Specific CIDEB gene of mammal and application of specific CIDEB gene in increasing oil content of rape

Abstract

The invention discloses a mammal specific CIDEB gene, and belongs to the technical field of gene engineering and biology. The nucleotide sequence of the CIDEB gene is shown as SEQ ID NO: 1, the CIDEB gene is composed of 657 bp, a lipid droplet localization protein is encoded, an agrobacterium-mediated genetic transformation method is used, an expression vector containing the CIDEB gene is transformed into a genome of rape, a transgenic rape variety is obtained, and it is found that the CIDEB gene can positively regulate and control accumulation of the oil content. The high-oil-content rape obtained by the invention has very important application value in high-oil breeding.

Description

Technical Field

[0001] The present invention belongs to the fields of genetic engineering and biotechnology, and particularly relates to a mammalian-specific CIDEB gene and its application in regulating the oil content of rapeseed. Background Art

[0002] Brassica napus is the third largest oil crop in the world, producing about 15% of the world's edible oil. In addition, rapeseed oil is also an important resource for extracting biodiesel. Therefore, improving the seed oil content of Brassica napus is a major goal for breeders. Although many genes involved in lipid biosynthesis have been reported, since seed oil content is a quantitative trait, only a few genes have been truly used to increase seed oil content in breeding. In the past decade, many genes have been verified to effectively increase the seed oil content of Brassica napus through genetic engineering. For example, the pyruvate transporter (BnaBASS2) in Brassica napus can significantly increase the seed oil content by 1.4% - 3.4%, and overexpression of the long non-coding RNA MSTRG.86004 increases the seed oil content by about 2% and changes the fatty acid composition.

[0003] Transferring animal genes into plants can increase plant resistance and improve quality. For example, transferring the human interferon gene into tobacco and rice makes the transgenic plants have antiviral properties; introducing the animal obesity gene FTO into rice can promote tillering and root growth of rice, enhance photosynthesis, and improve drought resistance; transferring the Bt gene of Bacillus thuringiensis into cotton to synthesize Bt insecticidal protein, and the transgenic insect-resistant cotton saves the country tens of billions or even hundreds of billions of yuan in economic losses every year; introducing a mammalian gene called CYP2E1 into the indoor plant ivy, and the CYP2E1 gene can encode plant cytochrome P450 2E1, and this enzyme can decompose volatile organic compounds, effectively improving the detoxification and air purification ability of plants; the antifreeze protein gene AFP from polar fish can help these fish survive in low-temperature environments, and introducing the fish AFP gene into rice can enhance cold tolerance. In short, introducing animal genes into plants can endow plants with unprecedented traits and provide more ways for the breeding of new varieties.

[0004] However, no animal gene has been reported to be used to increase the seed oil content of Brassica napus.

[0005] Cell death-inducing DFFA-like effector b (CIDEB) is a mammalian-specific protein. It has been previously reported that CIDEB induces apoptosis and DNA fragmentation when localized to mitochondria. CIDEB has a unique tissue expression pattern. Human CIDEB protein is highly expressed in the liver and lowly expressed in the small intestine, while mouse CIDEB is mainly expressed in the liver and kidney. CIDEB plays an important role in regulating lipid storage, LD formation, lipid degradation, and hepatic steatosis. However, the function of CIDEB in plants is still unclear.

[0006] Based on this, the present invention cloned a mammalian-specific protein gene CIDEB, and used the Napin promoter to initiate the expression of CIDEB. The oil content of the obtained rapeseed was significantly increased. The results of this study indicate that the CIDEB gene plays an important role in regulating the oil content of rapeseed and has considerable application prospects in creating new rapeseed germplasms with high oil content. Summary of the Invention

[0007] The object of the present invention is to provide a mammalian-specific CIDEB gene and its application in increasing the oil content of rapeseed.

[0008] The nucleotide sequence of the CIDEB gene is shown in SEQ ID NO:1 and consists of 657 bp; it encodes a lipid droplet-localized protein, and the protein sequence is shown in SEQ ID NO:2, encoding 219 amino acids. The gene of the present invention and any polynucleotide fragment of interest can be amplified from the vector of the synthetic gene by PCR technology.

[0009] The present invention further provides a method for obtaining rapeseed with high oil content: using the Agrobacterium-mediated genetic transformation method, transforming the expression vector containing the CIDEB gene into the genome of rapeseed to obtain transgenic rapeseed varieties.

[0010] When constructing the recombinant plant expression vector, a seed-specific expression promoter Napin is added before the transcriptional start nucleotide of the CIDEB gene. In addition, to facilitate the screening of transgenic plants, an antibiotic marker is also added to the expression vector.

[0011] The present invention also provides a recombinant plant expression vector containing the CIDEB gene and a transgenic rapeseed plant.

