CN117660499A - Glycococcus fatty acid thioesterase gene and its application in improving linoleic acid synthesis - Google Patents

Abstract

The invention discloses a coccoid fatty acid thioesterase gene and application thereof in improving linoleic acid synthesis, belonging to the technical field of microalgae biology. The coccum fatty acid thioesterase gene is COCSUDRAFT_4465 gene obtained under the condition of inducing the accumulation of coccum grease, the nucleotide sequence of the coccum fatty acid thioesterase gene is shown as SEQ ID NO.1, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 2. The invention discloses a new function of the gene for the first time, namely that the gene is transformed into the chlorella through homologous recombination and over-expressed, so that not only is the high specific growth rate of the transformed chlorella strain endowed, but also the growth period can be shortened, and the synthesis and accumulation amount of linoleic acid in algae cells can be synchronously enhanced, so that the linoleic acid product developed by the new variety of the transformed chlorella strain has high yield, low cost and good safety, and is a positive source of novel linoleic acid dietary raw materials.

Description

Glycococcus fatty acid thioesterase gene and its application in improving linoleic acid synthesis

Technical field

The invention belongs to the field of microalgae biotechnology, and specifically relates to the Glycococcus fatty acid thioesterase gene and its application in improving the synthesis of linoleic acid.

Background technique

Linoleic acid has the effects of lowering blood pressure and blood lipids, promoting metabolism, and losing weight. At present, the main dietary sources of linoleic acid are safflower seed oil and sunflower seed oil. Although they have high linoleic acid content, the output is difficult to meet the growing market demand due to limitations in vegetation planting. New linoleic acid is sought. Meal sources are imminent. Coccomyxa subbellipsoidea C-169 is a single-cell green alga with high lipid content, lack of rigid cell walls to simplify lipid extraction and genetic manipulation, can be cultivated outdoors on a large scale, and the entire genome sequence is known. Especially under certain induction conditions, the oil content of Glycococcus can reach 20% to 50% of the dry cell weight, and it is rich in linoleic acid and other unsaturated fatty acids. It has a high yield per unit area and is known as a new food-derived product. Oil microalgae.

In recent years, domestic and foreign scholars have conducted a large number of studies on the properties of linoleic acid synthesized by Glycococcus. For example, strategies such as changing the type and concentration of nitrogen sources, adjusting light intensity, temperature and pH, and adding external chemical regulators (such as Na ₂ SiO ₃ , NaNO ₃ ) and other environmental factors can induce microalgae to accumulate linoleic acid. Although the synthesis and accumulation of linoleic acid can be effectively induced through the regulation of environmental factors and the linoleic acid yield can be optimized as a whole, there is still a gap compared with the lipid yield of industrial applications. There are also effective strategies to improve the linoleic acid yield of Glycococcus Still required. With the development of genetic engineering technology, metabolic engineering has become one of the effective strategies to improve lipid synthesis and fatty acid synthesis and accumulation in microalgae. Unlike other stress methods that promote the accumulation of lipids and fatty acids in microalgae, genetic engineering methods increase the synthesis of fatty acids in microalgae without limiting their growth. Fatty acid thioesterase can catalyze the hydrolysis of fatty acyl-CoA and fatty acyl-ACP to generate CoA, fatty acyl carrier protein ACP, and other sulfhydryl-containing compounds, including proteins and peptides containing cysteine residues, while releasing free fatty acids. By strengthening the activity of thioesterase, it promotes the metabolism of fatty acyl-CoA, reduces its accumulation, promotes lipid synthesis and accumulation, thereby increasing fatty acid production. However, the role of fatty acid thioesterase genes in the synthesis of linoleic acid in microalgae has not been reported. .

Contents of the invention

In order to further improve the yield of linoleic acid, the present invention provides the Glycococcus fatty acid thioesterase gene and its application in improving the synthesis of linoleic acid.

The above-mentioned Glycococcus fatty acid thioesterase gene is COCSUDRAFT_4465, its nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2.

Further, the above-mentioned Glycococcus fatty acid thioesterase gene was obtained under conditions that induce Glycococcus lipid accumulation.

Further, the above-mentioned Gloeococcus is Coccomyxa subbellipsoidea C-169.

The above-mentioned Glycococcus COCSUDRAFT_4465 gene is used to improve the synthesis of linoleic acid. It is transformed into Glycococcus through homologous recombination and overexpressed to achieve the synthesis and accumulation of linoleic acid in algae cells, including PCR amplification and overexpression recombinant construction.

Further, the PCR amplification was based on the cDNA reverse transcribed from the total RNA of Glycococcus. The designed primers included the upstream primer sequence 5’-ATTCCACAACAGCCTTGCGA-3’ and the downstream primer sequence 5’-CCAGGTGGTGCGAGCC-3’.

