Chlamydomonas reinhardtii ABC transporter gene CrABCG, and the encoded protein and application thereof

Abstract

The application provides a Chlamydomonas reinhardtii ABC transporter gene CrABCG, a coding protein and application thereof, and the gene comprises CrABCG3 or CrABCG5, and the gene sequence comprises SEQ ID NO:1 or SEQ ID NO:2. It is found by the application that the CrABCG protein is related to carbon and nitrogen metabolism of Chlamydomonas reinhardtii, and preliminary researches show that the expression of the CrABCG3 gene of Chlamydomonas reinhardtii is increased after nitrogen limitation, while the expression of the CrABCG5 gene is first decreased and then increased, and the results show that the CrABCG protein influences the transport, synthesis and accumulation of lipid substances. Therefore, the engineering algae strains with high lipid content can be obtained by screening and modification of the algae strains with high expression of the CrABCG gene.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, specifically relating to a Chlamydomonas reinhardtii ABC transporter gene CrABCG, its encoded protein, and its applications. Background Technology

[0002] Biofuels, as renewable and sustainable alternative energy sources, are promising candidates to replace fossil fuels. Among various alternative energy sources, microalgae are considered a potential material for biofuel production due to their higher photosynthetic activity and biomass productivity. *Chlamydomonas reinhardtii*, as a haploid organism, readily exhibits loss-of-function phenotypes caused by gene mutations. In the constructed transgenic microalgae with high lipid content, transgenic *Chlamydomonas* strains account for approximately 27%, making them a highly regarded model microalga for studying lipid accumulation mechanisms in recent years.

[0003] In algae, de novo synthesis of fatty acids occurs in the plastid matrix, after which the fatty acids are transferred to the endoplasmic reticulum for the synthesis of substances such as triglycerides. Cr FAX1 and Cr FAX5 participate in the production of TAG (triglycerides) by functioning in the chloroplast and endoplasmic reticulum membranes, respectively. Some literature mentions that Cr FAX1 plays a transport role in the inner membrane of chloroplasts and Cr FAX5 plays a transport role in the endoplasmic reticulum.

[0004] The ABC protein superfamily is widely distributed in microorganisms, animals, and plants, and is one of the oldest and largest known protein families. It can utilize the energy released from the hydrolysis of adenine nucleoside triphosphate (ATP) to carry out transmembrane transport of various substrates such as heavy metal ions, fatty acids, and plant hormones, thereby participating in processes such as detoxification, prevention of pathogen invasion, and growth and development.

[0005] The ABC transporter family is large, widespread, and numerous. In the plant kingdom, the ABC family is divided into eight subfamilies: ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG, and ABCH. In Arabidopsis thaliana, AtABCA9 promotes fatty acid transport on the endoplasmic reticulum during seed development. In Chlamydomonas reinhardtii, CrABCA2, a protein homologous to AtABCA9, shows lower triglyceride accumulation in knockout strains and higher triglyceride content in overexpressing strains. In Arabidopsis thaliana, AtABCG9 and AtABCG31 are involved in the transport of precursors for pollen exwall sporopollenin synthesis, while corkyl resin monomers are transported by AtABCG1. Therefore, ABCG proteins in Arabidopsis thaliana are related to lipid transport. The structures and functions of the different subfamilies of plant ABC transporters are distinctly different, making structural prediction impossible. Whether Chlamydomonas reinhardtii, possessing the ABC transporter gene CrABCG, regulates lipid metabolism has not been reported.

[0006] The synthesis and metabolism of lipids occur in the cell's plastid matrix and endoplasmic reticulum. Transport proteins play an important role in enabling substances to move between different organelles, but research on lipid transport proteins in Chlamydomonas reinhardtii is currently insufficient.

[0007] ABCG proteins are widely present in lower and higher organisms and are relatively conserved in evolution. As transport proteins, some of them in Arabidopsis thaliana have been shown to be involved in lipid transport. However, there are few reports on Chlamydomonas reinhardtii. In-depth research on the role of ABCG proteins in Chlamydomonas reinhardtii is of great significance for the development of biofuels. Summary of the Invention

[0008] To address the shortcomings of existing technologies, the present invention aims to provide a Chlamydomonas reinhardtii ABC transporter gene CrABCG, its encoded protein, and its applications, particularly in regulating algal lipid metabolism. This invention constructs an overexpression vector for the CrABCG transporter gene to explore its role in lipid metabolism and lays the foundation for further research into the transport function of the CrABCG protein.

[0009] To achieve this objective, the present invention adopts the following technical solution:

[0010] In a first aspect, the present invention provides a Chlamydomonas reinhardtii ABC transporter gene CrABCG, wherein the Chlamydomonas reinhardtii ABC transporter gene includes: CrABCG3 or CrABCG5.

[0011] The nucleotide sequence of the gene CrABCG3 includes:

[0012] (A) The nucleotide sequence shown in SEQ ID NO:1;

[0013] Or (B) a nucleotide sequence that has more than 95% homology with the nucleotide sequence shown in SEQ ID NO:1 and encodes a protein with the same function;

[0014] The nucleotide sequence of the gene CrABCG5 includes:

[0015] (1) The nucleotide sequence shown in SEQ ID NO:2;

[0016] Or (2) a nucleotide sequence that has more than 95% homology with the nucleotide sequence shown in SEQ ID NO:2 and encodes a protein with the same function.

[0017] In a second aspect, the present invention provides a Chlamydomonas reinhardtii ABC transporter CrABCG, wherein the Chlamydomonas reinhardtii ABC transporter includes: CrABCG3 or CrABCG5.

