Application of miR1858 in the preparation and / or production of eicosapentaenoic acid

Abstract

The present invention relates to the field of bioengineering, and in particular to the application of miR1858 in the preparation and / or production of eicosapentaenoic acid. The present invention obtains the microchlorophyll miR1858 engineered algae strain through engineering metabolic modification, which has an extremely high level of EPA accumulation, and does not require optimization of the culture process, thereby reducing the culture cost. Different from the previous engineering metabolic means of improving EPA, the present invention performs genetic modification at the non-coding level, and the obtained engineered algae strain can efficiently and stably accumulate EPA. The EPA produced by the miR1858 engineered algae strain of the present invention has the characteristics of being green, environmentally friendly, and healthy, and the production of EPA by the engineered algae strain does not pollute the environment, nor does it damage the ecosystem, and is suitable for large-scale cultivation. Therefore, the engineered algae strain is a potential engineered algae strain for commercial production of EPA.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering, and in particular to the application of miR1858 in the preparation and / or production of eicosapentaenoic acid. Background Art

[0002] Eicosapentaenoic acid (EPA) is an essential fatty acid for the human body. Fatty acids are divided into saturated fatty acids and unsaturated fatty acids, among which unsaturated fatty acids are divided into monounsaturated fatty acids and polyunsaturated fatty acids; polyunsaturated fatty acids (PUFAs) can be divided into omega-3 and omega-6. The most beneficial (and most influential in industry) polyunsaturated fatty acids are omega-3 derivatives, represented by eicosapentaenoic acid (EPA, 20:5, ω3), docosahexaenoic acid (DHA, 22:6, ω3) and alpha-linolenic acid (ALA, 18:3, ω3). Polyunsaturated fatty acids are essential fatty acids for the human body, but the human body cannot synthesize polyunsaturated fatty acids such as ALA, EPA, and DHA through de novo synthesis, so they need to be supplemented by consuming foods rich in polyunsaturated fatty acids or health products.

[0003] EPA plays an important role in maintaining human health, including preventing cardiovascular disease, reducing inflammatory responses, and promoting brain development and function. Studies related to EPA's physiological functions have shown that it has the following benefits for human health: (1) preventing thrombosis and atherosclerosis, improving the cardiovascular system, and reducing the incidence of cardiovascular diseases such as hypertension and coronary heart disease; (2) reducing the production of inflammatory mediators such as leukotrienes and cytokines, thereby preventing inflammation and alleviating autoimmune diseases such as rheumatoid arthritis, ulcerative colitis, and type 1 diabetes; (3) affecting the development of neurodegenerative diseases and being a potential drug for the treatment or prevention of neurodegenerative diseases and nervous system diseases such as mood disorders and Parkinson's disease; (4) having anticancer activity, or synergizing with a variety of chemotherapy drugs to improve the metabolism and body composition of cancer patients. The above research results all show that EPA is closely related to human health and disease, and it has great application prospects as a health product and potential drug.

[0004] As people pay more and more attention to health issues, the demand for EPA is increasing. EPA mainly comes from single-cell phytoplankton and seaweed, and is passed to other species through the food chain. Humans can obtain EPA by eating fish (or fish oil) and other marine animals. However, in recent years, factors such as marine environmental pollution and overfishing have led to the depletion of marine resources, making it more difficult and insufficient to obtain polyunsaturated fatty acids such as EPA and DHA from marine animals. At the same time, global warming has caused the surface sea temperature to rise, and a large number of phytoplankton are slowly adapting to high temperatures, thereby reducing the synthesis of cold-resistant polyunsaturated fatty acids, which has led to a large reduction in the natural synthesis of EPA in the marine ecosystem. The latest survey shows that the total daily demand for EPA and DHA by the global population is 1.4 million tons, while aquaculture, fisheries and other marine sources can supply EPA and DHA annually. The above results show that the EPA supply market is facing a serious gap.

[0005] Marine microalgae are a type of photoautotrophic organisms with significant advantages such as simple cell structure, strong reproductive ability, and short growth cycle. They are an excellent base organism for industrial production and are therefore regarded as a natural factory with great potential for sustainable synthesis of EPA. Compared with marine animals, the cultivation conditions of microalgae are relatively simple, and the EPA production of microalgae can be significantly increased by optimizing cultivation conditions, developing efficient cultivation systems, and improving genetic traits, providing the possibility for large-scale production. Currently, marine microalgae have been widely used in renewable energy production, biomedicine, food industry, and other fields.

