Cre-FTO gene for improving carbon sequestration efficiency of chlamydomonas reinhardtii, related biological material and application

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a cre-FTO gene for improving the carbon sequestration efficiency of chlamydomonas reinhardtii, a related biological material and application. The cre-FTO gene disclosed by the invention is an RNA (Ribonucleic Acid) demethylase gene, and the nucleotide sequence of the cre-FTO gene is shown as SEQ ID NO: 1. The recombinant expression vector pDb124-cre-FTO containing the cre-FTO gene is transformed into the chlamydomonas reinhardtii, so that the photosynthetic efficiency, the carbon sequestration efficiency and the biomass of wild type chlamydomonas reinhardtii can be improved. The cre-FTO gene and related biological materials provided by the invention are helpful for improving the photosynthetic efficiency, starch conversion rate, pyruvic acid conversion rate and other carbon sequestration efficiencies of chlamydomonas reinhardtii, and have a more promising application value in improving the photosynthetic efficiency and the carbon sequestration efficiency.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a cre-FTO gene for improving the carbon fixation efficiency of Chlamydomonas reinhardtii, and related biological materials and applications. Background Art

[0002] Microalgae can efficiently fix carbon dioxide through the carbon dioxide concentrating mechanism (CCM), and are excellent organisms for realizing carbon dioxide capture, utilization and storage technology (CCUS). Their growth rate and carbon fixation ability are many times higher than those of terrestrial plants. Compared with plants, Chlamydomonas is easy to culture, has high photosynthetic efficiency, strong adaptability, and can achieve high-efficiency high-density culture. And through photosynthesis, light energy and CO2 are stored in carbohydrates, and high-value products such as fatty acids, pigments, and polysaccharides are produced through its own metabolism.

[0003] In recent years, research has mainly focused on two directions to improve the carbon fixation ability of microalgae. The breeding direction includes algal strain domestication and mutant screening, or the use of genetic engineering means to transform the metabolic pathway of Chlamydomonas. The engineering direction includes improving the culture medium, optimizing the culture conditions, and the transformation and design of photobioreactors. Among them, the use of genetic engineering means to directionally transform Chlamydomonas is one of the most effective ways to improve the carbon fixation ability of algal strains.

[0004] RNA demethylase (FTO) is the first discovered m6A demethylase. It was early considered a gene related to severe obesity in humans and was only found in vertebrates. Some studies have found that m6A modification exists in both Chlamydomonas reinhardtii and animals and plants. The modification mediated by the mammalian m6A RNA demethylase FTO regulates cell growth, proliferation and gluconeogenesis metabolism (involved in regulating the key gluconeogenesis gene G6PC), and affects cell growth and energy metabolism. The introduction of human FTO into monocotyledonous rice and dicotyledonous potato significantly improves photosynthetic efficiency, biomass and yield. However, no FTO homologues have been found in Chlamydomonas reinhardtii, and only the ALKBH5 homologous proteins CrALKBH1B and CrALKBH2B have been found. In addition, Chlamydomonas reinhardtii belongs to green algae. Although it can convert carbon dioxide into biomass through the photosynthesis system, its carbon fixation efficiency is low, and the conversion rate of converting light energy into starch after carbon fixation is about 3%. Therefore, studying how to improve the carbon fixation ability of Chlamydomonas reinhardtii and the technical means to improve the synthesis efficiency of biomass such as starch, organic acids, and lipids has great practical significance for CCUS technology. Summary of the Invention

[0005] In view of the above technical problems, the present invention provides a cre-FTO gene for improving the carbon fixation efficiency of Chlamydomonas reinhardtii, and related biological materials and applications.

[0006] The technical solution of the present invention is as follows:

[0007] The present invention provides a cre-FTO gene for improving the carbon fixation efficiency of Chlamydomonas reinhardtii, and the cre-FTO gene has the nucleotide sequence shown in SEQ ID NO: 1.

[0008] The present invention also provides a biological material related to the cre-FTO gene, and the biological material is any one of A1)-A2):

[0009] A1) A recombinant expression vector containing the cre-FTO gene as claimed in claim 1;

[0010] A2) An expression cassette containing the cre-FTO gene as claimed in claim 1.

[0011] As an implementation manner, the A1) recombinant expression vector is constructed by connecting the cre-FTO gene with the plasmid pDb124.

[0012] The present invention also provides an application of the cre-FTO gene and the biological material related to the cre-FTO gene in a preparation for improving the carbon fixation efficiency or photosynthetic efficiency of Chlamydomonas reinhardtii.

[0013] The present invention also provides a preparation for improving the carbon fixation efficiency of Chlamydomonas reinhardtii, which includes the cre-FTO gene or the biological material related to the cre-FTO gene.

[0014] The present invention also provides an application of the cre-FTO gene or the biological material related to the cre-FTO gene in algae breeding for improving the carbon fixation efficiency or photosynthetic efficiency of Chlamydomonas reinhardtii.

[0015] The present invention also provides a breeding method for improving the carbon fixation efficiency or photosynthetic efficiency of Chlamydomonas reinhardtii. Transfer the cre-FTO gene or the biological material related to the cre-FTO gene into Chlamydomonas reinhardtii, culture it in a culture medium, and screen for Chlamydomonas reinhardtii that can grow on an antibiotic medium or has a high OD 750 absorbance value.

[0016] As an implementation manner, the culture medium is a TAP medium or a TP medium.

[0017] As an implementation manner, CO2 is also introduced into the TAP medium or the TP medium.

[0018] As an implementation manner, the concentration of the introduced CO2 is 1%-10%.