Further, overexpression recombinant construction includes designing primers, amplification, obtaining recombinant plasmids, and obtaining overexpression algal strains.

Further, the primers are designed based on the COCSUDRAFT_4465 gene and pCAMBIA1303 vector information. The primer sequence includes the upstream primer sequence 5'- TATGACCATGATTACGAATTC ATTCCACAACAGCCTTGCGA-3' and the downstream primer sequence 5'- ACGACGGCCAGTGCCAAGCTT CCAGGTGGTGCGAGCC-3'. Both ends of the primer include parts of the vector respectively. The sequence (underlined, including straight and wavy lines) and restriction site (underlined) serve as homology arms.

Further, the amplification is PCR amplification, and the amplification product contains the partial sequence and restriction site of the pCAMBIA1303 vector.

Furthermore, the method for obtaining the recombinant plasmid is the homologous recombination method, and the COCSUDRAFT_4465 gene is constructed into the pCAMBIA1303 vector to obtain the recombinant plasmid.

Furthermore, the method of obtaining the overexpressing algae strain is to use electrotransformation method to transform the recombinant plasmid into Glycococcus, and screen out the recombinant Glycococcus that positively overexpresses COCSUDRAFT_4465 through antibiotic screening and expression level detection.

Further, the reaction system for PCR amplification is: 2.2~2.8 μL cDNA, 20~30 μL 2×Hieff Ultra-Rapid Hot Start PCR Master Mix (with Dye), 2.2~2.8 μL each of upstream and downstream primers, 17.2~12.8 μLddH ₂ O; PCR amplification program is: pre-denaturation 93~97 ℃ 2~4 min, denaturation 93~97 ℃ 25~35 s, annealing 55~65 ℃ 25~35 s, extension 68~74 ℃ 6~10 s , thoroughly extended for 8~12 min, 30~40 cycles; the PCR amplification product was purified and recovered by 0.5wt%~3wt% agarose gel electrophoresis, and the homologous recombination amplification product of the COCSUDRAFT_4465 gene was obtained.

Further, the homologous recombination method is: perform a homologous recombination reaction between the pCAMBIA1303 double enzyme digestion product and the COCSUDRAFT_4465 gene homologous recombination product. The reaction system is: 90~110 ng pCAMBIA1303 double enzyme digestion product, 90~110 ng COCSUDRAFT_4465 gene PCR. Source recombinant product, 1.5~2.5 μL Exnase II, 3~5 μL 5×CEII Buffer, 7~13 μL ddH ₂ O, react at 36~38°C for 25~35 min; transform the homologous product into E. coli, and identify the product containing Positive bacteria of pCAMBIA1303-COCSUDRAFT_4465 recombinant plasmid.

Preferably, the specific steps to obtain overexpressing algae strains are: mix 90~110 µL resuspended Collococcus cells with 3~5 µg pCAMBIA1303-COCSUDRAFT_4465 recombinant plasmid and 35~45 µg salmon sperm DNA, and incubate on ice for 8~12 minutes. Electroporation was performed. After electroporation, the Glycococcus cells were immediately transferred to a test tube containing 8 to 12 mL of Basal culture medium, recovered under dark conditions for 18 to 30 hours, and then transferred to normal light intensity for induction for 18 to 30 hours, and then 3500 Centrifuge at ~4500g for 8~12 minutes to collect the algal cells, then resuspend the algal cells in 0.5~0.8 mL Basal culture medium, and use hygromycin B to screen to obtain preliminary positive algae; then use expression detection to screen to obtain overexpressing COCSUDRAFT_4465 gene Positive recombinant Glycococcus strains.

Furthermore, the above application also includes the cultivation, collection, fatty acid extraction and linoleic acid content calculation of transformed Glycococcus strains. Specifically, the positive recombinant monoclonal Glycococcus strains overexpressing the COCSUDRAFT_4465 gene are selected and cultured in Basal medium. At the end of logarithmic stability, centrifuge the recombinant algae culture solution, clean the algae mud, obtain algae powder through drying (freeze drying or spray drying), and weigh the algae powder to obtain the recombinant algae biomass; at the same time, the recombinant algae powder will be obtained through organic Solvent extraction was used to obtain the oil, and the oil was saponified to obtain fatty acid methyl ester. The fatty acid composition was measured using the GC internal standard method, and the linoleic acid content was calculated.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The Glycococcus fatty acid thioesterase gene COCSUDRAFT_4465 was transformed into Glycococcus through homologous recombination and overexpressed. It can not only significantly enhance the synthesis and accumulation efficiency of linoleic acid in the algae cells, but also significantly shorten the growth cycle.

(2) The COCSUDRAFT_4465 gene is the own gene of Glycococcus and has good safety performance. The developed linoleic acid product has high yield and low cost, and has good industrial application value.

