Chlamydomonas reinhardtii ABC transporter protein gene CrABCG as well as encoding protein and application thereof

Abstract

The invention provides a chlamydomonas reinhardtii ABC transporter protein gene CrABCG as well as an encoding protein and application thereof. The chlamydomonas reinhardtii ABC transporter protein gene comprises CrABCG3 or CrABCG5, and the encoding protein of the chlamydomonas reinhardtii ABC transporter protein gene CrABCG is an ABC transporter protein The gene sequence is shown as SEQ ID NO: 1 or SEQ ID NO: 2. The invention finds that the CrABCG protein is related to carbon and nitrogen metabolism of chlamydomonas reinhardtii, preliminary research shows that the expression of the CrABCG3 gene of the chlamydomonas reinhardtii is improved after nitrogen limitation, the expression of the CrABCG5 gene is reduced and then improved, and the result shows that the CrABCG protein affects the transport, synthesis and accumulation of lipid substances. Therefore, an engineering algal strain with high lipid content can be obtained by screening and transforming the algal strain with high expression of the CrABCG gene.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and in particular relates to a Chlamydomonas reinhardtii ABC transporter gene CrABCG and a coded protein and application thereof. Background Art

[0002] Biofuels are renewable and sustainable alternative energy sources and are promising candidates to replace fossil fuels. Among various alternative energy sources, microalgae are considered to be potential materials for biofuel production due to their higher photosynthetic activity and biomass productivity. As a haploid organism, Chlamydomonas reinhardtii is easy to observe the loss-of-function phenotype caused by gene mutation. Among the transgenic microalgae with high oil content that have been constructed, transgenic Chlamydomonas strains account for about 27%, and have become a model microalgae that has attracted much attention in recent years to study the mechanism of lipid accumulation.

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

[0004] The ABC protein superfamily is widely present in microorganisms, animals and plants, and is one of the oldest and largest protein families known to date. It can use the energy released by hydrolyzing adenosine triphosphate (ATP) to transport heavy metal ions, fatty acids, plant hormones and other substrates across the membrane, thereby participating in detoxification, preventing pathogen invasion and growth and development.

[0005] The ABC transporter family is large, with a wide range of existence and a large number. In the plant kingdom, the ABC family is divided into 8 subfamilies: ABCA, ABCB, ABCC, ABCD, ABCE, ABCF, ABCG, and ABCH. Arabidopsis AtABCA9 promotes the transport of fatty acids in the endoplasmic reticulum during seed development, while in Chlamydomonas reinhardtii, CrABCA2, a protein homologous to AtABCA9, the CrABCA2 knockout strain accumulates less triglycerides, and the CrABCA2 overexpression strain has a higher triglyceride content. Arabidopsis AtABCG9 and AtABCG31 are involved in the transport of precursors for the synthesis of sporopollenin in the exine of pollen grains, and suberin monomers are transported by AtABCG1. Therefore, ABCG proteins in Arabidopsis are related to the transport of lipid substances. The structures and functions of each subfamily of plant ABC transporters are completely different and cannot be predicted by structure. There is no report on whether Chlamydomonas reinhardtii, which has the ABC transporter gene CrABCG, regulates lipid metabolism.

[0006] The anabolism of lipid substances occurs in the cell's plastid matrix and endoplasmic reticulum, and transport proteins play an important role in enabling substances to move between different organelles. However, there is currently insufficient research on Chlamydomonas reinhardtii lipid transport proteins.

