Application of BoSLIM1 protein in improving drought resistance in broccoli

Abstract

This invention discloses the application of BoSLIM1 protein in improving the drought resistance of broccoli, belonging to the field of plant breeding technology. To enhance the drought resistance of broccoli, this invention utilizes the BoSLIM1 protein, the sequence of which is shown in SEQ ID NO.2. This is of great significance for understanding the stress tolerance mechanism of broccoli and for breeding drought-resistant varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding technology, specifically relating to the application of BoSLIM1 protein in improving the drought resistance of broccoli. Background Technology

[0002] Broccoli (Brassica oleracea var. italica), also known as green cauliflower, is a variety of Brassica oleracea in the Brassicaceae family. Broccoli is rich in nutrients and medicinal value, containing abundant protein, vitamins, and minerals, as well as glucosinolates and various indole derivatives. During its vegetative growth period, broccoli is sensitive to water requirements; water shortage significantly impacts yield.

[0003] Currently, broccoli is classified as a semi-hardy vegetable with poor drought resistance. It fails to form flower heads at temperatures exceeding 30°C, and growth in dry soil results in stunted plants and premature formation of small flower heads. There are no effective methods to combat drought in broccoli. Therefore, studying the physiological responses of broccoli under drought stress is crucial for understanding its stress tolerance mechanisms and for cultivating drought-resistant varieties.

[0004] Broccoli thrives in moist environments and is highly sensitive to water availability during its vegetative growth stage. Water shortages negatively impact germination rates, transplant survival rates, and can even reduce yield and quality. Even mild drought can adversely affect broccoli yield; a 20% or 40% reduction in irrigation can lead to yield decreases of 29% and 41%, respectively. Current drought-resistance strategies include deep-rooted irrigation, utilizing available water for irrigation, and adjusting the broccoli's life cycle to match rainfall. Because this process depends on changes in the external environment, there is an urgent need to utilize genetic engineering to improve the drought resistance of broccoli. Summary of the Invention

[0005] The purpose of this invention is to improve the drought resistance of broccoli.

[0006] This invention provides an application of BoSLIM1 protein in improving the drought resistance of broccoli, the sequence of which is shown in SEQ ID NO.2.

[0007] This invention provides an application of the BoSLIM1 gene in improving the drought resistance of broccoli, the sequence of which is shown in SEQ ID NO.1.

[0008] This invention provides the application of a recombinant vector containing the BoSLIM1 gene in improving the drought resistance of broccoli, the sequence of which is shown in SEQ ID NO.1.

[0009] Further specifying, the starting vector for the recombinant vector is pCAMBIA1301-35S-EGFP.

[0010] This invention provides the application of recombinant microbial cells containing the BoSLIM1 gene in improving the drought resistance of broccoli, the sequence of which is shown in SEQ ID NO.1.

[0011] Further specifying, the starting microbial cell of the recombinant microbial cell is a prokaryotic microbial cell or a eukaryotic microbial cell.

[0012] This invention provides an application of broccoli overexpressing the BoSLIM1 gene in drought resistance, the sequence of which is shown in SEQ ID NO.1.

[0013] This invention provides a cultivation method for improving the drought resistance of broccoli, the specific steps of which are as follows:

[0014] Step 1: Ligate the BoSLIM1 gene with the pCAMBIA1301-35S-EGFP vector to obtain the recombinant vector;

[0015] Step 2: The recombinant vector obtained in Step 1 is transferred into Agrobacterium to obtain recombinant Agrobacterium;

[0016] Step 3: Infect broccoli with the recombinant Agrobacterium obtained in Step 2 to obtain transgenic broccoli.

[0017] To further specify, the primers used to amplify the BoSLIM1 gene in step 1 are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0018] To further specify, the sequence of the BoSLIM1 gene is shown in SEQ ID NO.1.

[0019] Beneficial effects: After drought treatment, the leaves of broccoli with transgenic BoSLIM1 showed less wilting than those with no transgenic leaves, and overexpression of BoSLIM1 in broccoli improved the drought resistance of the plants.

