Application of BnaA06. TINY gene, primer pair and recombinant vector

Abstract

The invention discloses application of a BnaA06. TINY gene, a primer pair and a recombinant vector. The gene is used for improving the drought resistance of brassica napus. The gene can significantly enhance the drought resistance of brassica napus by increasing the root length and root-shoot ratio of brassica napus plants, reducing the accumulation of H2O2 and superoxide ions in leaves, reducing the MDA content and increasing the content of soluble sugar, proline and soluble protein in the leaves. The invention also provides a specific primer pair for amplifying the gene and a recombinant vector containing the gene, and the specific primer pair can be used for genetic transformation of the brassica napus so as to improve the drought resistance of the brassica napus. Through an agrobacterium tumefaciens-mediated transformation method, the gene is introduced into brassica napus cells and is expressed, so that the viability of brassica napus under drought conditions can be effectively improved, and a new gene resource and an effective method are provided for cultivating a drought-resistant brassica napus variety.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and relates to an application of a Brassica napus drought stress resistance gene, a specific primer pair for amplifying the BnaA06.TINY gene, a recombinant vector containing the BnaA06.TINY gene, and a method for improving the drought resistance of Brassica napus. Background Art

[0002] As one of the main oil crops in my country, Brassica napus occupies an extremely important position in agricultural production and edible oil supply, especially in arid and semi-arid areas. It has a wide planting area and has the advantages of high yield, high-quality oil content, strong adaptability, cold resistance, moisture resistance and fertilizer resistance. It is the dominant variety in my country's southern winter rapeseed area, accounting for more than 80% of the planting area.

[0003] However, with global warming, abiotic stresses such as drought, high temperature, low temperature and soil water deficiency are becoming more frequent and intense, seriously restricting the growth, development, yield and quality of Brassica napus. Drought stress has become one of the key factors restricting the production of Brassica napus. Drought can lead to insufficient soil moisture, making it impossible for rapeseed plants to absorb enough water normally, which in turn affects their photosynthesis, nutritional metabolism and growth and development, ultimately causing slow plant growth, reduced yield and reduced quality, causing huge economic losses to agricultural production and posing a serious threat to the safety of my country's edible oil industry.

[0004] In the process of plant response to adverse stress, gene expression regulation plays a key role, among which transcription factors are important regulatory factors. The AP2 / ERF transcription factor family is one of the largest transcription factor families in plants, and its members play an important role in plant growth and development and stress response. As an important branch of the AP2 / ERF family, the DREB subfamily can be divided into six subgroups (A1-A6) according to sequence homology. Members of different subgroups can respond to different abiotic stresses, such as drought, low temperature, high salt, etc., and regulate the expression of downstream genes by binding to cis-acting elements such as ABRE, DRE / CRT, GCC box, etc., thereby enhancing the stress resistance of plants.

[0005] Currently, there is no literature reporting the drought resistance function and expression pattern of AP2 / ERF transcription factor BnaA06.TINY in Brassica napus. The members of the DREB subfamily in Brassica napus and their structural characteristics are still unclear. Summary of the invention

[0006] In view of this, the present invention provides an application of the BnaA06.TINY gene in improving the drought stress resistance of Brassica napus based on the newly discovered functional gene BnaA06.TINY gene. At the same time, the present invention also provides a specific primer pair for amplifying the BnaA06.TINY gene, a recombinant vector containing the BnaA06.TINY gene, and a method for improving the drought resistance of Brassica napus.

[0007] The inventors have continuously innovated and reformed through long-term exploration and attempts, as well as multiple experiments and efforts, to solve the above technical problems. The technical solution provided by the present invention is to provide a use of the BnaA06.TINY gene, wherein the nucleotide sequence of the BnaA06.TINY gene is as follows:

[0008] The nucleotide sequence shown in SEQ ID NO: 1 in the sequence listing; or

[0009] A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

[0010] The BnaA06.TINY gene is used to improve the drought resistance of Brassica napus.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] Through in-depth research, the present invention found that the BnaA06.TINY gene plays a unique and important role in improving the drought resistance of Brassica napus. Overexpression of this gene can significantly increase the root length and root-to-crown ratio of Brassica napus plants, making their root system more developed, thereby more effectively absorbing water and nutrients from the soil. At the same time, the BnaA06.TINY gene can reduce the H in the leaves of rapeseed plants. 2 O 2 The accumulation of superoxide ions and MDA content in leaves was reduced, indicating that it can reduce the oxidative damage and membrane lipid peroxidation degree of plant cells under drought stress. In addition, after drought treatment, the soluble sugar content, proline content and soluble protein content in the leaves of plants overexpressing the gene were significantly higher than those of the wild type. The increase of these substances helps the plant maintain the osmotic pressure balance of the cells and enhances its physiological adaptability under drought conditions. The introduction of the BnaA06.TINY gene into Brassica napus cells by Agrobacterium-mediated transformation can effectively improve the drought resistance of Brassica napus, providing new gene resources and theoretical basis for cultivating new varieties of Brassica napus with strong drought resistance. The present invention provides new ideas and methods for drought-resistant breeding of Brassica napus, and has important application value.