[0019] Compared with the prior art, the beneficial effects of the present invention:

[0020] 1. The RNA demethylase gene (cre-FTO gene) established by the present invention and related biological materials can increase the CO2 concentration in chloroplasts and have the functions of efficiently fixing CO2 and improving photosynthetic efficiency.

[0021] 2. The Chlamydomonas reinhardtii engineering bacteria containing the RNA demethylase gene established by the present invention can fix and convert CO2 into starch and pyruvate, increase the contents of products such as starch and pyruvate, help improve the biomass of Chlamydomonas reinhardtii and the synthesis of its metabolites, and have more promising application value in improving carbon fixation efficiency and the biomass of Chlamydomonas reinhardtii. Brief Description of the Drawings

[0022] Figure 1 It is the map of the pDb124-cre-FTO recombinant expression vector and the information of multiple cloning sites for constructing the Chlamydomonas reinhardtii expression system of the RNA demethylase cre-FTO gene of the present invention;

[0023] Figure 2 It is the growth curve of the Chlamydomonas reinhardtii engineering bacteria containing the recombinant expression vector pDb124-cre-FTO detected by the present invention under different culture conditions. Among them, A is cultured in TAP liquid medium, B is cultured in TAP liquid medium + CO2, C is cultured in TP liquid medium + CO2, WT represents wild-type Chlamydomonas reinhardtii bacteria, and FTO1 represents the Chlamydomonas reinhardtii engineering bacteria containing the recombinant expression vector pDb124-cre-FTO;

[0024] Figure 3 It is the detection of the dry weight, pyruvate content, starch content and glycerol content of the Chlamydomonas reinhardtii engineering bacteria containing the recombinant expression vector pDb124-cre-FTO and wild-type Chlamydomonas reinhardtii bacteria under different culture conditions. Among them, A is the dry weight of the bacteria, B is the pyruvate content, C is the starch content, D is the glycerol content, WT represents wild-type Chlamydomonas reinhardtii bacteria, and FTO1 represents the Chlamydomonas reinhardtii engineering bacteria containing the recombinant expression vector pDb124-cre-FTO. Detailed Embodiments

[0025] The present invention provides a cre-FTO gene for improving the carbon fixation efficiency of Chlamydomonas reinhardtii, and the cre-FTO gene is the nucleotide sequence shown in SEQ ID NO: 1. In the present invention, multiple nucleotide sequences are screened according to the codon preference of the FTO amino acid sequence, and it is found that the nucleotide sequence shown in SEQ ID NO: 1 has the best effect on improving the carbon fixation efficiency of Chlamydomonas reinhardtii, and the nucleotide sequence shown in SEQ ID NO: 1 is named the cre-FTO gene.

[0026] The present invention also provides a biological material related to the cre-FTO gene, and the biological material is any one of A1)-A2):

[0027] A1) A recombinant expression vector containing the cre-FTO gene recited in claim 1. In the present invention, the cre-FTO gene is ligated with a plasmid to construct a recombinant expression vector. In one embodiment, the plasmid is any one of plasmid pPha-TOPO-1, plasmid pChlamy-1, and plasmid pDb124. In one embodiment, the promoter of the recombinant expression vector is any one of the PsaD promoter, the Prbcs2 promoter, and the fcp promoter. In the present invention, the recombinant expression vector is pDb124-cre-FTO.

[0028] In the present invention, the recombinant expression vector pDb124-cre-FTO is linearized and then transformed. In one embodiment, the linearization includes restriction endonuclease digestion, chemical cleavage, and physical breakage; in one embodiment, the restriction endonuclease digestion method uses one or more of the restriction endonucleases Pml I, Nhe I, or NotI. In the present invention, the construction of the recombinant expression vector pDb124-cre-FTO includes: both ends of the cre-FTO gene are double-digested with the restriction endonucleases Pml I and Nhe I, and the pDb124 plasmid is digested with the same restriction endonucleases, and then ligated to obtain the recombinant expression vector pDb124-cre-FTO.

[0029] A2) An expression cassette containing the cre-FTO gene recited in claim 1. In the present invention, the expression cassette contains a specific promoter and a terminator. In one embodiment, the promoter is the PsaD promoter.

[0030] The present invention also provides the application of the cre-FTO gene or the biological material related to the cre-FTO gene in a preparation for improving the carbon fixation efficiency or photosynthetic efficiency of Chlamydomonas reinhardtii.

[0031] The present invention also provides a preparation for improving the carbon fixation efficiency of Chlamydomonas reinhardtii, which includes the cre-FTO gene or a biological material related to the cre-FTO gene.

[0032] The present invention also provides the application of the cre-FTO gene or the biological material related to the cre-FTO gene in algae breeding for improving the carbon fixation efficiency or photosynthetic efficiency of Chlamydomonas reinhardtii.

[0033] The present invention also provides a breeding method for improving the carbon fixation efficiency or photosynthetic efficiency of Chlamydomonas reinhardtii. The cre-FTO gene or related biological materials of the cre-FTO gene are transferred into Chlamydomonas reinhardtii and cultured in a medium, and Chlamydomonas reinhardtii that can grow on an antibiotic medium or has a high OD 750 absorbance value is screened.

[0034] In the present invention, the constructed pDb124-cre-FTO recombinant expression vector is transformed into Chlamydomonas reinhardtii competent cells. As an implementation method, the transformation methods include microinjection, gene gun method, electroporation, bead milling method, CaCl2 method, and PEG method. As an implementation method, the medium is any one of TAP medium, BBM medium, TP medium, MCM medium, SE medium, BG11 medium, and seTA medium. In one implementation method, the medium is TAP medium or TP medium.

