Brassica napus drought-tolerant gene bnac04ARL and use thereof

Abstract

Provided in the present invention are a Brassica napus drought-tolerant gene BnaC04ARL and the use thereof. The gene is expressed in a plant to improve drought tolerance of the plant. The gene comprises a nucleotide sequence selected from the group consisting of: a nucleotide sequence shown as SEQ ID NO: 1; and / or a nucleotide sequence encoding an amino acid sequence shown as SEQ ID NO: 2. The overexpression of the gene can improve the viability of Brassica napus under drought conditions, enhance root system development, and improve the thousand-grain weight, the grain yield per plant, the number of siliques per plant, dry weight per plant, silique length, etc.

Description

A drought-resistant gene BnaC04ARL in Brassica napus and its application Technical Field

[0001] This invention belongs to the field of biotechnology and relates to drought-resistant genes in Brassica napus and their application in improving the drought resistance of Brassica napus. Background Technology

[0002] In recent years, research on the molecular mechanisms by which plants alleviate drought has made some progress, and evaluating the physiological effects of drought-induced gene production has become a research hotspot and important direction in plant stress resistance molecular biology and genetic engineering. Seasonal droughts frequently occur in my country's main rapeseed producing areas, leading to poor seedling emergence, slow growth, and reduced yields. Because Brassica napus is relatively poor at drought and infertile soil, it requires a large amount of water throughout its growth cycle.

[0003] Therefore, identifying drought-resistant genes and superior drought-resistant germplasm resources in rapeseed is of great significance for breeding drought-resistant rapeseed varieties and ensuring the supply of vegetable oil. This applicant is funded by the National Key Research and Development Program of China – Major Project of Agricultural Bio-breeding, “Discovery and Breeding Value Evaluation of New Genes Controlling Plant Type, 2023ZD0406801”, to conduct research on discovering drought-resistant genes in Brassica napus, providing technical support for the development of drought-resistant rapeseed varieties. Summary of the Invention

[0004] Therefore, the present invention aims to provide a drought-resistant gene BnaC04ARL for Brassica napus, the protein encoded by the gene, and a method and application for using the gene to improve the drought resistance of Brassica napus.

[0005] Through long-term exploration and experimentation, as well as numerous trials and efforts, the inventors have continuously reformed and innovated to solve the above-mentioned technical problems. The technical solution provided by this invention is to provide a drought-resistant gene BnaC04ARL for Brassica napus, which is expressed in plants to improve their drought resistance. The BnaC04ARL gene contains nucleotide sequences selected from the following group:

[0006] A. The nucleotide sequence is as shown in SEQ ID NO:1;

[0007] B. The nucleotide sequence of the amino acid sequence shown in SEQ ID NO.2.

[0008] The technical advantages of this invention are as follows: The drought-resistant gene BnaC04ARL in Brassica napus was successfully cloned and identified. The discovery of this gene provides a new molecular tool for improving the drought resistance of rapeseed crops. Expressing the BnaC04ARL gene in plants can significantly enhance the survival ability of Brassica napus under drought conditions. This not only helps improve the yield stability of crops under adverse climatic conditions but also reduces dependence on water resources, which has important strategic significance for agricultural production in drought-prone areas.

[0009] The present invention also provides a protein encoded by the drought-resistant gene BnaC04ARL of the Brassica napus, the amino acid sequence of which is shown in SEQ ID NO.2.

[0010] The present invention also provides a primer pair for cloning the drought-resistant gene BnaC04ARL in Brassica napus, the base sequence of which is as follows:

[0011] BnaC04ARL-F: 5'-CGGGATCCATGATTCGTGAAATCTCTGG-3',

[0012] BnaC04ARL-R: 5'-CCCAAGCTTTTACATATAAGTTCTTGTTACATGTT-3'.