[0012] By obtaining transgenic rapeseed plants, the expression level, oil content, and lipidome of the transformed materials were measured, and the cytological sections of the seeds of the transformed materials were analyzed. It was found that the CIDEB gene positively regulates the accumulation of oil content. The rapeseed with high oil content obtained in the present invention has no obvious difference in the vegetative growth and reproductive growth cycles from normal plants, and this genetic resource has very important applications in rapeseed high-oil breeding.

[0013] The expression vector described in the present invention refers to any vector known in the prior art that can be expressed in plants. For example, the expression vectors suitable for constructing the expression vectors described in the present invention include but are not limited to, such as Napin-PBILoxP, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 : Plasmid map of the overexpression vector of CIDEB constructed.

[0015] Figure 2 : qRT-PCR detection of transgenic single plants. * indicates P < 0.05 in Student's t test, and ** indicates P < 0.01 in Student's t test.

[0016] Figure 3 : Phenotypic identification of CIDEB in rapeseed. The oil content and fatty acid content of rapeseed seeds were analyzed by gas chromatography - flame ionization detector (GC-FID). OE4, OE5, and OE6 are overexpression lines, and each line has 6 - 8 different single plants. * indicates P < 0.05 in Student's t test, and ** indicates P < 0.01 in Student's t test.

[0017] Figure 4 : Transmission electron microscopy observation of oil bodies in rapeseed seeds. (a) Transmission electron microscopy observation of seeds. (b) Number of lipid droplets and size of each lipid droplet in each picture under 4000 times magnification.

[0018] Figure 5 : Determination of total seed lipid TAG and each TAG. Each line has 6 different single plants. * indicates P < 0.05 in Student's t test, and ** indicates P < 0.01 in Student's t test. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] The present invention will be further explained below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions such as those described in the reference book "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory, 1989), or according to the methods recommended in the operation manuals provided by the manufacturers.

[0020] Example 1 Synthesis and Amplification of CIDEB Gene

[0021] (1) Synthesis of CIDEB Gene

[0022] Cell death-inducing DFFA-like effector b (CIDEB) is a mammalian-specific protein with no homologous genes in plants. Therefore, the CDS sequence (NP_034024) of this gene in mice was obtained from the NCBI website, directly synthesized by the company after optimizing the codons to be more suitable for plants, and ensuring that the amino acid sequence remained unchanged. The CDS sequence and the protein sequence are the sequences shown in SEQ ID NO:1 and SEQ ID NO:2 of the sequence listing, respectively. The CDS sequence includes a complete ORF reading frame and the start codon ATG, consisting of 657 bp and 219 amino acids respectively.

[0023] (2) Amplification of CIDEB gene

[0024] Primers were designed according to the CDS sequence obtained from the above analysis. Using the company's general synthetic vector plasmid as a template, the forward primer CIDEB-Asc1-F sequence is 5’-TTGGCGCGCCACCATGGAGTACCTCTCTGCTTT-3’, and the reverse primer CIDEB-BamH1-R sequence is 5’-CGGGATCCTTATTTGTCGTCGTCGTCCTTGTAGTCCATAGAGTGAAGGTGTCTTTGTCC-3’. A fragment containing the full-length CDS of CIDEB was amplified. I-5 TM 2×High-Fidelity Master Mix (TSINGKE Biologica technology) was used for PCR amplification. The PCR amplification system is as follows:

[0025]

[0026] PCR amplification program: Total denaturation at 98℃ for 1 min; denaturation at 98℃ for 15 sec, annealing at 55℃ for 15 sec, extension at 72℃ for 18 sec, 34 circles; total extension at 72℃ for 5 min.

[0027] The amplified product was detected by agarose gel electrophoresis. The full-length CIDEB of 657 bp was obtained by amplification. The product was gel-extracted using the Tiangen agarose gel extraction kit ( http: / / www.tiangen.com / ).

[0028] Example 2 Construction of CIDEB gene overexpression transformation vector

[0029] (1) The obtained CIDEB fragment was double-digested with the fast restriction enzymes Asc1 and BamHI. The double-digestion system is as follows:

[0030]

[0031] The digestion reaction was carried out in a 37°C water bath for 1 hour. The digested products were recovered using Tiangen's DNA purification kit.

[0032] (2) The digested products were ligated to the vector Napin-PBILoxP (kindly provided by the Oil Crops Research Institute, Chinese Academy of Agricultural Sciences), which contains a seed-specific expression promoter Napin and an antibiotic marker.

[0033] The ligation method is as follows:

[0034]

[0035] Ligation reaction conditions: Overnight at 4°C.