(3) Overexpression of the COCSUDRAFT_4465 gene can provide new algae strains for industrial utilization of microalgae to produce linoleic acid, which can further improve the efficiency of industrial application.

Description of drawings

Figure 1 is the electrophoresis diagram of the PCR amplification product of the Glycococcus COCSUDRAFT_4465 gene (Note: M is DNA Maker; 1 is the PCR amplification product of the Glycococcus COCSUDRAFT_4465 gene).

Figure 2 is the electrophoresis diagram of the PCR homologous recombination amplification product of the Glycococcus COCSUDRAFT_4465 gene (Note: The size of the PCR product fragment is the homologous part of the vector + EcoR I and Hind III enzyme cutting sites + COCSUDRAFT_4465 gene; M is DNAMaker; 1 is COCSUDRAFT_4465 Gene PCR homologous recombination amplification product).

Figure 3 is the electrophoresis diagram of pCAMBIA1303-COCSUDRAFT_4465 positive bacterial plasmid colony PCR product (Note: M is DNAMaker; 1 and 2 are pCAMBIA1303-COCSUDRAFT_4465 positive bacterial plasmid colony PCR product)

Figure 4 is a verification electrophoresis diagram of double enzyme digestion of pCAMBIA1303-COCSUDRAFT_4465 recombinant plasmid (Note: M is DNAMaker; 1 is the double enzyme digestion product of pCAMBIA1303-COCSUDRAFT_4465 recombinant plasmid).

Figure 5 is a diagram of colonies selected for hygromycin B resistance transformed by COCSUDRAFT_4465 gene.

Figure 6 shows the gene expression analysis in the COCSUDRAFT_4465 gene recombinant Glycococcus (note: the wild strain is the Glycococcus strain that has not been transformed with the COCSUDRAFT_4465 gene; the transformed strain is the COCSUDRAFT_4465 gene overexpression recombinant Glycococcus strain; the asterisks in the columns indicate statistical analysis) Significant difference (t-test, * p <0.05)).

Figure 7 is a comparison chart of the growth cycle and growth rate of COCSUDRAFT_4465 gene-overexpressing recombinant Glycococcus and wild-type Glycococcus (note: the wild strain is a Glycococcus strain that has not been transformed with the COCSUDRAFT_4465 gene; the transformed strain is the COCSUDRAFT_4465 gene-overexpressing recombinant Glycococcus Algal strains; column asterisks indicate significant differences in statistical analysis, t test, ** p <0.01).

Figure 8 is a comparison of the oil content of COCSUDRAFT_4465 gene overexpressed recombinant Glycococcus and wild-type Glycococcus (note: the wild strain is the Glycococcus strain that has not been transformed with the COCSUDRAFT_4465 gene; the transformed strain is the COCSUDRAFT_4465 gene

Overexpression of recombinant Glycococcus strain; column asterisk indicates significant difference in statistical analysis, t test, * p < 0.05, ** p < 0.01).

Figure 9 is a gas chromatogram of fatty acids from recombinant Glycococcus overexpressing the COCSUDRAFT_4465 gene.

Figure 10 is a gas chromatogram of fatty acids of wild-type Glycococcus.

Figure 11 is a comparison of the linoleic acid content and yield of the COCSUDRAFT_4465 gene overexpression recombinant Glycococcus and wild-type Glycococcus (note: the wild strain is the COCSUDRAFT_4465 gene overexpression recombinant strain) (Note: the wild strain is the COCSUDRAFT_4465 gene overexpression recombinant strain) Glycococcus strains; asterisks on columns indicate significant differences in statistical analysis, t test, * p < 0.05, ** p < 0.01).

Detailed ways

The present invention will be further described in detail below with reference to specific embodiments, but the present invention is not limited thereto.

Example 1 Acquisition of Glycococcus COCSUDRAFT_4465 gene

1. Obtaining the COCSUDRAFT_4465 gene of Glycococcus

A Glycococcus fatty acid thioesterase gene was screened using transcriptomics. The gene's ID number in NCBI's Genebank is COCSUDRAFT_4465. The full-length coding frame nucleotide sequence of the gene is 843 bp and consists of 281 amino acids. The encoding nucleotide sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

2. Cloning of Glycococcus COCSUDRAFT_4465 gene

Glycococcus cells cultured to the logarithmic phase were collected by centrifugation (the algae cell concentration was 1×10 ⁶ cells/mL). The total RNA of the Glycococcus cells was extracted using the Trizol method and reverse transcribed by reverse transcriptase to obtain the cDNA template.