[0007] ABCG proteins are widely present in lower and higher organisms and are relatively conservative in evolution. As transport proteins, they have been partially shown to be related to the transport of lipid substances in Arabidopsis, but there are few reports on Chlamydomonas reinhardtii. In-depth exploration of the role of ABCG proteins in Chlamydomonas reinhardtii is of great significance for the development of biofuels. Summary of the invention

[0008] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a Chlamydomonas reinhardtii ABC transporter gene CrABCG and its encoded protein and application, especially in regulating algae lipid metabolism. The present invention constructs an overexpression vector of the transporter gene CrABCG to explore its role in lipid metabolism and lays a foundation for further exploring the transport function of the CrABCG protein.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

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

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

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

[0013] or (B) a nucleotide sequence having 95% or more homology with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein with the same function;

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

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

[0016] or (2) a nucleotide sequence that has 95% or more 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 comprises: CrABCG3 or CrABCG5;

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

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

[0020] (b) an amino acid sequence derived from (a) with the amino acid sequence shown in SEQ ID NO: 3 being substituted, deleted, inserted or added and having the same activity as the amino acid sequence shown in SEQ ID NO: 3;

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

[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, after substitution, deletion, insertion or addition.

[0024] The present invention discovered a CrABCG protein and its gene homologous to Arabidopsis in Chlamydomonas reinhardtii. The study found that the CrABCG protein may be related to the carbon and nitrogen metabolism of Chlamydomonas reinhardtii. Preliminary studies have shown that after nitrogen limitation, the expression of the CrABCG3 gene of Chlamydomonas reinhardtii will increase, while the expression of the CrABCG5 gene will first decrease and then increase. The study shows that the CrABCG protein affects the transport, synthesis and accumulation of lipid substances. Therefore, an engineered algae strain with high lipid content can be obtained by screening and modifying an algae strain with high expression of the CrABCG gene.

[0025] In a third aspect, the present invention provides use 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 the present invention, the Chlamydomonas reinhardtii ABC transporter gene CrABCG and its protein participate in regulating lipid metabolism in the following two aspects: increasing the neutral lipid content of the algae strain and increasing the total fatty acid content of the algae strain.

[0027] In a fourth aspect, the present invention provides an engineered algae strain, wherein the engineered algae strain is obtained by using Chlamydomonas reinhardtii as a starting strain and overexpressing the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 or CrABCG5.

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

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

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

[0031] In a fifth aspect, the present invention provides an algae cultivation method, comprising: constructing an expression vector containing the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 or CrABCG5, and transferring the expression vector into Chlamydomonas reinhardtii to obtain an algae strain having a neutral lipid content and a total fatty acid content higher than that of the wild type.

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

[0033] (1) The pPHRB vector was double-digested with Eco RI and Eco R72 Ⅰ to produce a vector backbone, and the CrABCG3 gene was connected to 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 neutral lipid content higher than that of the wild type.

[0034] (2) The pPHRB vector was double-digested with Eco RI and Eco R72 Ⅰ to produce a vector backbone, and the CrABCG5 gene was connected to the expression vector backbone to obtain an overexpression vector of the CrABCG5 gene. The vector was then transferred into Chlamydomonas reinhardtii to obtain an algal strain with a neutral lipid content higher than that of the wild type.

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

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

[0037] In an eighth aspect, 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 include those shown in SEQ ID NO:18 and SEQ ID NO:19; the upstream and downstream primers for amplifying CrABCG5 include those 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] The present invention obtains a Chlamydomonas reinhardtii strain overexpressing CrABCG3 or CrABCG5 by constructing a vector overexpressing the gene CrABCG3 and / or CrABCG5. The CrABCG-overexpressing strain significantly increases the content of neutral lipids and total fatty acids, laying a good theoretical and application foundation for cultivating microalgae varieties with high oil content. In addition, the present invention finds that the overexpressing strain can more effectively increase lipid accumulation under nitrogen-limited conditions. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

[0043] Figure 4 This is the colony PCR result after constructing the CrABCG3 overexpression vector.

[0044] Figure 5 This is the colony PCR result after constructing the CrABCG5 overexpression vector.

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

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

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

[0048] Fig. 9These are the BODIPY staining results of CC-5325, CrABCG3-overexpressing algal strain oe3, and CrABCG5-overexpressing algal strain oe5 after 48 h of nitrogen-limited culture.