[0013] The technical advantage of this invention lies in its ability to precisely and efficiently amplify the target gene using PCR technology via the BnaC04ARL-F / R primer pair. The key to this specific primer design is ensuring that only the target gene BnaC04ARL is selectively amplified, thereby avoiding non-specific amplification. This is crucial for subsequent gene cloning, sequence analysis, and functional studies. By using the BnaC04ARL-F / R primer pair, researchers were able to successfully clone the drought-resistant gene BnaC04ARL from the genomic DNA of Brassica napus, laying the foundation for further genetic transformation and crop improvement efforts. Furthermore, this precise cloning technology helps protect and utilize plant genetic diversity, promotes the genetic improvement of drought-resistant traits, and ultimately enhances the crop's production potential under drought conditions.

[0014] This invention also provides a method for constructing a BnaC04ARL overexpression vector based on DsRed, the method comprising the following steps:

[0015] 1) The BnaC04ARL gene was ligated into the double-digested DsRed vector;

[0016] 2) Transform the successfully constructed vector plasmid into Agrobacterium tumefaciens GV3101;

[0017] 3) The target rapeseed variety was genetically transformed using Agrobacterium-mediated transformation to obtain BnaC04ARL overexpressing transgenic plants.

[0018] The technical advantages of this invention are as follows: By introducing the BnaC04ARL gene into Brassica napus using Agrobacterium-mediated genetic transformation, transgenic plants overexpressing this gene were successfully obtained. These transgenic plants exhibited better growth and drought resistance physiological indicators under drought conditions, including lower peroxide content, higher proline and soluble sugar content, and stronger root development, thus demonstrating the important role of the BnaC04ARL gene in improving plant drought tolerance.

[0019] This invention also provides a primer for positive identification of overexpression transgenic rapeseed lines, the primer comprising:

[0020] OE-BnaC04ARL-F: 5'-CGGGATCCATGATTCGTGAAATCTCTGG-3',

[0021] OE-BnaC04ARL-R: 5'-CCCAAGCTTTTACATATAAGTTCTTGTTACATGTT-3';

[0022] And the DsRed vector-specific primer pairs Dsred-F and Dsred-R.

[0023] Furthermore, the electrophoresis bands showed that the target band was positive and the absence of a target band indicated a negative result.

[0024] This invention also provides a method for positive identification of overexpressing rapeseed transgenic lines. Using DNA from the T0 / T1 generation of transgenic plants as a template, the obtained overexpressing rapeseed transgenic lines are positively identified using OE-BnaC04ARL-F / R primers, and finally positive plants are obtained.

[0025] The technical advantages of this invention are as follows: The positive identification primers for overexpression rapeseed transgenic lines provided by this patent ensure rapid confirmation of the successful insertion of the BnaC04ARL gene in transgenic plants through accurate PCR detection, thereby improving the efficiency and accuracy of transgenic technology. Furthermore, the application of DsRed vector-specific primer pairs further enhances the reliability of the identification process, providing a powerful molecular tool for the screening and research of transgenic lines.

[0026] This invention also provides a method for positive identification of overexpressing transgenic rapeseed lines. The method utilizes a red light labeling element on a vector and uses a red fluorescent protein excitation gun LUYOR-3415CG to irradiate and screen transgenic seedlings. Plants that emit red light from their roots are positive.

[0027] The technical advantage of this invention is that by using the carrier red light labeling element and the red fluorescent protein excitation gun LUYOR-3415CG for irradiation, rapid, intuitive and non-destructive screening of transgenic seedlings is achieved, thereby effectively improving the efficiency and accuracy of positive plant identification.

[0028] This invention also provides a primer pair for quantitative fluorescence analysis of the gene BnaC04ARL, the primer pair comprising:

[0029] BnaC04ARL-qPCR-F: 5'-TCTCACAGCATCGCTCTTG-3';

[0030] BnaC04ARL-qPCR-R: 5'-GGCATTAGAAGAAGAAGAAGAAGA-3'.