According to the COCSUDRAFT_4465 gene CDs sequence in NCBI’s Gene bank, primers were designed according to standard procedures: upstream primer sequence: 5’-ATTCCACAACAGCCTTGCGA-3’; downstream primer sequence: 5’-CCAGGTGGTGCGAG

CC-3'; The COCSUDRAFT_4465 gene amplification product was obtained through PCR amplification. The reaction system for PCR amplification was: 2.5 μL cDNA, 25 μL 2×Hieff Ultra-Rapid HotStart PCR Master Mix (with Dye), upstream and downstream primers respectively 2.5 μL, 17.5 μL ddH ₂ O (PCR amplification kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.). The PCR amplification program was: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 8 s, and complete extension for 10 min, 35 cycles.

The PCR amplification product of the COCSUDRAFT_4465 gene was verified by 1wt% agarose gel electrophoresis to verify its nucleotide fragment size. As shown in Figure 1, a band appeared at the 840 bp fragment size position. The amplified product was further cut and recovered and sent to a sequencing company. After sequencing, the sequence was 100% similar to the sequence (SEQ ID NO. 1) recorded in NCBI's Gene bank. This indicates that the PCR amplification of the gene product is successful and can be used in the next experiment.

Example 2 Obtaining COCSUDRAFT_4465 gene overexpression recombinant Glycococcus

1. Construction of pCAMBIA1303-COCSUDRAFT_4465 expression vector

Design primers based on the gene sequence of COCSUDRAFT_4465 and pCAMBIA1303 vector information: upstream primer sequence: 5'- TATGACCATGATTACGAATTC ATTCCACAACAGCCTTGCGA-3', downstream primer sequence: 5'- ACGACGGCC AGTGCCAAGCTT CCAGGTGGTGCGAGCC-3', and both ends of the primer include the partial sequence of the vector (underlined) Lines) and restriction enzyme cleavage sites (wavy lines) serve as homology arms.

The COCSUDRAFT_4465 gene homologous recombination product was obtained through PCR amplification. The PCR amplification reaction system was: 2.5 μL COCSUDRAFT_4465 gene amplification product (in Example 1), 25 μL 2×Hieff Ultra-Rapid HotStartPCR Master Mix (with Dye) (PCR The amplification kit was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd.), with 2.5 μL of upstream and downstream primers and 17.5 μL of ddH ₂ O; the PCR amplification program was: pre-denaturation at 95 °C for 3 min, denaturation at 95 °C for 30 s, Annealing at 60°C for 30 s, extension at 72°C for 8 s, complete extension for 10 min, 35 cycles. The size of the nucleotide fragment of the PCR amplification product was verified by 1wt% agarose gel electrophoresis. As can be seen from Figure 2, a band appears at the 885 bp fragment size position, indicating that the homologous recombination product of Glycococcus COCSUDRAFT_4465 gene (with pCAMBIA1303 vector partial sequence and enzyme cutting site) was successfully amplified by PCR. According to the operating instructions, the PCR amplification product was purified and recovered using a recovery kit (purchased from Sangon Bioengineering Shanghai Co., Ltd.) for the next experiment.

The pCAMBIA1303 vector was double digested with EcoR I and Hind III. The enzyme digestion system was: 25 μL pCAMBIA1303, 1 μL Quick Cut EcoR I, 1 μL Quick Cut Hind III, 10 μL 10×QuickCutBuffer (Nanjing Novizan Biotechnology Co., Ltd.) , 13 μL ddH ₂ O, digested at 37°C for 30 min. According to the operating instructions, the digested products were cleaned and recovered using a column DNA gel recovery kit (purchased from Sangon Bioengineering Shanghai Co., Ltd.). Then, perform a homologous recombination reaction between the pCAMBIA1303 double enzyme digestion product and the COCSUDRAFT_4465 homologous recombination product. The reaction system is: 2 μL pCAMBIA1303 double enzyme digestion product, 2 μL COCSUDRAFT_4465 PCR homologous recombination product, 2 μL Exnase II, 4 μL 5×CE II Buffer, 10 μL ddH ₂ O, react at 37°C for 30 minutes. The homologous product was transformed into Escherichia coli, and the positive bacterial plasmid containing pCAMBIA1303-COCSUDRAFT_4465 was obtained through kanamycin identification. Then, the pCAMBIA1303-COCSUDRAFT_4465 positive bacterial plasmid was verified by colony PCR, and the PCR product was verified by 1wt% agarose gel electrophoresis to verify its nucleotide fragment size. As shown in Figure 3, the electrophoresis chart of the PCR product of the pCAMBIA1303-COCSUDRAFT_4465 plasmid-positive bacterial colony shows that a band appears at the 885 bp fragment size position. In order to further verify whether the recombinant plasmid was successfully constructed, the recombinant plasmid was extracted from the bacterial liquid with a kit and double enzyme digested and then run on gel. As shown in Figure 4, two bands appeared at 13200 bp and 840 bp. The bacterial liquid was further It was sent to Sangon Bioengineering Shanghai Co., Ltd. for sequencing, and the nucleotide sequence was determined as shown in SEQ ID NO.3 (the "tatgaccatg attacgaattc" at the beginning of the sequence and the "aagctt ggcact ggccg tcgt " at the end of the sequence are homology arms sequence, and the rest is the COCSUDRAFT_4465 gene sequence, which is 100% similar to the sequence (SEQ ID NO. 1) recorded in NCBI's Gene bank), indicating that the pCAMBIA1303-COCSUDRAFT_4465 recombinant plasmid was successfully constructed and can be used for the next experiment.

