Specific CIDEA gene of mammal and application of CIDEA gene in increasing oil content of rape

Abstract

The invention discloses a mammal specific CIDEA gene, and belongs to the technical field of gene engineering and biology. The nucleotide sequence of the CIDEA gene is shown as SEQ ID NO: 1, the CIDEA gene is composed of 651bp, a lipid droplet localization protein is encoded, an agrobacterium-mediated genetic transformation method is used, an expression vector containing the CIDEA gene is transformed into a genome of rape, a transgenic rape variety is obtained, and the CIDEA gene is found to 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 invention belongs to the field of genetic engineering and biotechnology, and specifically relates to a mammal-specific CIDEA gene and its application in regulating the oil content of rapeseed. Background Art

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

[0003] Transferring animal genes into plants can increase plant resistance and improve quality. For example, transferring human interferon genes into tobacco and rice will result in transgenic plants with antiviral properties; introducing animal obesity genes FTO into rice can promote rice tillering and root growth, enhance photosynthesis, and improve drought resistance; transferring Bacillus thuringiensis Bt genes into cotton to synthesize Bt insecticidal proteins, and transgenic insect-resistant cotton reduces the country's economic losses by billions or even tens of billions of yuan each year; introducing a mammalian gene called CYP2E1 into the indoor plant ivy, the CYP2E1 gene can encode plant cytochrome P4502E1, which can decompose volatile organic compounds and effectively improve the plant's detoxification and air purification capabilities; the antifreeze protein gene AFP from polar fish can help these fish survive in low temperature environments, and introducing fish AFP genes into rice can enhance cold resistance. In short, introducing animal genes into plants can add unprecedented traits to plants and provide more ways to select and breed new varieties.

[0004] However, no animal genes have been reported to improve seed oil content in Brassica napus.

[0005] Cell death-inducing DFFA-like effector a (CIDEA) is a mammalian-specific protein that is enriched at LD-LD contact sites and mediates lipid transport between LDs. It has been previously reported that CIDEA induces apoptosis and DNA fragmentation when localized in mitochondria. The expression of CIDEA in adipocytes is unexpectedly high. Since then, an increasing number of studies have suggested that CIDEA protein is a key protein involved in lipid metabolic pathways and maintaining lipid homeostasis. CIDEA plays an important role in regulating lipid storage, LD formation, lipid degradation, and hepatic steatosis. CIDEA has a unique tissue expression pattern, such as expression in brown adipose tissue (BAT) in mice and humans. However, the conversion and function of CIDEA in plants are still unclear.

[0006] Based on this, the present invention cloned a mammalian-specific protein gene CIDEA, and used the Napin promoter to start the expression of CIDEA, and the oil content of the obtained rapeseed seeds was significantly increased. The results of this study indicate that the CIDEA gene plays an important role in regulating the oil content of rapeseed, and it has considerable application potential in creating new oil-rich rapeseed germplasm. Summary of the invention

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

[0008] The nucleotide sequence of the CIDEA gene is shown in SEQ ID NO: 1, which consists of 651 bp and encodes a lipid droplet localization protein. The protein sequence is shown in SEQ ID NO: 2, which encodes 217 amino acids. The gene of the present invention and any polynucleotide fragment of interest can be amplified from the vector of the synthetic gene using PCR technology.

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

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

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

[0012] By obtaining transgenic rapeseed plants, the expression level, oil content and lipid group of the transformation material were measured, and the cytological sections of the seeds of the transformation material were analyzed, and it was found that the CIDEA gene positively regulated the accumulation of oil content. The high-oil-content rapeseed obtained by the present invention has no obvious difference in its vegetative growth and reproductive growth cycle from normal plants, and this genetic resource has a very important application 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 vector suitable for constructing the expression vector described in the present invention includes but is not limited to Napin-PBILoxP and the like. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0015] Figure 2 : qRT-PCR detection of transformed individual strains. * indicates P < 0.05 in Student's t test, ** indicates P < 0.01 in Student's t test.

[0016] Figure 3 : Phenotypic identification of CIDEA in rapeseed. Oil content and fatty acid content of rapeseed seeds were analyzed by gas chromatography-flame ionization detector (GC-FID). OE1, OE2 and OE3 are overexpression lines, each with 6-8 different individuals, * indicates P < 0.05 in Student's t test, ** indicates P < 0.01 in Student's t test.

[0017] Figure 4 : Observation of oil bodies in rapeseed seeds under transmission electron microscopy. (a) Observation of seeds under transmission electron microscopy; (b) The number and size of lipid droplets in each image under a 4000x microscope.

[0018] Figure 5 :Determination of total TAG and individual TAG in seed lipids. Each strain has 6 different individuals, * indicates P < 0.05 in Student's t test, ** indicates P < 0.01 in Student's t test. DETAILED DESCRIPTION

[0019] The present invention is further explained below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to 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 operating manual provided by the manufacturer.