[0018] The amino acid sequence of the transporter protein CrABCG3 includes:

[0019] (a) The amino acid sequence shown in SEQ ID NO:3;

[0020] (b) An amino acid sequence derived from (a) having the same activity as the amino acid sequence shown in SEQ ID NO:3, by substitution, deletion, insertion or addition;

[0021] The amino acid sequence of the transporter protein CrABCG5 includes:

[0022] (I) The amino acid sequence shown in SEQ ID NO:4;

[0023] (II) An amino acid sequence derived from (I) having the same activity as the amino acid sequence shown in SEQ ID NO:4, by substitution, deletion, insertion or addition.

[0024] This invention discovered a CrABCG protein and its gene in *Chlamydomonas reinhardtii* that is homologous to *Arabidopsis thaliana*. Studies have shown that the CrABCG protein may be related to carbon and nitrogen metabolism in *Chlamydomonas reinhardtii*. Preliminary research indicates that after nitrogen restriction, the expression of the CrABCG3 gene in *Chlamydomonas reinhardtii* increases, while the expression of the CrABCG5 gene initially decreases and then increases. Research suggests that the CrABCG protein affects lipid transport, synthesis, and accumulation. Therefore, engineered algal strains with high lipid content can be obtained by screening and modifying algal strains that highly express the CrABCG gene.

[0025] Thirdly, the present invention provides the application of the Chlamydomonas reinhardtii ABC transporter gene CrABCG described in the first aspect and / or the Chlamydomonas reinhardtii ABC transporter CrABCG described in the second aspect in regulating algal lipid metabolism.

[0026] In this invention, the Chlamydomonas reinhardtii ABC transporter gene CrABCG and its protein participate in the regulation of lipid metabolism in the following two aspects: increasing the neutral lipid content of the algal strain and increasing the total fatty acid content of the algal strain.

[0027] Fourthly, the present invention provides an engineered algal strain, which is obtained by overexpressing the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 or CrABCG5, using Chlamydomonas reinhardtii as the starting strain.

[0028] Preferably, the overexpression method includes: linking the nucleotide sequence of the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 or CrABCG5 to the expression vector backbone to construct an overexpression vector, linearizing the overexpression vector, and transforming it into Chlamydomonas reinhardtii to obtain an engineered algal strain.

[0029] Preferably, the linearized overexpression vector is transformed into Chlamydomonas reinhardtii via glass bead method or electroporation method.

[0030] Preferably, the starting strain is Chlamydomonas reinhardtii CC-5325.

[0031] Fifthly, the present invention provides a method for cultivating algae, the method comprising: constructing an expression vector containing the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 or CrABCG5, transferring the expression vector into Chlamydomonas reinhardtii, and obtaining an algal strain with a higher content of neutral lipids and total fatty acids than the wild type.

[0032] In one specific embodiment of the present invention, the algae cultivation method is selected from (1) or (2):

[0033] (1) The pPHRB vector was digested with Eco RⅠ and Eco R72Ⅰ to produce a vector backbone. The CrABCG3 gene was ligated into the expression vector backbone to obtain an overexpression vector of the CrABCG3 gene. The vector was then transferred into Chlamydomonas reinhardtii to obtain an algal strain with a higher neutral lipid content than the wild type.

[0034] (2) The pPHRB vector was digested with Eco RⅠ and Eco R72Ⅰ to produce a vector backbone. The CrABCG5 gene was ligated into the expression vector backbone to obtain an overexpression vector of the CrABCG5 gene. The vector was then transformed into Chlamydomonas reinhardtii to obtain an algal strain with a higher neutral lipid content than the wild type.

[0035] In a sixth aspect, the present invention provides an expression vector comprising the Chlamydomonas reinhardtii ABC transporter genes CrABCG3 and / or CrABCG5 as described in the first aspect.

[0036] In a seventh aspect, the present invention provides a host cell comprising the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 and / or CrABCG5 as described in the first aspect; or the host cell comprising the expression vector as described in the sixth aspect.

[0037] Eighthly, the present invention provides primers for amplifying the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 or CrABCG5, wherein the upstream and downstream primers for amplifying CrABCG3 are shown in SEQ ID NO:18 and SEQ ID NO:19; and the upstream and downstream primers for amplifying CrABCG5 are shown in SEQ ID NO:20 and SEQ ID NO:21.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] This invention obtained Chlamydomonas reinhardtii strains overexpressing CrABCG3 or CrABCG5 by constructing vectors that overexpress either gene. The CrABCG3-overexpressing strains significantly increased the content of neutral lipids and total fatty acids, laying a solid theoretical and applied foundation for cultivating microalgae with high lipid content. Furthermore, this invention found that the overexpressing strains can more effectively increase lipid accumulation under nitrogen-limited conditions. Attached Figure Description

[0040] Figure 1 This is an evolutionary analysis diagram of the Chlamydomonas reinhardtii CrABCG protein.

[0041] Figure 2 It is the structure of the overexpression vector CrABCG3-pPHRB.

[0042] Figure 3 It is the structure of the overexpression vector CrABCG5-pPHRB.

[0043] Figure 4 These are the colony PCR results after constructing the CrABCG3 overexpression vector.

[0044] Figure 5 These are the colony PCR results after constructing the CrABCG5 overexpression vector.

[0045] Figure 6 The expression level of the CrABCG3 gene in the CrABCG3 overexpressing algal strain oe3 relative to that in CC-5325.

