Application of Aida gene in tolerance of Chlamydomonas reinhardtii to copper ion stress

Abstract

The application of Aida gene in the tolerance of Chlamydomonas reinhardtii to copper ion stress, the nucleotide sequence of Aida gene is shown as SEQ ID No 1, the nucleotide sequence of its coding region is shown as SEQ ID No 2; the amino acid sequence of Aida protein encoded by Aida gene is shown as SEQ ID No 3. The Chlamydomonas reinhardtii cell strain obtained after the mutation of Aida gene in the present invention, through the determination of copper ion growth curve and adsorption efficiency, found that the mutant algae strain has faster growth rate, higher adsorption efficiency, and higher copper ion tolerance. The present invention provides convenience for the subsequent in-depth study of how Aida gene affects various oxidative stress reactions and metal transport, the expression and activity of metabolism-related proteins and related stress organelle functions in Chlamydomonas reinhardtii, and will later lay a solid foundation for the theoretical study of the heavy metal copper metabolism mechanism of Chlamydomonas reinhardtii and the application research of heavy metal bioremediation.

Description

Technical Field

[0001] The invention relates to the fields of ecological environment, genetic engineering and molecular biotechnology, and in particular to application of an Aida gene in the tolerance of Chlamydomonas reinhardtii to copper ion stress. Background Art

[0002] In today's society where science and technology are developing rapidly, how to achieve economic development and environmental protection in tandem has become an urgent problem to be solved. In particular, the rapid development of heavy industry has made it difficult to effectively treat sewage in a timely manner, and the resulting environmental deterioration has attracted people's attention. On the one hand, industrial wastewater flows into rivers, lakes, and will cause the death of aquatic plants and animals. On the other hand, industrial wastewater may also penetrate into the ground and pollute the growing environment of crops. Therefore, solving such heavy metal pollution is a top priority.

[0003] Copper is one of the naturally occurring trace metals that helps maintain cell homeostasis. It is an essential micronutrient for plant growth and development, and an essential nutrient in the metabolism of organisms. It can participate in the activity regulation as a cofactor of many enzymes, such as cytochrome oxidase and superoxide dismutase. However, if there is an excess of copper in plants, it will disrupt many biological processes, such as protein and enzyme dysfunction, and damage the mineral homeostasis in the body. In addition, copper can also damage the structure of plant DNA and cause chromosomal aberrations. Excessive copper in the human body can cause severe oxidative stress and damage human tissues. Ingestion of copper-contaminated water can cause abdominal pain, nausea, headache, vomiting and other symptoms. When the human body ingests a large amount of copper at one time, causing copper overload in the tissue, it can cause Wilson's Disease, an autosomal recessive copper metabolism disease that causes copper-mediated oxidative damage and cell death. The leaked copper enters the blood and eventually accumulates in extrahepatic tissues, leading to liver failure. In addition, excessive copper in certain areas of the brain may lead to the occurrence of some neurodegenerative diseases, such as Alzheimer's Disease, Creutzfeldt-Jakob Disease, and Amyotrophic Lateral Sclerosis. In order to prevent the occurrence of various copper excess diseases, the copper content in the human body needs to be maintained at 100-150 mg. Due to the pollution of various heavy industries in the current living environment, copper excess occurs from time to time. In this case, the control of copper pollution is an urgent and critical issue. At present, there are some methods for the treatment of copper pollution in water bodies, such as physical adsorption, which uses solid substances with more pores to adsorb heavy metals in water bodies. Commonly used are activated carbon, nanomaterials, etc., but this material is often expensive and difficult to reuse, which is limited to a certain extent. In addition, chemical precipitation can also be used, using a certain precipitant to combine with metal ions in the water body to produce precipitation and then separate it by filtration, but the precipitate in this method may cause secondary pollution to the water body, and the effect is not significant when the concentration of heavy metal ions is low. In contrast, using bioaccumulation methods to treat heavy metal pollution can effectively solve the shortcomings of traditional methods. Not only can it be reused, it will not cause secondary pollution to the water body and it also reduces cost consumption to a certain extent.

[0004] Bioremediation can utilize the biological mechanisms of organisms themselves to remove environmental pollutants from ecosystems, including direct adsorption of heavy metals in the environment and redox reactions to reduce heavy metal pollution. Plants and microorganisms are both good species for bioremediation. Plants can seal and compartmentalize heavy metals in the soil and detoxify metals through cell complexation and metal chelation. Microorganisms have powerful cell metabolism. Through respiration and cometabolism, they can use toxic compounds in the environment as a source of energy to maintain survival. Microorganisms have evolved multiple ways to resist metal pollution. Bioremediation makes up for some of the shortcomings of physical adsorption and chemical precipitation. Studies have shown that microalgae use glutathione, plant chelators and metallothioneins to resist heavy metal toxicity.