2. Transformation of Gliococcus cells with recombinant plasmid

The above recombinant plasmid is transformed into Glycococcus cells through electroporation, and then the electroporated algae cells are poured into Basal medium for recovery, and then spread on a solid medium containing bleomycin, and placed in light culture Cultured in a box. Specific steps include the following:

Centrifuge the Gliococcus cells (4×10 ⁶ ~5×10 ⁶ cell/mL) that have been cultured to the logarithmic phase at 4°C and 8000g for 10 minutes, and resuspend the algae slurry cells in sterile frozen ultrapure water. 3 times, and finally resuspended in 100 μL ultrapure water to make the final concentration of algal cells 2×10 ⁹ cells/mL. Subsequently, extract the pCAMBIA1303-COCSUDRAFT_4465 recombinant plasmid, take 100 μL of resuspended algal cells, mix with 4 μg of pCAMBIA1303-COCSUDRAFT_4465 recombinant plasmid and 40 μg (10 μg/μL) of salmon sperm DNA (denatured by boiling in a water bath above 95°C for 1 min), and incubate on ice Incubate for at least 10 minutes, then transfer to a 2 mm electroshock cup, and use a Bio-rad electroconverter for electroporation. The parameter settings are: exponential decay, field strength 2000 V, capacitance 25 μF, and parallel resistance 200 Ω. After electrotransformation, the algae cells were immediately transferred to a test tube containing 10 mL Basal medium, recovered in a dark environment for 24 h, and then transferred to normal light intensity for culture for 24 h. The algal cells were collected by centrifugation at 4000 g for 10 min, resuspended in 0.5 mL Basal culture medium, and plated on solid Basal medium containing 15 μg/mL hygromycin B and cultured for several days.

3. Screening of positive transformed Gliococcus strains

As can be seen from Figure 5, after several days of cultivation of the COCSUDRAFT_4465 gene electroconverted Glycococcus strain, single algae colonies are clearly visible and the growth is good, and the next step of screening and identification of positive algae strains can be carried out. The specific steps are: pick the above-mentioned single algal colony, insert it into the liquid Basal medium and conduct recovery and culture for 4 to 5 days. After the single algal colony expands and grows, absorb 1 mL of algal liquid and add 10 mL of fresh algal solution containing 15 μg/mL tide. Continue culturing in Basal medium containing mycomycin B for 4 to 5 days. After the algae liquid grows and expands, it is screened three times repeatedly, and the wild-type algae strain is used as a control. When the positive clone of Glycococcus can be stably expressed in the resistant medium, 1 mL is added to 200 mL of fresh Basal medium to expand the culture for subsequent verification experiments. Take 50 mL of logarithmic phase algae liquid, extract genomic DNA, use hygromycin B gene-specific primers for PCR, and verify by 1wt% agarose gel electrophoresis.

Example 3 Verification of overexpression of COCSUDRAFT_4465 gene in transformed algal strains

Take another 50 mL of logarithmic phase algae liquid, centrifuge it at 4000 g for 5 min at 4°C, discard the supernatant, quickly freeze the obtained algae mud cells in liquid nitrogen, grind them into powder, and use the RNeasy Plant Mini Kit ( Total RNA was extracted using kits such as QIAGEN Inc., Valencia, CA, USA), Total RNA Kit I, and miRNA Isolation Kit, and the RNA was reverse transcribed using PrimeScript® RT Reagent Kit (Perfect Real Time) (TakaraBio, Otsu, Japan). cDNA, and dilute the cDNA sample to 50 ng/μL with TE buffer and store at -20 °C. qRT-PCR was performed on a LightCycler 96 Real-Time PCR system (Roche, Basel, Switzerland) using 2xSYBR Green I PCR Master Mix (Applied Biosystems, CA, USA). Upstream primer F: 5’-ATTCCACAACAGCCTTGCGAAGC-3’ and downstream primer R: 5’-AACAGCATGATTCCCAGCCACTTCC-3’. Thermal cycling conditions were as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 8 s, and complete extension for 10 min, 35 cycles.

Three replicates of qRT-PCR were performed on both wild strain and transformed strain samples, with the 54775 60S gene as the internal reference gene, and the gene expression level was calculated using the 2 ^-ΔΔCt method. As can be seen from Figure 6, the gene expression of the transgenic Gliococcus strain transferred into the pCAMBIA1303-COCSUDRAFT_4465 vector is significantly higher than that of the wild-type Glycococcus strain, indicating that the COCSUDRAFT_4465 gene is overexpressed successfully, and it is a COCSUDRAFT_4465 overexpression-positive Glycococcus strain.