[0046] Figure 7 The expression level of the CrABCG5 gene in the CrABCG5 overexpressing algal strain oe5 relative to that in CC-5325.

[0047] Figure 8 The results of BODIPY staining at mid-log intervals for CC-5325, CrABCG3 overexpressing algal strain oe3, and CrABCG5 overexpressing algal strain oe5.

[0048] Figure 9The results of BODIPY staining for CC-5325, CrABCG3 overexpressing algal strain oe3, and CrABCG5 overexpressing algal strain oe5 after 48 h of nitrogen-limited culture.

[0049] Figure 10 The content of lipids that can be extracted from each gram of freeze-dried algal powder after 48 hours of nitrogen-limited culture for the CC-5325, CrABCG3 overexpressing algal strain oe3, and CrABCG5 overexpressing algal strain oe5.

[0050] Figure 11 The content of various fatty acids and total fatty acid content per gram of algal powder for CC-5325, CrABCG3 overexpression algal strain oe3 and CrABCG5 overexpression algal strain oe5 after 48 h of nitrogen-limited culture. Detailed Implementation

[0051] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0052] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0053] The culture medium used in the following specific embodiments includes:

[0054] TAP medium: 10 mL / L Tris-Acetate (100×), 10 mL / L phosphate buffer (100×), 10 mL / L Liejerinck's solution (100×), 1 mL / L trace element solution (1000×).

[0055] TAP-N medium: 10 mL / L Tris-Acetate (100×), 10 mL / L phosphate buffer (100×), 10 mL / L Eijerinck's Solution-N (100×), 1 mL / L trace element solution (1000×).

[0056] LB medium: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L sodium chloride.

[0057] Tris-Acetate (100×): 242 g / L of tris-hydroxymethylaminomethane, 100 mL of glacial acetic acid.

[0058] Phosphate buffer (100×): K2HPO4 11.92 g / L, KH2PO4 6.05 g / L.

[0059] Beijerinck's solution (100×): NH4Cl 40 g / L, MgSO4·7H2O 10 g / L, CaCl2·2H2O 5 g / L.

[0060] Trace element solution (1000×): H3BO3 11.4 g / L, MnCl2·4H2O 5.6 g / L, ZnSO4·7H2O 22 g / L, FeSO4·7H2O 5 g / L, CoCl2·6H2O 1.61 g / L, CuSO4·5H2O 1.57 g / L, (NH4)6Mo7O 24 ·4H2O1.1g / L, Na2EDTA 50g / L.

[0061] Beijerinck's solution - N(100×): KCl 56 g / L, MgSO4·7H2O 10 g / L, CaCl2·2H2O 5 g / L.

[0062] Example 1

[0063] Evolutionary analysis of Chlamydomonas reinhardtii ABCG proteins

[0064] The amino acid sequences of 43 ABCG proteins from Arabidopsis were downloaded from the TAIR database (https: / / www.Arabidopsis.org / ). Using the Arabidopsis ABCG11 protein (AT1G17840) as the BLAST target, homologous ABCG proteins in Chlamydomonas reinhardtii were searched in NCBI (https: / / www.ncbi.nlm.nih.gov / ) and PHYTOZOME (https: / / phytozome-next.jgi.doe.gov / ), yielding 5 predicted homologous proteins. An phylogenetic tree was constructed by analyzing the evolutionary relationships of these 5 CrABCG proteins with ABCG proteins from other microalgae, plants, and other species.

[0065] Species names: Haematococcus lacustris (Hl), Haematococcus lacustris; Tetrabaena socialis (Ts), Tetrabaena socialis; Chlorella sorokiniana (Cs), Chlorella sorokiniana; Nannochloropsis gaditana (Ng), Nannochloropsis gaditana; Physcomitrella patens (Ppa), Physcomitrella patens; Volvox carteri (Vc), Volvox; Arabidopsis thaliana (At), Arabidopsis thaliana; Nicotiana benthamiana (Nb), Nicotiana benthamiana; Saccharomyces cerevisiae (Sc), Saccharomyces cerevisiae; Salmonella enterica ser. Typhymurium (St), Salmonella typhimurium; Escherichia coli (Eco), Escherichia coli; Micractinium conductrix (Mc), Micrium conductrix; Oryza sativa (Os), rice; Glycine max (Gm), soybean; Vitis vinifera (Vv), grape; Brassica napus (Bn), rapeseed; Prunus persica (Pp), peach.

[0066] The phylogenetic relationships of the five CrABCG proteins with ABCG proteins from other microalgae, plants, and other species were analyzed, and the phylogenetic tree constructed is as follows: Figure 1 As shown in the figure, CrABCG3 is closely related to Arabidopsis thaliana AtABCG11, AtABCG12 and AtABCG13 and rice OsABCG11 in terms of evolution, while CrABCG5 is in the same evolutionary branch as Arabidopsis thaliana AtABCG9, AtABCG14 and AtABCG26.

[0067] Example 2

[0068] Obtaining CrABCG3 and CrABCG5 genes and constructing overexpression vectors

[0069] 1. Extraction of total RNA from Chlamydomonas reinhardtii

[0070] (1) CrABCG3 and CrABCG5 are the target genes required for this experiment. First, their gene sequences must be obtained from Chlamydomonas reinhardtii. The relevant mRNA transcribed from Chlamydomonas reinhardtii itself can function as the target gene CDS. Total RNA was extracted from Chlamydomonas reinhardtii CC-5325 cultured to the logarithmic growth phase using the RNA extraction kit from Shanghai Feijie, and then its cDNA was reverse transcribed for transgenesis.