[0020] Example 1 CIDEA gene synthesis and amplification

[0021] (1) CIDEA gene synthesis

[0022] Cell death-inducing DFFA-like effector a (CIDEA) is a mammalian-specific protein with no homologous genes in plants. Therefore, the CDS sequence of this gene in mice (NP_031728) was obtained from the NCBI website. The company directly synthesized it after optimizing the codons to make it more suitable for plants, while ensuring that the amino acid sequence remained unchanged. The CDS sequence and protein sequence are the sequences shown in the sequence listing SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The CDS sequence includes a complete ORF reading frame and a start codon ATG, which are composed of 651 bp and 217 amino acids, respectively.

[0023] (2) CIDEA gene amplification

[0024] Primers were designed based on the CDS sequence obtained from the above analysis, and the company's universal synthetic vector plasmid was used as a template. The forward primer CIDEA-Asc1-F sequence was 5'-TTGGCGCGCCACCATGGAGACCGCTAGAGATT-3', and the reverse primer CIDEA-BamH1-R sequence was 5'-CGGGATCCTTATTTGTCGTCGTCGTCCTTGTAGTCCATCATGAACCAACCTTTGGTAGAAG-3'. The fragment containing the full-length CDS of CIDEA was amplified. TM 2× High-Fidelity Master Mix (TSINGKE Biologica technology) was used for PCR amplification. The PCR amplification system was as follows:

[0025]

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

[0027] The amplified product was detected by agarose gel electrophoresis, and the full-length CIDEA of 651 bp was obtained. The product was recovered by gel extraction using the Tiangen Agarose Gel Recovery Kit ( http: / / www.tiangen.com / ).

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

[0029] (1) The CIDEA fragment obtained above was double-digested with the rapid restriction endonucleases Asc1 and BamHI. The double-digestion system is as follows:

[0030]

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

[0032] (2) The digested product was connected to the vector Napin-PBILoxP (donated by the Oil Crops Research Institute, Chinese Academy of Agricultural Sciences), which contains the seed-specific expression promoter Napin and an antibiotic marker.

[0033] The connection method is as follows:

[0034]

[0035] Ligation reaction conditions: 4°C overnight.

[0036] (3) Transformation of Escherichia coli DH5α, the transformation method is 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 minutes;

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

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

[0040] d. According to the experimental needs, absorb different volumes of resuscitation solution and evenly spread it on LB culture medium containing corresponding antibiotics, and place the plate upside down in a 37℃ incubator for overnight culture.

[0041] Single clones were screened for positive identification, and three positive clones were selected for sequencing. The analysis results showed that the CDS sequence of the CIDEA gene was successfully connected to the vector, that is, the plant expression vector Napin-PBILoxP (such as Figure 1 shown).

[0042] (4) Introduce the correctly constructed recombinant plasmid vector into Agrobacterium strain GV3101, select positive single clones and store them in a -80°C refrigerator. The introduction method is as follows:

[0043] a. Clean the electric rotating cup: first wash it with pure water, then wash it with ultrapure water, pour it out, then wash it with anhydrous ethanol (blowing it with a 1ml pipette tip), pour out the anhydrous ethanol, and place it on a clean bench to dry;

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

[0045] c. Take 1 μl of the correctly constructed recombinant plasmid and add it to 50 μl of competent medium. Mix by gently pipetting to avoid bubbles.

[0046] d. Place the washed and dried electric rotating cup in ice to pre-cool, and then pour the above mixed solution into the cup wall;

[0047] e. Adjust the electric rotator to 1800V;

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

[0049] g. Place the electric rotating cup into the instrument and press the "push" button twice in succession. If you hear a "beep" sound after a few seconds, it is successful.

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

[0051] i. Activate at 28℃ for about 2h, apply 100μl to the plate containing double antibody, seal with sealing film, invert and culture in a 28℃ incubator for 2 days, and detect by spot picking.

[0052] (5) Agrobacterium colony detection

[0053] Select colonies and culture them in double-antibody LB at 28°C for 2 hours. Take an appropriate amount of bacterial solution for PCR detection and save the positive Agrobacterium bacterial solution.

[0054] Example 3 Genetic transformation experiment

[0055] (1) Genetic transformation of rapeseed

[0056] The constructed CIDEA overexpression vector was used for genetic transformation of rapeseed using Agrobacterium-mediated genetic transformation. The receptor used for rapeseed transformation in the present invention is Brassica napus Westar. The specific operation process is detailed in the reference: Anefficient Agrobacterium-mediated transformation method using hypocotyl asexplants for Brassica napus.