[0035] As an implementation method, the transferred Chlamydomonas reinhardtii is cultured on a medium containing antibiotics, and the colonies that can grow on the antibiotic-containing medium are screened, which are Chlamydomonas reinhardtii with high carbon fixation efficiency or photosynthetic efficiency. As an implementation method, the medium is a plate medium; in one implementation method, the antibiotic is any one or several of bleomycin, pingyangmycin, sanglermycin, and doxorubicin. In one implementation method, the antibiotic is bleomycin.

[0036] As an implementation method, the transferred Chlamydomonas reinhardtii is cultured in a liquid medium, and the Chlamydomonas reinhardtii with a high OD 750 absorbance value is screened, which is Chlamydomonas reinhardtii with high carbon fixation efficiency or photosynthetic efficiency. As an implementation method, CO2 is also introduced into the liquid medium; in one implementation method, the concentration of the introduced CO2 is 1%-10%; in one implementation method, the concentration of the introduced CO2 is 3%-8%; in one implementation method, the concentration of the introduced CO2 is 4%-6%.

[0037] To make the objectives, technical solutions, and advantages of the present invention clearer and more understandable, the present invention will be described in detail below with reference to embodiments. However, they should not be construed as limiting the protection scope of the present invention.

[0038] For the materials, reagents, etc. used in the following embodiments, unless otherwise specified, the reagents, consumables, etc. involved in the present invention can be obtained from commercial channels. If the specific usage conditions are not indicated, they are usually carried out under conventional conditions or according to the conditions recommended by the company.

[0039] In the present invention, the TAP medium: 10 mL of Tris-Acetate, 10 mL of phosphate buffer, 10 mL of Beijerinek’s solution, and 1 mL of trace elements.

[0040] Among them, Tris-Acetate (100×): Tris 242 g / L, glacial acetic acid 100 mL / L; phosphate buffer (100×): K2HPO4 (K2HPO4·3H2O) 11.92 g / L (15.5 g / L), KH2PO4 6.05 g / L; Beijerinek’s solution (100×): NH4Cl 40 g / L, MgSO4·7H2O 10 g / L, CaCl2·H2O (CaCl2) 5 g / L (3.8 g / L); trace elements (1000×): H3BO3 11.4 g, MnCl2·4H2O 5.06 g, ZnSO4·7H2O 22 g, FeSO4·7H2O 4.99 g, CoCl2·6H2O 1.61 g, CuSO4·5H2O 1.57 g, (NH4)Mo7O 24 ·4H2O 1.1 g.

[0041] TP medium: Tris 2.42 g, phosphate buffer 10 mL, Beijerinek’s solution 10 mL, trace elements 1 mL.

[0042] Among them, phosphate buffer (100×): K2HPO4 (K2HPO4·3H2O) 11.92 g / L (15.5 g / L), KH2PO4 6.05 g / L; Beijerinek’s solution (100×): NH4Cl 40 g / L, MgSO4·7H2O 10 g / L, CaCl2·H2O (CaCl2) 5 g / L (3.8 g / L); trace elements (1000×): H3BO3 11.4 g, MnCl2·4H2O 5.06 g, ZnSO4·7H2O 22 g, FeSO4·7H2O 4.99 g, CoCl2·6H2O 1.61 g, CuSO4·5H2O 1.57 g, (NH4)Mo7O 24 ·4H2O 1.1 g.

[0043] Starch content detection kit: Reagent 1 is a liquid 50 mL × 1 bottle, Reagent 2 is a liquid 20 mL × 1 bottle, Reagent 3 is a powder 50 mL × 1 bottle, and the standard product is a powder × 1 bottle, purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0044] Pyruvate detection kit Pyruvate Assay Kit, A081-1-1: Reagent 1, Reagent 2, Reagent 3, standard product, purchased from Nanjing Jiancheng Bioengineering Institute.

[0045] Glycerol Assay Kit, F005-2-1: Reagent 1, Reagent 2, Standard, purchased from Nanjing Jiancheng Bioengineering Institute.

[0046] Example 1 Obtaining the cre-FTO gene

[0047] 1. Obtaining the FTO gene through database retrieval

[0048] Using the reported protein sequence of human RNA demethylase (FTO, GeneBank accession number: NP_001073901.1).

[0049] 2. Analysis of the FTO gene

[0050] Using the online analysis of conserved domains (https: / / www.ncbi.nlm.nih.gov / Structure / cdd / wrpsb.cgi?INPUT_TYPE=live&SEQUENCE=NP_001073901.1) to analyze the protein domain of the deduced amino acid sequence of the FTO gene, it was found that the protein contains a catalytic AlkB-like domain (AlkB-like domain) of the FTO protein with 290 amino acids, which conforms to the characteristics of the RNA demethylase gene. It was also found that the protein contains a C-terminal domain (DNA demethylase domain) of the FTO protein with 170 amino acids, which conforms to the characteristics of the RNA demethylase gene.

[0051] 3. Codon optimization of the RNA demethylase gene

[0052] Using genetic engineering techniques, according to the codon preference of the FTO amino acid sequence, the RNA demethylase gene sequence was optimized and designed. Among them, the artificially synthesized cre-FTO gene obtained by codon optimization has the best effect on improving the carbon fixation efficiency of Chlamydomonas reinhardtii. The nucleotide sequence of the cre-FTO gene is shown in SEQ ID NO: 1, and the length of the nucleotide sequence is 1515 bases. The amino acid sequence corresponding to the RNA demethylase expressed by the cre-FTO gene of the present invention is the polypeptide shown in SEQ ID NO: 2.