[0031] The technical advantage of this invention is that by using primer pairs for quantitative fluorescence analysis of the gene BnaC04ARL, and through precise qPCR technology, the expression level of this gene can be accurately determined, providing a reliable molecular tool for assessing gene function and its role in drought resistance.

[0032] The present invention also provides a method for detecting the expression level of the gene BnaC04ARL in Brassica napus, the method comprising the following steps:

[0033] 1) Extract total RNA from the leaves of Brassica napus;

[0034] 2) The RNA is reverse transcribed into cDNA;

[0035] 3) The expression level of the BnaC04ARL gene was quantitatively determined by using the BnaC04ARL-qPCR-F / R primer pair for real-time PCR analysis.

[0036] This invention also provides the uses of the drought-resistant gene BnaC04ARL in Brassica napus, the uses including:

[0037] A. Improving the survival rate of Brassica napus under drought conditions;

[0038] B. Enhance root development in Brassica napus under drought conditions;

[0039] And / or,

[0040] C. To improve agronomic traits of rapeseed, including thousand-grain weight, single-plant grain yield, total number of pods, single-plant dry weight and / or pod length, under drought conditions.

[0041] The technical advantages of this invention are as follows: The application of the BnaC04ARL gene in improving agronomic traits of Brassica napus (Brassica napus) demonstrates this through field trials on overexpression lines. The results show that overexpression of the BnaC04ARL gene can improve key agronomic traits such as thousand-grain weight, single-plant grain yield, and total silique number, providing a technical basis for breeding drought-resistant and high-yielding new rapeseed varieties. Furthermore, the application of this gene may be extended to improving the drought resistance of other crops, exhibiting broad application prospects and commercial value. Attached Figure Description

[0042] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained from these drawings without creative effort.

[0043] Figure 1 is a schematic diagram of the BnaC04ARL gene structure, amino acid sequence, and vector construction of this invention. In Figure 1, a: Schematic diagram of the BnaC04ARL gene structure, with black representing exons. b: Multiple sequence alignment results of the open reading frames of the BnaC04ARL gene and three other homologous genes. Multiple alignment of the full-length coding frame sequence and the amino acid sequences of ARL homologous genes in Brassica napus. c: Schematic diagram of key elements of the overexpression vector. d: Schematic diagram of key elements of the interference vector.

[0044] Figure 2 shows the functional analysis of the BnaC04ARL transgenic lines in response to drought stress. In Figure 2, a: drought-induced changes in ARL gene expression levels. b: relative expression level of ARL in overexpressing transgenic plants. c: relative expression level of ARL in interfering transgenic plants. d: phenotypic changes of each genotype before, after, and after rehydration treatment (scale bar = 3 cm). e: infrared spectroscopy measurements of each genotype before, after, and after rehydration treatment. f: survival rate of each genotype after drought and rehydration. g: changes in leaf water loss rate of overexpressing transgenic plants. h: changes in leaf water loss rate of interfering transgenic plants. i: DAB and NBT staining of each genotype before and after drought treatment (scale bar = 1 cm). j: comparison of proline content in each genotype before and after drought. k: comparison of chlorophyll content in leaves of each genotype before and after drought. l: comparison of soluble sugar content in each genotype before and after drought. m: comparison of soluble protein content in each genotype before and after drought. n: Comparison of malondialdehyde (MDA) content in plants of different genotypes before and after drought. o: Comparison of hydrogen peroxide (H2O) content in plants of different genotypes before and after drought. n≥3, ** indicates extremely significant difference at the P<0.01 level, * indicates significant difference at the P<0.05 level.

[0045] Figure 3 shows the root development changes of BnaC04ARL transgenic lines under different stress treatments. In Figure 3, a: root changes of each transgenic plant after 21 days of growth in pure water, 10% (w / v) PEG 6000, 100mM Mannitol, and 100mM NaCl aqueous solution (scale bar = 3cm). b: Statistical count of lateral roots for each transgenic line under different treatments. c: Statistical count of fresh root weight for each transgenic line under different treatments. d: Statistical count of dry root weight for each transgenic line under different treatments. n≥3, ** indicates extremely significant difference at the P<0.01 level, * indicates significant difference at the P<0.05 level.