[0057] (2) Identification of overexpression transformed strains

[0058] The genomic DNA of the obtained rapeseed overexpression transformation plant was extracted, and the insertion of the exogenous gene fragment was detected by PCR. The overexpression backbone vector in the present invention was Napin-PBILoxP. The primer Primer-R (5'-TTTTACATTAGCCTCAAACATCC-3') was designed on the backbone vector, and the primer Primer-CIDEA-F (5'-ATGGAGACCGCTAGAGATT-3') was designed on the exogenous fragment. The transgenic seedlings were detected at the PCR level by matching the vector backbone primer with the exogenous fragment primer. The PCR system was: 2×Taq Master Mix 5μL, Primer-R 0.5μL, Primer-CIDEA-F 0.5μL, DNA 1μL, ddH20 3μL. PCR conditions: 95℃ total denaturation for 3min; 95℃ denaturation for 15sec, 55℃ annealing for 15sec, 72℃ extension for 40sec, 34circles; 72℃ total extension for 5min.

[0059] The transgenic rapeseed positive seedlings obtained by PCR were subjected to qRT-PCR to detect the gene expression level. RNA was extracted from seeds of individual transformed plants and cDNA was synthesized. Quantitative primers were designed using Primer 3 software. The product size was between 100 bp and 200 bp. The primer sequences were CIDEA-qpcr-F (5'-CGAGATGTACTCTGTTTCTTACGATAT CAG-3'), CIDE-qpcr-R (5'-TTATTTGTCGTCGTCGTCCTTGTAGT-3'), BnACTIN7-L (5'-CGCGCCTAGCAGCATGAA-3') and BnACTIN7-R (5'-GTTGGAAAGTGCTGAGA GATGCA-3') as internal reference primers for qRT-PCR in rapeseed (see Zhou et al 2012: BnMs3 is required for tapetal differentiation and degradation, microspore separation, and pollen-wall biosynthesis in Brassicanapus).

[0060] RNA extraction: Total RNA was extracted using Shanghai Promega Super Total RNA Isolation Kit (Cat. No. LS1040).

[0061] cDNA synthesis: Reverse transcription was performed using the full-stranded gold One-Step gDNA Removal and cDNA Synthesis SuperMix (Catalog No. AE311). Use 2 μg of total RNA as template, add 1 μl of Anchored Oligo(dT)18 Primer, 10 μl of 2×ES Reaction Mix, RT / RI Enzyme Mix 1μl, gDNARemover 1μl, RNase-free Water to 20μl. Gently mix the above system and place it at 42℃ for 30min. This step is to synthesize the first-strand cDNA and remove gDNA. Heat at 85℃ for 5 seconds to inactivate RT / RI and gDNA Remover. Add 100 μl RNase-free water to dissolve the synthesized cDNA and set aside.

[0062] The qRT-PCR reaction system is:

[0063]

[0064] The reaction program was as follows: 94°C for 30 s, 94°C for 10 s, 60°C for 15 s, 72°C for 30 s, and 45 cycles; the melting curve was drawn. qRT-PCR was performed in a Bio-Rad CFX96 Real-Time System.

[0065] The internal reference primers were used for standardization, and the quantitative variation between different replicates was calculated using the delta-deltaathreshold cycle relative quantification (2-ΔΔCT) method. Finally, the rapeseed overexpression transformed plants OE1, OE2, and OE3 ( Figure 2 ).

[0066] (3) Phenotypic analysis of transformed plants related to oil content

[0067] ① Determination of oil content and fatty acid content in rapeseed seeds by GC-FID

[0068] The oil content and fatty acid content of rapeseed seeds harvested at maturity were determined using gas chromatography-flame ionization detector to obtain seed oil content and fatty acid data. The measuring instruments were provided by the National Key Laboratory of Crop Genetic Improvement of Huazhong Agricultural University.

[0069] The results of oil content showed that the oil content of the receptor background material Westar was 40.11±0.78, and the oil contents of the three overexpression strains OE1, OE2 and OE3 were 43.85±1.46, 44.64±0.44 and 43.73±1.09, respectively, which were significantly increased by 3.74, 4.53 and 3.62 percentage points ( Figure 3 a), among which oleic acid (C18:1) and linolenic acid (C18:3) increased significantly ( Figure 3 b).

[0070] ② Determination of triglyceride content in rapeseed seeds by LC-MS / MS method

[0071] The lipids of rapeseed seeds harvested at maturity were extracted and analyzed using 6500plus Qtrap. The results showed that the TAG content of OE1, OE2 and OE3 increased by 14.36%, 14.55% and 16.94% respectively compared with the wild type. Figure 5 a), among which TAG-52:4, TAG-52:3 and TAG-54:3 had the largest increases ( Figure 5 b).

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

[0073] The cotyledon sections were taken and lipid droplets were analyzed 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 CIDEA gene in transgenic plants plays an important role in regulating the oil content of rapeseed.

Claims

1. An isolated mammal-specific CIDEA gene, characterized in that: The nucleotide sequence of the CIDEA gene is shown in SEQ ID NO: 1, which consists of 651 bp and encodes a lipid droplet localization protein. The amino acid sequence of the protein is shown in SEQ ID NO: 2, which contains 217 amino acids.

2. A recombinant plant expression vector, characterized in that: The invention comprises the CIDEA 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 CIDEA 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 CIDEA 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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