[0071] (2) Take 2 mL of algal solution into a 2 mL centrifuge tube free of RNase. Be careful when adding the solution to avoid spilling the solution. Centrifuge at 10,000 rpm for 1 min and discard the supernatant. An RNase-free environment must be maintained during RNA extraction.

[0072] (3) Add 500 μL of RA2 to the centrifuge tube and pipette until clear, about 6 times. The number of times should not be too many or too few. Too many times will cause RNA degradation, and too few times will not completely dissolve the precipitate.

[0073] (4) Place the adsorption column in a 2mL collection tube, take the liquid from the centrifuge tube and add it to the adsorption column. Centrifuge at 12000rpm for 1min. The liquid in the collection tube is waste liquid and should be discarded.

[0074] (5) Add 500 μL of rinsing buffer, centrifuge at 12000 rpm for 1 min, and discard the waste liquid. Repeat once.

[0075] (6) Centrifuge at 12000 rpm for 1 min to ensure that the liquid in the adsorption column is completely removed.

[0076] (7) Place the adsorption column in a sterile 1.5 mL centrifuge tube, add 50 μL of RNase-free water, let stand for 1 min, and then centrifuge at 12000 rpm for 1 min. Collect the RNA into the centrifuge tube.

[0077] (8) The concentration and purity of RNA were detected by Nanodrop2000 (A260 / A280, A260 / A230), and RNA integrity was detected by 1% non-denaturing agarose gel electrophoresis.

[0078] 2. RNA reverse transcription

[0079] The reverse transcription reaction was performed using the TransGen AE311 Reverse Transcription Kit according to the manufacturer's instructions. The reaction program was an incubation at 42°C for 30 min. Table 1 shows the reverse transcription system (for PCR).

[0080] Table 1

[0081]

[0082]

[0083] 3. Amplification of the target gene

[0084] Based on the sequences of SEQ ID NO:1 and SEQ ID NO:2, specific primers CrABCG3-F and CrABCG3-R, CrABCG5-F and CrABCG5-R (SEQ ID NO:8-SEQ ID NO:11) were designed. Using the cDNA obtained in the previous step as a template, amplification was performed according to the system shown in the table below. The reaction program was: 98℃ for 3 min; 98℃ for 10 s, 61℃ for 10 s, 72℃ for 2 min, 35 cycles; 72℃ for 5 min; and finally, held at 4℃. After the reaction program was completed, the amplified products of the corresponding fragments were obtained. Table 2 shows the PCR reaction system.

[0085] Table 2

[0086] reagents Added amount KOD One PCR reaction mixture 10μL Forward primer (10 μmol / L) 0.5μL Reverse primer (10 μmol / L) 0.5μL DNA 2μL <![CDATA[ddH2O]]> Make up to 20 μL

[0087] 4. Gel extraction and recovery of PCR products

[0088] After amplifying the target gene in the previous step, the products need to be verified by agarose gel electrophoresis. Prepare a 1.5% agarose gel and perform electrophoresis on all PCR products. Use a full-gold electrophoresis gel recovery kit to recover the PCR products.

[0089] (1) After electrophoresis, clear bands can be seen under ultraviolet light. Quickly cut off the band that matches the size of the target fragment with a knife. This is the gel containing the desired DNA fragment. Transfer it to an empty centrifuge tube and then chop the gel as finely as possible. Weigh the empty centrifuge tube beforehand, and then weigh the centrifuge tube containing the gel. The weight of the gel is the difference between the weight of the empty and the weight of the gel. In the experiment, 100 mg of gel is equivalent to 100 μL of gel.

[0090] (2) Add buffer solution GDP equal to the volume of the gel. Heat in a 50-55℃ metal bath for 10 minutes to completely dissolve the gel. During this time, you can invert the centrifuge tube to make the gel dissolve more thoroughly.

[0091] (3) Place the adsorption column on the collection tube, transfer 700 μL of gel solution into the adsorption column, and centrifuge at 12000 rpm for 1 min. If the gel solution exceeds 700 μL, it can be added to the adsorption column in multiple portions. Discard the filtrate and return the adsorption column to the collection tube.

[0092] (4) Add anhydrous ethanol to the buffer GW in advance and then label it. Add 700 μL of buffer GW to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Repeat this step once.

[0093] (5) Place the adsorption column back into the collection tube and centrifuge at 12000 rpm for 2 min to completely remove ethanol. After centrifugation, transfer the adsorption column to a 1.5 mL sterile centrifuge tube, open the cap to allow the ethanol to evaporate for 1 min, add 30 μL of elution buffer, and let it stand for 2 min. Centrifuge at 12000 rpm for 1 min. Collect the DNA solution in the centrifuge tube, then determine its concentration using a Nanodrop 2000 and store it at -20°C.

[0094] 5. Addition of homologous arms

[0095] Using the gel-recovered product of the target gene fragment as a template, new primers were designed based on the cleavage sites of the enzymes required for subsequent plasmid digestion. PCR was performed using these new primers to add homologous arms to the target gene fragment, facilitating seamless ligation with the digested plasmid. The primers used for adding the homologous arms were Ha3-F and Ha3-R(CrABCG3), and Ha5-F and Ha5-R(CrABCG5) (SEQ ID NO:12-SEQ ID NO:15). The PCR products were also gel-recovered.