[0005] Chlamydomonas reinhardtii is a unicellular green algae of the green algae lineage with a high cell division rate. It is one of the most developed reference organisms for studying algal biology. Chlamydomonas reinhardtii can also grow to high cell density in simple culture medium, and it is easy to make it metal-excessive or overloaded by various means, and then evaluate its growth status by monitoring the expression of certain metal-related genes. Chlamydomonas reinhardtii can reduce the toxicity of heavy metals to cells through intracellular heavy metal binding factors, including macromolecules such as phytochelatin, thioredoxin, and glutathione. In addition, the toxic effect of copper is generally believed to damage cells by producing reactive oxygen species, and Chlamydomonas reinhardtii can resist such damage through antioxidants. At present, the pathways for the enrichment of heavy metals in Chlamydomonas reinhardtii and the related regulatory methods have not been fully clarified, especially the analysis of proteins and regulatory mechanisms related to heavy metal resistance is still relatively rare in China.

[0006] As genomic research continues to advance, Chlamydomonas reinhardtii is widely used as a model organism in transgenic technology to construct new mutant varieties. Screening mutants to further identify genes related to heavy metal resistance has become an effective method for studying Chlamydomonas heavy metal resistance. Summary of the invention

[0007] The purpose of the present invention is to provide an application of the Aida gene in the tolerance of Chlamydomonas reinhardtii to copper ion stress, aiming to improve the tolerance of Chlamydomonas reinhardtii strains in heavy metal copper-containing wastewater in actual production, so as to increase its biomass and adsorption efficiency, thereby improving the ability of bioremediation of copper-containing wastewater. In addition, the present invention can also facilitate the subsequent in-depth study of how the Aida gene affects various oxidative stress reactions and metal transport and metabolism-related proteins in Chlamydomonas and the functions of related stress organelles, and will later lay a solid foundation for theoretical research on the heavy metal copper metabolism mechanism of Chlamydomonas reinhardtii and applied research on heavy metal bioremediation.

[0008] In order to achieve the above object, the technical solution adopted by the present invention is:

[0009] Application of the Aida gene in the tolerance of Chlamydomonas reinhardtii to copper ion stress, the nucleotide sequence of the Aida gene is shown in SEQ ID No 1, and the insertion mutation of the Aida gene can improve the tolerance of Chlamydomonas reinhardtii to copper ion stress.

[0010] Furthermore, the coding region of the Aida gene can encode the Aida protein, and the nucleotide sequence of the coding region is shown as SEQ ID No 2; the amino acid sequence of the Aida protein is shown as SEQ ID No 3.

[0011] Further, in order to realize the application of Aida gene in the tolerance of Chlamydomonas reinhardtii to copper ion stress, the implementation scheme includes the following steps:

[0012] (1) constructing a mutant library of wild-type Chlamydomonas reinhardtii and randomly integrating the resistance gene fragment aphVIII into the Chlamydomonas genome to cause random insertion mutations;

[0013] (2) Screening target algae strains through the copper ion tolerance / adsorption algae cell screening method and extracting the genome of mutant cells;

[0014] (3) The mutation site sequence was amplified by RESDA-PCR technology, sequenced and compared with the Chlamydomonas reinhardtii genome database, and it was found that the aphVIII fragment was inserted into the 3'UTR region of the Aida gene. The Chlamydomonas reinhardtii genome database is Chlamydomonas reinhardtii v5.6, and the website is https: / / phytozome-next.jgi.doe.gov / blast-search.

[0015] In this way, the Aida gene mutant Chlamydomonas reinhardtii strain of the present invention was obtained. Through the measurement of the copper ion growth curve and adsorption efficiency, it was found that the mutant strain had a faster growth rate and higher copper ion tolerance; at the same time, it was shown that the mutant strain had a certain improvement in the adsorption efficiency of copper ions.

[0016] Preferably, in step (1), the resistance gene fragment aphVIII is a conventional resistance screening gene fragment of Chlamydomonas reinhardtii, which has paromomycin resistance. This gene is a screening gene fragment widely used in Chlamydomonas reinhardtii cell lines.

[0017] Furthermore, in step (2), the method for screening algae cells for copper ion tolerance / adsorption is to 2+ The random mutant transformants were spread on TAP plates to screen candidate mutant algal strains.