[0006] The biosynthesis pathway of EPA in microalgae is mainly divided into two steps: (1) acetate is used as a precursor to synthesize stearic acid (18:0), then Δ9 desaturase catalyzes stearic acid to produce oleic acid (18:1 ω-9), and then oleic acid is catalyzed by Δ12 desaturase to form linoleic acid (18:2 ω-6), which is then converted to α-linolenic acid (18:3 ω-3) by Δ15 desaturase; (2) α-linolenic acid is converted into EPA through a series of stepwise desaturation and elongation steps. It has been found that many microalgae, including Nannochloropsis oceanica, Phaeodactylum tricornutum, and Chlorella minutissima, can synthesize EPA, but the yield cannot meet human needs. Therefore, there is an urgent need to develop process optimization or metabolic engineering strategies to increase the yield of EPA in microalgae.

[0007] In recent years, overexpression of key genes related to EPA synthesis to increase EPA production has been regarded as one of the most promising strategies. For example, overexpression of Δ5 or Δ12 desaturase in Nannochloropsis increased the proportion of EPA in total fatty acids by 25%; overexpression of Δ5 desaturase encoding genes in Phaeodactylum tricornutum increased EPA production by 58%, and overexpression of diacylglycerol acyltransferase (DGAT2) increased the content of EPA in fatty acids to 76.2%; heterologous overexpression of Δ6 desaturase encoding genes from Thalassiosirapseudonana in Dunaliella salina increased EPA production to 21.3 mg / L. The above strategies focus on genetic modification of key coding nodes for EPA synthesis, but there is no relevant technology for increasing EPA production by genetic modification at the non-coding level.

[0008] MicroRNA (miRNA) is a class of small single-stranded RNA with a length of 19-25 nucleotides. It belongs to endogenous conserved non-coding RNA and has the function of regulating gene expression. miRNA is an important post-transcriptional regulator. It forms a silencing complex with Argonaute family proteins. The complex is partially or completely complementary to the target mRNA, thereby mediating the degradation or translation inhibition of the target mRNA and achieving post-transcriptional silencing of the target mRNA. By regulating the post-transcriptional silencing of genes, miRNA participates in various biological processes such as cell proliferation and differentiation, body metabolism, and disease occurrence. Studies have shown that 48 miRNAs are involved in the response of Dunaliella salina to high light and high salt, and promote the accumulation of β-carotene. Transcriptome analysis showed that 83 miRNAs were involved in the response of Haematococcus pluvialis to high light, promoting the accumulation of lipids and astaxanthin. And the expression of phosphoenolpyruvate carboxylase was downregulated by artificial miRNA, thereby increasing the total fatty acid production of Chlamydomonas reinhardtii. The above studies suggest that miRNA plays an important regulatory role in the environmental response and synthesis of secondary metabolites (such as lipids) of microalgae.

[0009] In the prior art, wild-type Nannochloropsis can synthesize EPA, but the yield is low. Many marine microalgae can accumulate EPA, and Nannochloropsis is one of them. However, when Nannochloropsis oceanica CCMP1779 / CCAP 849 / 10, which has not been genetically modified, is cultured in f / 2 medium, the EPA obtained only accounts for 3%-5% of the total fatty acids, and the content in the dry weight of biomass is 40-50 mg / g, only 83-92 ng / 10 −6 cell. If large-scale industrial cultivation of non-genetically modified Nannochloropsis algae is carried out, more equipment costs will be required to obtain the required yield for supply. Therefore, in order to obtain higher EPA yields, it is not suitable to directly carry out large-scale industrial cultivation of wild-type Nannochloropsis algae. Although EPA from microalgae is a renewable resource, it faces a huge gap in global supply, and relying solely on the natural synthesis of microalgae is far from enough. Although the existing technology can improve the EPA yield by optimizing the cultivation process, it is costly and may cause environmental pollution. Without genetic modification, optimizing the cultivation process is one of the strategies to increase the EPA yield of Nannochloropsis algae. For example, changing the light source (such as using LED blue light for illumination), optimizing the composition of the culture medium (such as changing the NaNO3 concentration in the f / 2 culture medium or using BG-11 culture medium), and changing the cultivation method (such as heterotrophic cultivation) can all increase the EPA yield of Nannochloropsis algae. However, optimizing the cultivation process requires more investment. For example, if a light source with a specific wavelength is used for large-scale cultivation, the light source needs to be customized, which increases the capital cost; or if glucose is used as a carbon source for large-scale cultivation, the amount of glucose used also increases the cost. In addition, the cultivation process of increasing EPA production by adding heavy metals to the culture medium will not only cause serious pollution to the environment, but the heavy metals may enter the human body and cause heavy metal poisoning. In summary, the method of increasing EPA production by optimizing the cultivation process is costly and may cause environmental pollution. Summary of the invention