[0096] 6. Construction of overexpression vectors

[0097] The linearized overexpression vector backbone and the target gene fragment containing homologous arms were ligated together using a seamless cloning method. First, the vector pPHRB (SEQ ID NO:5) was double-digested according to Table 3 at 37°C for 30 min to ensure complete digestion and the formation of sticky ends. The PCR product with added homologous arms obtained in the previous step was also double-digested. After digestion, gel electrophoresis was performed to verify that the obtained vector was linearized and that its size met the requirements. After confirmation, the gel was excised and recovered. Based on the concentrations of the linearized vector and the target gene fragment with added homologous arms, they were added to the seamless cloning system (Table 4) in the specified proportions. After incubation at 50°C for 15 min, the overexpression vector containing the target gene fragment was obtained.

[0098] The structure of the overexpression vector CrABCG3-pPHRB is as follows: Figure 2 As shown, the structure of the overexpression vector CrABCG5-pPHRB is as follows: Figure 3 As shown.

[0099] Table 3

[0100]

[0101] Table 4

[0102] reagents Added amount <![CDATA[ddH2O]]> Up to 10 μL Linearized carrier 150ng Target gene fragment The molar ratio of the carrier to the carrier is 2:1. 5×OK Clon reaction mixture 2μL

[0103] 7. Escherichia coli transformation

[0104] (1) Take the competent cells Top10 out of the -80℃ freezer and place them on ice to wait for them to thaw. In the clean bench, add the reaction solution after seamless cloning to the competent cells, mix well, and incubate on ice for 15 minutes.

[0105] (2) First, preheat the water bath to 42°C, then heat the competent cells in a 42°C water bath for 60 seconds, and then quickly place them in an ice box for 2 minutes.

[0106] (3) Preheat LB liquid medium in 37°C water, then add 600 μL of preheated LB medium to competent cells and revive the cells at 37°C, 180 rpm for 45 min.

[0107] (4) After the recovery is complete, take 100 μL of bacterial solution and spread it on a plate containing ampicillin resistance. Incubate at 37°C upside down for 12-15 h.

[0108] (5) Then, use a sterile pipette tip to randomly pick bacteria that have grown on the plate, transfer them to an LB plate containing ampicillin resistance, and perform PCR verification.

[0109] 8. Colony PCR verification

[0110] Several positive clones were selected for colony PCR verification. The primers were CrABCG3-F and CrABCG3-R, CrABCG5-F and CrABCG5-R (SEQ ID NO:8-SEQ ID NO:11), which were the same as those mentioned above. Figure 4 To obtain the colony PCR results after constructing the CrABCG3 overexpression vector, the primers used were SEQ ID NO:8 and SEQ ID NO:9, and the fragment length should be 1869bp. Figure 5 To obtain the colony PCR results after constructing the CrABCG5 overexpression vector, primers SEQ ID NO:10 and SEQ ID NO:11 were used, with a fragment length of 2142 bp. Strains that tested positive by PCR were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0111] 9. Extraction of overexpression vectors

[0112] A portion of the positive clone was picked and added to LB broth containing ampicillin and incubated at 37°C for 15 hours. Then, plasmid extraction was performed. The plasmid was extracted using a fully gold-based plasmid extractor. The Plasmid MiniPrep Kit has the following specific steps.

[0113] (1) Add 2 mL of bacterial culture to a centrifuge tube and centrifuge at 10,000 rpm for 1 min. Discard the waste liquid and try to keep the liquid residue as small as possible.

[0114] (2) If bacterial precipitate appears in the centrifuge tube, add 250 μL of RB (containing RNase A) and mix well. Be sure to thoroughly resuspend the bacteria until no bacterial clumps are visible and the solution is clear.

[0115] (3) Add 250 μL of blue LB solution, invert the container about 6 times to mix thoroughly, and ensure complete lysis of the bacteria. Be careful not to be too rough and damage the plasmids. If the solution is viscous and clear, then the bacteria have been fully lysed.

[0116] (4) Add 350 μL of NB, mix gently 5-6 times, wait for yellow precipitate to form in the centrifuge tube, let stand for 2 min, and then centrifuge at 12000 rpm for 5 min.

[0117] (5) Place the adsorption column into a 2 mL collection tube. Take the supernatant from the previous step and transfer it to the adsorption column using a pipette. Note that you should only use the liquid and not the precipitate. Centrifuge at 12,000 rpm for 1 min. Discard the waste liquid and put the adsorption column back into the collection tube.

[0118] (6) Add 500 μL of WB to the adsorption column. Centrifuge at 12000 rpm for 1 min. Discard the waste liquid and return the adsorption column to the collection tube. Repeat once.

[0119] (7) Return the adsorption column to the collection tube and centrifuge at 12,000 rpm for 1 min to completely remove the residual liquid in the adsorption column.

[0120] (8) Discard the collection tube, take a 1.5 mL centrifuge tube, and place the adsorption column in it. Add 30-100 μL of elution buffer to the center of the membrane of the adsorption column, let it stand at room temperature for 2 min, and centrifuge at 12000 rpm for 1 min. The plasmid collected in the centrifuge tube is the plasmid.

[0121] (9) Store the plasmid at -20°C to prevent degradation.

[0122] Thus, the CrABCG3 and CrABCG5 overexpression vectors (SEQ ID NO:6 and SEQ ID NO:7) required for subsequent experiments were obtained, and these vectors will be used to conduct some exploratory experiments on the effects of ABCG proteins on lipid synthesis.

