Rapeseed bnaRING1b protein, encoding gene and application thereof

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a rapeseed BnaRING1b protein, its encoding gene, and its applications. The protein has one of the following amino acid residue sequences: 1) SEQ ID No: 1 in the sequence listing; 2) a protein that improves the cold resistance of rapeseed by aligning the amino acid residue sequence of SEQ ID No: 1 in the sequence listing. This invention also includes a recombinant expression vector containing the gene of this invention, a transgenic cell line, and engineered bacteria. Expression of the rapeseed BnaRING1b protein encoding gene in rapeseed significantly improves the cold resistance of rapeseed. This invention provides a novel cold-resistance gene resource for rapeseed cold resistance.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of enhancing rapeseed tolerance by silencing the BnaRNG1b gene in Brassica napus. Background Technology

[0002] Rapeseed is my country's most important oilseed crop, with domestically produced rapeseed oil accounting for approximately 50% of the country's total oil production, playing an irreplaceable strategic role in ensuring national edible oil supply security. However, my country's rapeseed production has long faced severe damage from low temperatures. Against the backdrop of intensifying global climate change and frequent extreme low-temperature events, low-temperature freezing damage is one of the major meteorological disasters currently facing rapeseed production in my country. The double blow of low temperatures to rapeseed yield and quality has seriously threatened my country's edible oil supply security, making the cultivation of drought-resistant rapeseed varieties an urgent need to ensure the stable development of the industry. However, in my country's main rapeseed producing area—the winter rapeseed region of the Yangtze River Basin—frequent cold waves and temperature drops every winter pose a serious threat to rapeseed production. When the average daily temperature drops below 5℃, rapeseed stops growing; at 0℃, slight freezing damage occurs; at -3℃ to -5℃, the leaves begin to freeze; and when extreme low temperatures of -8℃ to -10℃ occur, the freezing damage rate can reach 30% to 80%, resulting in extremely severe yield reduction. A cold-resistant material was obtained through germplasm selection of rapeseed, and a cold-resistant gene, BnaRING1b, was successfully located using this material. A mutation in this gene caused premature termination of transcription, resulting in loss of gene function. Using genetic engineering methods and RNAi technology, the BnaRING1b gene in Brassica napus was silenced, significantly improving the cold resistance of the rapeseed. This provides a new gene resource for cold-resistant rapeseed breeding. This invention, through genetic engineering methods, is the first to discover the BnaRING1b protein and its encoding gene, which can significantly improve the resistance of rapeseed to cold damage. Summary of the Invention

[0003] The purpose of this invention is to address the shortcomings of the prior art by providing a cold-resistant gene resource, the BnaRING1b protein, and its encoding gene, and to provide the application of this gene in cold resistance in rapeseed.

[0004] To achieve the above objectives, the technical solution adopted by this invention is: a BnaRING1b protein, derived from a rapeseed gene mutation, which is one of the following amino acid residue sequences: 1) SEQ ID No:1 in the sequence list; 2) Proteins that improve the cold resistance of rapeseed by substituting and / or deleting and / or adding one to five amino acid residues in the amino acid sequence of SEQ ID No:1 in the sequence listing.

[0005] The gene encoding the BnaRING1b protein of this invention (BnaRING1b) has one of the following nucleotide sequences: 1) The nucleotide sequence of SEQ ID No:2 in the sequence listing; 2) DNA encoding the protein sequence of SEQ ID No:1 in the sequence listing; 3) A nucleotide sequence that has more than 90% homology with the nucleotide sequence defined by SEQ ID No:2 in the sequence listing and encodes a protein with the same function; SEQ ID No:2 in the sequence listing consists of 987 bases, and its coding frame is from the 5' end, from the 1st to the 987th base, encoding a protein having the amino acid residue sequence of SEQ ID No:1 in the sequence listing.

[0006] The present invention also includes a recombinant expression vector containing the gene of the present invention, a transgenic cell line and engineered bacteria, and primer pairs for amplifying any fragment of the gene.

[0007] This invention also provides the application of the above-mentioned BnaRING1b protein gene in regulating the cold resistance of rapeseed.

[0008] The present invention also provides a method for transferring the gene encoding the above-mentioned BnaRING1b protein into rapeseed, and then cultivating it to obtain transgenic rapeseed. The cold resistance of the transgenic rapeseed is improved, thereby obtaining cold-resistant rapeseed germplasm resources.