[0010] In view of this, the present invention provides the use of miR1858 in the preparation and / or production of eicosapentaenoic acid. The present invention obtains the microalgae miR1858 engineered algae strain through engineering metabolic modification, which has an extremely high level of EPA accumulation and does not require optimization of the culture process, thereby reducing the culture cost. Different from the previous engineering metabolic means of increasing EPA, the present invention performs genetic modification at the non-coding level, and the obtained engineered algae strain can efficiently and stably accumulate EPA. The EPA produced by the miR1858 engineered algae strain of the present invention has the characteristics of being green, environmentally friendly, and healthy, and the production of EPA by the engineered algae strain does not pollute the environment, nor does it damage the ecosystem, and is suitable for large-scale cultivation. Therefore, the engineered algae strain is a potential engineered algae strain for commercial production of EPA.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides use of miR1858 in the preparation and / or production of eicosapentaenoic acid; the miR1858 has: a nucleotide sequence as shown in SEQ ID NO:1.

[0013] The present invention also provides the use of overexpressed miR1858 in the preparation and / or production of eicosapentaenoic acid; the miR1858 has: a nucleotide sequence as shown in SEQ ID NO:1.

[0014] The present invention also provides an expression cassette, comprising: the miR1858 in the above application, a resistance gene and an acceptable gene element; the resistance gene comprises: bleomycin.

[0015] The present invention also provides a plasmid, comprising: the above expression cassette.

[0016] The present invention also provides a host, and the plasmid is transformed and / or transfected into the chassis algae strain.

[0017] In some embodiments of the present invention, in the above-mentioned host, the chassis algae strain includes: Nannochloropsis sp.

[0018] The present invention also provides use of the expression cassette, the plasmid and / or the host in the preparation and / or production of eicosapentaenoic acid.

[0019] The present invention also provides the use of the expression box, the plasmid and / or the host in increasing the yield of eicosapentaenoic acid.

[0020] The nitrogen restriction treatment was applied to Nannochloropsis oceanica CCMP1779, which was purchased from the National Center for MarineAlgae and Microbiota, USA, to increase its lipid content. High-throughput sequencing and bioinformatics were used for omics association analysis, and it was found that miR1858 was involved in the regulation of lipid synthesis, and its potential predicted targets were ACCase2, LC-FACS, LPAAT, and DGAT2. Through genetic modification, miR1858 was overexpressed, and its overexpression caused the upregulation of stearoyl-CoA desaturase (Δ-9 desaturase) and omega-6 fatty acid desaturase (Δ-12 desaturase). Therefore, in terms of biological function, it promoted the conversion of saturated fatty acids to unsaturated fatty acids, thereby promoting the synthesis of unsaturated fatty acids. Through gas chromatography-mass spectrometry (GC-MS) analysis, it was found that overexpression of miR1858 caused changes in the fatty acid composition of microalgae, with a significant decrease in the content of saturated fatty acids and a significant increase in the content of unsaturated fatty acids. In the two engineered algae strains miR1858-1 and miR1858-2, the relative content of EPA increased by 5.5 times and 4.5 times compared with the wild type, respectively; the absolute content of EPA in the dry weight of biomass reached 159.6±3.5 mg / g and 143.3±2.6 mg / g, respectively, while the wild type only had 36.0±1.6 mg / g; the production of EPA per unit culture reached 32.3±1.2 mg / L and 29.2±0.4 mg / L, respectively, while the wild type only had 7.5±0.2 mg / L. The above results show that miR1858 engineered algae can efficiently synthesize EPA, which provides the possibility for large-scale production.

[0021] The present invention provides the use of miR1858 in the preparation and / or production of eicosapentaenoic acid.

[0022] The beneficial effects of the present invention include:

[0023] (1) The present invention develops an engineered microalgae that can efficiently synthesize eicosapentaenoic acid (EPA) and has the characteristics of high efficiency, greenness and environmental protection.

[0024] (2) The present invention utilizes marine Nannochloropsis algae engineered strains to efficiently synthesize EPA. The production is not restricted by natural waters, is less affected by the environment, and can be artificially cultivated on a large scale.

