Application of long-chain non-coding RNA lncRNA0410 in regulating and controlling drought resistance of brassica napus

Abstract

The invention relates to the field of plant genetic engineering, in particular to application of long-chain non-coding RNA lncRNA0410 in regulation and control of drought resistance of brassica napus. According to the invention, a 35S:: lncRNA0410 overexpression vector is constructed, and agrobacterium tumefaciens-mediated genetic transformation is utilized to obtain a transgenic rape material, so that the lncRNA0410 is successfully overexpressed. The drought resistance phenotype analysis is carried out on the overexpressed transgenic material, and the result shows that compared with the wild type, the lncRNA0410 overexpressed plant shows higher survival rate. The invention proposes and proves that the lncRNA0410 has a remarkable positive drought resistance regulation effect in the brassica napus for the first time. The gene can be widely applied to stress resistance improvement and related molecular breeding processes of oilseed rape, and has important theoretical value and application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to long non-coding RNA. lncRNA0410 Application in regulating drought resistance in Brassica napus. Background Technology

[0002] Drought is one of the most severe abiotic stresses in global agricultural production, particularly detrimental to rapeseed emergence, growth, flowering, and fruiting. Improving rapeseed drought resistance is a crucial goal for increasing and stabilizing yields. Long non-coding RNAs (lncRNAs) are defined as non-coding RNA molecules exceeding 200 nt in length, characterized by the lack of open reading frames, complex secondary structures, and the potential presence of functional domains. Based on genomic location, lncRNAs can be classified into five types: sense strand, antisense strand, bidirectional, intronic, and intergenic. LncRNAs are widely distributed in eukaryotes and play key regulatory roles in plant stress responses, regulating chromatin state, gene expression, small RNA production, and hormone signaling. However, functional studies in rapeseed remain relatively limited. In recent years, this field has primarily focused on coding genes, with few reports on lncRNA-mediated drought resistance mechanisms in rapeseed. Therefore, identifying lncRNAs in rapeseed and studying their molecular mechanisms in response to drought stress can deepen our understanding of rapeseed's adaptation mechanisms to drought stress, provide excellent drought-resistant gene resources, and contribute to the breeding of stress-resistant rapeseed varieties. Summary of the Invention

[0003] In view of this, the present invention provides a long non-coding RNA. lncRNA0410 Its application in regulating drought resistance in Brassica napus, through regulation lncRNA0410 The expression level of this substance can be used to achieve molecular regulation of drought resistance in Brassica napus, thereby promoting crop drought resistance and providing a new molecular tool for high-efficiency rapeseed breeding.

[0004] The specific plan is as follows: The first aspect of the present invention provides lncRNA0410 Use of genes or related biological materials in at least one of the following: A1) Regulate the drought resistance of Brassica napus or prepare products that regulate the drought resistance of Brassica napus; A2) Cultivate drought-resistant varieties of Brassica napus or prepare products that improve the drought resistance of Brassica napus; A3) Preparation of transgenic Brassica napus; The lncRNA0410 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0005] Furthermore, the biomaterial includes one or more of the following: A1)lncRNA0410 RNA transcribed from genes and / or containing the aforementioned lncRNA0410 Gene recombinant vectors, recombinant microorganisms, or transgenic Brassica napus cell lines; A2) Contains negative regulation lncRNA0410 Nucleic acid molecules expressing genes, recombinant vectors, recombinant microorganisms, or transgenic Brassica napus cell lines.

[0006] Furthermore, the regulation of drought resistance in Brassica napus includes: regulating the drought resistance of Brassica napus... BnaA03.MYB12 Gene expression.

[0007] Furthermore, the regulation of Brassica napus in... BnaA03.MYB12 Gene expression includes: through upregulation lncRNA0410 The expression of [a specific gene] positively regulates its downstream genes. BnaA03.MYB12 To improve the drought resistance of Brassica napus.

