Rapeseed bnaREV3 protein, gene encoding same and use thereof

Abstract

The application belongs to the technical field of biological gene engineering, and particularly relates to a rape BnaREV3 protein, a coding gene thereof and application. The protein is one of the following amino acid residue sequences: 1) SEQ ID No: 1 in the sequence table; 2) an amino acid residue sequence of SEQ ID No: 1 in the sequence table after substitution, deletion and / or addition of one to five amino acid residues and for a protein for improving cold resistance of rape. The application further includes a recombinant expression vector, a transgenic cell line and an engineering bacterium containing the gene. Expression of the coding gene of the rape BnaREV3 protein in rape can significantly improve the cold resistance of rape. The application provides a brand-new approach for cold resistance of rape.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological gene engineering, and particularly relates to a Brassica napus BnaREV3 protein, a coding gene thereof and application thereof in cold resistance. BACKGROUND

[0002] Brassica napus is an important economic crop. Rapeseed oil from Brassica napus is one of the main sources of human edible oil and is an important part of China's grain and oil security, and is one of China's important strategic materials. At present, nearly 60% of China's edible oil depends on imports. In order to improve China's edible oil security, expanding the acreage of Brassica napus is an important measure taken by the state. In the south of the middle and lower reaches of the Yangtze River, more than 60 million mu of winter idle land is not utilized, the main reason being that there is a conflict between rice and rapeseed planting in the rice-rapeseed cropping system. In order to solve this conflict, the growth period of rapeseed needs to be shortened, and the flowering of rapeseed needs to be advanced to January, which is the month with the lowest temperature in the winter idle land area. If rapeseed flowers in January, the young pods will freeze and cannot set seeds, resulting in no yield. A cold-tolerant material is obtained through breeding of Brassica napus germplasm, and a cold-tolerant gene BnaREV3 is successfully located using the material. Through genetic engineering, the cold-tolerant gene BnaREV3 is cloned, and an overexpression plasmid of the gene is constructed. Then, the BnaREV3 protein gene is transferred into Brassica napus that is not cold-tolerant, so as to obtain Brassica napus germplasm resources with cold resistance. The BnaREV3 protein and the coding gene thereof are first discovered through genetic engineering, and the gene can significantly improve the cold resistance of Brassica napus. SUMMARY

[0003] The purpose of the present application is to provide a cold-tolerant gene resource BnaREV3 protein and the coding gene thereof, and to provide the application of the gene in Brassica napus cold resistance.

[0004] To achieve the above purpose, the technical solution adopted by the present application is as follows: a BnaREV3 protein, which is derived from Brassica napus gene mutation and is one of the following amino acid residue sequences:

[0005] 1) the amino acid residue sequence of SEQ ID No: 1 in the sequence listing;

[0006] 2) a protein obtained by substituting, deleting and / or adding one to five amino acid residues in the amino acid residue sequence of SEQ ID No: 1 in the sequence listing and improving the cold resistance of Brassica napus.

[0007] The gene (BnaREV3) encoding the BnaREV3 protein of the present application is one of the following nucleotide sequences:

[0008] 1) the nucleotide sequence of SEQ ID No: 2 in the sequence listing;

[0009] 2) DNA encoding the protein sequence of SEQ ID No: 1 in the sequence listing;

[0010] 3) a nucleotide sequence having more than 90% homology with the nucleotide sequence defined in SEQ ID No: 2 in the sequence listing and encoding the same functional protein;

[0011] SEQ ID No: 2 in the sequence listing consists of 891 bases, the coding frame of which is the 1-891 bases from the 5' end, encoding a protein having the amino acid residue sequence of SEQ ID No: 1 in the sequence listing.

[0012] The present application also includes a recombinant expression vector containing the gene of the present application, a transgenic cell line and an engineered bacterium, and a primer pair for amplifying any fragment of the gene.

[0013] The present application also provides the application of the above-mentioned BnaREV3 protein gene in regulating the cold resistance of Brassica napus.

[0014] The present application also provides the introduction of the gene encoding the above-mentioned BnaREV3 protein into Brassica napus, and the cultivation of the transgenic Brassica napus, so that the cold resistance of the transgenic Brassica napus is improved, thereby obtaining cold-resistant Brassica napus germplasm resources.

[0015] The transgenic Brassica napus of the present application, which is overexpressing the BnaREV3 protein gene, has significantly improved low-temperature resistance. The present application provides a high-quality gene for improving the cold resistance of Brassica napus. The protein and its encoding gene of the present application have high practical application value and broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is an electrophoretogram of total RNA isolated from Brassica napus leaves.

[0017] Among them: M is a DNA molecular marker indicating the size of the DNA fragment, 1 is the extracted RNA; the RNA sample 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.

[0018] Figure 2 is an electrophoretogram of the PCR amplification product of the full-length CDS of the Brassica napus BnaREV3 protein gene.

[0019] Among them, M is a 100bp plus Ladder DNA marker indicating the size of the DNA fragment, and 1, 2 and 3 are the amplification products.