[0013] On the basis of the above technical solution, the present invention can also be improved as follows:

[0014] Further: the BnaA06.TINY gene improves the drought resistance of Brassica napus in at least the following ways:

[0015] Increase root length and root-to-shoot ratio of rapeseed plants;

[0016] Reducing H in rapeseed plant leaves 2 O 2 and accumulation of superoxide ions;

[0017] Reduce the MDA content in the leaves of rapeseed plants;

[0018] Improve the soluble sugar content, proline content and soluble protein content in the leaves of rapeseed plants after drought treatment.

[0019] Compared with the prior art, the beneficial effects of adopting the above further technical solution are:

[0020] The BnaA06.TINY gene significantly improved the drought resistance of Brassica napus by enhancing the root absorption capacity, reducing oxidative damage, and improving the plant's osmotic regulation ability, providing a more comprehensive and effective solution for breeding drought-resistant rapeseed varieties.

[0021] The present invention also provides a specific primer pair for amplifying the BnaA06.TINY gene, and the sequence of the primer pair is as follows:

[0022] TINY-F2: 5'-cgggatccatgacagcattagacagtacgttca-3';

[0023] TINY-R2: 5'-cccaagcttttaataattaaacagtcctgaaagatc-3'.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The specific primer pair provided by the present invention can efficiently and specifically amplify the BnaA06.TINY gene, providing a powerful tool for further research and application of the gene. The primer pair can accurately detect and quantify the BnaA06.TINY gene, which is helpful for in-depth research on its functional mechanism at the molecular level, and at the same time provides a convenient and reliable detection method for drought-resistant breeding of Brassica napus, promoting the rapid cultivation and promotion of drought-resistant varieties.

[0026] The present invention also provides a recombinant vector containing the BnaA06.TINY gene, wherein the recombinant vector is a BnaA06.TINY overexpression vector or an interference vector.

[0027] Furthermore, the overexpression vector is constructed by inserting the BnaA06.TINY gene into the Dsred expression vector; and the interference vector is constructed by inserting the BnaA06.TINY gene into the pFGC5941M vector.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] The recombinant vector provided by the present invention constructs an efficient overexpression and interference vector by inserting the BnaA06.TINY gene into the Dsred expression vector and the pFGC5941M vector, respectively, and provides a powerful tool for studying the function of the BnaA06.TINY gene. Using these vectors, the expression level of the BnaA06.TINY gene in Brassica napus can be accurately regulated, thereby providing an important means for in-depth exploration of the molecular mechanism of the gene in the drought resistance process of Brassica napus, and helping to promote the progress of drought-resistant breeding of Brassica napus.

[0030] The present invention also provides a method for improving the drought resistance of Brassica napus. The method comprises introducing the BnaA06.TINY gene into Brassica napus cells to express the gene in the Brassica napus cells.

[0031] Furthermore, the introduction is carried out by Agrobacterium-mediated transformation.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] The method for improving the drought resistance of Brassica napus provided by the present invention uses an Agrobacterium-mediated transformation method to introduce the BnaA06.TINY gene into Brassica napus cells, and can efficiently and stably achieve the expression of the gene in the rapeseed cells. The transgenic Brassica napus plants obtained by the method show significantly enhanced drought resistance, including a more developed root system, stronger antioxidant capacity and higher osmotic regulating substance content, thereby providing a practical technical solution for the drought resistance breeding of Brassica napus, helping to cultivate excellent varieties that adapt to drought environments and ensure the yield and quality of rapeseed. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 This is the BnaA06.TINY promoter cis-element analysis provided in the embodiments of the present invention. Figure 1 In the figure, dark red is marked as abscisic acid response element, bright red, dark yellow and bright green are light response elements, dark green is jasmonic acid response element, light blue is dehydration response element, gray is ethylene response element, orange is MYB binding site involved in drought induction, and purple is jasmonic acid response element.

