants, providing high-quality gene resources and technical support for the breeding of new salt-tolerant plant varieties and the efficient cultivation and utilization of saline-alkali land.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a plant stress resistance-related protein, BnabZIP12, wherein the protein BnabZIP12 is any one of the following:

[0009] a1) The amino acid sequence of this protein is that of SEQ ID No. 1;

[0010] a2) A protein that has more than 80% identity with and has the same function as the protein shown in a1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No. 1.

[0011] a3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2).

[0012] Preferably, the tags in a3) include: eGFP (enhanced green fluorescent protein) and His-tag protein.

[0013] A nucleic acid molecule encoding BnabZIP12, a protein associated with plant stress resistance, may be a DNA molecule as shown in b1) or b2) below:

[0014] b1) The coding region is the DNA molecule shown in SEQ ID NO: 2;

[0015] b2) A DNA molecule that hybridizes under strict conditions with the DNA molecule defined in (b1) and encodes the same protein with the same activity.

[0016] Preferably, b1) is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO: 2.

[0017] SEQ ID NO: 2 consists of 819 nucleotides, and the nucleotides of SEQ ID NO: 2 encode the amino acid sequence shown in SEQ ID NO: 1. Those skilled in the art should understand that, due to the degeneracy of codons, nucleotide sequences obtained by synonymous substitution, modification, or optimization of the nucleotide sequence shown in SEQ ID NO: 2 without altering the amino acid sequence of the BnabZIP12 protein can all encode the functionally identical BnabZIP12 protein. Therefore, all nucleotide sequences capable of encoding the BnabZIP12 protein of claim 1, i.e., the DNA molecule shown in b2) above, fall within the protection scope of this invention.

[0018] A method for cultivating transgenic plants includes the following steps: increasing the expression level of any of the proteins BnabZIP12 described above in the plant to obtain transgenic plants overexpressing BnabZIP12; or, mutating BnabZIP12 to obtain transgenic plants with homozygous knockout of BnabZIP12.

[0019] Preferably, the transgenic plants overexpressing BnabZIP12 have increased salt tolerance compared to wild-type plants; the transgenic plants with homozygous knockout of BnabZIP12 have decreased salt tolerance compared to wild-type plants.

[0020] Preferably, the method for mutating BnabZIP12 includes utilizing CRISPR / Cas9.

[0021] Preferably, the above-mentioned improvement in salt tolerance is manifested in an increase in root length and root weight of the plant after salt treatment;

[0022] Preferably, the improved salt tolerance is manifested in increased CAT activity and increased POD activity.

[0023] The present invention also provides the application of any of the above-described proteins BnabZIP12 or their encoding genes, wherein the application is at least one of C1)-C6):

[0024] C1) Improves the salt tolerance of plants;

[0025] C2) Developing transgenic plants with enhanced salt tolerance;

[0026] C3) Increases the SPAD content in plants;

[0027] C4) Cultivating SPAD-enhanced transgenic plants;

[0028] C5) Enhances the plant's ability to scavenge reactive oxygen species;

[0029] C6) Developing transgenic plants with reactive oxygen species scavenging capabilities;

[0030] Preferably, the plant is rapeseed.

[0031] A BnabZIP12 gene-specific quantitative primer, wherein the primers are BnaActin7-qRT-F, BnaActin7-qRT-R, qp-bZIP12-F, and qp-bZIP12-R; the nucleotide sequences are as shown in SEQ ID NO: 5-8.

[0032] Beneficial effects:

[0033] This invention is the first to verify the salt tolerance regulatory function of the protein BnabZIP12 in plants. Experiments have confirmed that this gene / protein can significantly improve crop salt stress tolerance. Overexpression of the BnabZIP12 gene in wild-type rapeseed can improve rapeseed salt tolerance from multiple dimensions, including morphology, physiology, and photosynthesis: First, it significantly increases the root length and root weight of rapeseed plants under salt stress, maintaining normal root growth and development and alleviating the inhibitory damage to the root system caused by salt stress; second, it significantly increases the activity of CAT and POD antioxidant enzymes in the plant, enhancing the plant's ability to scavenge reactive oxygen species and reducing oxidative stress damage caused by salt stress; third, it effectively alleviates chlorophyll loss caused by salt stress, maintains a high SPAD value in the plant, and ensures normal photosynthesis under salt stress. This invention fills the technical gap in the salt tolerance regulation of the rapeseed BnabZIP12 gene, providing a reliable gene target and technical support for molecular breeding of salt-tolerant rapeseed, high-efficiency cultivation of rapeseed in saline-alkali land, and the breeding of new salt-tolerant crop varieties. It has extremely high scientific research value and promising prospects for agricultural industrialization. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0035] Figure 1 The relative expression level of the BnabZIP12 gene in overexpressed rapeseed lines.