[0053] Example 2 Obtaining the FTO1 Chlamydomonas reinhardtii engineering bacteria

[0054] 1. Construction of the pDb124-cre-FTO recombinant expression vector

[0055] Restriction enzyme cleavage sites were designed and added to both ends of the codon-optimized synthetic cre-FTO gene in Example 1, namely Pml I and Nhe I. Then, Nanjing Genscript Biotech Co., Ltd. was commissioned for artificial gene synthesis and ligated into plasmid pDb124 at the Pml I and Nhe I sites as Figure 1 shown to construct the pDb124-cre-FTO recombinant expression vector. The plasmid pDb124 used was provided by the Aquatic Biology Laboratory of the College of Life and Marine Sciences, Shenzhen University.

[0056] 2. Transformation and screening of Chlamydomonas reinhardtii engineering bacteria FTO1

[0057] 2.1 Transformation of Chlamydomonas reinhardtii engineering bacteria FTO1

[0058] Chlamydomonas reinhardtii cells CC849 cultured to the logarithmic growth phase were collected by centrifugation at room temperature at 5000 rpm / min, and the cell concentration was adjusted to 1×10 8 cell / mL as competent cells. 500 μL of Chlamydomonas reinhardtii competent cells were pipetted into an EP tube containing 0.3 g of glass beads, and 1.5 μg of the pDb124-cre-FTO recombinant plasmid linearized with NotI was added thereto. The centrifuge tube was placed on a high-speed vortex mixer and vortexed at high speed for 25 sec to complete the transformation. In the present invention, the recombinant vector pDb124-cre-FTO was transformed into Chlamydomonas reinhardtii strain CC849 by the "bead milling method" transformation method. The plasmid map and multiple cloning site information of the pDb124-cre-FTO vector for constructing the Chlamydomonas reinhardtii expression system of the RNA demethylase cre-FTO gene are as Figure 1 shown. As Figure 1 can be seen, the recombinant expression vector pDb124-cre-FTO expressing the RNA demethylase used contains the essential genes crucial for FTO. The Chlamydomonas reinhardtii expression vector also contains the Blemycinresistance gene and has bleomycin resistance. Therefore, the successfully transformed Chlamydomonas reinhardtii engineering bacteria can grow on a plate containing bleomycin resistance.

[0059] 2.2 Screening of Chlamydomonas reinhardtii engineering bacteria FTO1

[0060] In a clean bench, add 10 mL of antibiotic-free TAP liquid culture medium to the centrifuge tube where the Chlamydomonas reinhardtii engineered bacteria were transformed, and resuscitate at 22°C and 110 rpm for 20 hours; centrifuge at 3000 rpm for 5 min, and remove the supernatant culture medium; resuspend the precipitated algae with 200 μL of fresh TAP liquid culture medium, apply it on a TAP plate containing bleomycin (concentration 10 mg / L) and ampicillin (concentration 100 mg / L), and culture at 22°C for 10 to 14 days; pick a single clone on the plate, transfer it to a new TAP plate containing bleomycin (concentration 10 mg / L) and ampicillin (concentration 100 mg / L) and culture it for 4-5 days, pick a single clone on the plate to ensure that the successfully transformed Chlamydomonas reinhardtii engineered bacteria FTO1 is screened out.

[0061] Example 3 Detection of Growth Indicators of Chlamydomonas reinhardtii Engineered Bacteria FTO1 in Different Culture Media

[0062] 1. Growth index detection of Chlamydomonas reinhardtii engineered bacteria FTO1 in TAP medium

[0063] The selected engineered Chlamydomonas reinhardtii FTO1 and wild-type Chlamydomonas reinhardtii were transferred to TAP liquid medium without antibiotics, cultured overnight at 22°C and 110rpm, and then 1mL of algae solution was taken from each bottle and inoculated into different triangular flasks containing 50mL of liquid TAP medium, and cultured at 22°C and 110rpm until the stationary phase. Every 24h, 200μL of algae solution was taken from the triangular flask and transferred to a 96-well plate, repeated three times, and shaken 3 times. The OD was detected by an ELISA reader. 750 , record OD 750 The absorbance values ​​are shown in Table 1, and the growth curves of the engineered Chlamydomonas reinhardtii strain FTO1 and the wild-type Chlamydomonas reinhardtii strain are plotted. Figure 2 As shown in A.

[0064] Table 1 Absorbance of engineered bacteria and wild-type bacteria in TAP medium

[0065] Day1 Day2 Day3 Day4 Day5 Day6 Day7 Day8 Engineered bacteria 0.057 0.19 0.569 0.721 0.896 0.968 1.041 1.315 Wild-type bacteria 0.066 0.227 0.205 0.257 0.322 0.378 0.412 0.394

[0066] From the experimental results, according to the absorbance values ​​in Table 1, it can be seen that when cultured in the culture medium for 8 days, the growth rate of the engineered Chlamydomonas reinhardtii FTO1 was significantly increased by 234% compared with the wild-type Chlamydomonas reinhardtii.

[0067] The selected engineered Chlamydomonas reinhardtii strain FTO1 and the wild-type Chlamydomonas reinhardtii strain were respectively transferred to antibiotic-free TAP liquid medium and cultured overnight with shaking at 22 °C and 110 rpm. Then, 1 mL of the algal solution was taken from each culture and inoculated into different Erlenmeyer flasks containing 50 mL of liquid TAP medium, and cultured with shaking at 22 °C and 110 rpm until the stationary phase. 50 mL of the algal solution was collected respectively, and the algal cells were collected by centrifugation at 5000 rpm for 5 min at 4 °C. After washing the cells twice with distilled water, they were freeze-dried in a freeze dryer until constant weight, and the dry weights of the engineered Chlamydomonas reinhardtii strain FTO1 and the wild-type Chlamydomonas reinhardtii strain were weighed, as Figure 3 shown in A. According to Figure 3 the dry weights weighed in A, the dry weight of the engineered Chlamydomonas reinhardtii strain FTO1 was 0.067 g, and the dry weight of the wild-type Chlamydomonas reinhardtii strain was 0.034 g, indicating that the engineered Chlamydomonas reinhardtii strain FTO1 can improve the CO2 conversion efficiency and efficiently convert CO2 into biomass.