[0123] Example 3

[0124] Obtaining overexpressed algal strains

[0125] 1. Linearization of overexpression vectors

[0126] The recombinant vector containing the target fragment was digested with enzymes. The enzyme digestion system is shown in Table 5 below. The reaction program was to incubate at 37℃ for 30 min, and then heat to 85℃ to inactivate the enzyme, thereby obtaining a linearized overexpression vector for transformation into Chlamydomonas reinhardtii.

[0127] Table 5

[0128] reagents Added amount <![CDATA[ddH2O]]> Up to 20 μL Overexpression vector 2μg KpnⅠ 2μL 10×FastDigest Green Buffer 2μL

[0129] 2. Glass bead method for converting Chlamydomonas reinhardtii

[0130] (1) The linearized overexpression vector was transformed into Chlamydomonas reinhardtii CC-5325 cells using the glass bead method. The algae were first cultured to the logarithmic growth phase, at which point the cell density was approximately (1-2) × 10⁻⁶ cells / year. 6 Centrifuge at 5000 rpm for 5 min, discard the supernatant, and keep the algal cells.

[0131] (2) Take 300 mg of glass beads with a diameter of 0.5 mm into a 1.5 mL centrifuge tube in advance and sterilize at 121 °C for 20 min. Add 1-2 mL of fresh TAP medium according to the algal solution concentration to resuspend the algal cells, and then take 300 μL of the resuspended algal solution into a centrifuge tube containing glass beads.

[0132] (3) Add 20 μL of the linearized carrier prepared according to Table 5 and mix well by pipetting at the liquid surface. Vortex at the maximum speed (2500 rpm) for 29 s. Add 10 mL of fresh TAP medium to a 50 mL centrifuge tube, then add the vortexed algal solution to it, and incubate overnight at 120 rpm in the dark.

[0133] (4) After culturing for 20-22 hours, take out a 50 mL centrifuge tube, centrifuge at 3000 rpm for 5 min, discard the supernatant, then add 100 μL of fresh TAP to resuspend the algal cells, and spread them on TAP solid medium containing bleomycin and invert them for culture. After two weeks of culture, transformants will grow on the plate.

[0134] 3. Screening of overexpressing algal strains

[0135] (1) DNA verification

[0136] Add 50 μL of 5% CHELEX solution (w / v) to a 0.2 mL centrifuge tube. In a clean bench, use a sterile pipette tip to pick up algal colonies and add them to the CHELEX solution. Vortex for 10 s. Incubate at 98 °C for 20 min, then cool on ice for 1 min. After vortexing for 10 s, centrifuge at 2200 rpm for 5 min. The supernatant is the extract containing genomic DNA. Perform PCR on the extracted genomic DNA using primer pairs CrABCG3-F and CrABCG3-R, and CrABCG5-F and CrABCG5-R (SEQ ID NO:8-SEQ ID NO:11) to verify whether the transformants contain the overexpression vector.

[0137] (2) qPCR verification

[0138] Take 2 mL of the logarithmic midline (4 × 10) 6 Algal cells (cells / mL) were collected by centrifuging at 10,000 rpm for 1 min in 2 mL RNase-free centrifuge tubes. RNA was extracted using an RNA extraction kit (Shanghai Feijie Biotechnology). cDNA was obtained by reverse transcription of the extracted RNA using an Aike Rui reverse transcription kit.

[0139] RNA reverse transcription was performed using the Akerui reverse transcription kit, as shown in Table 6.

[0140] Table 6

[0141]

[0142]

[0143] The reverse-transcribed cDNA was stored at -20°C.

[0144] The cDNA was diluted 10-fold with RNase-free water and set aside. Using RACK1 as the internal reference gene, the reaction system was prepared using the Aike Rui Real-Time PCR (qRT-PCR) kit. The reaction program was 95℃ for 10 min; 95℃ for 15 s, 60℃ for 1 min, 40 cycles; the melting curve was 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s. Primers used for detecting the internal reference gene were qRACK-F and qRACK-R (SEQ ID NO:16 and SEQ ID NO:17), primers used for detecting CrABCG3 were q3-F and q3-R (SEQ ID NO:18 and SEQ ID NO:19), and primers used for detecting CrABCG5 were q5-F and q5-R (SEQ ID NO:20 and SEQ ID NO:21). After the reaction, according to 2... -△△CtThe expression levels of the target gene in the transformants were relatively quantified. As a result, the CrABCG3 overexpressing algal strain oe3 and the CrABCG5 overexpressing algal strain oe5 were obtained.

[0145] The qRT-PCR reaction conditions were: 95℃, 10 min; 95℃, 15 s, 60℃, 1 min, 40 cycles. The melting curve program was: 95℃, 15 s; 60℃, 1 min, 95℃, 15 s.

[0146] Figure 6 The expression level of the CrABCG3 gene in the CrABCG3 overexpressing algal strain oe3 relative to that in CC-5325. Figure 7 The expression level of the CrABCG5 gene in the CrABCG5 overexpressing algal strain oe5 relative to that in CC-5325.

[0147] (3) BODIPY screening

[0148] Take 2 mL of the logarithmic midline (4 × 10) 6 Algal culture (cells / mL) was collected in 2 mL centrifuge tubes, centrifuged at 10,000 rpm for 1 min, and the algal cells were washed twice with 1×PBS to dilute the cells to a density of 2×10⁻⁶ cells / mL. 6 200 μL of the algal solution was taken from both a black opaque 96-well plate and a clear 96-well plate, respectively. For the black opaque 96-well plate, 2.6 μL of BODIPY505 / 515 solution (10 μg / mL, dissolved in DMSO) was added under light-protected conditions, and the mixture was thoroughly mixed and incubated at room temperature for 15 min. The fluorescence value of the algal solution in the black opaque 96-well plate (excitation wavelength 480 nm, emission wavelength 510 nm) was detected using a multi-mode microplate reader, and the OD value of the algal solution in the clear 96-well plate was also detected. 750nm Fluorescence intensity = fluorescence value / OD 750nm Fluorescence intensity can characterize the content of neutral lipids in algal cells.