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

[0050] Fig.11 The contents of various fatty acids per gram of algal powder and the total fatty acid content of CC-5325, CrABCG3-overexpressing algal strain oe3 and CrABCG5-overexpressing algal strain oe5 after 48 hours of nitrogen-limited culture. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0052] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular 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 Beijerinck'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 Beijerinck's solution-N (100×), 1 mL / L trace element solution (1000×).

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

[0057] Tris-Acetate (Tris(hydroxymethyl)aminomethane acetate) (100×): Tris(hydroxymethyl)aminomethane 242 g / L, glacial acetic acid 100 mL.

[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 ABCG proteins in Chlamydomonas reinhardtii

[0064] The amino acid sequences of 43 ABCG proteins in Arabidopsis were downloaded from the TAIR database (https: / / www.Arabidopsis.org / ), and the Arabidopsis ABCG11 protein (AT1G17840) was used 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 / ), and 5 predicted homologous proteins were obtained. The evolutionary relationship of the five CrABCG proteins was analyzed with ABCG proteins of other microalgae, plants and other species to construct an evolutionary tree.

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

[0066] The evolutionary relationship between the five CrABCG proteins and the ABCG proteins of other microalgae and plants was analyzed, and the evolutionary tree constructed was as follows: Figure 1 As shown in the figure, it can be seen that CrABCG3 has a close evolutionary relationship with Arabidopsis AtABCG11, AtABCG12 and AtABCG13 and rice OsABCG11, while CrABCG5 is located in the same evolutionary branch with Arabidopsis AtABCG9, AtABCG14, AtABCG26 and other proteins.

[0067] Example 2

[0068] Obtaining CrABCG3 and CrABCG5 genes and constructing their 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, we need to obtain their gene sequences in Chlamydomonas reinhardtii. The relevant mRNA transcribed by Chlamydomonas reinhardtii itself can serve as the target gene CDS. The total RNA of Chlamydomonas reinhardtii CC-5325 cultured to the logarithmic phase was extracted using the RNA extraction kit of Shanghai Feijie, and then its cDNA was reverse transcribed for transgenic use.

[0071] (2) Take 2 mL of algae solution and place it in a 2 mL centrifuge tube without RNase. Be careful not to spill the liquid out of the tube. Centrifuge at 10,000 rpm for 1 min and discard the supernatant. The 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 much or too little. Too much will cause RNA degradation, and too little will prevent the precipitate from being completely dissolved.

[0073] (4) Place the adsorption column in a 2 mL collection tube, aspirate the liquid in the centrifuge tube, add it to the adsorption column, and centrifuge at 12,000 rpm for 1 min. The liquid in the collection tube is waste liquid and should be discarded.

[0074] (5) Add 500 μL of washing buffer and centrifuge at 12,000 rpm for 1 min. Do not discard the waste liquid. Repeat this process.

[0075] (6) Centrifuge at 12000 rpm for 1 min to fully remove the liquid in the adsorption column.

[0076] (7) Place the adsorption column in a sterilized 1.5 mL centrifuge tube, add 50 μL of RNase-free water, let stand for 1 min, and then centrifuge at 12,000 rpm for 1 min. Collect the RNA into the centrifuge tube.

[0077] (8) Nanodrop2000 was used to detect RNA concentration and purity (A260 / A280, A260 / A230), and RNA integrity was detected using 1% non-denaturing agarose electrophoresis.

[0078] 2. RNA Reverse Transcription

[0079] The reverse transcription reaction was performed using the All-Gold AE311 reverse transcription kit according to the instructions. The reaction procedure was 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 target gene

[0084] According to 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. The cDNA obtained in the previous step was used as a template, and the primers were used for amplification according to the system in the table below. The reaction program was 98℃ for 3min; 98℃ for 10s, 61℃ for 10s, 72℃ for 2min for 35 cycles; 72℃ for 5min; and finally kept at 4℃. After the reaction program was completed, the amplified product of the corresponding fragment was obtained. Table 2 is the PCR reaction system.