[0020] Figure 3Restriction enzyme digestion detection results of overexpression plasmid pFGC5941-BnaREV3 CDS construction, 1-4 are different colonies extracted plasmid vector restriction enzyme digestion fragments, the size is consistent with the expected.

[0021] Figure 4 is BnaREV3 PCR verification electrophoretogram of transgenic plants.

[0022] Among them: M is a DNA molecular marker indicating the size of the DNA fragment, 1-14 are resistant plants, CK is a blank control, and the figure shows that there are 4 transgenic plants.

[0023] Figure 5 is BnaREV3 Cold resistance test of overexpression transgenic rapeseed.

[0024] Among them: with the extension of the treatment time in low temperature, the control 77gao is obviously harmed; BnaREV3 Overexpression transgenic rapeseed has not shown harmful symptoms, indicating BnaREV3 Overexpression can promote the cold resistance of rapeseed. DETAILED DESCRIPTION

[0025] The methods in the following examples are conventional methods unless otherwise specified. The primers and sequencing work used are completed by Genescript Biotech Co., Ltd.

[0026] Example 1. Isolation of total RNA from rapeseed and cloning of full-length CDS of BnaREV3 protein gene

[0027] I. Isolation of total RNA from rapeseed and synthesis of first complementary strand cDNA

[0028] Total RNA was extracted from rapeseed seedlings leaf blades using an RNA kit (Promega, Shanghai), and the centrifuge tube with total RNA precipitate was placed in a biological safety cabinet and blown dry until the precipitate was translucent, then 40 μL of RNase-free water was added to dissolve the RNA for about 5 min; the entire operation was performed with a mask and disposable gloves. 5 μL of 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 electrophoresis detection was performed using a 1.5% agarose gel (1×TAE buffer: 0.04 M Tris-acetate, 0.001 M EDTA, pH 7.8). Figure 1 The results showed that the 28S rRNA and 18S rRNA bands in lane 1 were obviously not degraded, indicating that the extracted S. sclerotiorum RNA was of good quality and high purity, and could be used for the next step of reverse transcription to synthesize cDNA.

[0029] 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. BnaREV3 PCR template for cloning the full-length CDS of the gene.

[0030] two, BnaREV3 Full-length CDS cloning

[0031] Search on the website https: / / yanglab.hzau.edu.cn / BnTIR BnaREV3 The reference sequence for the gene is BnaA10G0004700ZS. Based on the full-length CDS sequence, Primer Premier 5 software was used to design the gene. BnaREV3 The PCR primer pair for the full-length CDS gene is as follows: forward primer BnaREV3CDS-F: 5'- TCTAGA ATGAATATGAGCTTACCGGGTTTCAG-3' (SEQ ID No:3, underscore indicates) Xba I restriction site) and reverse primer BnaREV3CDS-R: 5'- CCATGG TCACTGTTGTATGATGATATTAACCTCCTCTG-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. BnaREV3 The full-length CDS sequence of the gene was obtained. 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 903 bp, consistent with the expected target fragment size.Figure 2 After 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-BnaREV3CDS containing the target fragment. After transformation, screening, and sequencing, the nucleotide sequence shown in SEQ ID No:2 is obtained. It consists of 891 bases, and its coding frame is from the 5' end, bases 1-891 (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 311 amino acid residues. The encoded protein is named BnaREV3.

[0032] Example 2. BnaREV3 Construction of gene overexpression plasmids

[0033] Select the correctly sequenced pEASY-BnaREV3CDS 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, ligate the two recovered target DNA fragments using T4 ligase, and transform the ligation product into E. coli using the heat shock method. Esherichia coli DH5α-transformed cells were revived in liquid LB medium at 37°C and 200 rpm for 1 h. 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 BnaREV3CDS-R (SEQ ID No. 4). The PCR amplification product showed an electrophoretic band between 1000 bp and 900 bp, consistent with the expected fragment size. Single colonies that passed PCR were selected for expansion culture, plasmids were extracted, and double digestion with FastDigest Enzyme NcoI and XbaI was performed for verification. (See [link to relevant documentation]). Figure 4The double-enzyme digestion fragment was slightly larger than 1000 bp, consistent with the expected fragment size. Both PCR and double-enzyme digestion results confirmed the successful construction of the overexpression plasmid pFGC5941-BnaREV3CDS.

[0034] Example 3. Transformation and Molecular Identification of Brassica napus and Cold Resistance Detection of Transgenic Rapeseed

[0035] I. Conversion of Brassica napus

[0036] 1) Preparation of explants

[0037] Approximately 100 intact and plump seeds of the susceptible rapeseed variety "77gao" 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 4-5 days. Once the rapeseed seedlings reached 4-5cm in height, they were transferred to light conditions and allowed to grow for 6-8 hours until the cotyledons turned green. The cotyledon petioles were used as explants for transformation.

[0038] (ii) Preparation of Agrobacterium

[0039] When the seedlings reach 1-2 cm in height, prepare the bacterial culture. Agrobacterium LBA4404 strain, each carrying the pFGC5941-BnaREV3CDS 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.