[0036] Figure 2 The invention provides the acquisition and identification of the BnaA06.TINY overexpressing rapeseed provided in the embodiments of the present invention. Figure 2 In the figure, a: BnaA06.TINY overexpression vector; b: molecular identification, M is a 2000 bp marker; c: positive plant identification; d: RNA expression level identification: ** indicates extremely significant difference at the P<0.01 level.

[0037] Figure 3 The invention provides the acquisition and identification of the BnaA06.TINY interference plant provided in the embodiments of the present invention. Figure 3 In the figure, a: BnaA06.TINY interference vector; b: molecular identification: A. Primers 35S-F and RNAi-F, B. Primers RNAi-R and BnPAPI2-R, C. Primers BnPAPI2-F and RNAi-R, D. Primers RNAi-F and OCS-R, M is a 2000 bp marker; c: RNA expression level identification: ** indicates extremely significant difference at the P<0.01 level.

[0038] Figure 4 The drought phenotype and root growth of BnaA06.TINY overexpressed rapeseed provided by the embodiments of the present invention. Figure 4 a: Observation of taproot length phenotype before drought; b: Observation of taproot length phenotype after drought; c: Observation of phenotype after drought treatment; d: Observation of phenotype after rewatering; e: Statistics of plant survival rate after drought treatment; f: Statistics of taproot length before and after drought; g: Statistics of root-to-crown ratio before and after drought: ** indicates extremely significant difference at the P<0.01 level.

[0039] Figure 5 The BnaA06.TINY provided in the embodiment of the present invention interferes with the drought phenotype of rapeseed. Figure 5 In the figure, a: phenotypic observation before drought; b: phenotypic observation after drought rewatering; c: statistical data of plant survival rate after drought. ** indicates extremely significant difference at the P<0.01 level.

[0040] Figure 6 This is the physiological index determination of BnaA06.TINY overexpressing rapeseed under drought stress provided by the embodiments of the present invention. Figure 6a: DAB staining; b: NBT staining; c: malondialdehyde content; d: soluble sugar content; e: proline content; f: soluble protein content: ** indicates extremely significant difference at the P<0.01 level.

[0041] Figure 7 The embodiments of the present invention provide the experimental results of water loss rate of detached leaves of BnaA06.TINY overexpressing rapeseed. Figure 7 In the figure, a: wilting of detached leaves; b: statistical graph of water loss rate. ** indicates extremely significant difference at the P<0.01 level.

[0042] Figure 8 This is the root length observation of BnaA06.TINY overexpressing rapeseed after ACC and ethephon treatment provided by the embodiment of the present invention. Figure 8 In the figure, a: Observation of root length phenotype after ACC treatment; b: Observation of root length phenotype after ethephon treatment; c: Statistics of taproot length before and after ACC treatment; d: Statistics of taproot length before and after ethephon treatment: ** indicates extremely significant difference at the P<0.01 level. DETAILED DESCRIPTION

[0043] The following describes the invention in conjunction with specific embodiments.

[0044] In order to make the purpose, 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 combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work 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 invention claimed for protection, but merely represents selected embodiments of the present invention.

[0045] In the present invention, unless otherwise specified, all equipment and raw materials can be purchased from the market or are commonly used in the industry. The methods in the following embodiments, unless otherwise specified, are conventional methods in the art.

[0046] In this embodiment, the nucleotide sequence of the BnaA06.TINY gene is shown in the sequence listing SEQ ID NO: 1, and the amino acid sequence encoded based on the nucleotide sequence is shown in the sequence listing SEQ ID NO: 2.

[0047] Example 1

[0048] This example describes the cloning of the BnaA06.TINY gene.

[0049] Fresh leaves of Brassica napus (variety J9709) were selected, quick-frozen in liquid nitrogen and ground into powder, and total plant RNA was extracted using the Trizol method.

[0050] Genomic DNA contamination was removed using a gDNA removal kit (Nanjing Novozymes Corporation).

[0051] The gDNA removal system is: Total RNA 1.0μg, gDNA Eraser 1.0μL, 5×gDNA EraserBuffer 2.0μL, RNase-Free ddH 2 O to 10.0 μL, place the sample in a PCR instrument at 42°C for 2 min, and place on ice for the next step.

[0052] The reverse transcription reaction system is: gDNA removal system 10.0μL, 5× Buffer 2 (for RealTime) 4.0μL, RT Primer Mix 1.0μL, RT Enzyme Mix 1 1.0μL, RNase-FreeddH 2 O 4.0μL, place the sample in a PCR instrument, treat it according to the program of 37℃15min, 85℃5s, and place it on ice for the next step.