[0020] After treatment with 10% PEG6000 for 1 hour, the stomatal aperture of broccoli transgenic with 35S::BoSLIM1 and the control transgenic with empty vector both decreased significantly, indicating that the stomata closed. The greater degree of closure in the transgenic plants suggests that BoSLIM1 participates in the defense against drought stress by regulating stomatal aperture. Attached Figure Description

[0021] Figure 1The process of obtaining hairy roots from BoSLIM1 gene-transgenic broccoli is illustrated in the following diagrams: A: Broccoli at 15 days old; B: Infection with Agrobacterium ATCC15834; C: Broccoli removed from vermiculite and lateral roots cut off; D: Broccoli cultured for another 7 days in a 1:1 mixture of nutrient soil and vermiculite; E: Obtaining hairy roots from BoSLIM1 gene-transgenic broccoli.

[0022] Figure 2 Figure 1 shows the results of drought resistance analysis of hairy roots of BoSLIM1-transgenic broccoli; A is the phenotypic diagram of broccoli after 24 hours of drought treatment; B is the stomatal diagram of broccoli after 1 hour of drought treatment; C is the stomatal aperture after 1 hour of drought treatment.

[0023] Figure 3 The results show the identification of BoSLIM1 gene overexpression. Detailed Implementation

[0024] Example 1. BoSLIM1 gene amplification and construction of recombinant vector

[0025] 1. Target gene name: BoSLIM1 (broccoli sulfate restriction 1) gene sequence: SEQ ID NO.1