[0009] This invention provides a transgenic rapeseed strain that exhibits significantly enhanced low-temperature resistance through overexpression of the BnaRING1b protein gene. This invention offers a high-quality gene for improving the cold resistance of rapeseed. The protein and its encoding gene of this invention have high practical application value and broad application prospects. Attached Figure Description

[0010] Figure 1 This is an electrophoresis diagram of total RNA isolated from rapeseed leaves.

[0011] Wherein: M is a DNA molecular marker indicating the size of the DNA fragment, and 1 is the extracted RNA; the RNA from the sample source has obvious 18S rRNA and 28S rRNA bands, indicating that the RNA quality is reliable and can be used for the next step of reverse transcription to synthesize cDNA.

[0012] Figure 2 This is an electrophoresis image of the PCR amplification product of the full-length CDS of the rapeseed BnaRING1b protein gene.

[0013] Where M is a 100bp plus Ladder DNA marker representing the molecular weight of the DNA fragment, 1, 2, 3, and 4 are amplification products, and - is a negative control.

[0014] Figure 3 The enzyme digestion detection results of the overexpression plasmid pFGC5941-BnaRING1b CDS construction are as follows: 1 shows the enzyme digestion fragments of the plasmid vector extracted from different colonies, and the size is consistent with the expectation; 2 shows the undigested vector.

[0015] Figure 4 yes BnaRING1b Electrophoresis image of PCR verification of transgenic plants.

[0016] Where: M is a DNA molecular marker indicating the size of DNA fragments, 1-8 are resistant plants, + is a positive control, and - is a negative control. The figure shows 4 transgenic plants.

[0017] Figure 5 yes BnaRING1b Cold tolerance test of transgenic rapeseed overexpressing transgenic rapeseed.

[0018] Among them: as the treatment time at low temperature increased, the control group Xiangyou 15 was significantly damaged; while BnaRING1b The overexpression of genetically modified rapeseed has not yet shown any harmful symptoms, indicating that... BnaRING1b Overexpression can promote cold resistance in rapeseed. Detailed Implementation

[0019] Unless otherwise specified, the methods described in the following examples are conventional methods. The primers and sequencing work were performed by Qingke Biotechnology Co., Ltd.

[0020] Example 1. Isolation of total RNA from rapeseed and cloning of the full-length CDS of the BnaRING1b protein gene I. Isolation of Total RNA from Rapeseed and Synthesis of First Complementary Strand cDNA Total RNA was extracted from rapeseed seedling leaves using an RNA kit (Promag, Shanghai). Centrifuge tubes containing the total RNA precipitate were dried in a biosafety cabinet until the precipitate became translucent. Then, 40 μL of RNase-free water was added to dissolve the RNA for approximately 5 minutes. Masks and disposable gloves were worn throughout the procedure. 5 μL of the RNA solution was mixed with 1× loading buffer (6× loading buffer: 30 mM EDTA, 36% (v / v) glycerol, 0.05% (w / v) bromophenol blue), and the mixture was analyzed by electrophoresis on a 1.5% agarose gel. Figure 1 The results showed that the 28S rRNA and 18S rRNA bands in lane 1 were distinct and showed no degradation, indicating that the extracted Sclerotinia sclerotiorum RNA was of good quality and high purity, and could be used for the next step of reverse transcription to synthesize cDNA.

[0021] Aspirate 1 μg RNA into a sterile 200 μL PCR tube free of nucleases. Add 1 μL of Oligd(T) and bring the total volume to 11 μL with ddH2O. Incubate at 65°C for 5 min, then place on ice for 3 min. Next, add reagents according to the dosage and reaction mixture of the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Shanghai). Mix gently and centrifuge briefly. Incubate in a PCR instrument at 42°C for 1 hr, then terminate the reaction at 70°C for 5 min. Finally, store the reverse-transcribed cDNA at -20°C for the next step. BnaRING1b PCR template for cloning the full-length CDS of the gene.