[0085] Table 2

[0086] Reagents Addition amount KOD One PCR Reaction Mix 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 excision and recovery of PCR products

[0088] After the target gene is amplified in the previous step, the product needs to be verified by agarose gel electrophoresis. Prepare 1.5% agarose gel to electrophoress all PCR products, and use the full gold electrophoresis gel recovery kit to recover the PCR product.

[0089] (1) After electrophoresis, clear bands can be seen under ultraviolet light. Use a knife to quickly cut off the band that matches the size of the target fragment. This is the gel containing the desired DNA fragment. Put it into an empty centrifuge tube and then cut the gel into pieces as much as possible. Use a balance to weigh the empty centrifuge tube in advance, and then weigh the centrifuge tube containing the gel. The weight of the gel is obtained by subtracting the weight of the former from the latter. In the experiment, 100 mg of gel is equivalent to 100 μL of gel.

[0090] (2) Add a volume of GDP buffer equal to the volume of gel. Place the gel in a 50-55°C metal bath for 10 min to completely dissolve the gel. During this time, the centrifuge tube can be turned upside down to allow the gel to dissolve more fully.

[0091] (3) Place the adsorption column on the collection tube, transfer 700 μL of the gel solution to the adsorption column, and centrifuge at 12,000 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 place the adsorption column back into the collection tube.

[0092] (4) Add anhydrous ethanol to buffer GW in advance and then mark it on the label. 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 12000rpm for 2min to fully remove the ethanol. After centrifugation, transfer the adsorption column to a 1.5mL sterilized centrifuge tube, open the lid to allow the ethanol to evaporate for 1min, add 30μL elution buffer, and let it stand for 2min. Centrifuge at 12000rpm for 1min. Collect the DNA solution in the centrifuge tube, measure its concentration using Nanodrop2000, and place it in a -20℃ refrigerator.

[0094] 5. Adding homology arms

[0095] The gel-cutting recovery product of the target gene fragment is used as a template, and new primers are designed according to the cleavage site of the enzyme required for the subsequent plasmid digestion. The new primers are used for PCR reaction to add homology arms to the target gene fragment, so that it can be connected to the plasmid after digestion by seamless cloning. The primers for adding homology arms are Ha3-F and Ha3-R (CrABCG3), Ha5-F and Ha5-R (CrABCG5) (SEQ ID NO: 12-SEQ ID NO: 15). The PCR product is also cut and recovered.

[0096] 6. Overexpression vector construction

[0097] Use the seamless cloning method to connect the linearized overexpression vector backbone and the target gene fragment containing homology arms. First, the vector pPHRB (SEQ ID NO: 5) should be double-digested according to Table 3, and digested at 37°C for 30 minutes to completely digest and produce sticky ends. The PCR product with homology arms added obtained in the previous step should also be double-digested. After the digestion is completed, gel electrophoresis is performed to verify whether the obtained vector is a linearized vector and whether the size meets the requirements. After confirmation, the gel is cut and recovered. According to the DNA concentration of the linearized vector and the target fragment with homology arms added, the two are added to the seamless cloning system (Table 4) in proportion, and the overexpression vector containing the target gene fragment is obtained after reacting at 50°C for 15 minutes.

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

[0099] Table 3

[0100]

[0101] Table 4

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

[0103] 7. E. coli transformation

[0104] (1) Take out the competent cells Top10 from the -80℃ refrigerator and place them on ice. Wait for them to thaw. Add the reaction solution after seamless cloning to the competent cells in the clean bench, mix well, and place on ice for 15 minutes.

[0105] (2) Preheat the water bath to 42°C, heat shock 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 the LB liquid medium in 37°C water, then add 600 μL of preheated LB medium to the competent cells and incubate at 37°C, 180 rpm, for 45 min to revive the cells.