[0036] (3) Transformation of Escherichia coli DH5α was carried out as follows:

[0037] a. Take 100 μL of competent cells melted on ice, add the target ligation product, mix gently, and let stand on ice for 30 min;

[0038] b. Heat shock in a 42°C water bath for 45 s, quickly transfer to an ice bath, and let stand for 2 min;

[0039] c. Add 400 μL of sterile liquid LB medium without antibiotics to the centrifuge tube, mix well, and recover at 37°C and 200 rpm for 60 min;

[0040] d. According to the experimental needs, pipette different volumes of the recovered liquid and spread it evenly on LB medium containing the corresponding antibiotics. Invert the plate and incubate it overnight in a 37°C incubator.

[0041] Screen monoclonal colonies for positive identification. Select 3 positive clones for sample submission and sequencing. The analysis results show that the CDS sequence of the CIDEB gene was successfully ligated to the vector, that is, the plant expression vector Napin-PBILoxP of the transformed plant was successfully constructed (as Figure 1 shown).

[0042] (4) The correctly constructed recombinant plasmid vector was introduced into the Agrobacterium tumefaciens strain GV3101, and positive monoclonal colonies were selected and stored in an -80°C refrigerator. The introduction method is as follows:

[0043] a. Wash the electroporation cuvette: First wash with pure water, then with ultrapure water, pour out, then wash with anhydrous ethanol (pipette with a 1 ml pipette tip), pour out the anhydrous ethanol, and place it in a laminar flow hood to dry;

[0044] b. Take 50 μl of Agrobacterium tumefaciens competent cells GV3101;

[0045] c. Take 1 μl of the correctly constructed recombinant plasmid, add it to 50 μl of competent cells, gently pipette and mix well to avoid generating bubbles;

[0046] d. Place the washed and dried electroporation cuvette in ice for pre-cooling, and then pipette the above-mentioned mixture along the wall of the cuvette;

[0047] e. Adjust the electroporator to 1800V;

[0048] f. Take out the electroporation cuvette from the ice and wipe the outer wall of the electroporation cuvette clean with absorbent paper;

[0049] g. Put the electroporation cuvette into the instrument and press the "push" key twice continuously. If you hear a "beep" sound after a few seconds, it is successful;

[0050] h. After successful electroporation, add 400 μl of antibiotic-free LB to the electroporation cuvette, pipette a few times, and transfer it to a sterile centrifuge tube;

[0051] i. Activate at 28 °C for about 2 h, take 100 μl and spread it on a plate containing double antibiotics. Seal it with a sealing film and incubate it upside down in a 28 °C incubator for 2 days, then pick colonies for detection.

[0052] (5) Agrobacterium colony detection

[0053] Pick colonies into double-antibiotic LB, culture at 28 °C for 2 hours, take an appropriate amount of bacterial liquid for PCR detection, and preserve the positive Agrobacterium bacterial liquid.

[0054] Example 3 Genetic transformation experiment

[0055] (1) Genetic transformation of rapeseed

[0056] Perform genetic transformation of the constructed CIDEB overexpression vector on rapeseed using the Agrobacterium-mediated genetic transformation method. The receptor used for rapeseed transformation in the present invention is Brassica napus Westar. For the specific operation procedure, please refer to the reference: An efficient Agrobacterium-mediated transformation method using hypocotyls as explants for Brassica napus.

[0057] (2) Identification of overexpression transgenic individuals

[0058] Genomic DNA of the obtained overexpressing transgenic rapeseed plants was extracted, and the insertion of the exogenous gene fragment was detected by PCR. In this invention, the backbone vector for overexpression was Napin-PBILoxP. Primers Primer-R (5’-TTTTACATTAGCCTCAAACATCC-3’) were designed on the backbone vector, and primers Primer-CIDEB-F (5’-ATGGAGTACCTCTCTGCTTT-3’) were designed on the exogenous fragment. Transgenic seedlings were detected at the PCR level by pairing the vector backbone primers with the exogenous fragment primers. The PCR system was as follows: 5 μL of 2×Taq Master Mix, 0.5 μL of Primer-R, 0.5 μL of Primer-CIDEB-F, 1 μL of DNA, and 2 0.3 μL of ddH

[0059] qRT-PCR was performed on the positive rapeseed transgenic seedlings obtained by PCR to detect the gene expression level. RNA of the transgenic single plant seeds was extracted and cDNA was synthesized. The quantitative primers were designed using Primer 3 software, and the product size was between 100 bp and 200 bp. The primer sequences were CIDEB-qpcr-F (5’-CCAAGGTGTTGGACCTAAAAGAGTT C-3’), CIDE-qpcr-R (5’-TTATTTGTCGTCGTCGTCCTTGTAGT-3’), BnACTIN7-L (5’-CGCGCCTAGCAGCATGAA-3’), and BnACTIN7-R (5’-GTTGGAAAGTGCTGAGAGA TGCA-3’), which were used as internal reference primers for rapeseed qRT-PCR (see Zhou et al 2012: BnMs3 is required for ta petal differentiation and degradation, microspore separation, and pollen-wall biosynthesis in Bra ssica napus).