[0025] (3) The present invention utilizes optimized engineered microalgae to produce high-value-added EPA, thereby achieving high-level production and filling the gap of current insufficient supply.

[0026] (4) The production of EPA by the present invention has the characteristics of low energy consumption and high yield, and can be widely used in the pharmaceutical, food, cosmetics and other industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0028] Figure 1 Figure 2 shows the laser confocal microscopy observation of wild-type Nannochloropsis in normal culture medium and nitrogen-deficient culture medium. The cells were stained with Nile red. The white spheres in the cells are lipid droplets stained with Nile red. The larger the spheres and / or the more the number, the higher the lipid content. The scale bar is 5 μm.

[0029] Figure 2 Schematic diagram of pNA19 vector; wherein: Phsp20: heat shock protein 20 promoter; TMV Ω: enhancer; EYFP: yellow fluorescent protein; TfcpA: fcpA terminator; Pgapdh: glyceraldehyde-3-phosphate dehydrogenase promoter; ble: bleomycin resistance gene; Thsp20: heat shock protein 20 terminator; AmpR: ampicillin gene; Ori: replication origin;

[0030] Figure 3 pNA19-premiR1858 gene and bleomycin resistance gene expression cassette; wherein: premiR1858: miR1858 precursor gene; Phsp20: heat shock protein 20 promoter; Ω: enhancer; TtcpA: fcpA terminator; Pgapdh: glyceraldehyde-3-phosphate dehydrogenase promoter; ble: bleomycin resistance gene; Thsp20: heat shock protein 20 terminator;

[0031] Figure 4 The figure shows the molecular verification of miR1858 engineered algae at the genome level; where: M: marker; 1: WT; 2: miR1858-1; 3: miR1858-2;

[0032] Figure 5 The relative transcription level analysis of miR1858 in two miR1858 engineered algae strains is shown; the data label above each column represents the mean of the group, and the significant difference between the groups was determined by t-test, P < 0.05 ( * ) or P<0.01( ** ) levels indicate significant differences, and error bars represent the mean ± SD of three independent experiments;

[0033] Figure 6 The peak diagram of fatty acid composition of algae cells analyzed by GC-MS is shown;

[0034] Figure 7 The relative quantitative analysis of each fatty acid component in algae cells is shown; the data label above each column represents the mean of the group, and the significant difference between groups was determined by t-test, P < 0.05 ( * ) or P<0.01 ( ** ) levels indicate significant differences; error bars represent the mean ± SD of three independent experiments;

[0035] Figure 8 Figure 2 shows the analysis of eicosapentaenoic acid content in wild-type and miR1858-transformed algae; A shows the percentage of eicosapentaenoic acid in total fatty acids; B shows the absolute content of eicosapentaenoic acid in biomass dry weight; C shows the eicosapentaenoic acid content per million cells; D shows the eicosapentaenoic acid yield per unit volume; the data label above each column represents the mean of the group; significant differences between groups were determined by t-test, P < 0.05 ( * ) or P<0.01 ( ** ) levels indicate significant differences; error bars represent the mean ± SD of three independent experiments. DETAILED DESCRIPTION

[0036] The invention discloses application of miR1858 in the preparation and / or production of eicosapentaenoic acid.

[0037] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0038] The use of the terms "comprising", "having" or "containing", including their grammatical synonyms, should generally be understood as open and non-restrictive, for example not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0039] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0040] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0041] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, values ​​and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0042] When the model microalga Nannochloropsis oceanica CCMP1779 / CCAP 849 / 10 was subjected to nitrogen restriction treatment to promote its lipid synthesis, high-throughput sequencing and bioinformatics analysis revealed that miR1858 can participate in the regulation of lipid synthesis, and its potential regulatory targets are acetyl-CoA carboxylase 2 (Acetyl-CoAcarboxylase 2, ACCase2), long-chain fatty acyl-CoA synthetase (Long-Chain Fatty Acyl-CoASynthetase, LC-FACS), lysophosphatidic acidacyltransferases (LPAAT), and diacylglycerol acyltransferase2 (Diacylglycerol Acyltransferase2, DGAT2). When miR1858 was overexpressed, the expression of protein encoding genes such as ACCase2, LC-FACS, LPAAT, and DGAT2 in the overexpressed algae was significantly downregulated, while the expression of two desaturase encoding genes, stearoyl-CoA Desaturase (SCD, Δ-9 desaturase) and omega-6 fatty acid desaturase (FAD, Δ-12desaturase), was significantly upregulated, thereby significantly promoting the synthesis of EPA.