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

[0108] (5) Then, use a sterile pipette tip to randomly pick the bacteria 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, and 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 above. Figure 4 For 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 1869 bp. Figure 5 The results of colony PCR after constructing the CrABCG5 overexpression vector were shown in Figure 1. The primers used were SEQ ID NO: 10 and SEQ ID NO: 11, and the fragment length should be 2142 bp. The strains that were verified as positive by PCR were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0111] 9. Extraction of overexpression vector

[0112] The positive clones were selected and added to LB liquid medium containing ampicillin and cultured at 37°C for 15 hours. Then the plasmid was extracted. The plasmid was extracted using the full-form gold Plasmid MiniPrep Kit, the specific steps are as follows.

[0113] (1) Take 2 mL of bacterial solution and add it to a centrifuge tube. Centrifuge at 10,000 rpm for 1 min. Pour away the waste liquid and try to minimize the amount of liquid left.

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

[0115] (3) Add 250 μL of blue LB and mix by inverting the tube about 6 times to fully lyse the bacteria. Be careful not to be too rough and destroy the plasmid. If the solution is viscous and clear, the bacteria have been fully lysed.

[0116] (4) Add 350 μL of NB and mix gently for 5-6 times. Wait for a yellow agglutinate to form in the centrifuge tube. Let stand for 2 min and then centrifuge at 12,000 rpm for 5 min.

[0117] (5) Place the adsorption column in a 2 mL collection tube. Pipette the supernatant from the previous step and transfer it to the adsorption column using a pipette. Be careful to only take the liquid and not the precipitate. Centrifuge at 12,000 rpm for 1 min. Pour away the waste liquid and place the adsorption column back into the collection tube.

[0118] (6) Add 500 μL WB to the adsorption column. Centrifuge at 12,000 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 minutes, and centrifuge it at 12,000 rpm for 1 minute. The plasmid is collected in the centrifuge tube.

[0121] (9) Store the plasmid in a -20°C freezer 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 overexpression algae strains

[0125] 1. Linearization of overexpression vector

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

[0127] Table 5

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

[0129] 2. Transformation of Chlamydomonas reinhardtii by glass bead method

[0130] (1) The linearized overexpression vector was transferred into Chlamydomonas reinhardtii CC-5325 using the glass bead method. First, the algae was cultured to the logarithmic phase, at which time the cell density was approximately (1-2)×10 6 cells / mL, centrifuge at 5000rpm for 5min, discard the supernatant and keep the algal cells.

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

[0132] (3) Add 20 μL of the linearized vector prepared according to Table 5, and mix well by blowing at the liquid surface with a pipette. Vortex at the maximum speed (2500 rpm) for 29 seconds. Add 10 mL of fresh TAP medium to a 50 mL centrifuge tube, then pipette the shaken algae solution into it, and culture overnight at 120 rpm in the dark.

[0133] (4) After culturing for 20-22 hours, take out the 50 mL centrifuge tube, centrifuge at 3000 rpm for 5 minutes, 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 culture them upside down. After two weeks of culture, transformants will grow on the plate.

[0134] 3. Screening of overexpression algae strains

[0135] (1) DNA verification

[0136] Add 50 μL of 5% CHELEX solution (w / v) to a 0.2 mL centrifuge tube, pick up algae with a sterilized pipette tip in a clean bench, add to the CHELEX solution, and vortex for 10 seconds. Incubate at 98°C for 20 minutes and cool on ice for 1 minute. Vortex for 10 seconds and centrifuge at 2200 rpm for 5 minutes. The supernatant is the extract containing genomic DNA. Use the two pairs of primers CrABCG3-F and CrABCG3-R, CrABCG5-F and CrABCG5-R (SEQ ID NO: 8-SEQ ID NO: 11) to perform PCR reaction on the extracted genomic DNA to verify whether the transformant contains the overexpression vector.

