Rape BnaREV3 protein as well as coding gene and application thereof

Abstract

The invention belongs to the technical field of biological genetic engineering, and particularly relates to a rape BnaREV3 protein as well as a coding gene and application thereof. The protein is one of the following amino acid residue sequences: 1) SEQ ID No: 1 in a sequence table; and 2) a protein which is obtained by substituting and / or deleting and / or adding one to five amino acid residues in the amino acid residue sequence of SEQ ID No: 1 in the sequence table and has the effect of improving the cold resistance of the rape. The invention also comprises a recombinant expression vector containing the gene, a transgenic cell line and an engineering bacterium. The expression of the coding gene of the rape BnaREV3 protein in the rape can significantly improve the cold resistance of the rape. The invention provides a brand-new way for cold resistance of oilseed rape.

Description

Technical Field

[0001] The invention belongs to the technical field of biological gene engineering, and specifically relates to a rapeseed BnaREV3 protein and a coding gene thereof and applications in cold resistance. Background Art

[0002] Rapeseed is an important cash crop. Rapeseed oil from rapeseed is one of the main sources of edible oil for human beings. It is an important part of my country's grain and oil security and one of my country's important strategic materials. At present, nearly 60% of my country's edible oil depends on imports. In order to improve my country's edible oil safety, expanding the area of ​​rapeseed germplasm is an important measure taken at the national level. In the south of the middle and lower reaches of the Yangtze River, more than 60 million mu of winter fallow fields are not used. The main reason is that there is a conflict between rice and rapeseed planting under the rice-oil farming method. In order to solve this conflict, it is necessary to shorten the growth period of rapeseed and advance the flowering of rapeseed to January. January is the month with the lowest temperature in the winter fallow field area. If rapeseed blooms in January, the young pods of rapeseed will freeze to death and cannot bear fruit, resulting in no yield. A cold-resistant material was obtained through the breeding of rapeseed germplasm, and a cold-resistant gene BnaREV3 was successfully located using this material. Through genetic engineering methods, the cold-resistant gene BnaREV3 is first cloned and an overexpression plasmid of the gene is constructed; then the BnaREV3 protein gene is transferred into Brassica napus, which is not cold-resistant, in order to obtain cold-resistant rapeseed germplasm resources. The present invention discovers the BnaREV3 protein and its encoding gene for the first time through genetic engineering methods, and the gene can significantly improve the resistance of rapeseed to cold damage. Summary of the invention

[0003] The purpose of the present invention is to provide a cold-resistant gene resource BnaREV3 protein and its encoding gene in view of the above-mentioned deficiencies in the prior art, and to provide the application of the gene in rapeseed cold resistance.

[0004] To achieve the above object, the technical solution adopted by the present invention is: a BnaREV3 protein, derived from rapeseed gene mutation, is one of the following amino acid residue sequences:

[0005] 1) SEQ ID No: 1 in the sequence listing;

[0006] 2) A protein having an effect of improving the cold resistance of rapeseed by replacing and / or deleting and / or adding one to five amino acid residues of the amino acid residue sequence of SEQ ID No: 1 in the sequence list.

[0007] The gene encoding the BnaREV3 protein of the present invention (BnaREV3) 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 SEQ ID No: 1 in the sequence list;

[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 a protein with the same function;

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

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

[0013] The present invention also provides application of the BnaREV3 protein gene in regulating rapeseed cold resistance.

[0014] The present invention also provides the method of transferring the gene encoding the BnaREV3 protein into rapeseed to obtain transgenic rapeseed through cultivation, wherein the cold resistance of the transgenic rapeseed is improved, thereby obtaining cold-resistant rapeseed germplasm resources.

[0015] The invention is a transgenic rapeseed with overexpression of the BnaREV3 protein gene, which significantly improves the low temperature resistance. The invention provides a high-quality gene for improving the cold resistance of rapeseed. The protein and the encoding gene of the invention have high practical application value and broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the electrophoresis diagram of total RNA isolated from rapeseed leaves.

[0017] Among them: M is a DNA molecular marker indicating the size of the DNA fragment, 1 is the extracted RNA; the RNA from the 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 This is the electrophoresis diagram of the PCR amplification product of the full-length CDS of the rapeseed BnaREV3 protein gene.