Example 4 Detection of linoleic acid production ability of Glycococcus strain transformed with COCSUDRAFT_4465 gene

For visual comparison, the culture, growth cycle, growth rate, oil yield and linoleic acid production of wild-type Glycococcus strains were used as comparison indicators.

1. Culture of COCSUDRAFT_4465 gene-transformed Glycococcus strain

Select single colonies of the COCSUDRAFT_4465 gene overexpressing Glycococcus and wild-type Glycococcus strains and insert them into the liquid Basal medium. Place the inoculated liquid culture bottle into a constant temperature incubator with a rotation speed of 160 rpm. Set the culture temperature to 28± 1 ℃, light intensity 50 µmol/m ² /s, photoperiod 12 h light: 12 h dark and incubate for 144 h to obtain the inoculated seed solution. Take the above seed liquid and insert it into a culture bottle containing 100 mL Basal medium at an inoculum volume of 10%. The temperature is 28 ± 1°C, the light intensity is 50 µmol/m ² /s, and the photoperiod is 12 h light: 12 h dark and rotating speed. Cultivate in a constant temperature incubator at 160 rpm for several days.

2. Determination of the growth cycle and growth rate of COCSUDRAFT_4465 gene-transformed Glycococcus strains

First, the initial inoculation OD value of the seed solution of the wild strain Glycococcus and the transformed Glycococcus strain was measured using a UV spectrophotometer to ensure that the inoculation density of the seed solution was roughly consistent. The wild strain and the transformed strain were respectively inserted into the Basal liquid medium according to the measured initial inoculation amount. The wild strain and the transformed strain were each set up in 3 parallels, and finally transferred to the incubator for culture. The culture temperature was 28 ± 1 ℃, and the light intensity was 2800±200 lux, light-dark ratio 12 h:12 h, rotation speed 120 rpm/min. During the cultivation process, collect 3 bottles each of wild strain and transformed strain culture medium at 24-h intervals, centrifuge at 8000 rpm/min for 10 min, discard the culture medium, and then wash the algae mud 2 to 3 times with sterilized distilled water. The collected algae mud is quickly frozen in liquid nitrogen and then put into a vacuum freeze dryer to freeze-dry to collect algae powder. The quality of the algae powder is obtained by weighing it with a precision electronic balance. The growth cycle of microalgae is determined based on the weight changes of the algae powder in different time periods.

Collect the algae liquid that has been cultured to the end of the stable stage, centrifuge it at 8000g for 10 minutes, discard the supernatant, wash the algae slurry 2-3 times with distilled water, freeze it in a -80°C ultra-low temperature refrigerator for 10 hours, and then freeze it in a freeze dryer. Algae powder was obtained in 48 h. Weigh the freeze-dried algae powder and calculate the growth rate according to formula (1).

V _x (g/h)=(X ₁ -X ₀ )/(T ₁ -T ₀ ) (1)

In the _{formula ,} V _​ ​ _​ ​ _​

3. Determination of intracellular lipid content of COCSUDRAFT_4465 gene-transformed Gliococcus strains

Accurately weigh 0.02 g ( W ₁ ) of the above freeze-dried algae powder, add 0.5 mL water/chloroform/methanol (v/v/v=0.5:2:1), mix thoroughly and shake for 20 min, centrifuge at 10000 rpm for 10 min, and collect For the chloroform layer, repeat the extraction 3-5 times. Collect the chloroform layer into a 10 mL centrifuge tube that has been weighed ( W ₂ ). Combine all the cleaning solutions and the chloroform layer, vacuum dry to constant weight ( W ₃ ), and use formula (2) and (3) calculate the total lipid content and yield.

C _Lipid (%) =(W ₃ -W ₂ )/W ₁ (2)

P _Lipid (g/L/d)= P _Biomass × C _Lipid (%) (3)

In the formula, P _Lipid - total lipid yield (g/L/d); W ₁ - weight of algae powder (g); W ₂ - weight of centrifuge tube (g); W ₃ - total weight of centrifuge tube and oil; C _lipid —Total lipid content (%).