[0008] Furthermore, the regulation of Brassica napus in... BnaA03.MYB12 Gene expression includes: through downregulation lncRNA0410 The expression of [a specific gene] negatively regulates downstream genes. BnaA03.MYB12 To reduce the drought resistance of Brassica napus.

[0009] Furthermore, the aforementioned lncRNA0410 The nucleotide sequence of the RNA transcribed from the gene contains the sequence shown in SEQ ID NO.2.

[0010] Furthermore, the containing lncRNA0410 Gene recombination vectors include basic vectors and lncRNA0410 The basic carrier includes pCAMBIA1300 Carrier.

[0011] Furthermore, the containing lncRNA0410 Recombinant microorganisms include Agrobacterium tumefaciens GV3101.

[0012] The second aspect of the invention provides positive regulation lncRNA0410 The application of gene-expressing biomaterials in cultivating drought-resistant Brassica napus. lncRNA0410 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0013] Further, the biomaterial includes: lncRNA0410 The ribonucleic acid transcribed from the gene and / or contains positive regulatory elements. lncRNA0410 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic rapeseed cell lines.

[0014] Furthermore, the improved drought resistance includes: by increasing... lncRNA0410 The expression of [a specific gene] positively regulates its downstream genes. BnaA03.MYB12The expression.

[0015] A third aspect of the present invention provides a method for improving the drought resistance of Brassica napus, comprising: Positive regulation lncRNA0410 Gene-expressing biological materials were transferred into Brassica napus.

[0016] Furthermore, the method of transferring the biological material into rapeseed includes Agrobacterium-mediated transformation.

[0017] A fourth aspect of the present invention provides a method for cultivating drought-resistant Brassica napus, comprising: Positive regulation lncRNA0410 Gene-expressing biological materials were transferred into Brassica napus and cultivated.

[0018] Furthermore, the method of transferring the biological material into rapeseed includes Agrobacterium-mediated transformation.

[0019] Furthermore, the aforementioned lncRNA0410 The nucleotide sequence of the RNA transcribed from the gene contains the sequence shown in SEQ ID NO.2.

[0020] Furthermore, the containing lncRNA0410 Gene recombination vectors include basic vectors and lncRNA0410 The basic carrier includes pCAMBIA1300 Carrier.

[0021] Furthermore, the containing lncRNA0410 Recombinant microorganisms include Agrobacterium tumefaciens GV3101.

[0022] Furthermore, the variety of Brassica napus is Westar.

[0023] Furthermore, the growth characteristics of the Brassica napus are enhanced, including: hypocotyl elongation and enhanced root development.

[0024] Furthermore, the flavonol synthesis indicators of the rapeseed are improved, including: increased total flavonol content and increased flavonol synthase activity.

[0025] Furthermore, the drought resistance of the rapeseed is improved, including increased plant survival rate.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention reveals long non-coding RNA lncRNA0410 It is a key positive regulator of drought resistance in Brassica napus. After constructing a 35S-driven overexpression material, the drought resistance of the plants was significantly enhanced, and this was verified through interaction experiments. lncRNA0410 Through positive regulation BnaA03.MYB12This study enhances plant drought resistance, deepens the understanding of the molecular regulatory network of drought resistance in rapeseed, and provides a new technical solution for rapeseed breeding. Attached Figure Description

[0027] Figure 1 In Example 1 lncRNA0410 Phylogenetic tree of cruciferous plants.

[0028] Figure 2 In Example 1 lncRNA0410 and its target genes BnaA03.MYB12 Phylogenetic analysis of different genomes of Brassica napus.

[0029] Figure 3 In Example 4 lncRNA0410 (A) and BnaA03.MYB12 (B) In wild type (WT) and lncRNA0410 Expression levels in overexpression lines.

[0030] Figure 4 For example 5, wild type (WT) and lncRNA0410 Root length in overexpressing lines.