[0019] Among them, M is the 100bp plus ladder DNA marker of the molecular weight of the DNA fragment, and 1, 2, and 3 are the amplification products.

[0020] Figure 3The restriction enzyme detection results of the overexpression plasmid pFGC5941-BnaREV3CDS construction. 1-4 are the restriction enzyme fragments of plasmid vectors extracted from different colonies, and the sizes are consistent with expectations.

[0021] Figure 4 This is the PCR verification electrophoresis diagram of BnaREV3 transgenic plants.

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

[0023] Figure 5 This is a cold resistance test of BnaREV3 overexpressing transgenic rapeseed.

[0024] Among them: as the treatment time in low temperature increased, the control 77gao was obviously damaged; while the BnaREV3 overexpression transgenic rapeseed did not show any damage symptoms, indicating that BnaREV3 overexpression can promote rapeseed's cold resistance. DETAILED DESCRIPTION

[0025] The methods in the following examples are all conventional methods unless otherwise specified. The primers and sequencing work were completed by Qingke Biotechnology Co., Ltd.

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

[0027] 1. Isolation of total RNA from rapeseed and synthesis of first complementary strand cDNA

[0028] Total RNA from rapeseed seedling leaves was extracted using an RNA kit (Promega, Shanghai). The centrifuge tube with total RNA precipitate was placed in a biosafety cabinet and blown dry until the precipitate became translucent. Then 40 μL of RNase-free water was added to dissolve the RNA for about 5 minutes. Masks and disposable gloves were worn during the entire operation. 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 detected by electrophoresis using 1.5% agarose gel ( Figure 1 The results showed that the 28S rRNA and 18S rRNA bands in lane 1 were obvious and not degraded, indicating that the extracted RNA of Sclerotinia sclerotiorum was of good quality and high purity and could be used for the next step of reverse transcription to synthesize cDNA.

[0029] Pipette 1 μg RNA into a sterile nuclease-free 200 μL PCR tube, add 1 μL Oligod(T) first, and then use ddH 20 to a total volume of 11 μL, incubate at 65°C for 5 min, and then place on ice for 3 min; then add each reagent according to the system of the reverse transcription kit RevertAidFirst Strand cDNA Synthesis Kit (ThermoFisher Scientific, Shanghai), mix gently, and centrifuge briefly. Then put it in a PCR instrument for 1 hour at 42°C and incubate at 70°C for 5 min to terminate the entire reaction. Finally, the cDNA obtained by the reverse transcription was stored at -20°C and used as a PCR template for cloning the full-length CDS of the BnaREV3 gene in the next step.

[0030] 2. Cloning of the full-length CDS of BnaREV3 gene

[0031] The reference sequence of BnaREV3 gene was found on the website https: / / yanglab.hzau.edu.cn / BnTIR, which was BnaA10G0004700ZS. Based on the reference CDS full-length sequence, Primer Premier 5 software was used to design the PCR primer pair for the full-length CDS of BnaREV3 gene. The primer sequences were forward primer BnaREV3CDS-F: 5'- TCTAGA ATGAATATGAGCTTACCGGGTTTCAG-3'(SEQ ID No:3, the underline indicates the XbaI restriction site) and reverse primer BnaREV3CDS-R: 5'- CCATGG TCACTGTTGTATGATGATATTAACCTCCTCTG-3' (SEQ ID No: 4, the underline indicates the NcoI restriction site). The full-length CDS sequence of the BnaREV3 gene was amplified by high-fidelity PCR using the cDNA reverse transcribed above as a template. The 50μl PCR reaction system contained: 2μl template, 1μl high-fidelity enzyme Phusion High-fidelity DNA Polymerase (Invitrogen), 5μl 10× buffer, 8μl 2.5uM dNTP, 1μl each of 20μM forward and reverse primers, and 32μl water. The reaction conditions were: pre-denaturation at 94℃ for 2 minutes; denaturation at 94℃ for 30 seconds, annealing at 57℃ for 30 seconds, and extension at 68℃ for 1 minute, for a total of 30 cycles. After the reaction, the PCR product was detected by 1.5% agarose gel electrophoresis, and the result showed that a 903bp DNA fragment was amplified, which was 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 Quanshijin Company) to obtain the recombinant plasmid pEASY-BnaREV3CDS containing the target fragment. After transformation and screening, sequencing was performed. The result is shown in the nucleotide sequence shown in SEQ ID No: 2, which consists of 891 bases, and its coding frame is the 1st to 891th bases from the 5' end (the last three bases are the stop codon TGA), encoding 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. Construction of BnaREV3 gene overexpression plasmid