[0053] Use the reverse transcription reaction system to synthesize cDNA using the total RNA as a template using a reverse transcription kit (Beijing Qingke Company). Design specific primers:

[0054] TINY F2: 5'-cgggatccatgacagcattagacagtacgttca-3'

[0055] TINY R2: 5'-cccaagcttttaataattaaacagtcctgaaagatc-3'

[0056] PCR reaction conditions: 95°C for 1 min, 95°C for 20 s, (Tm-5)°C for 20 s, 72°C for 2 kb / min [40 cycles of 95°C, (Tm-5)°C, and 72°C], 72°C for 10 min, and 12°C for 1 h for PCR amplification.

[0057] PCR reaction system: cDNA 1.0μL, 5×TransStart FastPfu Buffer 10.0μL, TransStartFastPfu DNA Polymerase 1.0μL, forward primer 1.0μL, 2.5mM dNTPs 4.0μL, reverse primer 1.0μL, ddH 2 O to 50.0 μL. The amplified samples were detected by agarose gel electrophoresis.

[0058] After PCR amplification, gel recovery was performed using the FastPure Gel DNA Extraction Mini Kit from Beijing Quanshijin Biotechnology Co., Ltd. The steps are as follows:

[0059] Cut the target band obtained in the previous step from the gel, place it in a 2mL centrifuge tube, add 3 times the volume of GDP solution of the gel, and heat it in a 50℃ water bath for 10 minutes until the gel is completely dissolved. After adding 100μL of isopropanol, transfer the sample to the adsorption column, centrifuge at 12000rpm for 1min, and discard the filtrate. Add 700μL of rinse solution (with anhydrous ethanol added) to the adsorption column, centrifuge at 12000rpm for 1min, discard the filtrate, and repeat the above operation. Put the adsorption column back into the collection tube, centrifuge at 12000rpm for 2min, transfer the adsorption column to a new 1.5mL centrifuge tube, and let it stand for 3-5min. Add 20μL of ddH2O to the adsorption column. 2 O, stand at room temperature for 1 min, then centrifuge at 12000 rpm for 1 min to obtain the gel recovery product, which can be temporarily stored in a 4°C refrigerator.

[0060] After gel recovery, enzyme digestion and ligation are performed. The operation steps are as follows:

[0061] According to the designed primer restriction sites, add the corresponding restriction endonucleases to the vector and target fragment. Taking the construction of overexpression vector as an example, perform restriction digestion according to the double restriction digestion reaction system. Place the centrifuge tube with the sample on a PCR instrument for restriction digestion at 37°C for 30 minutes, then perform agarose gel electrophoresis on the digested sample and recover the gel. Connect the products obtained above according to the connection system. Place the mixed system on a PCR instrument at 25°C for 2 hours, and then proceed to the next transformation operation with the connected sample. Double restriction digestion reaction system: BamHI 3.0μL, SpeI 3.0μL, gel recovery product / plasmid 3.0μg, Buffer 3.0μL, ddH 2O to 30.0 μL. Ligation system: cloned gene fragment 1.0-7.0 μL, linearized vector 1.0 μL, 10×T4 DNA Ligase Buffer 1.0 μL, T4 DNA Ligase 1.0 μL, ddH 2 Add 3% HO to 10.0 μL.

[0062] Finally, E. coli was transformed and plasmid was extracted, and the gene sequence was sequenced to verify the correctness. The nucleotide sequence is shown in the sequence list SEQ ID NO: 1. The amino acid sequence encoded by the nucleotide sequence is shown in the sequence list SEQ ID NO: 2.

[0063] Example 2

[0064] This example describes the construction of Brassica napus overexpression vector, interference vector and analysis of cis-elements of the BnaA06.TINY gene promoter.

[0065] Construction of BnaA06.TINY overexpression vector:

[0066] Specific amplification primers were designed according to the downloaded CDS sequence of the BnaA06.TINY gene (SEQ ID NO: 1), and the primer sequences are as follows:

[0067] TINY-F2: 5'-cgggatccatgacagcattagacagtacgttca-3';

[0068] TINY-R2: 5'-cccaagcttttaataattaaacagtcctgaaagatc-3'.

[0069] The BnaA06.TINY gene was inserted into the Dsred expression vector, and the BamHI+HindIII restriction enzyme combination was used, and PCR amplification was performed with the designed primers, and then the expression vector of the BnaA06.TINY gene was obtained through the above-mentioned vector construction process.