[0043] (1) Construction of pNA19-premiR1858 vector: pNA19 is a vector (plasmid) that can be used for genetic modification of Chlorella vulgaris. The target gene can be overexpressed in Chlorella vulgaris using this vector. pNA19 is double-digested with SwaI and HpaI enzymes, and then the cloned target gene (premiR1858) can be connected to the gap of the restriction site. The ligation product is transferred into the competent Escherichia coli by heat shock, and then screened on a solid culture medium containing ampicillin antibiotics. Finally, nucleic acid sequencing is used to confirm that the vector connection is correct.

[0044] (2) Construction of miR1858 engineered algae strains: The constructed pNA19-premiR1858 was introduced into microalgae cells using an electroporator; monoclonal microalgae were screened on f / 2 solid culture medium containing bleomycin; after obtaining the monoclonal engineered algae strains, polymerase chain reaction (PCR) was used to verify whether pNA19-premiR1858 was integrated into the microalgae genome ( Figure 4 ); The relative transcription level of miR1858 was verified by real-time fluorescence quantitative PCR (qPCR) ( Figure 5 ).

[0045] (3) Fatty acid analysis of miR1858 engineered algae strains: Use an ultrasonic disruptor to disrupt the cells, then add 2M potassium hydroxide methanol solution and incubate at 75°C for 10 min; after cooling and stratification, transfer the supernatant to a new centrifuge tube, add an equal volume of 2M hydrochloric acid methanol solution, mix thoroughly, and incubate at 75°C for 10 min; after cooling and stratification, transfer the supernatant to a new centrifuge tube, add 15 mL of n-hexane to extract fatty acid methyl esters; add 10 mg / mL nonadecanoic acid methyl ester as an internal standard; then use nitrogen to concentrate the liquid to 1 mL; use GC-MS to analyze the fatty acid composition ( Figure 6 ), and the molecular formula and chemical structure were analyzed in the standard fatty acid mass spectrum library.

[0046] (4) Analysis of EPA content: The relative content of each fatty acid component was analyzed by the peak area of ​​the chromatogram ( Figure 7 and Figure 8 The absolute content of EPA was calculated by internal standard methyl nonadecanoate, and the content of EPA in the dry weight of algae was calculated by combining with the dry weight of algae ( Figure 8 B in the figure); calculate the content of EPA per unit cell by the number of cells ( Figure 8 C in ); and calculate the EPA yield per unit volume ( Figure 8 D in.

[0047] In Examples 1 to 5 of the present invention, all the raw materials and reagents used can be purchased from the market.

[0048] The present invention will be further described below in conjunction with embodiments:

[0049] Example 1 Based on nitrogen deficiency treatment and transcriptome sequencing analysis, it was found that miR1858 may be involved in the regulation of lipid synthesis

[0050] Nitrogen-deficient culture conditions can promote the synthesis and accumulation of microalgae lipids. Wild-type Nannochloropsis algae were cultured in normal culture medium and nitrogen-deficient culture medium. The cells were collected and stained with Nile red (Nile red can specifically stain lipid droplets), and then observed using a laser confocal microscope. The results showed that compared with cells cultured in normal culture medium, cells in nitrogen-deficient culture medium synthesized a large number of lipid droplets (such as Figure 1 In order to explore the non-coding regulatory level of lipid synthesis in Nannochloropsis, miRNA sequencing analysis was performed, and miR1858 was found to be the most significantly differentially expressed miRNA (as shown in Table 1). Combined with the above results, it is speculated that miR1858 may be a key miRNA involved in the lipid synthesis pathway of microalgae.

[0051] Table 1 The top five significantly differentially expressed miRNAs

[0052]

[0053] Example 2 Construction of an overexpression vector of miR1858 precursor gene based on pNA19 vector

[0054] Wild-type Nannochloropsis is sensitive to Bleomycin (ble), so a genetic expression vector can be constructed by using bleomycin as a selective resistance marker for Nannochloropsis. Figure 2 As shown in the figure, it contains two eukaryotic expression cassettes: the EYFP target gene expression cassette and the bleomycin resistance gene expression cassette. In this study, this vector was named pNA19. The pNA19 vector was linearized by double restriction digestion (SwaI and HpaI), and the miR1858 precursor gene fragment was cloned into the pNA19 vector using a ligase to obtain the miR1858 precursor gene overexpression vector: pNA19-premiR1858 (as shown in Figure 3 as shown).