[0040] (iii) Agrobacterium-mediated transformation of cotyledons and screening of resistant seedlings

[0041] 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.

[0042] 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 transplanted into soil, resulting in 14 glufosinate-resistant seedlings. These resistant seedlings were used for subsequent PCR transgenic detection and identification.

[0043] (iv) Identification of transgenic resistant seedlings and BnaREV3 Gene expression detection

[0044] Total DNA was extracted from the leaf tissues of resistant seedlings transformed with the pFGC5941-BnaREV3CDS plasmid using the CTAB method. Using the total DNA as a template, and 35SF (SEQ ID No. 5) and BnaREV3CDS-R (SEQ ID No. 4) as primers, PCR detection was performed (amplified fragment size 1915 bp). The results showed that four plants had amplified fragments of the same size as the positive control plasmid, indicating that a total of four plants were obtained. BnaREV3 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.

[0045] II. Cold Resistance Testing of Genetically Modified Rapeseed

[0046] The receptor rapeseed 77gao was used as a control, and the control was compared with... BnaREV3Transgenic plants overexpressing the gene were subjected to low-temperature treatment at -4℃ for 6, 12, and 24 hours, respectively. The results showed... BnaREV3 The cold resistance of transgenic plants overexpressing the gene was significantly higher than that of the control. Figure 5 ).

[0047] Related sequences

[0048] SEQ ID No. 1, rapeseed BnaREV3 protein

[0049] MNMSLPGFSTTLPHSKTTMPVSARSHTMSFSEDPTKKIRKPYTITKSRENWTEQEHDKFIEALHLFDRDWKKIEAFVGSKTVIQIRSHAQKYFLKVQKNGTNEHLPPPRPKRKANHPYPQKASKSVALTTSNALLEHEYLYPTDPQPV ISTPNHGLMRCNVITPIPVIKEELGVLENCCSTSRSRSIRDKMRTRTVTETDDQRVMPNFAEVYSFIGSVFDPKTTGHVQRLKQMDPINLETVLLLMKNLSVNLSSPEFEEQVSVTHRYTKFVKSSFFGLRGMIGLFYTEEVNIIIQQ

[0050] SEQ ID No. 2

[0051] ATGAATATGAGCTTACCGGGTTTCAGTACTACTCTTCCCCACTCGAAGACAACGATGCCTGTTTCTGCACGGAGCCATACGATGTCGTTCAGCGAGGATCCAACAAAGAAGATTAGAAAGCCATACACAATCACCAAGTCTAGAGAGAACTGGACGAGCAAGAACACGACAAGTTCATTGAAGCTCTCCATTTGTTTGACCGTGATTGGAAGAAAATAGAG GCCTTGTTGGATCAAAAACAGTTATCCAGATACGAAGCCACGCGCAGAAATACTTTCTAAAGGTTCAGAAGAATGGGACTAACGAACATCTTCCTCCTCCTCGACCAAAGAGGAAAGCTAATCATCCCTATCCACAAAAGGCTTCCAAAAGTGTGGCTCTTACAACTTCAAACGCATTGCTTGAACATGAGTACTTGTACCCCACTGATCCACAACCGGTGA TTAGTACTCCTAATCACGGATTAATGCGTTGCAATGTTATTACGCCAATTCCAGTGATCAAAGAGGAATTGGGTGTCCTAGAGAACTGTTGCAGCACTAGTCGTAGTAGGAGTATTAGAGATAAGATGAGGACGAGAACAGTTACGGAGACAGATGACCAGAGAGTGATGCCGAATTTCGCTGAAGTTTACAGCTTTATAGGAAGTGTATTCGATCCAAAAAC AACAGGTCATGTCCAGAGGTTAAAGCAAATGGATCCTATCAATCTAGAAACGGTCCTCTTATTGATGAAAAACTTGTCTGTAAACCTGTCAAGCCCCGAGTTTGAAGAACAAGTAAGTGTTACTCATCGCTACACAAAGTTTGTTAAATCTTCATTTTTTGGCTTACGGGGCATGATTGGTCTGTTTTATACAGAGGAGGTTAATATCATCATACAACAGTGA

[0052] SEQ ID No. 3

[0053] 5'- TCTAGAATGAATATGAGCTTACCGGGTTTCAG-3'(BnaREV3CDS-F,underscore indicates Xba I restriction site)

[0054] SEQ ID No. 4

[0055] 5'- CCATGG TCACTGTTGTATGATGATATTAACCTCCTCTG-3'(BnaREV3CDS-R,underscore indicates Nco I restriction site)

[0056] SEQ ID No. 5

[0057] 5'-CTATCCTTCGCAAGACCCTTC-3' (35SF)

Claims

1. Application of rapeseed BnaREV3 protein with amino acid sequence as shown in SEQ ID No:1 in improving the cold resistance of rapeseed.

2. Application of the gene encoding the BnaREV3 protein with the nucleic acid sequence 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 of 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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