[0070] BnaA06.TINY interference vector construction:

[0071] Specific amplification primers were designed according to the CDS sequence of the BnaA06.TINY gene. The primer sequences are as follows:

[0072] TINY-RNAi-F: 5'-ggatccgacgtcctctcaaagctgcacacatggaaat-3';

[0073] TINY-RNAi-R: 5'-tctagaccatggggtgaggtggataaacccaaaagtc-3'.

[0074] The BnaA06.TINY gene was inserted into the pFGC5941M vector, and the BamHI+XbaI restriction enzyme combination was used, and PCR amplification was performed with the designed primers, and then the BnaA06.TINY gene interference expression vector connected with the sense fragment was obtained through the above vector construction process. The vector was digested with NcoI and AatII, and the obtained linear vector was connected with the gene fragment digested with the same enzymes to obtain the BnaA06.TINY gene interference expression vector connected with both the sense fragment and the antisense fragment.

[0075] Analysis of cis-elements in the promoter of BnaA06.TINY gene:

[0076] To understand the potential function of the BnaA06.TINY gene and the regulatory network it may be involved in, the promoter region 2000 bp upstream of ATG was analyzed using the PlantCare website. Figure 1 As shown in the figure, the promoter of this gene contains a large number of hormone response elements, such as abscisic acid response element ABRE, ethylene response element ERE, jasmonic acid response element CGTCA-motif and TGACG-motif, etc.; dehydration response element DRE core; cis-acting element TC-richrepeats in defense and stress; multiple light response elements; MYB binding site MBS, which is involved in drought induction. Therefore, it is speculated that the BnaA06.TINY gene may be involved in the ABA and ethylene pathways and play an important role under adverse stress. Figure 1 The distribution of various hormone and environmental response elements in the promoter region of the BnaA06.TINY gene was displayed, indicating that this gene may play a role in a variety of plant hormone and environmental stress response pathways, providing a theoretical basis for the study of its function under drought stress.

[0077] Example 3

[0078] This example describes the genetic transformation of Brassica napus and the screening of positively identified transgenic plants.

[0079] In this embodiment, the culture media used are:

[0080] M0 medium: weigh 2.2 g MS powder and 30 g sucrose, and mix with ddH 2 After O is fully dissolved, the volume is adjusted to 1L, the pH is adjusted to 5.84-5.88, 8g of agar powder is added, and the mixture is sterilized at 121℃ and high temperature and high pressure for 15min. The mixture is cooled to room temperature and poured into plates when the temperature drops to about 50℃. After the culture medium solidifies in the culture dish, it is sealed and stored at 4℃.

[0081] M1 medium: weigh 4.4 g MS powder, 18 g mannitol, 30 g sucrose, 0.5 mL 2,4-D (1 mg / mL), 0.5 mL KT (0.3 mg / mL), and use ddH 2 After O is fully dissolved, the volume is adjusted to 1L, the pH is adjusted to 5.84-5.88, 8g of agar powder is added, and the mixture is sterilized at 121℃ and autoclaved for 15min. The mixture is cooled to room temperature, and 1mL of AS (100μmol / mL) is added to the culture dish after it solidifies and then stored at 4℃.

[0082] M2 medium: weigh 4.4 g MS powder, 18 g mannitol, 30 g sucrose, 0.5 mL 2,4-D (1 mg / mL), 0.5 mL KT (0.3 mg / mL), and use ddH 2 After O is fully dissolved, the volume is adjusted to 1L, the pH is adjusted to 5.84-5.88, 8g of agar powder is added, and the mixture is sterilized at 121℃ and autoclaved for 15min. The mixture is cooled to room temperature and 1mL TMT (200mg / mL) and 0.5mL Hyg (50mg / mL) are added to the culture dish after it solidifies and then stored at 4℃.

[0083] M3 medium: weigh 4.4 g MS powder, 0.25 g xylose, 10 g glucose, 0.6 g MES, and use ddH 2 After O is fully dissolved, the volume is adjusted to 1L, the pH is adjusted to 5.84-5.88, 8 g of agar powder is added, and the mixture is sterilized at 121°C and autoclaved for 15 min. The mixture is cooled to room temperature and cooled to about 50°C. 1 mL of TMT (200 mg / mL), 0.5 mL of Hyg (50 mg / mL), 1 mL of ZT (2 mg / mL), 0.2 mL of IAA (0.5 mg / mL), and 0.15 mL of silver nitrate (20 mg / mL) are added, mixed well, and poured onto a plate. After the culture medium solidifies in the culture dish, it is sealed and stored at 4°C.