[0068] 2. Detection of growth indexes of engineered Chlamydomonas reinhardtii strain FTO1 in TAP medium + CO2

[0069] The selected engineered Chlamydomonas reinhardtii strain FTO1 and the wild-type Chlamydomonas reinhardtii strain were respectively transferred to antibiotic-free TAP liquid medium, and 5% CO2 was introduced. They were cultured overnight with shaking at 22 °C and 110 rpm. Then, 1 mL of the algal solution was taken from each culture and inoculated into different Erlenmeyer flasks containing 50 mL of liquid TAP medium, and 5% CO2 was introduced. They were cultured with shaking at 22 °C and 110 rpm until the stationary phase. Every 24 h, 200 μL of the algal solution was taken from the Erlenmeyer flask and transferred to a 96-well plate, with three replicates. After shaking 3 times, the OD 750 was measured using a microplate reader, and the OD 750 absorbance values were recorded. The results are shown in Table 2, and the growth curves of the engineered Chlamydomonas reinhardtii strain FTO1 and the wild-type Chlamydomonas reinhardtii strain were plotted, as Figure 2 shown in B.

[0070] Table 2 Absorbance of the engineered strain and the wild-type strain in TAP medium + CO2

[0071] Day1 Day2 Day3 Day4 Day5 Day6 Day7 Day8 Day9 Engineered bacteria 0.063 0.43 0.995 1.477 1.702 1.903 2.612 3.376 3.32 Wild-type bacteria 0.015 0.157 0.513 0.9 0.789 1.081 1.419 1.639 1.629

[0072] From the experimental results, it can be seen that according to the absorbance values in Table 2, when cultured in the medium for 9 days, the growth rate of the engineered Chlamydomonas reinhardtii strain FTO1 was significantly increased by 104% compared with the wild-type Chlamydomonas reinhardtii strain.

[0073] Transfer the selected engineered Chlamydomonas reinhardtii strain FTO1 and wild-type Chlamydomonas reinhardtii strain into antibiotic-free TAP liquid medium respectively, introduce 5% CO2, and culture them overnight at 22 °C with shaking at 110 rpm. Then, pipette 1 mL of each algal solution and inoculate them into different Erlenmeyer flasks containing 50 mL of liquid TAP medium respectively, and culture them at 22 °C with shaking at 110 rpm until the stationary phase. Collect 50 mL of algal solution respectively, centrifuge at 4 °C and 5000 rpm for 5 min to collect the algal bodies, wash the cells twice with distilled water, and then place them in a freeze dryer to freeze-dry until constant weight. Weigh the dry weights of the engineered Chlamydomonas reinhardtii strain FTO1 and wild-type Chlamydomonas reinhardtii strain, as Figure 3 shown in A below. According to Figure 3 the dry weights weighed in A below, the dry weight of the engineered Chlamydomonas reinhardtii strain FTO1 is 0.13 g, and the dry weight of the wild-type Chlamydomonas reinhardtii strain is 0.08 g, indicating that the engineered Chlamydomonas reinhardtii strain FTO1 can improve the conversion efficiency of CO2 and efficiently convert CO2 into biomass.

[0074] 3. Detection of growth indicators of engineered Chlamydomonas reinhardtii strain FTO1 in TP medium + CO2

[0075] Transfer the selected engineered Chlamydomonas reinhardtii strain FTO1 and wild-type Chlamydomonas reinhardtii strain into antibiotic-free TP liquid medium respectively, introduce 5% CO2, and culture them overnight at 22 °C with shaking at 110 rpm. Then, pipette 1 mL of each algal solution and inoculate them into different Erlenmeyer flasks containing 50 mL of liquid TP medium respectively, introduce 5% CO2, and culture them at 22 °C with shaking at 110 rpm until the stationary phase. Every 24 h, pipette 200 μL of algal solution from the Erlenmeyer flask into a 96-well plate, with three replicates, shake 3 times, and use a microplate reader to detect OD 750 , record OD 750 absorbance values. See Table 3, and draw the growth curves of the engineered Chlamydomonas reinhardtii strain FTO1 and wild-type Chlamydomonas reinhardtii strain, as Figure 2 shown in C below.

[0076] Table 3 Absorbance of engineered strain and wild-type strain in TP medium + CO2

[0077] Day1 Day2 Day3 Day4 Day5 Day6 Day7 Day8 Engineered bacteria 0.045 0.144 0.301 0.464 0.64 0.813 1.005 0.865 Wild-type bacteria 0.013 0.09 0.174 0.305 0.403 0.604 0.753 0.552

[0078] It can be seen from the experimental results that according to the absorbance values in Table 3, when cultured in the medium for 8 days, the growth rate of the engineered Chlamydomonas reinhardtii strain FTO1 is significantly increased by 56.7% compared with the wild-type Chlamydomonas reinhardtii strain.

[0079] The selected engineered Chlamydomonas reinhardtii FTO1 and wild-type Chlamydomonas reinhardtii were transferred to TP liquid culture medium without antibiotics, and 5% CO2 was introduced. The culture was shaken at 22°C and 110rpm overnight, and then 1mL of algae liquid was taken from each of them and inoculated into different triangular flasks containing 50mL of liquid TP culture medium, and shaken at 22°C and 110rpm until the stationary phase. 50mL of algae liquid was collected respectively, and the algae were collected by centrifugation at 4°C and 5000rpm for 5min. After washing the algae twice with distilled water, they were placed in a freeze dryer and freeze-dried to constant weight. The dry weight of engineered Chlamydomonas reinhardtii FTO1 and wild-type Chlamydomonas reinhardtii was weighed, as shown in Table 1. Figure 3 As shown in A. Figure 3 From the dry weight measured in A, we can see that the dry weight of the engineered Chlamydomonas reinhardtii FTO1 is 0.054g, and the dry weight of the wild-type Chlamydomonas reinhardtii is 0.039g, indicating that the engineered Chlamydomonas reinhardtii FTO1 can improve the conversion efficiency of CO2 and efficiently convert CO2 into biomass.