[0022] two, BnaRING1b Full-length CDS cloning Search on the website https: / / yanglab.hzau.edu.cn / BnIR BnaRING1b The reference sequence for the gene is BnaA10T0021400ZS. Based on the full-length CDS sequence, Primer Premier 5 software was used to design the gene. BnaRING1b The PCR primer pair for the full-length CDS gene is as follows: forward primer BnaRING1bCDS-F: 5'- TCTAGA ATGCCTGCGTTGAACAACTTTTCC-3' (SEQ ID No:3, underscore indicates) Xba I restriction site) and reverse primer BnaRING1bCDS-R: 5'- CCATGG CTAGACACTGTTACTCTCTAAGGTAC-3' (SEQ ID No:4, underlined indicates ) Nco I restriction site). Using the aforementioned reverse-transcribed cDNA as a template, high-fidelity PCR amplification was performed. BnaRING1b The full-length CDS sequence of the gene. A 50 μl PCR reaction mixture contained: 2 μl template, 1 μl Phusion High-fidelity DNA Polymerase (Invitrogen), 5 μl 10× buffer, 8 μl 2.5 μM dNTPs, 1 μl each of 20 μM forward and reverse primers, and 32 μl water. The reaction conditions were: 94℃ pre-denaturation for 2 minutes; 94℃ denaturation for 30 seconds, 57℃ annealing for 30 seconds, and 68℃ extension for 1 minute, for a total of 30 cycles. After the reaction, the PCR product was detected by 1.5% agarose gel electrophoresis. The result showed an amplified DNA fragment of 996 bp, consistent with the expected target fragment size. Figure 2After the amplified fragment was recovered and purified, it was cloned into the vector pEASY-Blunt Cloning (purchased from TransGen Biotech) to obtain the recombinant plasmid pEASY-BnaRING1bCDS containing the target fragment. After transformation, screening, and sequencing, the nucleotide sequence shown in SEQ ID No:2 is obtained. It consists of 987 bases, and its coding frame is from the 5' end, bases 1-987 (the last three bases are the stop codon TGA), which encodes a protein with the amino acid residue sequence of SEQ ID No:1, which is 328 amino acid residues. The encoded protein is named BnaRING1b.

[0023] Example 2. BnaRING1b Construction of gene overexpression plasmids Select the correctly sequenced pEASY-BnaRING1bCDS recombinant plasmid and simultaneously run FastDigest Enzyme on both plasmids. Nco I and Xba I. Two plasmids (Fermetans) were double-digested at 37℃ for 30-40 min. The reaction volume was 40 μL (1 μg plasmid DNA, 2 μL 10×FastDigest Green Buffer, 0.5 μL FastDigest Green Buffer). Nco I, 0.5 μL FastDigest Xba I. Add water to 40 μL, following the Fermetans kit instructions; after separation of the enzyme digestion product by 1.5% agarose gel electrophoresis, cut off the target band, purify and recover it using a gel extraction kit, and ligate the two recovered target DNA fragments using T4 ligase; the ligation product is then transformed into E. coli using the heat shock method. Esherichia coli DH5α strain cells were transformed and revived in LB liquid medium at 37°C and 200 rpm for 45 min. The revived cells were then evenly spread onto LB solid culture dishes containing kanamycin (50 μg / mL) and incubated at 37°C for 16 h. Single colonies from the LB solid medium were picked and identified by colony PCR using primers 35SF: 5'-CTATCCTTCGCAAGACCCTTC-3' (SEQ ID No. 5) and BnaRING1bCDS-R (SEQ ID No. 4). The PCR amplification products showed electrophoretic bands between 2000 bp and 1000 bp, consistent with the expected fragment size. Single colonies that passed PCR were selected for amplification, plasmids were extracted, and double digestion with Fast Digest Enzyme NcoI and XbaI was performed for verification. (See below) Figure 4The double-digested fragment was slightly larger than 1000 bp, consistent with the expected fragment size. Both PCR and double-digestion results confirmed the successful construction of the overexpression plasmid pFGC5941-BnaRING1bCDS.

[0024] Example 3. Transformation and Molecular Identification of Brassica napus and Cold Resistance Detection of Transgenic Rapeseed I. Conversion of Brassica napus 1) Preparation of explants Approximately 100 complete and plump seeds of the rapeseed variety "Xiangyou 15" were selected and placed in a 150mL Erlenmeyer flask. 20-30mL of 75% ethanol was added to the flask, and the mixture was shaken for 30 seconds to sterilize. The ethanol was then discarded. Next, 20mL of 0.1% HgCl2 and 1 drop of TWEEN-20 disinfectant solution were added, and the mixture was shaken vigorously until foaming occurred. The mixture was allowed to stand for 20 minutes, and the disinfectant solution was discarded. The seeds were rinsed repeatedly with sterile water 3-5 times to remove the foam. 50mL of sterile water (sterilized at high temperature) was added to soak the seeds for 1 hour. The sterile water was then discarded, and the seeds were evenly spread on half of MS solid medium and incubated in the dark at 22℃ for 5-6 days. Once the rapeseed seedlings reached 7-9cm in height, they were transferred to light conditions and allowed to grow for 6-8 hours until the cotyledons turned green. The hypocotyls were cut into 1cm sections to serve as explants for transformation.