[0031] Figure 5 The wild type (WT) in Example 5 and lncRNA0410 Physiological indicators related to flavonol synthesis in overexpression lines; among which Figure 5 A represents DPBA staining and quantitative analysis of fluorescence intensity in leaves. Figure 5 B represents the activity of flavonol synthase. Figure 5 C represents the determination of total flavonol content.

[0032] Figure 6 For example 5, wild type (WT) and lncRNA0410 Survival rate of overexpression lines after 10 days of simulated drought treatment with 20% PEG-6000.

[0033] Figure 7 For example 5, wild type (WT) and lncRNA0410 Phenotypic comparison of overexpression lines treated with 45% PEG-6000 for 12 hours at 40-day seedling age.

[0034] Figure 8 This is a schematic diagram (A) of the reporter vector and effector vector in the instantaneous co-infiltration experiment of tobacco leaves in Example 6, showing the reporter gene ( pCambia2300-GUS , pBnaA03.MYB12-GUS ) and effector genes ( pCambia1300 , pCambia1300- lncRNA0410 GUS staining diagram of co-infiltrated tobacco leaves (B).

[0035] Figure 9The diagram shows the reporter vector and effector vector for dual-luciferase detection in Example 6 (A), the results of dual-luciferase detection in Arabidopsis protoplasts (B), and the quantitative statistical graph of luciferase activity (C). Detailed Implementation

[0036] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of protection of the present invention. Unless otherwise specified, the experimental materials, reagents, instruments, etc., used in the examples of the present invention are all commercially available; unless specifically specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0037] Long non-coding RNA in this invention lncRNA0410 The nucleotide sequence of the encoding gene is shown in SEQ ID NO.1; long non-coding RNA. lncRNA0410 The RNA sequence is shown in SEQ ID NO.2.

[0038] SEQ ID NO.1: 5'--3'。

[0039] SEQ ID NO.2: 5'--3'.

[0040] In this invention, M0 medium is a plant growth medium; LB medium is beef extract peptone medium used for bacterial culture; DM liquid medium is a liquid induction medium used for infection and co-culture stages; M1 medium is an induction medium used for inducing callus formation from explants; M2 medium is a screening medium used for callus screening and proliferation; M3 medium is a differentiation medium used for shoot differentiation and shoot proliferation; and M4 medium is a regeneration medium used for rooting or plant regeneration.

[0041] (1) M0 medium (solid) The components of M0 medium are as follows (per 1L): MS basal salt 0.44 g / L (Murashige and Skoogmedium, MS is a commonly used basal medium for plants); sucrose 30 g / L; agarose 2.4 g.

[0042] (2) LB medium (solid or liquid) The components of LB (Luria-Bertani) medium (per 1L) are as follows: 10 g peptone; 5 g yeast extract; 10 g sodium chloride (NaCl). For solid-state culture, add 15 g / L agar.

[0043] (3) DM liquid culture medium The components of DM medium are as follows (per 1L): MS basal salt 0.88 g / L; sucrose 30 g / L; acetosyringone (AS) 100 μM, added to 200 μL after sterilization.

[0044] (4) M1 medium (solid) The components of M1 medium are as follows (per 1L): MS basal salt 6 g / L; sucrose 30 g / L; mannitol 18 g / L; 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg / L) 400 μL; kinetin (KT, 0.3 mg / L) 400 μL; acetylsyringone (AS, 100 μM) 400 μL; agarose 2.4 g; pH of the medium adjusted to 5.8.

[0045] (5) M2 medium (solid) The components of M2 medium are as follows (per 1L): MS basal salt 6 g / L; sucrose 30 g / L; mannitol 18 g / L; 2,4-D (1.0 mg / L) 400 μL; kinetin (KT, 0.3 mg / L) 400 μL; agarose 2.4 g; silver thiosulfate (STS) 60 μL; kanamycin (50 mg / mL) 150 μL (Kanamycin is an aminoglycoside antibiotic); TMT solution 300 μL; pH of the medium adjusted to 5.8.