[0149] Figure 8 The BODIPY staining results at mid-log phase for CC-5325, the CrABCG3 overexpressing algal strain oe3, and the CrABCG5 overexpressing algal strain oe5 can characterize the neutral lipid content within algal cells. Figure 8 It can be seen that the neutral lipid content of OE3 and OE5 is 0.2 times and 0.1 times higher than that of CC-5325, respectively.

[0150] Figure 9 The BODIPY staining results for CC-5325, CrABCG3 overexpressing algal strain oe3, and CrABCG5 overexpressing algal strain oe5 after 48 h of nitrogen-limited culture can characterize the neutral lipid content in algal cells. Figure 9It can be seen that after nitrogen restriction for 48 hours, the neutral lipid content of OE3 and OE5 was 0.1 times and 0.2 times higher than that of CC-5325, respectively.

[0151] Example 4

[0152] Changes in lipid content of overexpressing algal strains under nitrogen-limited conditions

[0153] 1. Nitrogen-limited culture method

[0154] Algal culture was inoculated into TAP and incubated in a light incubator (25℃, 120 rpm, 100 μE·m). -2 ·s -1 After culturing to the logarithmic growth phase, collect algal cells by centrifugation. Wash the algal cells twice with TAP-N medium, centrifuge to discard the waste liquid, then resuspend the cells in TAP-N and inoculate them into Erlenmeyer flasks containing 150 mL of TAP-N, allowing the algal solution to rise from 2 × 10⁻⁶ cells / day. 6 The cells / mL were cultured and placed in a continuous light incubator for 48 hours. Samples were then taken for BODIPY analysis to determine the total lipid content and fatty acid content.

[0155] 2. BODIPY staining

[0156] Collect 2 mL of algal culture after 48 h of nitrogen-limited culture into a 2 mL centrifuge tube, centrifuge at 10000 rpm for 1 min to collect algal cells, wash twice with 1×PBS, and dilute the algal cells to a cell density of 2×10⁻⁶. 6 Cells / mL, 200 μL were taken into black opaque 96-well plates and clear 96-well plates, respectively. For the black opaque 96-well plates, 2.6 μL of LODIPY505 / 515 solution (10 μg / mL) was added under light-protected conditions, mixed thoroughly by pipetting, and incubated at room temperature for 15 min. The fluorescence value of the algal solution in the black opaque 96-well plates was detected using a multi-mode microplate reader (excitation wavelength 480 nm, emission wavelength 510 nm), and the OD value of the algal solution in the clear 96-well plates was also detected. 750nm Fluorescence intensity = fluorescence value / OD 750nm Fluorescence intensity can characterize the content of neutral lipids in algal cells.

[0157] Figure 10 The content of lipids that can be extracted from each gram of freeze-dried algal powder after 48 hours of nitrogen-limited culture for CC-5325, CrABCG3 overexpression algal strain oe3 and CrABCG5 overexpression algal strain oe5.

[0158] Figure 10 This indicates that both CrABCG3 overexpression and CrABCG5 overexpression can increase TAG accumulation in Chlamydomonas under nitrogen-limited conditions.

[0159] 3. Total lipid extraction rate

[0160] (1) Collect algal cells by centrifugation at 8000 rpm for 5 min, freeze them quickly with liquid nitrogen and then put them into a vacuum freeze dryer to remove cell moisture to obtain algal powder. According to the method of Supakorn et al., extract the total lipids in the algal cells.

[0161] (2) Weigh 10 mg of algae powder into a 10 mL digestion tube and add 4 mL of chloroform-methanol solution (2:1, v / v).

[0162] (3) Vortex for 5 min, add 1 mL of 0.9% sodium chloride solution (w / v), and vortex again for 5 min.

[0163] (4) After centrifugation at 1000 rpm for 5 min, the bottom organic phase liquid was collected into a 5 mL glass tube, dried under nitrogen, and weighed. The total lipid extraction rate was the dry weight of crude lipids that could be extracted per gram of algal powder, while CrABCG3 overexpression and CrABCG5 overexpression had no significant effect on the total lipid content.

[0164] 4. Fatty acid content detection

[0165] (1) Weigh 10 mg of lyophilized algae powder into a 10 mL digestion tube and add 50 μL of methyl nonadecanoate internal standard (500 μg / mL).

[0166] (2) Add 1 mL of sodium hydroxide-methanol solution (2 mol / L), and then incubate on a shaker (110 rpm, 1 h).

[0167] (3) Incubate in a 75°C water bath for 15 minutes, gently shaking the digestion tube every 5 minutes to mix the reaction solution. After incubation, cool to room temperature.

[0168] (4) Add 1 mL of hydrochloric acid-methanol solution (4 mol / L), then add 500 μL of concentrated hydrochloric acid to lower the pH of the reaction solution, and then bathe in a 75°C water bath for 15 min. During this period, gently shake the digestion tube every 5 min to mix the reaction solution. After the incubation is completed, the methyl esterification reaction of fatty acids is completed. The reaction solution is placed at room temperature to cool.