4. Determination of intracellular linoleic acid content of COCSUDRAFT_4465 gene-transformed Glycococcus strains

Weigh an appropriate amount of algae powder into a flask, add 2 mL of 95% ethanol and 4 mL of water and mix well, then add 10 mL of 8.3 mol/L hydrochloric acid solution and mix well. Place the flask into a 80°C water bath for hydrolysis for 40 minutes, and shake the flask at intervals of 10 minutes to mix the particles adhering to the wall of the flask into the solution. After hydrolysis is completed, take out the sample and cool it to room temperature. Add 10 mL of 95% ethanol and mix. Extract the fat three times with 100 mL diethyl ether-petroleum ether mixture, combine the extracts into a 100 mL flat-bottomed flask, and evaporate the petroleum ether-diethyl ether layer to dryness to obtain the fat. To the fat extract, continue to add 4 mL of 2wt% sodium hydroxide-methanol solution and 4 mL of 14wt% boron trifluoride-methanol solution. Place in a 45°C water bath for 20 minutes. After cooling to room temperature, add 3 mL of n-hexane. alkane, shake and extract for 2 minutes, let stand and separate. The supernatant was filtered through a 0.45 μm filter membrane and analyzed by gas chromatography. The gas chromatography conditions are as follows: chromatographic column TG-FAME (50 m×0.25 mm×0.20 μm); temperature rising program (80 ℃ held for 1 min, heated to 160 ℃ at a rate of 20 ℃/min, kept for 1.5 min, and then heated to 5 ℃ /min rate to 230 ℃ and hold for 6 minutes); inlet temperature 260 ℃; carrier gas flow rate 0.63 mL/min; split ratio 100:1; mass spectrometry conditions: ion source temperature 280 ℃; transfer line temperature 240 ℃; solvent delay Time 4 min; ion source: EI source 70 eV. According to the calculation method given by the national standard GB5009.168-2016, the linoleic acid content is calculated according to the retention time and corresponding peak area and formula (4):

W=(C*V*N)/m*k*10 ^-4 (4)

In the formula: W—the linoleic acid content in the sample, in g/100g; C—the concentration of methyl linoleate in the sample measurement solution, in mg/L; V—the constant volume, in mL; k —Conversion coefficient for converting methyl linoleate into linoleic acid; N—dilution factor; 10 ^-4 —Unit conversion coefficient; m—sample mass, in g.

It can be seen from the results in Figure 7 that the COCSUDRAFT_4465 gene overexpression recombinant Glycococcus strain grows better than the wild-type Glycococcus strain. Its growth cycle is 48 h shorter than that of the wild strain, and its growth rate is 1.58 times higher than that of the wild strain. Simultaneously, lipids are synthesized and accumulated. The efficiency is also significantly higher than that of the wild-type Glycococcus strain, with the oil content and oil yield increased to 1.61 times and 2.36 times (shown in Figure 8), indicating that overexpression of the COCSUDRAFT_64904 gene can significantly promote the growth and oil accumulation of the recombinant Glycococcus strain. More importantly, by comparing the fatty acids extracted from recombinant Glycococcus and wild-type Glycococcus through gas chromatography analysis (as shown in Figures 9 and 10), it was found that the relative content of linoleic acid in the recombinant Glycococcus strain was higher. Significantly improved (shown in Table 1). Specifically, the COCSUDRAFT_4465 gene overexpression recombinant Glycococcus strain has a higher proportion of linoleic acid, and the content and yield are 3.23 times and 3.05 times that of the wild strain respectively (shown in Figure 11). From the above, it can be seen that overexpression of COCSUDRAFT_4465 gene not only effectively promotes the growth of transformed Glycococcus, but also actively regulates the synthesis and accumulation of intracellular lipids, especially linoleic acid.

Table 1 Comparison of the relative fatty acid content of recombinant Glycococcus overexpressing COCSUDRAFT_4465 gene and wild-type Glycococcus

Note: The wild strain is the Glycococcus strain that has not been transformed with the COCSUDRAFT_4465 gene; the transformed strain is the recombinant Glycococcus strain that overexpresses the COCSUDRAFT_4465 gene; asterisks indicate significant differences in statistical analysis, t test, ** p <0.01.

Claims (10)

1. the coccum fatty acid thioesterase gene is characterized in that the coccum fatty acid thioesterase gene is coccum UDRAFT_4465, the nucleotide sequence of the coccum fatty acid thioesterase gene is shown as SEQ ID NO.1, and the amino acid sequence of the encoded protein of the coccum fatty acid thioesterase gene is shown as SEQ ID NO. 2.

2. The coccoid fatty acid thioesterase gene according to claim 1, wherein said coccoid fatty acid thioesterase gene is obtained under conditions that induce accumulation of coccoid grease.

3. The coccoid algae fatty acid thioesterase gene according to claim 1, wherein said coccoid algae is coccoid algaeCoccomyxa subbellipsoidea C-169。

4. Use of a coccoid fatty acid thioesterase gene according to any one of claims 1 to 3 for increasing linoleic acid synthesis, wherein the accumulation of algal cell linoleic acid synthesis is achieved by overexpression into coccoid by homologous recombination transformation, including PCR amplification and overexpression recombinant construction.