[0046] (6) M3 medium (solid) The components of M3 medium are as follows (per 1L): MS basal salt 76 g / L; sucrose 30 g / L; glucose 10 g / L; xylose 0.25 g / L; MES buffer (2-(N-morpholino)ethanesulfonic acid) 0.6 g / L; agarose 2.4 g; auxin IAA (indole-3-acetic acid, 0.1 mg / L) 40 μL; TMT (300 mg / L) 400 μL; TZ (2 mg / L) 400 μL; kanamycin (100 mg / L) 400 μL; pH adjusted to 5.8.

[0047] (7) M4 medium (solid) The components of M4 medium are as follows (per 1L): MS basal salt 1.76 g / L; sucrose 30 g / L; agarose 2.4 g; pH adjusted to 5.8.

[0048] All plants in this embodiment of the invention were cultivated in a greenhouse under the following conditions: light intensity of 100 μmol·m⁻¹. -2 ·s -1 A photoperiod of 16 / 8h (light / dark) and a constant temperature of 22°C.

[0049] Example 1: Long non-coding RNA lncRNA0410 Sequence acquisition and evolutionary relationships 1.1 Chromatin-rich antisense long non-coding RNAs were identified in hybrid populations. lncRNA0410 Positioned in CP12-1 The 5' end region of the gene has the nucleotide sequence shown in SEQ ID NO.1.

[0050] 1.2 Sequence conservation analysis shows that, lncRNA0410 The genome sequence shows high conservation in cruciferous plants, but lower conservation in other species. Figure 1 Phylogenetic results show that... lncRNA0410 exist B.napus Chinese rapeseed (and mustard-type rapeseed) B.juncea ) and Ethiopian mustard ( B.carinata ) forms a closely related evolutionary branch, while with black mustard ( B.nigra ),Chinese cabbage( B.rapa ) and cabbage ( B.oleracea The phylogenetic relationship is relatively distant. Further comparative analysis of the genomes of 11 Brassica napus varieties revealed that... lncRNA0410 With target genes BnaA03.MYB12 The sequence collinearity relationship exhibits intraspecific variation, mainly manifested as: lncRNA0410Sequence mapping varies among different varieties. Figure 2 ).

[0051] Example 2: Construction of long non-coding RNA lncRNA0410 Overexpression vector Through specific primer pairs lncRNA0410 The full-length DNA sequence (708 bp) was amplified. The forward primer sequence used for amplification is shown in SEQ ID NO.3, and the reverse primer sequence is shown in SEQ ID NO.4.

[0052] SEQ ID NO.3: 5'-ATCCATCGATAGTACTGTCGACAAATAAACATCTTCGCGTAACCGG-3'; SEQ ID NO.4: 5'-TCCTCGCCCTTGCTCACCATGGTACCAAATCGTGATCACAGAGTTCTGTATTTATCT-3'.

[0053] The amplified target DNA fragment was subcloned into a vector after enzyme digestion and ligation. pCAMBIA1300- mCherry-flag In the process, a recombinant expression vector was constructed. p35S:lncRNA0410-mCherry-flag .

[0054] The obtained positive plasmids were introduced into the rapeseed variety Westar via Agrobacterium-mediated transformation, and hypocotyls were used as explants for genetic transformation. The specific steps were as follows: First, rapeseed hypocotyl explants were transformed using Agrobacterium tumefaciens (AGC)... Agrobacterium tumefaciens The explants were immersed in the infection buffer for 20 min; then, they were placed in M1 medium (containing 30.0 g / L sucrose, 18.0 g / L mannitol, 1.0 mg / L 2,4-D, 0.3 mg / L kinetin (KT), 100 μM ferulic acid ester (AS), 6.0 g / L agarose, and pH adjusted to 5.8) and cultured in the dark for 48 h.