[0046] Figure 4 shows the agronomic traits of transgenic lines under drought treatment in pots. In Figure 4, a: phenotype of each line after natural drought treatment, scale bar = 11 cm. b: Seed display of each transgenic line after natural drought treatment, scale bar = 2 mm. c: Silique length of each transgenic line under natural drought treatment, scale bar = 3 cm. d: 1000-seed weight of each transgenic line under natural drought treatment. e: Seed yield per plant of each transgenic line under natural drought treatment. f: Total number of siliques of each transgenic line under natural drought treatment. g: Silique length of each transgenic line under natural drought treatment. h: Dry weight per plant of each transgenic line under natural drought treatment. i: Plant height of each transgenic line under natural drought treatment. j: Number of seeds per silique of each transgenic line under natural drought treatment. n ≥ 3, ** indicates extremely significant difference at the P < 0.01 level, * indicates significant difference at the P < 0.05 level. Detailed Implementation

[0047] The following description is based on specific embodiments.

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to represent selected embodiments of the present invention.

[0049] To avoid redundancy, this embodiment provides simultaneous explanations and descriptions of the drought-resistant gene BnaC04ARL in Brassica napus, the protein encoded by the gene BnaC04ARL, the primer pairs for cloning the BnaC04ARL gene, the method for constructing BnaC04ARL overexpression lines, the primers for positive identification of overexpression transgenic rapeseed lines, the method for positive identification of overexpression transgenic rapeseed lines, the primer pairs for quantitative fluorescence analysis of the BnaC04ARL gene, the method for detecting expression levels, and its applications.

[0050] In this embodiment, the material used for cloning the BnaC04ARL gene was a leaf of the rapeseed variety “Zhongshuang 11”. The overexpression transgenic vector was DsRed, the interference transgenic vector was pFGC5941M, and the transgenic recipient material was the rapeseed variety “J9709”. The transgenic plant culture conditions were 16h light / 24℃ and 8h darkness / 20℃.

[0051] 1. Cloning of the BnaC04ARL gene

[0052] Total RNA was extracted from the leaves of rapeseed cultivar “Zhongshuang 11” during the seedling stage using the Trizol method. cDNA synthesis was performed using the reverse transcription kit according to the instructions of Hifair® III Reverse Transcriptase from Yisheng Biotechnology Co., Ltd. (the reverse transcription system of this kit is 15 μL, and a maximum of 1 μg of total RNA can be used. Therefore, the following system is based on the requirement of 1 μg of total RNA). The specific steps are shown in Table 1 and Table 2.

[0053] (1) Removal of genomic DNA (15 μL system)

[0054] Table 1. Genomic DNA Removal Reaction System

[0055]

[0056] After preparing the reaction solution for the first step, incubate it in a PCR instrument at 42°C for 2 minutes. After that, proceed to the second step.

[0057] (2) Reverse transcription reaction system (20 μL system)

[0058] Table 2 Reverse transcription reaction system

[0059]

[0060] After preparing the reverse transcription reaction system, set the program to 25℃ for 5 min; 55℃ for 15 min; 85℃ for 5 min. The resulting product is cDNA.

[0061] Using the synthesized cDNA as a template, PCR amplification of the BnaC04ARL gene was performed using primer pairs (BnaC04ARL-F: 5'-CGGGATCCATGATTCGTGAAATCTCTGG-3'; BnaC04ARL-R: 5'-CCCAAGCTTTTACATATAAGTTCTTGTTACATGTT-3') (Tables 3 and 4). The amplified target gene fragment was purified and recovered, ligated into a TA cloning vector, transformed into DH5α competent cells, and sequenced using primers (M13-F: 5'-GTAAAACGACGGCCAGT-3'; M13-R: 5'-CAGGAAACAGCTATGAC-3'). Plasmids from correctly sequenced *E. coli* were extracted and stored at -20°C. The sequence of the BnaC04ARL gene is shown in SEQ ID NO:1.