[0084] M4 medium: weigh 4.4 g MS powder, 30 g sucrose, and use ddH 2 After O is fully dissolved, the volume is adjusted to 1L, the pH is adjusted to 5.84-5.88, 8g of agar powder is added, and the mixture is sterilized at 121℃ and high temperature and high pressure for 15min. The mixture is cooled to room temperature, and when the temperature drops to about 50℃, 100μL of NAA (0.5mg / mL) is added, mixed and poured into a glass bottle. After the culture medium solidifies, it is sealed and stored at room temperature.

[0085] DM solution: weigh 4.4g MS powder and 30g sucrose, use ddH 2After O is fully dissolved, the volume is adjusted to 1 L, the pH is adjusted to 5.84-5.88, and sterilized at 121°C for 15 min. The mixture is cooled to room temperature. When the temperature drops to about 50°C, 1 mL of AS (100 μmol / mL) is added and mixed. After cooling, the mixture is sealed at 4°C.

[0086] YEB liquid medium (Yeast Extract Mannitol Broth) (1L): 1g YE, 5g beef extract, 5g peptone, 5g sucrose, 0.5g MgSO4·7H 2 O, dissolve with pure water and make up to 1L, sterilize in an autoclave at 121℃ for 15min, add required antibiotics after cooling to room temperature and store in a refrigerator at 4℃.

[0087] The steps for obtaining transgenic plants by genetic transformation of rapeseed are as follows:

[0088] Select full and rounded seeds of Brassica napus J9709, disinfect them with 75% ethanol for 2 minutes, 50% 84 disinfectant for 5 minutes, and wash them with sterile water for 3 to 5 times. Sow the seeds in a seeding box containing M0 medium, with about 25 seeds in each seeding box, and culture the seeding box in a dark environment for 7 days. After 5 days of dark culture, the overexpression and interference vector Agrobacterium liquid of BnaA06.TINY gene was cultured overnight with YEB liquid medium containing antibiotics, and then cultured twice according to the concentration of the bacterial liquid, and a concentration gradient culture was set to obtain the optimal OD value of 0.4. Open the rapeseed seeding box that has been cultured in the dark for 7 days in the clean bench, and cut the hypocotyl of about 1 cm in length with sterile scissors and place it in a sterile culture dish containing 18mL DM solution. The cultured Agrobacterium liquid was aspirated into a 2mL centrifuge tube and centrifuged at 10000rpm for 1min to collect the bacteria. After resuspending the bacteria with 2mL DM solution, pour it into the culture dish containing the hypocotyl. Gently shake the culture dish to allow the bacterial solution to fully contact the hypocotyls. The infection process lasts for 10 minutes. When the infection lasts for 8 minutes, start to use a pipette to suck the infection solution in the culture dish and discard it. Then use sterile tweezers to place the hypocotyls on sterile filter paper and absorb the residual infection solution. Use sterile tweezers to pick up the hypocotyls on the filter paper and place them evenly on the M1 medium. Culture them in the dark for 2 days. Transfer the hypocotyls in M1 to the M2 medium, and culture the medium under normal conditions for 20 days. Use sterile tweezers to pick up the explants on the M2 medium and transfer them to the M3 medium. Similarly, place the medium in the tissue culture room. Replace the M3 medium every 14 days until the explants grow normal green leaves. Use sterile tweezers to pick up the explants with green leaves and transfer them to the M4 medium for rooting. When the leaves are covered, remove the rapeseed plants from the M4 medium and culture them hydroponically. Positive identification is performed during the hydroponic process, and then the positive plants are transplanted into the soil for cultivation.

[0089] The overexpressed rapeseed in hydroponics was sampled, and leaf DNA was extracted and used as a template. PCR amplification was performed using the above-mentioned gene primers and the universal primers on the vector. The PCR products were then detected by agarose gel electrophoresis. The strains with bands for both pairs of primers were positive plants. The interfered rapeseed in hydroponics was sampled, and leaf DNA was extracted and used as a template. PCR amplification was performed using the above-mentioned gene primers and the universal primers on the vector (35-S+TINY-RNAi-F, TINY-RNAi-R+BnPAPI2-R, BnPAPI2-F+TINY-RNAi-R, TINY-RNAi-F+OSC-R). The PCR products were then detected by agarose gel electrophoresis. The strains with bands for all four pairs of primers were positive plants ( Figure 2 (b)