[0036] Figure 2 Identification of BnabZIP12 knockout strains.

[0037] Figure 3 Salt-tolerant phenotype of BnabZIP12 transgenic plants.

[0038] Figure 4 Root length data of BnabZIP12 transgenic plants.

[0039] Figure 5 Root weight data of BnabZIP12 transgenic plants.

[0040] Figure 6 SPAD data for BnabZIP12 transgenic plants.

[0041] Figure 7 POD data for BnabZIP12 transgenic plants.

[0042] Figure 8 CAT data for BnabZIP12 transgenic plants. Detailed Implementation

[0043] This invention provides a protein BnabZIP12 and its encoding gene, as well as their application in improving the salt tolerance of plants.

[0044] The amino acid sequence of protein BnabZIP12 is shown in SEQ ID No:1:

[0045] MGSLRGNVEEPIHQSLPRQNSLYNLKLHEVQNHLGSSSKPLGSMNLDELLKSVLSAEANHPPEEGTEEGITRQGSLTLPRGLSIKTVNEVWRDIQHGERLDNPNKQPTLGEITLEDLLMKAGVVTESMTVPQNVVNVA SNGQWAQYPQQQHQGFMPYPVCDMQEMVPPTALMMGGLSETQQVHGRKRVASSEGEFVERIVERKQKRMIKNRESASRSRARKQAYTQELEIKVSSLEEENQKLRRLMEVEKILPSEPPPEPKWKLRRTSTASF (SEQ ID NO:1).

[0046] The nucleic acid molecule encoding the protein BnabZIP12 is shown in SEQ ID No:2:

[0047] (SEQ IDNO:2).

[0048] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0049] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0050] The various instruments, equipment, raw materials, or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through legitimate commercial channels and can be prepared according to conventional methods well known to those skilled in the art. Some of the reagents or raw materials are sourced as follows:

[0051] The Agrobacterium tumefaciens GV3101 used in the following examples was purchased from Beijing Coolplay Technology Co., Ltd.

[0052] In the following examples, the wild-type rapeseed material used is the Brassica napus variety Westar, hereinafter referred to as WT.

[0053] The vectors used in the following examples are all from Hainan Nixing Biotechnology Co., Ltd.

[0054] The POD detection kits in the following examples are from Beijing Box Biotechnology Co., Ltd., catalog number AKAO005M

[0055] The CAT detection kits in the following examples are from Suzhou Jeres Biotechnology Co., Ltd., catalog number G0101W48.

[0056] The RNA extraction kits used in the following examples were from Nanjing Novizan Biotechnology Co., Ltd., catalog number RC122-01.

[0057] Example 1: Cloning of the BnabZIP12 gene

[0058] 1. Total RNA extraction and cDNA synthesis from rapeseed leaves: Young leaves of the rapeseed variety Westar (WT) were taken, and total RNA was extracted using conventional RNA extraction methods. Then, cDNA from the rapeseed Westar leaves was synthesized through reverse transcription.

[0059] 2. PCR amplification of the target gene: Using the cDNA obtained above as a template, PCR amplification was performed using primer pairs pNC-bZIP12-F and pNC-bZIP12-R:

[0060] pNC-bZIP12-F: agtggtctctgtccagtcctATGGGTTCTCTTAGAGGAAACGTT (SEQ ID NO:3),

[0061] pNC-bZIP12-R:ggtctcagcagaccacaagtGTGAATCTTGTTTGCCAGATT (SEQ ID NO:4).

[0062] After the reaction was completed, a specific DNA fragment of approximately 817 bp was recovered.

[0063] 3. Sequencing and sequence verification of the amplified products: The recovered PCR products were sequenced and analyzed. The results showed that the amplified fragment contained the nucleotide sequence shown in SEQ ID NO: 2, with a full-length coding region of 817 bp, which was named the BnabZIP12 gene. Further analysis showed that the protein BnabZIP12 encoded by the BnabZIP12 gene has the amino acid sequence shown in SEQ ID NO: 1.

[0064] Example 2: Construction of BnabZIP12 overexpression vector

[0065] The PCR product recovered in Example 1 was ligated into the overexpression vector pNC-1304-35S with a 35S promoter. Nimble cloning was employed.

[0066] Example 3: Construction of rapeseed material overexpressing the BnabZIP12 gene

[0067] 1. The recombinant vector pNC-Cam1304-35S (35S:BnabZIP12) containing the BnabZIP12 gene constructed in Example 2 was transformed into Agrobacterium GV3101 competent strain, and positive Agrobacterium strains were obtained by PCR identification.