[0055] Subsequently, explants were transferred to M2 medium (containing 30 g / L sucrose, 18 g / L mannitol, agarose, 1.0 mg / L 2,4-D, 0.3 mg / L kinetin, 300 mg / L Timentin, and 50 mg / mL ampicillin, pH 5.8) to induce callus formation under appropriate antibiotic selection pressure. The resulting callus was further inoculated into M3 medium (containing 10 g / L glucose, 0.25 g / L xylose, 0.6 g / L MES, 2 mg / L zeatin, 0.1 mg / L IAA, 300 mg / L Timentin, and 25 mg / L Kanamycin, pH 5.8) to induce shoot differentiation; subsequently, the induced shoots were transferred to M4 medium (containing agarose and sucrose, pH 5.8) to promote root formation and achieve complete plant regeneration.

[0056] The obtained regenerated plants were identified as positive by RT-qPCR molecular detection. The forward primer sequence used for the detection is shown in SEQ ID NO.5, and the reverse primer sequence is shown in SEQ ID NO.6. The identified positive plants were named... lncRNA0410 OE .

[0057] SEQ ID NO.5: 5'-GCACCATACGAACCAAACCAAA-3'; SEQ ID NO.6: 5'-TCCTTTGGAAGAATACTGCAGTG-3'.

[0058] Example 3 lncRNA0410 Transformation of the vector into the hypocotyl of Brassica napus 3.1 Seed sterilization and sowing: Select plump "Westar" rapeseed seeds and surface disinfect them by soaking them in a 75% ethanol solution for 1 minute. After discarding the ethanol, soak them in a 50% sodium hypochlorite (NaClO) solution for 8–10 minutes for thorough sterilization. Then rinse them with sterile water 3–5 times, soaking for 5 minutes each time. Sow the sterilized seeds on MO medium and incubate them in the dark at 25°C for 5–6 days.

[0059] 3.2 Preparation of bacterial culture: 3–4 days after seed sowing, take out Agrobacterium GV3101 strain containing the target vector stored at -80℃, streak it on LB agar plates containing triple antibodies (kanamycin, gentamicin, and rifampin), and incubate upside down at 28℃ for 48 h. Pick a single colony and inoculate it into 5 mL of LB liquid medium containing the same triple antibodies, and incubate at 28℃ with shaking at 180 rpm for 24 h. Transfer 100 μL of the above bacterial culture to an Erlenmeyer flask containing 100 mL of LB liquid medium containing triple antibodies, and continue to incubate at 28℃ with shaking until OD reaches 0.5%. 600 The value is 0.5–0.8 (approximately 12–14 h). Collect the bacterial cells by centrifugation at 4000 r / min for 10 min, discard the supernatant, resuspend the bacterial cells in an equal volume of DM liquid medium (supplemented with acetylsyl syringone (AS) to a final concentration of 0.05 mmol / L), mix well, and pour into a sterile culture dish for later use.

[0060] 3.3 Infection and Co-culture: After 6 days of dark culture, sterile seedlings were removed. Using sterilized forceps and a scalpel in a laminar flow hood, the hypocotyl was excised and immediately placed in a DM solution containing Aspergillus subtilis (AS) for hydration. The hypocotyl should be quickly and vertically cut, ensuring a clean cut and a length of 0.8–1.0 cm. The explants were transferred to the Agrobacterium tumefaciens culture prepared in step 2.2 and gently shaken for 8–10 min. After inoculation, the culture was poured out, and the explants were spread evenly on sterile filter paper to absorb excess culture. The explants were then evenly placed on M1 medium using forceps, ensuring both ends were in full contact with the medium surface, and incubated in the dark at 25°C for 36–48 h.