[0137] (2) QPCR verification

[0138] Take 2 mL of mid-log phase (4×10 6 cells / mL) algae solution was placed in a 2 mL centrifuge tube without RNase, and the algae cells were collected by centrifugation at 10,000 rpm for 1 min. RNA was extracted using an RNA extraction kit (Shanghai Feijie Biotechnology). The extracted RNA was reverse transcribed using an Acre Reverse Transcription Kit to obtain cDNA.

[0139] RNA reverse transcription The extracted RNA was reverse transcribed using the Acryl reverse transcription kit, as shown in Table 6.

[0140] Table 6

[0141]

[0142]

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

[0144] Dilute cDNA ten-fold with RNase-free water for later use. RACK1 was used as the internal reference gene, and the reaction system was prepared using the Acryl real-time fluorescence quantitative PCR (qRT-PCR) kit. The reaction procedure was 95°C for 10 min; 95°C for 15 s, 60°C for 1 min, and 40 cycles; the melting curve was 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. The primers used to detect the internal reference gene were qRACK-F and qRACK-R (SEQ ID NO: 16 and SEQ ID NO: 17), the primers used to detect CrABCG3 were q3-F and q3-R (SEQ ID NO: 18 and SEQ ID NO: 19), and the primers used to detect CrABCG5 were q5-F and q5-R (SEQ ID NO: 20 and SEQ ID NO: 21). After the reaction, according to 2 -△△CtThe expression level of the target gene in the transformant was relatively quantified by the method. Thus, the CrABCG3 overexpression algae strain oe3 and the CrABCG5 overexpression algae strain oe5 were obtained.

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

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

[0147] (3) BODIPY screening

[0148] Take 2 mL of mid-log phase (4×10 6 cells / mL) algae solution in a 2 mL centrifuge tube, centrifuge at 10000 rpm for 1 min to collect algae cells, and wash them twice with 1× PBS. Dilute the algae cells to a cell density of 2×10 6 cells / mL, take 200μL into a black opaque 96-well plate and a transparent 96-well plate respectively, add 2.6μL BODIPY505 / 515 solution (10μg / mL, dissolved in DMSO) to the black opaque 96-well plate under light-proof conditions, mix well by pipetting, and incubate at room temperature for 15min. Use a multifunctional microplate reader to detect the fluorescence value of the algae solution in the black opaque 96-well plate (excitation wavelength is 480nm, emission wavelength is 510nm), and detect the OD of the algae solution in the transparent 96-well plate 750nm , fluorescence intensity = fluorescence value / OD 750nm The fluorescence intensity can characterize the neutral lipid content in algal cells.

[0149] Figure 8 The BODIPY staining results of CC-5325, CrABCG3 overexpression strain oe3 and CrABCG5 overexpression strain oe5 at mid-logarithmic phase can characterize the neutral lipid content in 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] Fig. 9 The results of BODIPY staining of CC-5325, CrABCG3 overexpressing strain oe3 and CrABCG5 overexpressing strain oe5 after 48h of nitrogen-limited culture can characterize the neutral lipid content in algal cells. Fig. 9It can be seen that after 48 hours of nitrogen limitation, the neutral lipid contents of oe3 and oe5 were 0.1 times and 0.2 times higher than that of CC-5325, respectively.

[0151] Example 4

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

[0153] 1. Nitrogen-limited culture method

[0154] The algae solution was inoculated into TAP and incubated in a light incubator (25°C, 120 rpm, 100 μE·m -2 ·s -1 ) was cultured to the logarithmic phase and the algal cells were collected by centrifugation. The algal cells were washed twice with TAP-N medium, the waste liquid was discarded by centrifugation, and then the cells were resuspended with TAP-N and inoculated into a conical flask containing 150 mL of TAP-N to increase the algal liquid from 2×10 6 cells / mL and culture was started in a continuous light incubator for 48 h before sampling for BODIPY detection, total lipid extraction and fatty acid content determination.