[0080] Example 4 Transformation rate of Chlamydomonas reinhardtii engineered bacteria FTO1 in different culture media

[0081] 1. Pyruvate conversion rate of Chlamydomonas reinhardtii engineered strain FTO1 in different culture media

[0082] 1.1 Pyruvate conversion rate of Chlamydomonas reinhardtii engineered strain FTO1 in TAP medium

[0083] The engineered Chlamydomonas reinhardtii and wild-type Chlamydomonas reinhardtii were transferred to TAP liquid culture medium without antibiotics, and cultured at 22°C and 110rpm with shaking until the logarithmic growth phase; 50mL of algae liquid was respectively aspirated, and the algae were collected by centrifugation at 4°C and 5000rpm for 5min, and the algae were washed twice with PBS buffer, and then placed in a freeze dryer for freeze drying overnight; 0.1g of dried algae powder was weighed and placed in a 2.0mL centrifuge tube, and 9 times the volume of PBS buffer (0.9mL) was added at a ratio of weight (g): volume (mL) = 1:9 for full homogenization; centrifuged at 2500rpm / min for 10min, and the supernatant was taken for testing (part of the supernatant was taken to measure the protein concentration by BCA method);

[0084] Detection was carried out using a pyruvate detection kit. Pipette 0.1 mL of Reagent 1, 0.5 mL of Reagent 2, and 0.1 mL of double-distilled water into a blank tube; pipette 0.1 mL of Reagent 1, 0.5 mL of Reagent 2, and 0.2 μmol / mL pyruvate standard into a standard tube; pipette 0.1 mL of Reagent 1, 0.5 mL of Reagent 2, and 0.1 mL of 10% supernatant to be tested into a measurement tube; after mixing the blank tube, standard tube, and measurement tube, react in a water bath at 37 °C for 10 min, then add 2.5 mL of Reagent 3 respectively and mix well; after standing at room temperature for 5 min, measure the absorbance OD value of each tube at 505 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and zero with double-distilled water. At this time, the absorbance value A of each well measured by the ELISA reader 标准 、A 空白 and A 测定 ,ΔA = A 标准 - A 空白 ,ΔA’ = A 测定 - A 空白 ; Calculate the pyruvate content according to Formula 1, and the pyruvate content result is as shown in B in Figure 3 .

[0085] Y = ΔA’÷ΔA×C 标准 ÷Cpr Formula 1;

[0086] where Y represents the pyruvate content of the sample (μmol / mgprot), C 标准 represents the standard solution concentration (0.2 μmol / mL), and Cpr represents the concentration of the supernatant to be tested (mgprot / L).

[0087] From the experimental results, the pyruvate content of wild-type Chlamydomonas reinhardtii is 0.094 μmol / mgprot, and the pyruvate content of Chlamydomonas reinhardtii engineering strain FTO1 is 0.361 μmol / mgprot, indicating that Chlamydomonas reinhardtii engineering strain FTO1 can improve the pyruvate conversion rate in biomass.

[0088] 1.2 Pyruvate conversion rate of Chlamydomonas reinhardtii engineering strain FTO1 in TAP medium + CO2

[0089] The method for measuring the pyruvate conversion rate is the same as that in 1.1 above. The difference is that the Chlamydomonas reinhardtii engineering strain and the wild-type Chlamydomonas reinhardtii are respectively transferred to an antibiotic-free TAP liquid medium, and 5% CO2 is introduced, and cultured with shaking at 22 °C and 110 rpm until the logarithmic growth phase. Calculate the pyruvate content according to Formula 1, and the pyruvate content result is as shown in B in Figure 3 .

[0090] From the experimental results, the pyruvate content of wild-type Chlamydomonas reinhardtii is 0.048 μmol / mgprot, and the pyruvate content of the engineered Chlamydomonas reinhardtii strain FTO1 is 0.312 μmol / mgprot, indicating that the engineered Chlamydomonas reinhardtii strain FTO1 can improve the pyruvate conversion rate in biomass.

[0091] 1.3 Pyruvate conversion rate of the engineered Chlamydomonas reinhardtii strain FTO1 in TP medium + CO2

[0092] The method for measuring the pyruvate conversion rate is the same as that in 1.1 above. The difference is that the engineered Chlamydomonas reinhardtii strain FTO1 and wild-type Chlamydomonas reinhardtii are respectively transferred to an antibiotic-free TP liquid medium, and 5% CO2 is introduced. The culture is shaken at 22 °C and 110 rpm until the logarithmic growth phase. The pyruvate content is calculated according to Formula 1, and the pyruvate content results are as Figure 3 shown in B below.

[0093] From the experimental results, the pyruvate content of wild-type Chlamydomonas reinhardtii is 0.175 μmol / mgprot, and the pyruvate content of the engineered Chlamydomonas reinhardtii strain FTO1 is 0.333 μmol / mgprot, indicating that the engineered Chlamydomonas reinhardtii strain FTO1 can improve the pyruvate conversion rate in biomass.

[0094] 2. Starch conversion rate of the engineered Chlamydomonas reinhardtii strain FTO1 in different media

[0095] Using the standard product in the starch content detection kit, the standard curve of starch content is y = 4.1301X - 0.0035, R 2 = 0.9934.