[0061] 3.4 Selection Culture and Callus Induction: After co-culture, explants were transferred to M2 medium containing selection pressure and callus-inducing components. M2 medium contained 12.5 mg / L hygromycin as a selection marker, and 300 mg / L timentin and an appropriate amount of silver thiosulfate were added to inhibit Agrobacterium tumefaciens overgrowth and explant browning. Explants were cultured at 25°C under a photoperiod of 16 h light / 8 h dark for 2–3 weeks to induce callus formation.

[0062] 3.5 Redifferentiation: The surviving explants after screening were transferred to M3 differentiation medium and cultured every 2 weeks. Browned and dead materials were discarded in time, and explants with swollen ends and green dotted structures on the surface were selected for further differentiation culture.

[0063] 3.6 Bud Growth and Rooting: After the differentiated buds have developed into complete plant morphology, the buds are cut with a sterile scalpel and inserted into M4 rooting medium for 4 weeks. After the root system has fully developed, the plants are first transferred to a light incubator for hardening off, and then transferred to a greenhouse for further cultivation to complete the entire genetic transformation and regeneration process.

[0064] Example 4: Molecular Identification of Gene-Edited Plants 4.1 Extraction of DNA from Gene-Edited Plants: Fresh, young tissue from gene-edited plants was collected and placed in 2 mL centrifuge tubes. A steel ball and 250 μL of cetyltrimethylammonium bromide (CTAB) extraction buffer were added, and the mixture was homogenized using a tissue homogenizer for 6 min. Then, 500 μL of CTAB buffer was added, and the centrifuge tubes were incubated in a 65°C water bath for 30–60 min, gently shaking every 5–10 min. After incubation, the samples were cooled to room temperature, and an equal volume of chloroform-isoamyl alcohol mixture (24:1) was added. The mixture was vortexed for 10–15 min, followed by centrifugation at 12000 rpm for 10 min. Approximately 400 μL of the supernatant was carefully transferred to a new 1.5 mL centrifuge tube, avoiding aspiration of the intermediate layer or precipitate. An equal volume of pre-chilled anhydrous ethanol was added to the supernatant, and the mixture was gently mixed and incubated at -20°C for 20–30 min. The sample was then centrifuged at 12000 rpm for 3–5 min, the supernatant was discarded, and the precipitate was washed with 500 μL of 75% ethanol. After standing for 5–6 min, the sample was centrifuged again and the ethanol was discarded. The centrifuge tube containing DNA was dried at room temperature until the ethanol had completely evaporated. 200 μL of ddH2O was added to dissolve the DNA, and the sample was left to stand at room temperature overnight to allow it to fully dissolve, thus obtaining the genomic DNA sample.

[0065] 4.2 Identification of positive seedlings: First, approximately 100 mg of tender leaves from the regenerated plants obtained through transformation were collected, and genomic DNA was extracted using a standard plant genomic DNA extraction kit. Then, using the obtained DNA as a template, PCR or RT-qPCR amplification reactions were performed using specific identification primers. The forward primer sequence used for positive identification is shown in SEQ ID NO. 5, and the reverse primer sequence is shown in SEQ ID NO. 6.

[0066] SEQ ID NO.5: 5'-GCACCATACGAACCAAACCAAA-3'; SEQ ID NO.6: 5'-TCCTTTGGAAGAATACTGCAGTG-3'.

[0067] The PCR reaction system was prepared using standard procedures and amplified according to the following program: pre-denaturation at 95°C for 3 min; followed by 35 cycles (95°C for 30 s, 58°C for 30 s, 72°C for 30 s); extension at 72°C for 5 min. The amplified products were then detected by 1.0% agarose gel electrophoresis. If a specific band consistent with the positive control appeared at the corresponding position, the plant was considered a PCR-positive plant.

[0068] For RT-qPCR identification, total RNA from regenerated plants was extracted using the TRIzol method, reverse transcribed to generate cDNA, and then subjected to qPCR using the primers described above. If a specific amplification curve and the expected melting peak were detected, the plant was identified as exogenous. lncRNA0410 Successfully expressed.