[0169] (5) Add 1 mL of n-hexane, vortex for 30 s to extract fatty acid methyl esters from the reaction solution, centrifuge at 1000 rpm for 5 min, then use a 1 mL syringe to aspirate the upper organic phase and pump it into a 5 mL glass tube through an organic phase filter.

[0170] (6) After drying the sample with nitrogen, the total fatty acid methyl esters extracted are obtained. The extract is resuspended in 500 μL of dichloromethane, transferred to a chromatographic vial, and then analyzed by the instrument.

[0171] (7) The extracted fatty acid methyl ester samples were detected using gas chromatography-mass spectrometry (GC-MS). The GC column was an Agilent 19091S-433HP-5MS (30m × 250μmol / L × 0.25μmol / L). The injection volume was 1μL, with helium as the carrier gas, a split ratio of 1:10, a purge flow rate of 1.5mL / min, and an injection port temperature of 250℃. The column oven temperature program was as follows: 80℃ for 2 min, increased to 195℃ at 25℃ / min, increased to 205℃ at 3℃ / min, increased to 250℃ at 7℃ / min and held for 6 min, followed by a run at 250℃ for 2 min. The mass spectrometry detection mode was full scan mode, with a scan range of 50-650 m / z. In the chromatographic results, the residence time of each fatty acid methyl ester peak was referenced to the residence time of 37 fatty acid methyl ester standards, and integral analysis was performed using Agilent MSDChemstation software.

[0172] Figure 11 The content of various fatty acids and total fatty acid content per gram of algal powder for CC-5325, CrABCG3 overexpression algal strain oe3 and CrABCG5 overexpression algal strain oe5 after 48 h of nitrogen-limited culture.

[0173] Figure 11 The results showed that the C16:4, C16:2, C16:0, C18:3, C18:2, C18:1, and C18:0 fatty acids of CrABCG3-overexpressing algal strain oe3 and CrABCG5-overexpressing algal strain oe5 were all higher than those of CC-5325, and the total fatty acid content increased by 0.19-fold and 0.28-fold, respectively, reaching 152.625 mg / g and 163.953 mg / g. Therefore, overexpression of CrABCG3 or CrABCG5 can enhance fatty acid accumulation in Chlamydomonas reinhardtii under nitrogen-limited conditions.

[0174] In summary, this invention constructs a CrABCG overexpression vector to explore its role in lipid metabolism and lays the foundation for further investigation of the transport function of CrABCG.

[0175] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A gene encoding an ABC transporter from Chlamydomonas reinhardtii. CrABCG Its application in improving lipid metabolism in Chlamydomonas reinhardtii is characterized by... The Chlamydomonas reinhardtii ABC transporter gene is: CrABCG3 or CrABCG5 ; The improvement of lipid metabolism in *Chlamydomonas reinhardtii* is achieved by overexpressing the *Chlamydomonas reinhardtii* ABC transporter gene. CrABCG3 or CrABCG5 accomplish; The gene CrABCG3 The nucleotide sequence is as shown in SEQ ID NO:1; The gene CrABCG5 The nucleotide sequence is as shown in SEQ ID NO:

2.

2. The application of a Chlamydomonas reinhardtii ABC transporter CrABCG in improving lipid metabolism in Chlamydomonas reinhardtii, characterized in that, The Chlamydomonas reinhardtii ABC transporter protein is either CrABCG3 or CrABCG5. The amino acid sequence of the transporter protein CrABCG3 is as shown in SEQ ID NO:3; The amino acid sequence of the transporter protein CrABCG5 is as shown in SEQ ID NO:

4. The improvement in lipid metabolism in Chlamydomonas reinhardtii is achieved by overexpressing the gene encoding the Chlamydomonas reinhardtii ABC transporter CrABCG3 or CrABCG5.

3. The application of the gene encoding the Chlamydomonas reinhardtii ABC transporter CrABCG according to claim 1 in improving lipid metabolism in Chlamydomonas reinhardtii, characterized in that, The overexpression method includes: transexpressing the Chlamydomonas reinhardtii ABC transporter gene. CrABCG3 or CrABCG5 The nucleotide sequence was linked to the expression vector backbone to construct an overexpression vector. The overexpression vector was linearized and transformed into Chlamydomonas reinhardtii to obtain an engineered algal strain.

4. The application of the gene encoding the Chlamydomonas reinhardtii ABC transporter CrABCG according to claim 3 in improving lipid metabolism in Chlamydomonas reinhardtii, characterized in that, The linearized overexpression vector was transformed into Chlamydomonas reinhardtii via glass bead method or electroporation method.

5. The application of the gene encoding the Chlamydomonas reinhardtii ABC transporter CrABCG according to claim 4 in improving lipid metabolism in Chlamydomonas reinhardtii, characterized in that, The engineered algal strain was Chlamydomonas reinhardtii as the starting strain. The originating strain was Chlamydomonas reinhardtii CC-5325.

6. A method for cultivating algal strains with higher neutral lipid content and total fatty acid content than wild-type algae, characterized in that, The method includes: constructing a gene containing the Chlamydomonas reinhardtii ABC transporter protein as described in claim 1. CrABCG3 or CrABCG5 The overexpression vector was used to transform Chlamydomonas reinhardtii, resulting in algal strains with higher neutral lipid and total fatty acid contents than the wild type.

7. The application of an overexpression vector in enhancing lipid metabolism in Chlamydomonas reinhardtii, characterized in that, The overexpression vector contains the Chlamydomonas reinhardtii ABC transporter gene as described in claim 1. CrABCG3 and / or CrABCG5 .

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