[0090] Construction of BnaA06.TINY overexpression vector ( Figure 2 a), and transformed rapeseed variety J9709 by Agrobacterium-mediated method to obtain rapeseed seedlings. DNA extraction and PCR positive identification were performed on the obtained plants to obtain BnaA06.TINY overexpression positive plants ( Figure 2 b). After germination, the roots of the seedlings were illuminated with a red fluorescence excitation gun, and it was found that the roots of the overexpression plants showed a distinct bright red color, while the roots of the wild-type plants showed a dark red color with no fluorescence, further indicating that the vector transformation was successful and transgenic plants had been obtained ( Figure 2 Then the positive lines were multiplied to obtain two homozygous lines for subsequent experiments. The expression level of the homozygous lines was detected, and the results showed that the expression level of the BnaA06.TINY gene in the overexpressed plants was significantly higher than that in the wild type ( Figure 2 (d) Figure 2 Figure d in the middle shows that compared with the wild type (WT), the expression level of the BnaA06.TINY gene in the overexpressed plants of OETINY-1 and OETINY-2 was significantly increased, among which OETINY-2 had the highest expression level, reaching about 15 times that of the wild type, and the expression level of OETINY-1 was about 4 times that of the wild type. This shows that the overexpression of the BnaA06.TINY gene was successfully achieved through transgenic operation, providing a basis for subsequent research on the function of this gene.

[0091] Similarly, the BnaA06.TINY interference vector ( Figure 3 In a), the rapeseed genetic transformation method was used to obtain candidate interference rapeseed plants of BnaA06.TINY. DNA molecular identification was performed to determine whether both the positive and negative fragments on the interference vector were present in the plants, and positive plants were obtained ( Figure 3 b). Verify the expression level of the interference strain, see Figure 3Figure c shows the relative expression levels of the BnaA06.TINY gene in the wild type (WT) and three RNAi plants (TINY-RNAi-1, TINY-RNAi-2, TINY-RNAi-3). Compared with the wild type (WT), the expression levels of the BnaA06.TINY gene in the three RNAi lines were significantly reduced, among which the expression levels of TINY-RNAi-1 and TINY-RNAi-3 were significantly lower than those of the wild type. This shows that the expression of the BnaA06.TINY gene in Brassica napus was successfully inhibited by RNA interference technology, providing an important reverse genetics tool for studying the function of this gene.

[0092] Example 4

[0093] This example describes the expression analysis of BnaA06.TINY and the analysis of drought tolerance of transgenic plants.

[0094] Wild-type and BnaA06.TINY overexpressing rapeseed seeds were sown on MS medium. After 2 days of growth, the root length was qualitatively and quantitatively observed. It was found that the root length of the overexpressing plants was significantly longer than that of the wild-type.

[0095] The overexpressed rapeseed seeds were placed in a petri dish to germinate and grow normally, and then transplanted to soil for normal culture, and the root length and root-to-crown ratio were measured. The results showed that the root length and root-to-crown ratio of the BnaA06.TINY overexpressing plants were significantly higher than those of the wild type ( Figure 4 A and Figure 4 Medium f and Figure 4 (g).

[0096] In order to explore the response of overexpression rapeseed lines to drought stress, rapeseed plants that had grown normally for 14 days were subjected to natural drought treatment and rehydrated after 7 days. The root length and root-to-crown ratio of the plants were then measured 3 days after rehydration. The results showed that the root length and root-to-crown ratio of BnaA06.TINY overexpression plants were significantly higher than those of the wild type ( Figure 4 Medium b and Figure 4 Medium f and Figure 4 g), and the survival rate after drought was significantly higher than that of wild-type plants ( Figure 4 Medium c and Figure 4 Medium D and Figure 4 (e)

[0097] The experiment showed that overexpression of BnaA06.TINY gene may increase the root length and root-to-shoot ratio of rapeseed plants, making the plants more drought-resistant.

[0098] The disturbed plants that had grown normally for 21 days were subjected to natural drought treatment. After 7 days, the survival rate of the plants was observed after rehydration. It was found that the survival rate of the disturbed plants was significantly lower than that of the wild type ( Figure 5 a, b, c).

[0099] After 7 days of drought treatment, rapeseed leaves were treated with DAB (3,3'-Diaminobenzidine) Figure 6 a) and NBT (Nitrotetrazolium blue chloride) Figure 6 The results of staining in (b) showed that the leaves of the overexpression plants were lighter in staining and had fewer spots than those of the wild type, indicating that less H accumulated in the leaves of the overexpression plants. 2 O 2 and superoxide ions, which indicates that the plants were less damaged by drought stress and the plants were more drought tolerant.