[0069] The positive plants obtained through identification are determined to be the constructs of this invention. lncRNA0410 Overexpressed material, and named lncRNA0410 OE The positive plants identified were used for subsequent phenotypic observation and functional analysis studies.

[0070] Molecular detection was performed on two stably inherited gene-edited lines. RT-qPCR results showed that in both lines... lncRNA0410 The expression level was 4-4.5 times that of wild-type (WT). Figure 3 A), its downstream target gene BnaA03.MYB12 The expression level was approximately twice that of WT ( Figure 3 B). The significant increase in expression levels indicates that the gene-editing construct successfully achieved the expected upregulation effect in plants.

[0071] Example 5: Phenotypic Identification of Gene-Edited Plants Firstly, in terms of morphological phenotype, compared with WT, the resulting gene-edited plants exhibited significantly enhanced growth characteristics, including marked hypocotyl elongation and enhanced root development. Specifically, the taproot length of T1 plants increased by approximately 30-40% compared to WT. Figure 4 Further examination of physiological indicators related to flavonol synthesis revealed that the flavonol fluorescence signal intensity, total flavonol content, and flavonol synthase activity of the gene-edited plants were significantly increased compared to the wild type. Figure 5 AC), Explanation lncRNA0410 Upregulation can significantly enhance flavonol metabolism.

[0072] Regarding drought resistance phenotype, drought resistance was assessed by applying 20% ​​PEG6000 osmotic stress to the seedling stage. After stress treatment, the seedling survival rate of the gene-edited lines was significantly higher than that of the WT line, with an increase of approximately 40%. Figure 6 In addition, wild-type (WT) and lncRNA0410 OE After 40 days of treatment with 35% PEG-6000 for 12 hours, the WT strain showed significant water loss and wilting. Figure 7 The above results indicate that by regulating... lncRNA0410 Expression can further positively regulate its downstream genes. BnaA03.MYB12 This significantly enhances the drought resistance of plants. In summary, lncRNA0410 In regulation [[ID=It plays an important role in enhancing the drought resistance of plants and provides an effective way to enhance the stress resistance of rapeseed through gene editing.

[0073] Example 6 ​ and ​ Verification of the regulatory relationship Validation was achieved by constructing effect vectors and report vectors. ​ Co-expressed genes BnaA03.MYB12 The regulatory relationship between them, the specific steps are as follows: lncRNA0410 Full-length sequence cloned to CaMV35S Startup driver pCambia1300 From the expression vector, the effect vector is obtained. pCambia1300-lncRNA0410 ;Will BnaA03.MYB12 The promoter sequence of the gene was cloned into the reporter vector. pCambia2300-GUS In the middle, the construction is obtained pBnaA03.MYB12-GUS ( Figure 8 A).

[0074] Subsequently, the report carrier ( pCambia2300-GUS or pBnaA03.MYB12-GUS ) and effector ( pCambia1300 or pCambia1300-lncRNA0410 Different combinations of these substances were injected into tobacco leaves to detect... lncRNA0410 right BnaA03.MYB12 The effect on promoter activity. The results show ( Figure 8 B), compared with the negative control group ( pCambia1300 + pCambia2300-GUS )compared to, pCambia1300 and pBnaA03.MYB12-GUS Only weak GUS activity was detected in the co-transformation combination; while when lncRNA0410 and pBnaA03.MYB12-GUS A significantly enhanced GUS activity was observed during co-transformation, indicating that lncRNA0410 Able to improve BnaA03.MYB12 Transcriptional activity of the promoter. Primer sequences determined by GUS are shown in SEQ ID NO.7 and SEQ ID NO.8.

[0075] SEQ ID NO.7: 5'-ATCCATCGATAGTACTGTCGACAAATAAACATCTTCGCGTAACCGG-3'; SEQ ID NO.8: 5'-TCCTCGCCCTTGCTCACCATGGTACCAAATCGTGATCACAGAGTTCTGTATTTATCT-3'.