[0096] 2.1 Starch conversion rate of the engineered Chlamydomonas reinhardtii strain FTO1 in TAP medium

[0097] Transfer the selected Chlamydomonas reinhardtii engineered strain FTO1 and wild-type Chlamydomonas reinhardtii strain to antibiotic-free TAP liquid medium respectively, and culture them at 22 °C with shaking at 110 rpm until the logarithmic growth phase; respectively pipette 50 mL of the algal solution, centrifuge at 5000 rpm for 5 min at 4 °C to collect the algal bodies, wash the cells twice with distilled water, and then place them in a freeze dryer to freeze-dry overnight. Weigh 0.1 g of the dried algal powder into 2.0 mL centrifuge tubes respectively, add 1 mL of Reagent 1 in the starch content detection kit and homogenize thoroughly, extract in a water bath at 80 °C for 30 min; at room temperature, centrifuge at 8000 rpm for 5 min to collect the algal bodies; add 0.5 mL of distilled water to resuspend, gelatinize in a boiling water bath for 15 min, after cooling, add 0.35 mL of Reagent 2 in the starch content detection kit, extract at room temperature for 15 min, and shake 3 - 5 times; add 0.85 mL of double-distilled water, mix well, centrifuge at 3000 rpm at room temperature for 10 min; take 0.2 mL of the supernatant and 1 mL of Reagent 3 in the starch content detection kit into an EP tube, incubate in a water bath at 95 °C for 10 min, cool naturally to room temperature, and record the absorbance values A of the Chlamydomonas reinhardtii engineered strain FTO1 and wild-type Chlamydomonas reinhardtii strain at a wavelength of 620 nm respectively. Calculate the starch content in the Chlamydomonas reinhardtii engineered strain FTO1 and wild-type Chlamydomonas reinhardtii strain according to the starch content standard curve, as Figure 3 shown in C. From the experimental results, the starch content of the wild-type Chlamydomonas reinhardtii strain is 0.09 g / 100 g, and the starch content of the Chlamydomonas reinhardtii engineered strain FTO1 is 13.18 g / 100 g of algal dry weight, indicating that the Chlamydomonas reinhardtii engineered strain FTO1 can improve the starch conversion rate in biomass.

[0098] 2.2 Starch conversion rate of Chlamydomonas reinhardtii engineered strain FTO1 in TAP medium + CO2

[0099] The method for measuring the starch conversion rate is the same as that in 2.1 above, except that the selected Chlamydomonas reinhardtii engineered strain FTO1 and wild-type Chlamydomonas reinhardtii strain are transferred to antibiotic-free TAP liquid medium respectively, and 5% CO2 is introduced, and cultured at 22 °C with shaking at 110 rpm until the logarithmic growth phase. Calculate the starch content in the Chlamydomonas reinhardtii engineered strain FTO1 and wild-type Chlamydomonas reinhardtii strain according to the starch content standard curve, as Figure 3 shown in C. From the experimental results, the starch content of the wild-type Chlamydomonas reinhardtii strain is 1.59 g / 100 g, and the starch content of the Chlamydomonas reinhardtii engineered strain FTO1 is 19.77 g / 100 g of algal dry weight, indicating that the Chlamydomonas reinhardtii engineered strain FTO1 can improve the starch conversion rate in biomass.

[0100] 2.3 Starch conversion rate of Chlamydomonas reinhardtii engineered strain FTO1 in TP medium + CO2

[0101] The method for measuring the starch conversion rate is the same as that in 2.1 above. The difference is that the selected Chlamydomonas reinhardtii engineered strain FTO1 and the wild-type Chlamydomonas reinhardtii strain are respectively transferred to an antibiotic-free TP liquid medium, and 5% CO2 is introduced. The cultures are shaken at 22 °C and 110 rpm until the logarithmic growth phase. The starch contents in the Chlamydomonas reinhardtii engineered strain FTO1 and the wild-type Chlamydomonas reinhardtii strain are calculated according to the starch content standard curve, as Figure 3 shown in C. From the experimental results, the starch content of the wild-type Chlamydomonas reinhardtii strain is 0.33 g / 100 g, and the starch content of the Chlamydomonas reinhardtii engineered strain FTO1 is 15.67 g / 100 g of algal dry weight, indicating that the Chlamydomonas reinhardtii engineered strain FTO1 can improve the starch conversion rate in the biomass.

[0102] 3. Triglyceride conversion rate of Chlamydomonas reinhardtii engineered strain FTO1 in different media

[0103] Glycerol is the hydrolysis product of triglyceride, and the glycerol content is a reliable detection index for the triglyceride hydrolysis reaction. By measuring the existing glycerol content, the triglyceride content in the cells at this state can be reflected, and the detection is more convenient.

[0104] 3.1 Glycerol conversion rate of Chlamydomonas reinhardtii engineered strain FTO1 in TAP medium

[0105] The selected Chlamydomonas reinhardtii engineered strain FTO1 and the wild-type Chlamydomonas reinhardtii strain are respectively transferred to an antibiotic-free TAP liquid medium, and the cultures are shaken at 22 °C and 110 rpm until the logarithmic growth phase; appropriate amounts of algal liquid are collected respectively, and the lysis solution is added at a ratio of 1×10 6 cells plus 0.1 mL of lysis solution, and mixed well for lysis; 500 μL of the lysis solution is transferred to a 1.5 mL centrifuge tube, and the remaining lysis solution is used for protein quantification by the BCA method to determine the protein concentration; the lipase is inactivated by a water bath at 70 °C for 10 min, and flocculent precipitates may appear during the process; centrifuge at 5000 rpm / min at room temperature for 5 min, and take the supernatant for detection;

[0106] Detection is carried out using a glycerol detection kit. Take 4 mL of reagent R1 and 1 mL of reagent R2 and mix them evenly as the working solution, which is prepared according to the number of samples and used immediately. Take 10 μL of distilled water and 190 μL of the working solution in the blank tube; take 10 μL of the standard product and 190 μL of the working solution in the standard tube; take 10 μL of the sample to be tested and 190 μL of the working solution in the determination tube. After mixing the blank tube, the standard tube, and the determination tube, place them at 37 °C for 10 min. After the reaction is balanced, place the 96-well plate in an enzyme-labeling instrument, measure the absorbance OD value of each tube at 550 nm, zero with double-distilled water, and correct with the protein concentration or the number of cells to measure the glycerol content, as Figure 3 shown in D.