[0033] The pEASY-BnaREV3CDS recombinant plasmid with correct sequencing was selected, and the pFGC5941 plasmid was double-digested with FastDigest Enzyme NcoI and XbaI (Fermetans) respectively, at a digestion temperature of 37°C, a digestion time of 30-40 min, and a reaction system of 40 μL (1 μg plasmid DNA, 2 μL 10×FastDigest Green Buffer, 0.5 μL FastDigestNcoI, 0.5 μL FastDigestXbaI, and water was added to 40 μL), and the specific steps were carried out according to the instructions of the Fermetans company kit; the digestion products were separated by 1.5% agarose gel electrophoresis, the target band was cut out, purified and recovered with a gel recovery kit, the two recovered target DNA fragments were connected by T4 ligase, and the connected products were transformed into Escherichia coli by heat shock method (Esherichia coli)DH5α, and the transformed cells were revived in liquid LB medium at 37°C and 200rpm for 1 hour; the recovered cells were evenly spread on LB solid culture dishes containing kanamycin (50μg / mL) and cultured at 37°C for 16 hours. Single colonies on LB solid culture 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), showing that the electrophoresis band of the PCR amplification product was between 1000bp and 900bp, which was consistent with the expected fragment size. The single colony with the correct PCR detection was selected for expansion culture, plasmid was extracted, and double enzyme digestion verification was performed with FastDigest Enzyme NcoI and XbaI, see Figure 4 The double enzyme digestion fragment was slightly larger than 1000bp, which was consistent with the expected fragment size. Both PCR identification and double enzyme digestion identification results showed that the overexpression plasmid pFGC5941-BnaREV3CDS was successfully constructed.

[0034] Example 3. Transformation and molecular identification of Brassica napus and detection of cold resistance of transgenic rapeseed

[0035] 1. Transformation of Brassica napus

[0036] 1) Explant preparation

[0037] Select about 100 complete and full seeds of the susceptible Brassica napus variety "77gao" and place them in a 150mL Erlenmeyer flask. Add 20-30mL of 75% ethanol to the Erlenmeyer flask containing the seeds, shake and sterilize for 30s, and discard the alcohol; then add 20mL of 0.1% HgCl 2 Add 1 drop of TWEEN-20 disinfectant, shake vigorously until foaming, let stand for 20 minutes, discard the disinfectant, rinse repeatedly with sterile water 3-5 times to wash off the foam; add 50mL of high-temperature sterilized sterile water to soak the seeds for 1 hour; pour out the sterile water, spread the seeds evenly on 1 / 2MS solid culture medium, and culture in the dark at 22℃ for 4-5 days. When the rapeseed seedlings grow to 4-5cm, transfer them to light conditions and grow for 6-8 hours to make the cotyledons turn green. The cotyledon petioles are used as explants for transformation.

[0038] II) Preparation of Agrobacterium

[0039] When the seedlings grow to 1-2 cm, start preparing the bacterial solution. Streak the Agrobacterium LBA4404 strain carrying the pFGC5941-BnaREV3CDS plasmid on YEB solid culture medium containing 50 mg / mL kanamycin, 50 mg / mL streptomycin and 100 mg / mL rifampicin, and culture at 28°C for 3 days. Pick a single colony and place it in 10 mL of YEB liquid culture medium containing 50 mg / mL kanamycin, 50 mg / mL streptomycin and 100 mg / mL rifampicin, and expand the culture at 28°C and 200 rpm for 16-18 hours. Pipette 1 mL of the bacterial solution and add it to 50 mL of YEB liquid culture medium containing 50 mg / mL kanamycin, 50 mg / mL streptomycin and 100 mg / mL rifampicin, and expand the culture until the bacterial solution OD 600 Reach 0.4-0.8.