[0076] Furthermore, the regulatory relationship was further validated using a dual-luciferase (LUC / REN) reporter system. The primer sequences for the dual-luciferase analysis are shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12.

[0077] SEQ ID NO.9: 5'-CCGCTCTAGAACTAGTGGATCCAAATAAACATCTTCGCGTAACCGG-3'; SEQ ID NO.10: 5'-CGGGCCCCCCCTCGAGGTCGACAAATCGTGATCACAGAGTTCTGTATTTATCT-3'; SEQ ID NO.11: 5'-GGTCGACGGTATCGATAAGCTTAAAATGTCTTTTTAATTCAATTTAACATTCAATTA-3'; SEQ ID NO.12: 5'-CCGCTCTAGAACTAGTGGATCCTTTTCTCGACGGTTTTATGTA-3'.

[0078] Furthermore, dual-luciferase reporter gene assays also confirmed this result. In this assay system, the reporter vector contains... BnaA03.MYB12 The promoter-driven firefly luciferase gene and the kidney luciferase (REN) gene driven by the cauliflower mosaic virus 35S promoter; while [[ID= It is then constructed in an independent effect vector, also by ​ Startup sub-driver ( ​ A). By measuring the luciferase activity under different combinations of reporter and effector vectors. ​ Transcription activated ​ The expression ( ​ B). Compared to individual expressions ​ In comparison, co-rotation ​ When used as an effector, firefly luciferase activity is increased approximately 3-fold. ​ C), indicating ​ Positive regulation ​ The expression.

Claims

1. lncRNA0410 Use of genes or related biological materials in at least one of the following: A1) Regulate the drought resistance of Brassica napus or prepare products that regulate the drought resistance of Brassica napus; A2) Cultivate drought-resistant varieties of Brassica napus or prepare products that improve the drought resistance of Brassica napus; A3) Preparation of transgenic Brassica napus; The lncRNA0410 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The biomaterials include one or more of the following: A1) lncRNA0410 RNA transcribed from the gene and / or containing the above lncRNA0410 Gene recombinant vectors, recombinant microorganisms, or transgenic Brassica napus cell lines; A2) Contains negative regulation lncRNA0410 Nucleic acid molecules expressing genes, recombinant vectors, recombinant microorganisms, or transgenic Brassica napus cell lines.

3. The application as described in claim 1 or 2, characterized in that, The regulation of drought resistance in Brassica napus includes: regulating the drought resistance of Brassica napus... BnaA03.MYB12 Gene expression.

4. The application as described in claim 3, characterized in that, The regulation of Brassica napus BnaA03.MYB12 Gene expression includes: through upregulation lncRNA0410 The expression of [a specific gene] positively regulates its downstream genes. BnaA03.MYB12 To improve the drought resistance of Brassica napus; Or, by lowering lncRNA0410 The expression of [a specific gene] negatively regulates downstream genes. BnaA03.MYB12 To reduce the drought resistance of Brassica napus.

5. Positive regulation lncRNA0410 The application of gene-expressing biomaterials in cultivating drought-resistant Brassica napus is characterized by, The lncRNA0410 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The application as described in claim 5, characterized in that, The biomaterial includes: lncRNA0410 The ribonucleic acid transcribed from the gene and / or contains positive regulatory elements. lncRNA0410 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic rapeseed cell lines.

7. The application as described in claim 5, characterized in that, The improved drought resistance includes: by increasing... lncRNA0410 The expression of [a specific gene] positively regulates its downstream genes. BnaA03.MYB12 The expression.

8. A method for improving the drought resistance of Brassica napus, characterized in that, include: Positive regulation lncRNA0410 Gene-expressing biological materials were transferred into Brassica napus.

9. A method for cultivating drought-resistant Brassica napus, characterized in that, include: Positive regulation lncRNA0410 Gene-expressing biological materials were transferred into Brassica napus.

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