[0155] 2. BODIPY staining

[0156] Take 2 mL of algae solution after 48 h of nitrogen-limited culture in a 2 mL centrifuge tube, centrifuge at 10000 rpm for 1 min to collect algae cells, and wash them twice with 1× PBS to dilute the algae cells to a cell density of 2×10 6 cells / mL, take 200μL into a black opaque 96-well plate and a transparent 96-well plate respectively, add 2.6μL BODIPY505 / 515 solution (10μg / mL) to the black opaque 96-well plate under light-proof conditions, mix well by pipetting, and incubate at room temperature for 15min. Use a multifunctional microplate reader to detect the fluorescence value of the algae solution in the black opaque 96-well plate (excitation wavelength is 480nm, emission wavelength is 510nm), and detect the OD of the algae solution in the transparent 96-well plate 750nm , fluorescence intensity = fluorescence value / OD 750nm The fluorescence intensity can characterize the neutral lipid content in algal cells.

[0157] Fig.10 The oil content that can be extracted from each gram of freeze-dried algal powder of CC-5325, CrABCG3-overexpressing algal strain oe3 and CrABCG5-overexpressing algal strain oe5 when cultured in nitrogen-limited culture for 48 hours.

[0158] Fig.10 These results indicate that overexpression of CrABCG3 and CrABCG5 can both increase TAG accumulation in Chlamydomonas when nitrogen is limited.

[0159] 3. Total fat extraction rate

[0160] (1) Algal cells were collected by centrifugation at 8000 rpm for 5 min, quickly frozen with liquid nitrogen, and placed in a vacuum freeze dryer to remove cell moisture to obtain algal powder. The total lipids in the algal cells were extracted according to the method of Supakorn et al.

[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 organic phase liquid at the bottom was aspirated into a 5 mL glass tube, dried with nitrogen, and weighed. The total lipid extraction rate is the dry weight of crude lipid that can be extracted per gram of algal powder, and overexpression of CrABCG3 and CrABCG5 had no significant effect on the total lipid content.

[0164] 4. Fatty acid content detection

[0165] (1) Weigh 10 mg of freeze-dried 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 incubate on a shaker (110 rpm, 1 h).

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

[0168] (4) Add 1 mL of hydrochloric acid-methanol solution (4 mol / L), and then add 500 μL of concentrated hydrochloric acid to lower the pH value of the reaction solution. Then, place the solution 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 the fatty acids is completed, and the reaction solution is cooled at room temperature.

[0169] (5) Add 1 mL of n-hexane and vortex for 30 seconds to extract fatty acid methyl esters in the reaction solution. Centrifuge at 1000 rpm for 5 minutes, then draw out the upper organic phase with a 1 mL syringe and pump it into a 5 mL glass tube through an organic phase filter.

[0170] (6) After drying the sample with nitrogen, the extracted total fatty acid methyl ester sample was obtained. 500 μL of dichloromethane was added to resuspend the extract, and the extract was transferred to a chromatographic bottle and tested on a chromatography machine.

[0171] (7) The extracted fatty acid methyl ester samples were detected by gas chromatography-mass spectrometry, and the gas chromatography column was Agilent 19091S-433HP-5MS (30m×250μmol / L×0.25μmol / L). The injection volume was 1μL, helium was used as the carrier gas, the split ratio was 1:10, the purge flow rate was 1.5mL / min, and the injection port temperature was 250°C. The column box temperature program was: 80°C for 2min, increased to 195°C at 25°C / min, increased to 205°C at 3°C / min, increased to 250°C at 7°C / min, and maintained for 6min, the post-operation temperature was 250°C, and the post-operation time was 2min. The mass spectrometry detection mode was full scan mode, and the scanning range was 50-650m / z. In the chromatographic results, the retention time of each fatty acid methyl ester peak was referenced to the retention time of 37 fatty acid methyl ester standards, and Agilent MSD Chemstation software was used for integration analysis.