[0068] 2. Select positive single clones and inoculate them into LB liquid medium containing 50 mg / L rifampin (Rif) and 50 mg / L kanamycin (Kan), and culture overnight with shaking at 28°C and 220 rpm. When the OD600 of the bacterial culture reaches 0.8~1.0, collect the bacterial cells by centrifugation at 5000×g for 10 min.

[0069] 3. The collected bacterial cells were resuspended in 2 mL of transformation solution, which consisted of 1 / 2 MS medium, 5% sucrose, and 0.02% Silwet L-77. Hypocotyls of sterile rapeseed (Brassica napus L. cv. Westar) seedlings were selected as recipient materials. The hypocotyl explants were immersed in Agrobacterium infection solution for 10–15 min, and then the excess bacterial solution was aspirated. The infected explants were placed on co-culture medium and cultured in the dark at 22–25 °C for 2–3 days to complete Agrobacterium-mediated genetic transformation. After co-culture, the explants were transferred to differentiation medium containing hygromycin screen for selection culture to induce adventitious shoot differentiation. When the resistant shoots reached 2–3 cm in length, they were cut and transferred to rooting medium for culture. This process continued until transgenic rapeseed seedlings differentiated, obtaining transgenic rapeseed plants overexpressing the BnabZIP12 gene.

[0070] Example 4: Real-time quantitative PCR detection of the relative expression level of the BnabZIP12 gene in rapeseed transgenic BnabZIP12.

[0071] 1. Leaf tissues of wild-type Westar rapeseed and rapeseed seedlings transgenic with the BnabZIP12 gene with uniform growth were selected respectively. Total RNA was extracted from each sample. After detecting the integrity and purity of the RNA, the first-strand cDNA was synthesized by reverse transcription. The obtained cDNA product was used as the amplification template for real-time fluorescence quantitative PCR.

[0072] 2. Designed BnabZIP12 gene-specific quantitative primers, and selected a rapeseed internal reference gene as a control gene to correct for sample loading errors. Amplification reaction systems were prepared using a real-time quantitative PCR kit, and amplification reactions were performed according to standard real-time PCR procedures. Three biological replicates were set for each sample to ensure the reliability of experimental data. Primers are as follows:

[0073] BnaActin7-qRT-F:CTATCCTCCGTCTCGATCTCGC (SEQ ID NO:5)

[0074] BnaActin7-qRT-R:CTTAGCCGTCTCCAGCTCTTGC (SEQ ID NO:6)

[0075] qp-bZIP12-F:CTGAGTCAATGACAGTCCCTC (SEQ ID NO:7)

[0076] qp-bZIP12-R:CCCATGCACTTGTTGTGTT (SEQ ID NO:8)

[0077] Test results are shown Figure 1 qPCR results showed that, compared with wild-type rapeseed (Westar, WT), the relative expression level of the BnabZIP12 gene was significantly upregulated in the BnabZIP12 overexpressing rapeseed lines.

[0078] Example 5: Identification of BnabZIP12 knockout lines

[0079] 1. Gene editing target sites were designed targeting the coding region of the BnabZIP12 gene. Target site sequence:

[0080] T1:GGGAAGTTCCTCAAAACCGT (SEQ ID NO:9)

[0081] T2: GGAAGCTTGACTTTGCCTCG (SEQ ID NO:10)

[0082] 2. Design primers based on target sites to identify homozygous knockout lines:

[0083] bZIP12-T1T2-A4-F: CACAGTCTGTTCTGCTTATGCC (SEQ ID NO: 11)

[0084] bZIP12-T1T2-A4-R: CTTGGAGTTGTTGCTGAGGC (SEQ ID NO: 12)

[0085] bZIP12-T1T2-C4-1-F:TGTTCTGCTCGCGGCAATG (SEQ ID NO:13)

[0086] bZIP12-T1T2-C4-1-R:CCCTTGGTGTTGTTGCTGAGG (SEQ ID NO:14)

[0087] bZIP12-T3T4-C4-2-F: TTATACTGTCCTGCTCGTAGTGG (SEQ ID NO: 15)

[0088] bZIP12-T3T4-C4-2-R:TATCTCCTCCTGATGGAGCCG (SEQ ID NO: 16)

[0089] bZIP12-T3T4-A5-F:TCTCCCAGTGATGCTTAGAACAAC (SEQ ID NO:17)

[0090] bZIP12-T3T4-A5-R: CTGAGGATGATGATACTGAATCCAC (SEQ ID NO: 18).

[0091] 3. Obtain sequencing results and match them; the test results are shown below. Figure 2 The test results showed that all three copies of BnabZIP12 knockout rapeseed lines had base insertions, resulting in 3 knockout lines.

[0092] Example 5: Detection of salt tolerance in rapeseed with overexpression and knockout of the BnabZIP12 gene.