[0107] From the experimental results, the triglyceride content of wild-type Chlamydomonas reinhardtii is 89.18 μmol / gprot, and the triglyceride content of the engineered Chlamydomonas reinhardtii strain FTO1 is 67.21 μmol / gprot, indicating that the engineered Chlamydomonas reinhardtii strain FTO1 reduces the conversion rate of triglycerides in biomass in TAP medium.

[0108] 3.2 Triglyceride conversion rate of the engineered Chlamydomonas reinhardtii strain FTO1 in TAP medium + CO2

[0109] The method for measuring the triglyceride conversion rate is the same as that in 3.1 above. The difference is that the picked engineered Chlamydomonas reinhardtii strain and wild-type Chlamydomonas reinhardtii strain are respectively transferred to antibiotic-free TAP liquid medium, and 5% CO2 is introduced. The cultures are shaken at 22 °C and 110 rpm until the logarithmic growth phase. The glycerol content is measured by correcting with protein concentration or cell number, as shown in Figure 3 D shown in

[0110] From the experimental results, the triglyceride content of wild-type Chlamydomonas reinhardtii is 227.59 μmol / gprot, and the triglyceride content of the engineered Chlamydomonas reinhardtii strain FTO1 is 158.83 μmol / gprot, indicating that the engineered Chlamydomonas reinhardtii strain FTO1 reduces the conversion rate of triglycerides in biomass in TAP medium + CO2.

[0111] 3.3 Triglyceride conversion rate of the engineered Chlamydomonas reinhardtii strain FTO1 in TP medium + CO2

[0112] The method for measuring the triglyceride conversion rate is the same as that in 3.1 above. The difference is that the picked engineered Chlamydomonas reinhardtii strain and wild-type Chlamydomonas reinhardtii strain are respectively transferred to antibiotic-free TP liquid medium, and 5% CO2 is introduced. The cultures are shaken at 22 °C and 110 rpm until the logarithmic growth phase. The glycerol content is measured by correcting with protein concentration or cell number, as shown in Figure 3 D shown in

[0113] From the experimental results, the triglyceride content of wild-type Chlamydomonas reinhardtii is 89.39 μmol / gprot, and the triglyceride content of the engineered Chlamydomonas reinhardtii strain FTO1 is 295.96 μmol / gprot, indicating that the engineered Chlamydomonas reinhardtii strain FTO1 can increase the conversion rate of triglycerides in biomass in TP medium + CO2.

[0114] In summary, it can be seen that the cre-FTO gene and related biological materials provided by the present invention contribute to improving the carbon fixation efficiency such as the photosynthetic efficiency, starch conversion rate, and pyruvate conversion rate of Chlamydomonas reinhardtii, and have more promising application values in improving photosynthetic efficiency and carbon fixation efficiency.

[0115] The above are only embodiments of the present invention, and do not thus limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the content of the specification of the present invention, or directly or indirectly applied in other related technical fields, shall similarly be included within the patent protection scope of the present invention.

Claims

1. A cre-FTO gene for improving the carbon fixation efficiency of Chlamydomonas reinhardtii, characterized in that, The cre-FTO gene is the nucleotide sequence shown in SEQ ID NO:

1.

2. The biomaterial related to the cre-FTO gene according to claim 1, characterized in that, The biological material is any one of A1)-A2): A1) A recombinant expression vector containing the cre-FTO gene recited in claim 1; A2) An expression cassette containing the cre-FTO gene recited in claim 1.

3. The biomaterial according to claim 2, wherein The recombinant expression vector of A1) is constructed by ligating the cre-FTO gene recited in claim 1 with the plasmid pDb124.

4. Use of the cre-FTO gene recited in claim 1 or the biological material recited in claim 2 in a preparation for improving the carbon fixation efficiency or photosynthetic efficiency of Chlamydomonas reinhardtii.

5. A preparation for improving the carbon fixation efficiency of Chlamydomonas reinhardtii, characterized in that, The preparation comprises the cre-FTO gene recited in claim 1 or comprises the biological material recited in claim 2.

6. Use of the cre-FTO gene recited in claim 1 or the biological material recited in claim 2 in algal breeding for improving the carbon fixation efficiency or photosynthetic efficiency of Chlamydomonas reinhardtii.

7. A breeding method for improving the carbon fixation efficiency or photosynthetic efficiency of Chlamydomonas reinhardtii, characterized in that, Transfer the cre-FTO gene described in claim 1 or the related biological material described in claim 2 into Chlamydomonas reinhardtii, culture it in a medium, and screen for Chlamydomonas reinhardtii that can grow on an antibiotic medium or has a high OD 750 absorbance value.

8. The breeding method according to claim 7, characterized in that, The culture medium is TAP medium or TP medium.

9. The breeding method according to claim 8, characterized in that, CO2 is also introduced into the TAP medium or TP medium.

10. The breeding method according to claim 9, characterized in that, The concentration of the introduced CO2 is 1%-10%.

Images