[0025] (ii) Preparation of Agrobacterium When the seedlings reach 1-2 cm in height, prepare the bacterial culture. Agrobacterium LBA4404 strain, each carrying the pFGC5941-BnaRING1bCDS plasmid, is streaked onto YEB solid medium containing 50 mg / mL kanamycin, 50 mg / mL streptomycin, and 100 mg / mL rifampin, and incubated at 28°C for 3 days. Single colonies are picked and added to 10 mL of YEB liquid medium containing 50 mg / mL kanamycin, 50 mg / mL streptomycin, and 100 mg / mL rifampin, and cultured at 28°C and 200 rpm for 16-18 hours. 1 mL of the bacterial culture is then added to 50 mL of YEB liquid medium containing 50 mg / mL kanamycin, 50 mg / mL streptomycin, and 100 mg / mL rifampin, and cultured until the bacterial concentration reaches OD500. 600 It reaches 0.4-0.8.

[0026] (iii) Agrobacterium-mediated transformation of cotyledons and screening of resistant seedlings Cut the cotyledons of rapeseed seedlings from the branch above the growing point and transfer them to BM solution. Transfer the expanded Agrobacterium suspension from the Erlenmeyer flask to a 50 mL centrifuge tube, centrifuge at 6000 rpm for 10 min, discard the supernatant, add 25 mL of BM solution to the centrifuge tube, and vortex to resuspend the Agrobacterium; then add 10 μL β-mercaptoethanol and 20 μL acetylsyleugenone, shake well, and pour the resulting working solution into sterilized Petri dishes. Transfer the cut cotyledons to this working solution and soak for 10 min. After soaking, transfer the cotyledons to sterilized absorbent paper. Use tweezers to gently agitate the cotyledons to absorb any remaining working solution from the surface. Finally, evenly spread the cotyledons on co-culture medium (1 L MS + 1 mg 6-benzylaminopurine + 30 g sucrose + 1.6 g plant gel) and co-culture in the dark at 22 °C for 36 h. After co-culture, the explants were transferred to selection medium (1 L MS + 2 mg 6-benzylaminopurine + 20 mg glufosinate + 500 mg cephalosporin + 30 g sucrose + 1.6 g plant gel). The medium was incubated at 22°C under long-day conditions; the selection medium was changed every 10-14 days.

[0027] After several clusters of resistant adventitious shoots differentiated from the explants, the adventitious shoots were cut and separated, and then cultured on rooting medium (1 L 1 / 2 MS + 20 mg glufosinate + 10 g sucrose + 1.6 g plant gel). Once the differentiated seedlings had developed sufficient roots, they were removed from the rooting medium and the roots were rinsed with sterile water to remove any remaining medium. The seedlings were then transferred to vermiculite and hardened off for 10 days. After hardening off, they were transferred to soil, resulting in 28 glufosinate-resistant seedlings. These resistant seedlings were used for subsequent PCR transgenic detection and identification.

[0028] (iv) Identification of transgenic resistant seedlings and BnaRING1b Gene expression detection Total DNA was extracted from the leaf tissues of resistant seedlings transformed with the pFGC5941-BnaRING1bCDS plasmid using the CTAB method. Using the total DNA as a template, and 35SF (SEQ ID No. 5) and BnaRING1bCDS-R (SEQ ID No. 4) as primers, PCR detection was performed (amplified fragment size 2011 bp). The results showed that 12 seedlings had amplified fragments of the same size as the positive control plasmid, indicating that a total of 12 seedlings were obtained. BnaRING1b Gene overexpression transgenic plants (see) Figure 4 After the transgenic plants flower and bear fruit, the seeds are collected, and seed number 11 is selected for cold resistance testing.

[0029] II. Cold Resistance Testing of Genetically Modified Rapeseed The recipient rapeseed variety Xiangyou 15 was used as a control, and the control was compared with... BnaRING1b Transgenic plants overexpressing the gene were subjected to low-temperature treatment at -4℃ for 6 hours. The results showed... BnaRING1b The cold resistance of transgenic plants overexpressing the gene was significantly higher than that of the control. Figure 5 ).