[0093] 1. The salt tolerance of Brassica napus was evaluated using hydroponics. Six-day-old seedlings were fixed above 4 liters of full-concentration Hoagland solution using cotton plugs. After 14 days, the plants were treated with 200 mM sodium chloride, or continued to be placed in full-concentration Hoagland solution as a control. The nutrient solution was changed weekly, and samples were collected from day 6 to 9 after treatment. Three independent experiments were conducted: three biological replicates were set for each line under each condition, with 15 seedlings per replicate. Phenotypic results after salt treatment are shown below. Figure 3 .

[0094] 2. Root length was analyzed using ImageJ, and root weight was measured using a balance. Results are as follows: Figure 4 , Figure 5 .

[0095] 3. SPAD values ​​were measured using a SPAD-502 Plus chlorophyll meter. In the salt stress experiment, the second-to-last fully expanded true leaf from each treatment group was selected for SPAD value measurement; five measurements were taken for each leaf, and the average value was recorded. The results are as follows: Figure 6 .

[0096] 4. Enzyme activity assay: Three biological replicates were used for each treatment, with each replicate containing a mixed tissue sample from 15 plants. All samples were tested under identical conditions. POD and CAT were measured strictly according to the reagent instructions. Results are shown below. Figure 7 , Figure 8 .

[0097] The results showed that under salt stress, the growth status and various stress resistance physiological indicators of the overexpressing lines were significantly better than those of the wild-type lines; the salt tolerance of the corresponding BnabZIP12 knockout lines was significantly reduced, and the plants were more sensitive to salt stress.

[0098] In summary, this invention, through overexpression and knockout of the endogenous gene BnabZIP12 in rapeseed, confirms that the BnabZIP12 protein and its encoding gene can positively regulate the salt stress tolerance of plants. Increasing the expression of BnabZIP12 protein in the target plant significantly enhances its salt tolerance and effectively strengthens its adaptability to high-salt stress environments.

[0099] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A plant stress resistance-related protein, BnabZIP12, characterized in that, The protein BnabZIP12 is any one of the following: a1) The amino acid sequence of this protein is that of SEQ ID No. 1; a2) A protein that has more than 80% identity with and has the same function as the protein shown in a1) obtained by substituting and / or deleting and / or adding amino acid residues of the amino acid sequence shown in SEQ ID No.

1. a3) A fusion protein with the same function is obtained by attaching a tag to the N-terminus and / or C-terminus of a1) or a2).

2. A nucleic acid molecule encoding BnabZIP12, a protein related to plant stress resistance, characterized in that, It can be a DNA molecule as shown in b1) or b2) below: b1) The coding region is the DNA molecule shown in SEQ ID NO: 2; b2) A DNA molecule that hybridizes under strict conditions with the DNA molecule defined in (b1) and encodes the same protein with the same activity.

3. The nucleic acid molecule according to claim 2, characterized in that, b1) is a DNA molecule with a nucleotide sequence as shown in SEQ ID NO:

2.

4. A method for cultivating transgenic plants, characterized in that, The process includes the following steps: increasing the expression level of any of the proteins BnabZIP12 described above in plants to obtain transgenic plants overexpressing BnabZIP12; or, mutating BnabZIP12 to obtain transgenic plants with homozygous knockout of BnabZIP12.

5. The method according to claim 4, characterized in that, The transgenic plants overexpressing BnabZIP12 showed increased salt tolerance compared to wild-type plants; the transgenic plants with homozygous knockout of BnabZIP12 showed decreased salt tolerance compared to wild-type plants.

6. The method according to claim 4, characterized in that, The method for mutating BnabZIP12 includes using CRISPR / Cas9.

7. The method according to claim 4, characterized in that, The improved salt tolerance is manifested in the increased root length and root weight of the plants after salt treatment.

8. The method according to claim 4, characterized in that, The improved salt tolerance is manifested in increased CAT activity and increased POD activity.

9. The application of the protein BnabZIP12 or its encoding gene as described in claim 1, characterized in that, The application is at least one of C1)-C6): C1) Improves the salt tolerance of plants; C2) Developing transgenic plants with enhanced salt tolerance; C3) Increases the SPAD content in plants; C4) Cultivating SPAD-enhanced transgenic plants; C5) Enhances the plant's ability to scavenge reactive oxygen species; C6) Develop transgenic plants with reactive oxygen species scavenging capabilities.

10. A BnabZIP12 gene-specific quantitative primer, characterized in that, The primers are BnaActin7-qRT-F, BnaActin7-qRT-R, qp-bZIP12-F, and qp-bZIP12-R; the nucleotide sequences are as shown in SEQ ID NO: 5-8.

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