[0026] ATGGGTGGGGGCGATCTAGCTTTGTCCGTTGCTGACATCAGGATGGAGAATGAGCAGC

[0027] CTGATGACTTGGCTAGCGATACCGTCGCTGAGATCGATGTGAGTGACGAAGAGATTGAT

[0028] GCAGAAGACTTAGAGAGACGTATGTGGAAAGACCGAGTCAGGCTCAAAAGAATCAAA

[0029] GAGCGTCAAAAAGGAGATTCTCAAGGACCTCAGGCGAAGGAGCCTCCCAAGAAGATC

[0030] TCTGACCAAGCGCAGAGGAAGAAGATGTCTAGAGCACAAGACGGTATACTCAAGTACA

[0031] TGCTGAAGCTAATGGAAGTCTGCAAAGTACGCGGCTTTGTGTACGGCATCATACCTGAA

[0032] AAAGGTAAGCCGGTGAGCGGCTCCTCCGACAATATAAGAGCTTGGTGGAAAGAGAAG

[0033] GTGAAGTTTGATAAAAACGGTCCTGCAGCGATTGCTAAATACGAAGAGGAGTGTTTGG

[0034] CGTTTGGGAAGTCAGACGGGAATAGAAACTCGCAGTTTGTGCTTCAAGACTTGCAGGA

[0035] CGCTACTCTAGGGTCTTTGTTGTCTTCTTTGATGCAGCATTGTGATCCTCCGCAGAGGAA

[0036] GTATCCGTTGGAGAAAGGGACGCCTCCTCCCTGGTGGCCGACGGGGAAGGAAGAGTG

[0037] GTGGGTGAAGCTCGGCTTGCCGCAGAGCCAGAGTCCTCCTTACAGGAAGCCTCATGAT

[0038] CTCAAGAAGATGTGGAAAGTTGGGGTGTTGACGGCTGTGATTAACCATATGTCTCCTGA

[0039] TATTGCTAAGATCAAGAGGCATGTTCGTCAGTCTAAGTGTTTGCAGGACAAGATGACGG

[0040] CGAAAGAGAGCGCGATTTGGTTGGCGGTTTTGAACCAGGAGGAGTCGCTTATTCAGCA

[0041] GCCGAGTAGTGATAATGGGACGTCTAATGTAACTGAGACGCACCGGAGGGGGAATAAC

[0042] GCTGACAGGAGGAAGACTGTGATTAATAGTGATAGTGATTATGATGTTGATGGGACTGA

[0043] GGAGGCTTCAGGCTCGGTTTCTTCTAAAGACAGTAGAAGAAATCAAGTTCCAGCAGCC

[0044] ACTTCACAACAACCAGTAAGAGATCAAGATAAAGCTGGGAAACATAAGAGAAGGAAA

[0045] AGACCTAGAATCAGATCCGGAACTCTTAATGTACAAGATGAAGAACAAGTAGAAGCTG

[0046] AAGGGAGAAATGTTTTACCTGATATGAATCATGTTGAGGCTCCTATGCTAGATTATAACA

[0047] TCAACGGTACTACTAATCACCATGAGGAAGGTGTTTTAGAACCAAACATCTCCTTAGGA

[0048] CCAGAAGAGAACGGTCTGGAACTGGTGGTTCCTGAGTTTGATAGCAACTATACTTATCT

[0049] TCCTCCTGTTGATGGACAAGCTATGATGCCTGTAGACGAAAGACCAATGCTTTACGGAG

[0050] CAAACCCCAACCAGGAGCTGCAATTTGGGTCAGGTTATAACTACTATAATACTTCTGCA

[0051] GTGTTTGTGCATAATCAGGAAGAAGACCTTATCCATACACAGATAGAGATGAACTCACA

[0052] AGCACCGCCTCACAGCAACGGGTTCGATGGCCAAGGAGGAGTACTTCAACCCCATGGG

[0053] AATGAAGAGGTGGGTGTAGCAGGAAGAGATATGCCTCCTCAGTTTCAGAGTGACCAAG

[0054] ATAAACTCTTGGACAGTAACATCCTATCTCCATTCAATGACTTGCCGTTTGATAGCAGCA

[0055] CCTTTTACTCTGGGTTTGATTCCTTTGGTGCATTTGATGATGACTACTCATGGTTTGGAGCTTAG;

[0056] Protein sequence of BoSLIM1 (Broccoli Sulfate Limitation 1): SEQ ID NO.2

[0057] MGGGDLALSVADIRMENEQPDDLASDTVAEIDVSDEEIDAEDLERRMWKDRVRLKRIKER

[0058] QKGDSQGPQAKEPPKKISDQAQRKKMSRAQDGILKYMLKLMEVCKVRGFVYGIIPEKGKP

[0059] VSGSSDNIRAWWKEKVKFDKNGPAAIAKYEEECLAFGKSDGNRNSQFVLQDLQDATLGSL

[0060] LSSLMQHCDPPQRKYPLEKGTPPPWWPTGKEEWWVKLGLPQSQSPPYRKPHDLKKMWKVG

[0061] VLTAVINHMSPDIAKIKRHVRQSKCLQDKMTAKESAIWLAVLNQEESLIQQPSSDNGTSN

[0062] VTETHRRGNNADRRKTVINSDSDYDVDGTEEASGSVSSKDSRRNQVPAATSQQPVRDQDK

[0063] AGKHKRRKRPRIRSGTLNVQDEEQVEAEGRNVLPDMNHVEAPMLDYNINGTTNHHEEGVL

[0064] EPNISLGPEENGLELVVPEFDSNYTYLPPVDGQAMMPVDERPMLYGANPNQELQFGSGYN

[0065] YYNTSAVFVHNQEEDLIHTQIEMNSQAPPHSNGFDGQGGVLQPHGNEEVGVAGRDMPPQF

[0066] QSDQDKLLDSNILSPFNDLPFDSSTFYSGFDSFGAFDDDYSWFGA;

[0067] Using 7-day-old broccoli seedlings as templates, PCR was performed using BoSLIM1 upstream and downstream primers according to the PCR reaction system in Table 1 to amplify the BoSLIM1 gene sequence. The target band was obtained after electrophoresis.

[0068] BoSLIM1 upstream primer: GAGCTCGGTACCCGGGGATCCATGGGTGGGGGCGATCTA (SEQ ID NO.3);

[0069] BoSLIM1 downstream primer: GGTGTCGACTCTAGAGGATCCAGCTCCAAACCATGAGTAGTC (SEQ ID NO.4);

[0070] PCR procedure:

[0071] Table 1 PCR reaction system

[0072] system volume dNTP Mix 2μl 10×Ex Taq Buffer 2μl Ex Taq Enzyme 0.3μl template 1μl upstream primer 1μL Downstream primer 1μL <![CDATA[RNase Free H2O]]> Up to 20μl

[0073] PCR program: 94°C 10 min → (94°C 30 s → 58°C 30 s → 72°C 2 min) × 30 → 72°C 10 min → 4°C.

[0074] 2. Recovery of the target fragment and carrier conjugation

[0075] Recovery of the target fragment: Recovery and purification were performed using the SPARK easy Gel DNA Extraction Kit, as follows:

[0076] (1) Under UV light, cut the gel block with the target band with a blade and put it into a 1.5mL EP tube.