[0172] Fig.11 The contents of various fatty acids per gram of algal powder and the total fatty acid content of CC-5325, CrABCG3-overexpressing algal strain oe3 and CrABCG5-overexpressing algal strain oe5 after 48 hours of nitrogen-limited culture.

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

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

[0175] The applicant declares that the above is only a specific implementation mode 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 thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A Chlamydomonas reinhardtii ABC transporter gene CrABCG, characterized in that: The Chlamydomonas reinhardtii ABC transporter gene includes: CrABCG3 or CrABCG5; The nucleotide sequence of the gene CrABCG3 comprises: (A) the nucleotide sequence shown in SEQ ID NO: 1; or (B) a nucleotide sequence having 95% or more homology with the nucleotide sequence shown in SEQ ID NO: 1 and encoding a protein with the same function; The nucleotide sequence of the gene CrABCG5 comprises: (1) the nucleotide sequence shown in SEQ ID NO: 2; or (2) a nucleotide sequence that has 95% or more homology with the nucleotide sequence shown in SEQ ID NO: 2 and encodes a protein with the same function.

2. A Chlamydomonas reinhardtii ABC transporter CrABCG, characterized in that The Chlamydomonas reinhardtii ABC transporter protein includes: CrABCG3 or CrABCG5; The amino acid sequence of the transporter CrABCG3 comprises: (a) the amino acid sequence shown in SEQ ID NO: 3; (b) an amino acid sequence derived from (a) with the amino acid sequence shown in SEQ ID NO: 3 being substituted, deleted, inserted or added and having the same activity as the amino acid sequence shown in SEQ ID NO: 3; The amino acid sequence of the transporter CrABCG5 comprises: (I) the amino acid sequence shown in SEQ ID NO:4; (II) An amino acid sequence derived from (I) having the same activity as the amino acid sequence shown in SEQ ID NO: 4, wherein the amino acid sequence shown in SEQ ID NO: 4 is substituted, deleted, inserted or added.

3. Use of the Chlamydomonas reinhardtii ABC transporter gene CrABCG according to claim 1 and / or the Chlamydomonas reinhardtii ABC transporter CrABCG according to claim 2 in regulating algae lipid metabolism.

4. An engineered algae strain, characterized in that: The engineered algae strain is obtained by taking Chlamydomonas reinhardtii as a starting strain and overexpressing the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 or CrABCG5.

5. The engineered algae strain according to claim 4, characterized in that: The overexpression method includes: connecting the nucleotide sequence of the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 or CrABCG5 with the expression vector skeleton, constructing the overexpression vector, linearizing the overexpression vector, and transforming it into Chlamydomonas reinhardtii to obtain the engineered algae strain.

6. The engineered algae strain according to claim 4 or 5, characterized in that: The linearized overexpression vector is transformed into Chlamydomonas reinhardtii by glass bead method or electroporation method; Preferably, the starting strain is Chlamydomonas reinhardtii CC-5325.

7. A method for growing algae, characterized in that: The method comprises: constructing an expression vector containing the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 or CrABCG5, and transferring the expression vector into Chlamydomonas reinhardtii to obtain an algae strain with a higher neutral lipid content and a higher total fatty acid content than a wild type.

8. An expression vector, characterized in that The expression vector comprises the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 and / or CrABCG5 according to claim 1.

9. A host cell, characterized in that The host cell comprises the Chlamydomonas reinhardtii ABC transporter gene CrABCG3 and / or CrABCG5 according to claim 1; or the host cell comprises the expression vector according to claim 8.

10. A primer for amplifying Chlamydomonas reinhardtii ABC transporter gene CrABCG3 or CrABCG5, characterized in that: The upstream and downstream primers for amplifying CrABCG3 include those shown in SEQ ID NO:18 and SEQ ID NO:19; the upstream and downstream primers for amplifying CrABCG5 include those shown in SEQ ID NO:20 and SEQ ID NO:21.

Images