[0062] Table 3 Target Fragment Amplification Reaction System

[0063]

[0064] Table 4 PCR reaction procedure

[0065]

[0066] A schematic diagram of the gene structure was drawn, as shown in Figure 1a. The results show that the gene has one exon, no introns or UTR regions, and a full-length coding frame of 408 bp, encoding 135 amino acids. The amino acid sequence is shown in SEQ ID NO:2, belonging to the ARGOS gene family. Additionally, the amino acid sequences of five homologous genes of BnaC04ARL in Brassica napus were downloaded, and the conserved domains of these genes were visualized using GeneDoc software. The results are shown in Figure 1b.

[0067] 2. Obtaining the BnaC04ARL overexpression line

[0068] See Figure 1c. The DsRed vector (overexpression vector) and the TA cloning vector containing the target fragment were double-digested with BamHI and HindIII. The digested BnaC04ARL target gene fragment was ligated to the DsRed vector using T4 ligase. The plasmid with the correct sequenced results was transformed into Agrobacterium tumefaciens GV3101 using primers OE-BnaC04ARL-F+Dsred-R and OE-BnaC04ARL-R+Dsred-F. Then, using rapeseed variety J9709 as the recipient material, the hypocotyl of rapeseed was genetically transformed using Agrobacterium-mediated transformation to finally obtain BnaC04ARL overexpressing transgenic plants.

[0069] Using DNA from the T0 / T1 generation of transgenic plants as templates, the obtained overexpressing rapeseed transgenic lines were positively identified using primers OE-BnaC04ARL-F+Dsred-R and OE-BnaC04ARL-R+Dsred-F. Positive plants were ultimately obtained (those showing the target band in the electrophoresis image were considered positive). Alternatively, a red fluorescent protein excitation gun (LUYOR-3415CG) was used to irradiate transgenic seedlings with the vector's red labeling element; plants emitting red light from their roots were considered positive.

[0070] Primers for positive identification of overexpression transgenic plants:

[0071] OE-BnaC04ARL-F: 5'-CGGGATCCATGATTCGTGAAATCTCTGG-3',

[0072] OE-BnaC04ARL-R: 5'-CCCAAGCTTTTACATATAAGTTCTTGTTACATGTT-3',

[0073] Dsred-F: 5'-TGGAGAGGACACTAGTGGATCC-3',

[0074] Dsred-R: 5'-TCATGCGATCATAGGCGTCTC-3'.

[0075] 3. Obtaining the BnaC04ARL interference line

[0076] See Figure 1d. The CDS sequences of the homologous genes of BnaC04ARL were downloaded from the BnIR website (http: / / yanglab.hzau.edu.cn / BnIR). Sequence alignment was performed using Alignment software, and the fragment with the highest sequence similarity (100-200 bp) among several homologous genes was selected as the interference fragment. Primers (ARL-RNAi-F: 5'-GGATCCGACGTCCGCTCTTCAAGCAGCAACA-3'; ARL-RNAi-R: 5'-TCTAGACCATGGAAGGCATTAAAGCAAGAACC)-3' were designed using Primer Premier5 software. PCR amplification was performed using cDNA from the Brassica napus variety "Zhongshuang 11" as a template. The target fragment was recovered, purified, and ligated into the pFGC5941M vector (interference vector). The ligation method for the interference vector involved double digestion of the vector at Aat11 and NcoI sites. The forward fragment was first ligated to the interference vector, followed by ligation to the reverse fragment. The correctly sequenced plasmid was transformed into Agrobacterium tumefaciens GV3101, and then, using rapeseed variety J9709 as the recipient material, Agrobacterium-mediated transformation was employed to obtain transgenic plants with impaired BnaC04ARL function.