[0060] RNA extraction: The total RNA was extracted using the Super Total RNA Extraction Kit (Catalog No. LS1040) from Promega Shanghai.

[0061] cDNA synthesis: The reverse transcription was performed using TransGen One-Step gDNA Removal and cDNA Synthesis SuperMix (Catalog No. AE311). Using 2 μg of total RNA as a template, sequentially add 1 μl of Anchored Oligo(dT)18 Primer, 10 μl of 2×ES Reaction Mix, 1 μl of RT / RI Enzyme Mix, 1 μl of gDNA Remover, and supplement with RNase-free Water to 20 μl. Gently mix the above system and place it at 42 °C for 30 min. This step synthesizes the first-strand cDNA and removes gDNA. Inactivate RT / RI and gDNA Remover by heating at 85 °C for 5 seconds. Add 100 μl of RNase-free Water to dissolve the synthesized cDNA for later use.

[0062] The qRT-PCR reaction system is as follows:

[0063]

[0064] Reaction program: 94 °C for 30 s; 94 °C for 10 s, 60 °C for 15 s, 72 °C for 30 s, for 45 cycles; draw the melting curve. qRT-PCR is performed on a Bio-Rad CFX96 Real-Time System.

[0065] Normalize according to the internal reference primers, and calculate the quantitative variation between different replicates using the delta-delta threshold cycle relative quantification (2-ΔΔCT) method. Finally, analyze and obtain the overexpressed transgenic single plants OE4, OE5, and OE6 of rapeseed ( Figure 2 ).

[0066] (3) Analysis of the phenotypes related to oil content of the transformed plants

[0067] ① Use GC-FID to determine the oil content and fatty acid content of rapeseed seeds

[0068] Using gas chromatography - flame ionization detector, determine the oil content and fatty acid content of rapeseed seeds harvested at maturity to obtain the oil content and fatty acid data of the seeds. The measuring instrument is provided by the National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University.

[0069] The oil content results show that the oil content of the receptor background material Westar is 40.11 ± 0.78, and the oil contents of the three overexpressed lines OE4, OE5, and OE6 are 43.94 ± 0.46, 43.96 ± 1.13, and 44.51 ± 2.53 respectively (Figure 3 a), which significantly increased by 3.83, 3.85, and 4.40 percentage points. This may be due to the energy-saving mechanism evolved by organisms over a long period, resulting in functional redundancy in gene expression. There was no significant difference in oil content between OE4 and OE5, OE6. The detection data of the content of each fatty acid are shown in Figure 3 b.

[0070] ②Determine the lipid content of rapeseed by LC-MS / MS method

[0071] Using 6500plus Qtrap, lipid extraction and analysis were performed on rapeseed harvested at maturity. The results showed that compared with the wild type, the TAG content of OE4, OE5, and OE6 increased by 13.77%, 12.45%, and 14.54% respectively ( Figure 5 a).

[0072] ③Observation of oil bodies in rapeseed by transmission electron microscopy

[0073] Take seed cotyledon sections and analyze lipid droplets under a transmission electron microscope. The results showed that the total area of lipid droplets and the area of each lipid droplet in OE plants were larger than those in the wild type, while the number of lipid droplets was smaller ( Figure 4 ).

[0074] In summary, the gene CIDEB in transgenic plants plays an important role in regulating rapeseed oil content.

Claims

1. An isolated mammal-specific CIDEB gene, characterized in that: The nucleotide sequence of the CIDEB gene is shown in SEQ ID NO: 1, which consists of 657 bp and encodes a lipid droplet localization protein. The amino acid sequence of the protein is shown in SEQ ID NO: 2, which contains 219 amino acids.

2. A recombinant plant expression vector, characterized in that: The invention comprises the CIDEB gene according to claim 1, and a seed-specific expression promoter Napin is connected before the transcription start nucleotide of the gene.

3. The recombinant plant expression vector according to claim 2, characterized in that: The vector also contains an antibiotic marker for screening transgenic plants.

4. A method for obtaining rapeseed with high oil content, characterized in that: The expression vector containing the CIDEB gene according to claim 1 is transformed into the genome of rapeseed by using an Agrobacterium-mediated genetic transformation method to obtain a transgenic rapeseed variety.

5. The method according to claim 4, characterized in that: The expression vector is the recombinant plant expression vector according to claim 2 or 3.

6. A rapeseed plant, characterized in that: The rapeseed plant is a transgenic plant containing the CIDEB gene of claim 1 obtained by an Agrobacterium-mediated genetic transformation method, and the oil content of the rapeseed plant is significantly higher than that of a normal plant.

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