[0077] (2) Add 600 μL of sol and heat in a 65°C metal bath until completely melted. Invert the container every 2 minutes to accelerate the melting process.

[0078] (3) Add the solution to the adsorption column EC, place it at room temperature for 1 min, and then centrifuge. Set the centrifuge speed to 13800×g and the time to 1 min. Discard the effluent.

[0079] (4) Add 600 μL of WB rinsing solution and centrifuge at 13800 × g for 1 min. Discard the effluent. (Repeat once)

[0080] (5) Place the adsorption column EC back into the collection tube, centrifuge, set the speed to 13800×g, and the time to 2min.

[0081] (6) Place the adsorption column EC into a new collection tube, add 20 μL of RNase Free H2O to the middle of the adsorption membrane, place at room temperature for 2 min, and then centrifuge. Set the centrifuge speed to 13800×g and the time to 1 min. The elution product is the purified target fragment.

[0082] Vector ligation: The pCAMBIA1301-35S-BoSLIM1-EGFP expression vector was constructed using homologous recombination. The recombination system is as follows:

[0083] Table 2 Construction of pCAMBIA1301-35S-BoSLIM1-EGFP expression vector

[0084] system volume Exnase II 2μl 5×CEII Buffer 4μl Inserted fragment products 1μl Linearized carrier 3μl <![CDATA[ddH2O]]> Up to 20μl

[0085] React at 37℃ for 30 minutes.

[0086] Example 2. Obtaining recombinant bacteria

[0087] Take 2 μL of the constructed plasmid pCAMBIA1301-35S-BoSLIM1-EGFP and add it to 50 μL of thawed Agrobacterium rhizogenes competent cells ATCC15834. Mix well by pipetting and then place on ice. Set the electroporation instrument program: C=25μF, PC=200Ω, V=2400V. Add the mixture after the ice bath to the pre-cooled electroporation cuvette, cover the cuvette, wipe it dry, and insert it into the groove of the electroporator. Start electroporation. After completion, quickly transfer to an EP tube and add 1 mL of TY medium. Incubate at 28℃ for 3 h, then spread on medium containing Kana and Rif. Incubate for 48 h before colony PCR identification. The correct colony can be used for subsequent hairy root transformation.

[0088] Example 3. Obtaining hairy roots from transgenic broccoli

[0089] 1. (1) When the broccoli has grown to 2 weeks, select healthy seedlings with consistent growth status for infection. The infection process is as follows: make a slanted cut 2cm below the cotyledon node, scrape the Agrobacterium rhizogenes from the slanted cut surface, and quickly insert it into a flowerpot containing only vermiculite to absorb OD. 600 The Agrobacterium tumefaciens bacterial solution obtained in Example 2 (=0.8) was injected into the roots, and a transparent plastic cup was immediately placed over it to retain moisture and ensure sufficient water.

[0090] (2) After one week of growth, numerous hairy roots appeared on the cut surface. The roots, except those on the oblique cut surface, were cut off and replanted in the mixed soil. The resulting hairy roots of 35S::BoSLIM1 were obtained, such as... Figure 1 As shown.

[0091] II. Identification

[0092] Green fluorescence detection and gene expression assays were performed on the hairy roots to obtain positive broccoli hairy root chimeras.

[0093] Identification primer: BoSLIM1-qpcr-upstream: CGTCAGTCTAAGTGTTTGCAG (SEQ ID NO.5);

[0094] BoSLIM1-qpcr downstream: AGTTCCGGATCTGATTCTAGG (SEQ ID NO.6), after identification, the results are as follows: Figure 3 As shown, the broccoli that overexpressed the BoSLIM1 gene was indeed overexpressing the BoSLIM1 gene.

[0095] Example 4. Analysis of drought resistance by overexpressing BoSLIM1 in the hairy roots of broccoli

[0096] 1. To elucidate the effect of BoSLIM1 overexpression on drought resistance in broccoli, drought resistance phenotypic analysis was performed on broccoli seedlings with hairy roots transfected with 35S::BoSLIM1 and those transfected with hairy roots from an empty vector. Normally growing broccoli seedlings were transferred to a Hoagland solution containing 10% PEG6000 for hydroponic cultivation to simulate drought stress for 24 hours, with seedlings transferred to a solution without PEG6000 serving as a control.