[0100] The MDA content of overexpression and wild-type plants before and after drought treatment was measured, and it was found that the MDA content of plants grown under normal conditions was almost the same, but after drought treatment, the MDA content of overexpression plants was significantly lower than that of wild-type plants ( Figure 6 c). The soluble sugar content, proline content and soluble protein content of the overexpression and wild-type plants before and after drought treatment were tested. It was found that there was almost no difference in the soluble sugar content, proline content and soluble protein content of the plants grown under normal conditions, while the soluble sugar content, proline content and soluble protein content of the overexpression plants after drought treatment were significantly higher than those of the wild type ( Figure 6 This further demonstrates that the overexpressing plants have stronger drought resistance.

[0101] Drought stress can cause the rapid accumulation of ABA in plants, which then enhances the drought tolerance of plants by regulating leaf transpiration.

[0102] In this example, a water loss rate experiment was conducted on detached leaves of BnaA06.TINY overexpressing rapeseed. The results showed that all leaves wilted as time went by ( Figure 7 However, the water loss rate of detached leaves of overexpressing rapeseed was significantly slower than that of wild-type plants ( Figure 7 (b) This indicates that under the same environment, the water retention capacity of the overexpressing rapeseed leaves is stronger than that of the wild type.

[0103] BnaA06.TINY overexpressing rapeseed and wild type were sown on a medium containing ACC and cultured in the dark for 10 days. The results showed that the root length of overexpressing rapeseed was significantly shorter than that of wild type, which was opposite to the root length results on ordinary MS medium, indicating that ACC restricted the root elongation of BnaA06.TINY overexpressing rapeseed to a greater extent ( Figure 8 A and Figure 8In this example, overexpression and wild-type rapeseed grown normally were treated with ethephon for 7 days, and then the root length phenotype of rapeseed was observed. The results showed that the root length of overexpression plants was significantly shorter than that of wild-type plants ( Figure 8 Medium b and Figure 8 (d)

[0104] The above results indicate that ethylene limits the root growth of BnaA06.TINY-promoted overexpressing Arabidopsis to a greater extent.

[0105] In the description of the present invention, it is to be understood that "-" and "~" represent a range between two values, and the 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.

[0106] In the description of the present invention, the term "and / or" herein is merely a description of the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B may represent three situations: A exists alone, A and B exist at the same time, and B exists alone.

[0107] In the description of the invention, the numerical values ​​of time, temperature, ratio, mass, etc. involved may be based on actual measurements, standard parameters of equipment, simplified rounding results, or within an acceptable error range, ensuring the practicability and repeatability of the invention.

[0108] In the description of the present invention, the term "about" or "approximately" is used to express the approximate value of a numerical value or range, allowing a certain error to ensure the flexibility and practicality of the description while remaining within an acceptable error range, with the maximum error range not exceeding 10% of the corresponding numerical value or numerical range.

[0109] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, 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 regarded as the protection scope of the present invention.

Claims

1. A use of the BnaA06.TINY gene, characterized in that: The nucleotide sequence of the BnaA06.TINY gene is as follows: A. The nucleotide sequence shown in SEQ ID NO: 1; or B. The nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO.2 in the sequence table; The BnaA06.TINY gene is used to improve the drought resistance of Brassica napus.

2. The use according to claim 1, characterized in that The BnaA06.TINY gene improves the drought resistance of Brassica napus in at least the following ways: 1) Increase the root length and root-to-crown ratio of rapeseed plants; 2) Reduce the accumulation of H2O2 and superoxide ions in the leaves of rapeseed plants; 3) Reduce the MDA content in rapeseed plant leaves; 4) Increase the soluble sugar content, proline content and soluble protein content in the leaves of rapeseed plants after drought treatment.

3. A specific primer pair for amplifying the BnaA06.TINY gene according to claim 1, characterized in that: The sequences of the primer pairs are as follows: TINY-F2: 5'-cgggatccatgacagcattagacagtacgttca-3'; TINY-R2: 5'-cccaagcttttaataattaaacagtcctgaaagatc-3'.

4. A recombinant vector containing the BnaA06.TINY gene according to claim 1, characterized in that: The recombinant vector is a BnaA06.TINY overexpression vector or an interference vector.

5. The recombinant vector according to claim 4, characterized in that The overexpression vector is constructed by inserting the BnaA06.TINY gene into the Dsred expression vector; the interference vector is constructed by inserting the BnaA06.TINY gene into the pFGC5941M vector.

6. A method for improving the drought resistance of Brassica napus, characterized in that: The method comprises introducing the BnaA06.TINY gene of claim 1 into Brassica napus cells, so that the gene is expressed in the Brassica napus cells.

7. The method according to claim 6, characterized in that The introduction is carried out by Agrobacterium-mediated transformation.

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