[0055] Example 3 Two miR1858 engineered algae strains were successfully obtained by electroporation

[0056] The pNA19-premiR1858 plasmid was introduced into wild-type Nannochloropsis by electroporation, and then screened on solid medium containing bleomycin. After obtaining monoclonal algae, we performed genome-wide analysis of the two miR1858 engineered algae strains (e.g. Figure 4 ) and transcriptional levels (as shown Figure 5 The results showed that the plasmid had been successfully integrated into the genome (as shown in Figure 4At the transcriptional level, the relative transcription level of miR1858 in the engineered algae strain miR1858-1 was slightly higher than that in the engineered algae strain miR1858-2, but the difference was not statistically significant, and both engineered algae strains achieved overexpression of miR1858 (as shown in Figure 5 as shown).

[0057] Example 4 Analysis of fatty acid composition of miR1858 engineered algae strains

[0058] The fatty acid composition of wild-type Nannochloropsis and miR1858 engineered algae in the stationary phase was analyzed by GC-MS (e.g. Figure 6 As shown in the figure, the peak time of EPA was 42 min, and by comparing the peak graph analysis, it was found that the peaks corresponding to EPA in the engineered algae strains miR1858-1 and miR1858-2 were significantly higher than those in the wild type, and the peak areas were also significantly larger than those in the wild type, indicating that the EPA content of the two miR1858 engineered algae strains was higher than that of the wild type. The relative content of each fatty acid component was further calculated by the peak area (as shown in the figure). Figure 7 As shown in Figure 2, the contents of tetradecanoic acid (C14:0) and hexadecanoic acid (C16:0) in the two engineered algae strains were significantly lower than those in the wild type, while the content of octadecanoic acid was significantly increased; the content of saturated fatty acids was significantly reduced, and the content of long-chain polyunsaturated fatty acids was significantly increased; the content of EPA was significantly higher than that of the wild type. The above results indicate that miR1858 overexpression can promote the conversion of saturated fatty acids to unsaturated fatty acids in engineered algae strains, especially the accumulation of EPA.

[0059] Example 5 Analysis of the absolute content of EPA in miR1858 engineered algae strains

[0060] Analysis of the content of EPA in total fatty acids found that the EPA in the engineered algae strains miR1858-1 and miR1858-2 was 5.5 times and 4.5 times that of the wild type, respectively (e.g. Figure 8 A in the figure). Using methyl nonadecanoate as an internal standard, an absolute quantitative analysis of EPA was performed, and it was found that the contents of EPA in the dry weight of biomass in the engineered algae strains miR1858-1 and miR1858-2 reached 159.6 mg / g DCW and 143.3 mg / g DCW, respectively (e.g. Figure 8 B); the content of EPA in the dry weight of a single cell reached 793.0 ng / 10 6 cells and 754.4 ng / 10 6 cells (such as Figure 8 C in the figure); the EPA production per unit volume of cell culture reached 32.3 mg / L and 29.2 mg / L respectively, while the wild type only had 7.5 mg / L (such as Figure 8 The above results show that the EPA content in the miR1858 engineered algae strain is significantly increased, which is the genetically engineered algae strain with the highest EPA production at present and can be used as an excellent algae species for industrial production of EPA.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of overexpression of miR1858 in Nannochloropsis sp. in the preparation and / or production of eicosapentaenoic acid; The sequence of miR1858 is: the nucleotide sequence shown in SEQ ID NO: 1; The overexpression of miR1858 is carried out by constructing a miR1858 precursor gene overexpression vector to obtain an engineered algae strain, thereby increasing the yield of eicosapentaenoic acid.

2. An expression cassette, characterized in that include: The miR1858, resistance gene and acceptable gene element used in the application of claim 1; The resistance genes include: bleomycin.

3. A plasmid, characterized in that include: The expression cassette of claim 2.

4. A host, characterized in that The plasmid as claimed in claim 3 is transformed and / or transfected into the chassis algae strain.

5. The host according to claim 4, characterized in that The chassis algae strains include: Nannochloropsis.

6. Use of the expression cassette according to claim 2, the plasmid according to claim 3 and / or the host according to claim 4 or 5 in the preparation and / or production of eicosapentaenoic acid.

7. Use of the expression cassette according to claim 2, the plasmid according to claim 3 and / or the host according to claim 4 or 5 in increasing the production of eicosapentaenoic acid.

Images