[0097] like Figure 2 As shown, without drought treatment, there was no difference in growth between all plants converted to empty vector and those converted to 35S::BoSLIM1. After drought treatment, all plants showed varying degrees of wilting, but the leaves of the transgenic BoSLIM1 broccoli showed less wilting than those converted to empty vector. The wilting rate of three broccoli plants converted to 35S::BoSLIM1 and three broccoli plants converted to empty vector were measured, and the formula is:

[0098]

[0099] The wilting rate of transgenic plants was found to be 47.63% ± 7.25%, while the wilting rate of transgenic broccoli was 69.41% ± 5.39%. These results indicate that overexpression of BoSLIM1 in broccoli improves the drought resistance of the plants.

[0100] 2. To further clarify whether the improved drought resistance of broccoli due to BoSLIM1 overexpression is related to stomatal sensitivity, PEG6000 simulated drought was applied to broccoli plants overexpressing BoSLIM1 and control plants. Changes in stomatal aperture were observed and recorded. Leaves from the same position in both 35S::BoSLIM1 transgenic broccoli and control broccoli were taken, the epidermis was peeled off with tape, and then immersed in a stomatal opening solution (30 mmol / L KCl, 0.1 mmol / L CaCl2, 10 mmol / L MES-KOH, pH 6.15) and placed under sufficient light for 2.5 h. Microscopic examination was then performed. Images were taken of stomata with normal morphology and large aperture. The slides were returned to the stomatal opening solution, and PEG6000 was added to a final concentration of 10% (experimental group). No PEG6000 was added (control). After being placed back into a well-lit environment for 1 hour, the broccoli was removed and soaked in 95% alcohol for shaping. After shaping, the residual alcohol on the surface was gently blotted off with tissue paper. The broccoli was then placed under an optical microscope with a 10x eyepiece and a 40x objective lens for observation and photographing. The length and width of the stomata were measured using ImageJ software. The ratio of the length to the width of the stomata was used to represent the stomatal opening. The length and width were measured three times and the average of the ratios was used for counting. 100 stomata were collected from both the 35S::BoSLIM1 transgenic broccoli and the control group for statistical analysis.

[0101] The results showed that after treatment with 10% PEG6000 for 1 h, the stomatal aperture of broccoli transgenic with 35S::BoSLIM1 and the control transgenic with no vector was significantly reduced, indicating that stomatal closure occurred, and the degree of closure was greater in the transgenic plants than in the control. Figure 2 (C) This indicates that BoSLIM1 participates in drought stress defense by regulating stomatal aperture.

Claims

1. The use of overexpression of BoSLIM1 protein in improving drought resistance of Brassica oleracea, characterized in that, The sequence of the BoSLIM1 protein is shown as SEQ ID NO.

2.

2. The use of overexpression of BoSLIM1 gene in improving drought resistance of Brassica oleracea, characterized in that, The sequence of the BoSLIM1 gene is shown as SEQ ID NO.

1.

3. Use of a recombinant vector containing the BoSLIM1 gene for improving drought resistance of Brassica oleracea, characterized in that, The sequence of the BoSLIM1 gene is shown as SEQ ID NO.

1.

4. Use according to claim 3, characterized in that, The starting vector of the recombinant vector is pCAMBIA1301-35S-EGFP.

5. Use of broccoli overexpressing the BoSLIM1 gene in drought resistance, characterized in that, The sequence of the BoSLIM1 gene is shown as SEQ ID NO.

1.

6. A method for breeding broccoli to improve drought tolerance, comprising the steps of, The specific steps of the method are as follows: Step 1: connecting the BoSLIM1 gene with the pCAMBIA1301-35S-EGFP vector to obtain a recombinant vector; the sequence of the BoSLIM1 gene is shown as SEQ ID NO. 1; Step 2: transferring the recombinant vector obtained in step 1 into Agrobacterium to obtain a recombinant Agrobacterium; Step 3: infecting broccoli with the recombinant Agrobacterium obtained in step 2 to obtain transgenic broccoli.

7. The breeding method according to claim 6, characterized by, The primers for amplifying the BoSLIM1 gene in step 1 are shown as SEQ ID NO. 3 and SEQ ID NO. 4.

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