[0040] III) Agrobacterium transformation of cotyledon petioles and screening of resistant seedlings

[0041] Cut the cotyledon petiole of rapeseed seedlings from the branch above the growth point and transfer it to BM solution. Transfer the expanded Agrobacterium solution from the triangular flask to a 50mL centrifuge tube, centrifuge at 6000rpm / min for 10min, remove the supernatant, add 25mL BM solution to the centrifuge tube, shake to resuspend the Agrobacterium; then add 10μL β-mercaptoethanol and 20μL acetosyringone, shake well and pour into a sterilized plate as working solution. Transfer the cut cotyledon petiole to the working solution and soak for 10min. After soaking, transfer the cotyledon petiole to sterilized absorbent paper. Use tweezers to turn the cotyledon petiole so that the residual working solution on the surface of the cotyledon petiole is absorbed by the absorbent paper. Finally, evenly spread the cotyledon petiole on the co-cultivation medium (1 liter MS + 1mg 6-benzylaminopurine + 30g sucrose + 1.6g plant gel) and co-cultivate in the dark at 22℃ for 36h. After co-cultivation, the explants were transferred to screening medium (1 liter MS + 2 mg 6-benzylaminopurine + 20 mg glufosinate + 500 mg cephalosporin + 30 g sucrose + 1.6 g phytagel) at 22°C with long daylight; the screening medium was changed every 10-14 days.

[0042] When the explant differentiates into several clusters of resistant adventitious buds, the adventitious buds are cut and separated and placed in rooting medium (1 liter 1 / 2MS + 20 mg glufosinate + 10 g sucrose + 1.6 g plant gel) for further cultivation. After the differentiated seedlings grow enough roots, they are taken out of the rooting medium and the culture medium remaining on the roots is cleaned with sterile water. The differentiated seedlings are then moved into vermiculite and hardened for 10 days. After hardening, they are transferred to soil, and a total of 14 glufosinate-resistant seedlings are obtained. The resistant seedlings are used for the next step of PCR transgenic detection and identification.

[0043] IV) Identification of transgenic resistant seedlings and detection of BnaREV3 gene expression

[0044] Total DNA was extracted from the leaf tissue of resistant seedlings transformed with pFGC5941-BnaREV3CDS plasmid using the CTAB method. PCR detection was performed using total DNA as a template and 35SF (SEQ ID No. 5) and BnaREV3CDS-R (SEQ ID No. 4) as primers (amplified fragment was 1915 bp). The test results showed that 14 strains had the same amplified fragment size as the plasmid as the positive control, indicating that a total of 4 BnaREV3 gene overexpressing transgenic plants were obtained (see Figure 4 After the transgenic plants bloomed and bore fruit, the seeds were collected and No. 11 was selected for cold resistance identification.

[0045] 2. Cold resistance test of genetically modified rapeseed

[0046] The recipient rapeseed 77gao was used as the control. The control and BnaREV3 gene overexpressing transgenic plants were subjected to low temperature treatment at -4°C for 6, 12 and 24 h, respectively. The results showed that the cold tolerance of BnaREV3 gene overexpressing transgenic plants 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, the underline indicates the XbaI restriction site)

[0054] SEQ ID No.4

[0055] 5' - CCATGG TCACTGTTGTATGATGATATTAACCTCCTCTG-3'(BnaREV3CDS-R, the underline indicates the NcoI restriction site)

[0056] SEQ ID No.5

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

Claims

1. A rapeseed BnaREV3 protein, which is a protein consisting of the amino acid sequence shown in SEQ ID No:

1.

2. A gene encoding the protein according to claim 1, wherein the nucleotide sequence is shown in SEQ ID No:

2.

3. A protein containing the rapeseed BnaREV3 protein of claim 1 with 1 to 5 amino acid residues substituted and / or deleted and / or added, and having the effect of improving the cold resistance of rapeseed.

4. A recombinant expression vector, an engineered bacterium and a transgenic cell line containing the gene according to claim 2.

5. Use of the protein according to claim 1 in rapeseed cold resistance.

6. Use of the gene according to claim 2 in plant resistance.

7. A method for cultivating rapeseed with improved cold resistance, characterized in that: The gene encoding the protein according to claim 1 or the gene according to claim 2 is transferred into rapeseed to obtain transgenic rapeseed after cultivation, and the resistance of the transgenic rapeseed to bacterial sclerotinia is improved.

Images