[0077] Given the complexity of the interference vector, it is necessary to perform DNA level identification of the forward and reverse fragments of the transgenic plants separately. The primers used for reverse fragment identification are ARL-RNAi-F+35S and ARL-RNAi-R+PAP-R; the primers used for forward fragment identification are ARL-RNAi-F+OCSR and ARL-RNAi-R+PAP-F.

[0078] Interfering with positive identification of plants:

[0079] ARL-RNAi-F:5'-GGATCCGACGTCCCGCTCTTCAAGCAGCAACA-3',

[0080] ARL-RNAi-R:5'-TCTAGACCATGGAAGGCATTAAAGCAAGAACC-3',

[0081] PAP-F:5'-GACGTCAGGTTTACATTCAAGACACA-3',

[0082] PAP-R:5'-GGATCCACCTAAGCATGCATTTGAAAA-3',

[0083] 35S:5'-GGAAGTTCATTCATTTGGAGAG-3',

[0084] OCSR:5'-GCTCAGGTTTTTTACAACGTGCAC-3'.

[0085] In this embodiment, the selected interference fragment is shown in the sequence list SEQ ID NO.3.

[0086] 4. Quantitative analysis of transgenic lines

[0087] RNA was extracted from leaves of transgenic plants at the same growth stage using the Trizol method. The cDNA obtained by reverse transcription was used to quantitatively analyze the target gene BnaC04ARL using primers (BnaC04ARL-qPCR-F: 5'-TCTCACAGCATCGCTCTTG-3'; BnaC04ARL-qPCR-R: 5'-GGCATTAGAAGAAGAAGAAGAAGA-3'). Real-time quantitative PCR was performed according to the instructions of the 2×TSINGKE® Master qPCR Mix (SYBR1) from Beijing Qingke Company, using the CFX96 Real-Time PCR Detection System. A standard three-step amplification procedure was adopted, as detailed below.

[0088] Table 5 qPCR reaction system

[0089]

[0090] Table 6. qPCR Three-Step Amplification Procedure

[0091]

[0092] After quantitative analysis, the overexpression lines OE-1 and OE-2, which showed good overexpression and interference effects, and the interference lines Ri-1 and Ri-2 were selected for subsequent experiments. WT was the wild-type control plant.

[0093] 5. Assessment of drought tolerance in BnaC04ARL transgenic plants

[0094] (1) Phenotypic changes and comparison of various physiological indicators of transgenic plants before and after drought treatment

[0095] To verify the response pattern of BnaC04ARL to drought stress, natural drought treatment was applied to the Shuang 11 variety of Brassica napus when it reached the three-leaf stage. RNA was extracted from leaves after 0, 24, 48, 72, and 96 hours of drought treatment, and the expression of BnaC04ARL was quantitatively analyzed. As shown in Figure 2a, the expression level of BnaC04ARL increased with the extension of drought treatment time, indicating that its expression was induced by drought. Plump seeds from each line were sown normally in glass dishes lined with moist filter paper. After the cotyledons emerged, the seedlings were transplanted into the soil. Transgenic plants overexpressing and interfering with BnaC04ARL, as well as wild-type controls, were subjected to natural drought and rehydration treatments during the seedling stage, and their phenotypes were observed. Multiple physiological indicators, DAB and NBT staining observations, stomatal observations, and leaf water loss rate measurements were performed on each line after drought treatment.