5. The use according to claim 4, wherein the PCR amplification is performed using cDNA reverse transcribed from total RNA of the genus Chlorella as a template, and the designed primers comprise an upstream primer sequence 5'-ATTCCACAACAGCCTTGCGA-3' and a downstream primer sequence 5'-CCAGGTGGTGCGAGCC-3'; the over-expression recombinant construction comprises the steps of designing a primer, amplifying, obtaining a recombinant plasmid and obtaining an over-expression algae strain.

6. The use according to claim 5, wherein the primers are designed based on the COCSUDRAFT_4465 gene and pCAMBIA1303 vector information, and the primer sequences comprise the upstream primer sequence 5'TATGACCATGATTACGAATTCATTCCACAACAGCCTTGCGA-3' and downstream primer sequence 5ACGACGGCCAGTGCCAAGCTTCCAGGTGGTGCGAGCC-3', the two ends of the primer respectively comprise a partial sequence of the vector shown in the dash line and an enzyme cutting site shown in the wavy line as homology arms; the amplification is PCR amplification, and an amplification product has a partial sequence and an enzyme cutting site of a pCAMBIA1303 vector; the method for obtaining the recombinant plasmid is a homologous recombination method, and the COCSUDRAFT_4465 gene is constructed and enters a pCAMBIA1303 vector to obtain the recombinant plasmid; the method for obtaining the over-expression algae strain is to convert the recombinant plasmid into the chlorella by adopting an electrotransformation method, and obtain the recombinant chlorella of positive over-expression COCSUDRAFT_4465 by screening antibiotics and detecting the expression quantity.

7. The use according to claim 6, wherein the reaction system for PCR amplification is: 2.2 to 2.8. Mu.L of cDNA,20 to 30. Mu.L of 2 XHieff Ultra-Rapid Hot Start PCR Master Mix (with Dye), 2.2 to 2.8. Mu.L of each of the upstream and downstream primers, 17.2 to 12.8. Mu.L of ddH ₂ O; the PCR amplification procedure was: pre-denaturation is carried out for 2-4 min at 93-97 ℃, denaturation is carried out for 25-35 s at 93-97 ℃, annealing is carried out for 25-35 s at 55-65 ℃, extension is carried out for 6-10 s at 68-74 ℃, and complete extension is carried out for 8-12 min for 30-40 cycles; and (3) purifying and recovering the PCR amplification product by using 0.5-3 wt% agarose gel electrophoresis to obtain the homologous recombination amplification product of the COCSUDRAFT_4465 gene.

8. The use according to claim 6, wherein the homologous recombination method is to carry out homologous recombination reaction on the double digestion products of pCAMBIA1303 and the homologous recombination products of COCSUDRAFT_4465 gene, and the reaction system is as follows: 90~110 ng pCAMBIA1303 double enzyme digestion products, 90~110 ng COCSUDRAFT_4465 gene PCR homologous recombination products, 1.5-2.5 mu L of Exnase II, 3-5 mu L of 5 XCE II Buffer, 7-13 mu L of ddH ₂ O, reacting for 25-35 min at 36-38 ℃; the homologous product is transformed into escherichia coli, and positive bacteria containing pCAMBIA1303-COCSUDRAFT_4465 recombinant plasmid are obtained through identification.

9. The use according to claim 6, wherein said obtaining an overexpressed strain of algae is in particular: uniformly mixing 90-110 mu L of heavy suspension chlorella cells with 3-5 mu g of pCAMBIA1303-COCSUDRAFT_4465 recombinant plasmid and 35-45 mu g of salmon sperm DNA, performing electroporation after incubating on ice for 8-12 min, immediately transferring the electroporated chlorella cells into a test tube containing 8-12 mL of Basal culture solution, resuscitating for 18-30 h under dark conditions, transferring to normal light intensity for induction for 18-30 h, centrifuging 3500-4500 g for 8-12 min to collect algae cells, and then re-suspending the algae cells with 0.5-0.8 mL of Basal culture solution, and screening by hygromycin B to obtain primary positive algae; and screening and obtaining the positive recombinant chlorella strain which overexpresses the COCSUDRAFT_4465 gene by an expression quantity detection mode.

10. The use according to any one of claims 4 to 9, further comprising culturing, harvesting, fatty acid extraction and linoleic acid content calculation of transformed coccoid strains, in particular: selecting a positive recombinant monoclonal coccoid strain which overexpresses COCSUDRAFT_4465 gene, inoculating a Basal culture medium to culture until the end of logarithmic stabilization, centrifuging recombinant algae culture solution, cleaning algae mud, drying to obtain algae powder, and weighing the algae powder to obtain recombinant algae biomass; synchronously extracting the obtained recombinant algae powder with an organic solvent to obtain grease, saponifying the grease to obtain fatty acid methyl ester, measuring the fatty acid composition by adopting a GC internal standard method, and calculating to obtain the linoleic acid content.