[0030] This patent application was supported by the Sino-French Joint Laboratory for Plant Epigenetics (2024YFE0105100). Related sequences SEQ ID No. 1, rapeseed BnaRING1b protein MPALNNFSAAEKEDDQLGRTTRAEEKEEDPENMDVMEEEGSKERSPSSTSEEQSESEFIEGIDLEDIRKYVQCPICLGIIRKTRTFMECLHRFCQECIDKSMRFGNHECPACRKHVPSRRSLRPDPKFDAFIAAIFGNVDSNEEQDLAFDEDELARNKQFQATI AQVSQRQSEALVKSSRKDAGVLPRSQPSGSGSRRRRNSRNMEHDTSQAAHDDDGDNNRVNASSSAEILPRKRNRRSATRSTAQPSSSSCPSNNDNNCANNVTEEAHHRDSRGIAHGFAWGKGGRRSNARQANNNQGASSSKSVRNARLNRLVDYLSTLESNSV SEQ ID No. 2 ATGCCTGCGTTGAACAACTTTTCCGCCGCCGAGAAAGAAGACGACCAACTTGGTCGTACCACACGAGCTGAGGAGAAAGAAGAAGATCCGGAAAACATGGATGTAATGGAAGAAGAAGGTTCCAAGGAGAGAAGTCCATCTTCTACATCAGAAGAGCAATCAGAATCAGAATTTATAGAAGGTATTGATCTAGAGGATATCCGTAAGTACGTGCAAGTGTCCTATATGTTTAGGAATTATAAGGAAAACAAGGACTTTCATGGAATGTCTCCATCGGTTTTGTCAAGAGTGCATTGATAAGTCAATGAGATTTGGGAACCATGAGTGTCCTGCTTGTAGAAAACATGTACCCAGCCGACGTTCATTAAGACCCGACCCAAAATTCGATGCTTTTATTGCAGCTATATTCGGAAATGTCGATAGTAATGAGGAGCAGGATTTGGCTTTTGATGAAGATGAGTTGGCTCGTAATAAGCAGTTTCAAGCAACTATAG CTCAAGTATCGCAGAGACAATCTGAGGCCCTTGTGAAGTCTTCTCGTAAAGATGCAGGGGTCTTACCGAGATCACAGCCTAGTGGTAGTGGCTCCAGAAGGAGGAGGAGGAACAGCAGAAACATGGAACATGATACATCACAAGCCGCCCATGATGATGATGTGATAACAACAGAGTCAACGCTTCGTCTTCTGCTGAAATACTGCCGAGAAAAAGAAATAGGCGCTCTGCAACTCGTTCTACAGC TCAGCCTTCTTCTTCTTCATGTCCAAGCAACAATGATAATAACTGCGCTAATAATGTGACAGAGGAGGCGCATCATAGAGACAGCAGAGGCATAGCACATGGGTTTGCATGGGGAAAAGGCGGCAGAAGGAGTAACGCAAGGCAAGCGAATAATAACCAAGGAGCTAGTAGTAGTAAGAGGTGTTAGGAATGCTCGCTTGAACAGACTTGTGGATTATCTCAGTACCTTAGAGAGTAACAGTGTCTAG SEQ ID No. 3 5'- TCTAGA ATGCCTGCGTTGAACAACTTTTCC-3'(BnaRING1bCDS-F,underscore indicates Xba I restriction site) SEQ ID No. 4 5'- CCATGG CTAGACACTGTTACTCTCTAAGGTAC-3'(BnaRING1bCDS-R,underscore indicates Nco I restriction site) SEQ ID No. 5 5'-CTATCCTTCGCAAGACCCTTC-3' (35SF).

Claims

1. Application of amino acid sequences as shown in SEQ ID No:1 in improving the cold resistance of rapeseed.

2. Application of the gene encoding the BnaRING1b protein, whose nucleic acid sequence is shown in SEQ ID No:2, in improving the cold resistance of rapeseed.

3. A method for cultivating and improving the cold resistance of rapeseed, characterized in that: The gene encoding the protein described in claim 1 is transferred into rapeseed to cultivate transgenic rapeseed that overexpresses the gene, thereby improving the cold resistance of rapeseed.

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