[0096] Referring to Figure 2d, before drought treatment, there was no significant difference in leaf freshness between the BnaC04ARL overexpressing plants and the interference plants and the control. One week after drought treatment, the overexpressing plants showed mild wilting, the wild type showed moderate wilting, and the interference transgenic plants showed severe wilting. After 3 days of rehydration treatment, the BnaC04ARL overexpressing plants had the highest survival rate, while the interference transgenic plants had the lowest survival rate (Figure 2f). These results indicate that overexpression of the BnaC04ARL gene can enhance the drought resistance of Brassica napus. Referring to Figure 2io, after 5 days of drought treatment on seedlings of various genotypes, the proline, chlorophyll, soluble sugar, and soluble protein contents of the BnaC04ARL overexpressing transgenic lines were significantly higher than those of the wild type and the interference lines, while the malondialdehyde and hydrogen peroxide contents were significantly lower; the DAB and NBT staining was lighter, indicating a lower degree of drought stress, suggesting that the overexpressing plants have stronger drought resistance. In addition, by measuring the water loss rate of normal detached leaves and irradiating rapeseed with infrared spectra before and after drought and after rehydration to determine the differences in stomatal water loss of different lineages based on leaf surface temperature (e, g, h in Figure 2), it was found that there were no obvious differences or patterns of change among the different genotypes, indicating that the enhanced drought resistance of BnaC04ARL overexpression transgenic plants is not closely related to the aboveground parts.

[0097] (2) Changes in root development of transgenic plants after stress treatment

[0098] Root morphology was determined using hydroponics. Materials of each genotype were hydroponically cultured in 10% (w / v) PEG 6000, 100 mM Mannitol, and 100 mM NaCl solutions to simulate drought, with pure water serving as a control. The treatment lasted for 3 weeks. As shown in Figure 3, under all simulated drought conditions, the number of lateral roots in the BnaC04ARL overexpression transgenic lines was significantly higher than that in the wild-type and interference lines, and their underground fresh and dry weights were also significantly higher. This indicates that the enhanced drought tolerance of BnaC04ARL overexpression transgenic plants is related to the root development characteristics of different lines.

[0099] (3) Investigation of agronomic traits of each genotype line after drought treatment

[0100] Each genotype line was transplanted to the field for cultivation and management. Normal watering was applied before flowering, followed by a drought treatment 10 days after flowering (watering once every 15 days) until maturity. Multiple agronomic traits were investigated, and relevant data were processed using SPSS 26 software and plotted using Graph Pad Prism 9 software.

[0101] Referring to Figure 4, the seeds of the BnaC04ARL overexpressing transgenic plants after drought treatment were rounder and fuller (Figure 4b). Their thousand-seed weight, single-plant seed yield, total number of siliques, silique length, single-plant dry weight, and plant height were all significantly higher than those of the wild-type and interference lines (Figure 4di). Conversely, the above indicators for the BnaC04ARL interference transgenic lines were all lower than those of the wild-type and overexpressing lines. There was no significant difference in the number of seeds per silique between the overexpressing and interference plants and the wild-type (Figure 4j).

[0102] In the description of this invention, it should be understood that "-" and "~" represent a range of two values, and this range includes the endpoints. For example, "AB" represents a range greater than or equal to A and less than or equal to B. "A~B" represents a range greater than or equal to A and less than or equal to B.

[0103] In the description of this invention, the term "and / or" is merely a description of the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can represent three cases: A exists alone, A and B exist simultaneously, and B exists alone.

[0104] In the description of the invention, the numerical values ​​of time, temperature, ratio, and mass involved can be based on actual measurements, standard equipment parameters, simplified rounding results, or within an acceptable error range, ensuring the practicality and repeatability of the invention.

[0105] In the description of this invention, the terms “about” or “approximately” are used to express approximate values ​​or ranges, allowing for a certain degree of error to ensure the flexibility and practicality of the description, while remaining within an acceptable range of error, with the maximum error not exceeding 10% of the corresponding value or range.

[0106] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the drought-resistant gene BnaC04ARL in Brassica napus, characterized in that, The nucleotide sequence of the BnaC04ARL gene is shown below: A. The nucleotide sequence shown in SEQ ID NO:1; B. The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2; The purpose is as follows: A. Improving the survival rate of Brassica napus under drought conditions; B. Enhance root development in Brassica napus; And / or, C. Improve agronomic traits of rapeseed, including thousand-grain weight, single-plant grain yield, total number of pods, single-plant dry weight and / or pod length.

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