[0018] Further, in step (3), the amplification primers used in the RESDA-PCR technique are: the primers used in the first round of PCR reaction are the specific primer outer-R and four degenerate primers DegAlu I, DegTaq I, DegSac II, and DegPst I; the primers used in the second round of PCR reaction are the specific primer inner-R and the primer Q0 for the specific binding site on the degenerate primer; the nucleotide sequences of primer outer-R, primer DegAlu I, primer DegTaq I, primer DegSac II, primer DegPst I, primer inner-R, and primer Q0 are shown in SEQ ID No 4, SEQ ID No 5, SEQ ID No 6, SEQ ID No 7, SEQ ID No 8, SEQ ID No 9, and SEQ ID No 10, respectively. The degenerate primers were designed according to literature reports, and the specific primers were designed by Snapgene software. The above primers were all synthesized by the company.

[0019] The first round PCR reaction program set by the RESDA-PCR technology is:

[0020]

[0021]

[0022] The second round PCR reaction program set by the RESDA-PCR technology is:

[0023]

[0024] This program effectively achieves the amplification of the gene sequence at the mutation site.

[0025] The insertion mutation of the Aida gene of the present invention improves the tolerance of Chlamydomonas reinhardtii to copper ion stress, that is, improves the tolerance of Chlamydomonas reinhardtii strains in heavy metal copper-containing wastewater in actual production, so as to increase its biomass and adsorption efficiency, thereby improving the ability to bioremediate copper-containing wastewater. In addition, the present invention also facilitates the subsequent in-depth study of how the Aida gene affects various oxidative stress reactions and metal transport and metabolism-related proteins in Chlamydomonas and the functions of related stress organelles, and will later lay a solid foundation for theoretical research on the heavy metal copper metabolism mechanism of Chlamydomonas reinhardtii and applied research on heavy metal bioremediation. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 A schematic diagram of the Aida (Cre12.g487450) gene structure and mutation location;

[0027] Figure 2 The wild-type cell line WT (a) and the mutant cell line Aida (b) were respectively 2+ Cell growth rate graph for concentrations ranging from 0 to 500 μM;

[0028] Figure 3 The wild-type cell line WT and the mutant cell line Aida were respectively 2+ Plot of cell adsorption efficiency at concentrations ranging from 0-500 μM. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0030] Example 1

[0031] This embodiment provides an application of an Aida gene in the tolerance of Chlamydomonas reinhardtii to copper ion stress. The gene is numbered Cre12.g487450 in the Chlamydomonas reinhardtii genome database (Chlamydomonas reinhardtii v5.6), the gene length is 3259bp, the coding region CDS length is 891bp, and it can encode an Aida protein with a length of 296 amino acids. The nucleotide sequence of the Aida gene is shown in SEQ ID No 1, and the insertion mutation of the Aida gene can improve the tolerance of Chlamydomonas reinhardtii to copper ion stress. The coding region of the Aida gene can encode the Aida protein, and the nucleotide sequence of the coding region is shown in SEQ ID No 2; the amino acid sequence of the Aida protein is shown in SEQ ID No 3.

[0032] The application of the Aida gene in the tolerance of Chlamydomonas reinhardtii to copper ion stress includes the following steps:

[0033] (1) constructing a mutant library of wild-type Chlamydomonas reinhardtii and randomly integrating the resistance gene fragment aphVIII into the Chlamydomonas genome to cause random insertion mutations;

[0034] (1-1) Obtaining the resistance gene fragment aphVIII: The plasmid pJMG-aphVIII (6682 bp) containing the fragment was digested with EcoRI, and aphVIII was purified and isolated, about 2114 bp in size, and the concentration was measured for later use.

[0035] (1-2) Electroporation of wild-type Chlamydomonas reinhardtii cells: The aphVIII fragment obtained above was randomly inserted into the wild-type Chlamydomonas genome by electroporation. The parameters for electroporation are: BTX ECM830 electroporator, voltage 500V, electroporation time 4ms, electroporation band 6-7 times, each electroporation time interval 100ms, and ice bath for 10 minutes after electroporation. The Chlamydomonas cell solution after electroporation was obtained and slowly recovered under weak light in a night shaker. On the second day, it was spread on a solid TAP medium plate containing 10μg / ml paromomycin. Under Chlamydomonas culture conditions (23±0.5℃, 8000Lx, 14 / 10h photoperiod), it was cultured for about 7 days to obtain a large number of transformant cells.

[0036] (2) Screening target algae strains through the copper ion tolerance / adsorption algae cell screening method and extracting the genome of mutant cells;

[0037] (2-1) Screening of algal cells with copper ion tolerance / adsorption: The transformants obtained above were selected to contain 400 μM Cu. 2+ Copper ion tolerant cell lines were screened on TAP plates, and the WT cell group was selected and inoculated on the same plate as the control group. They were cultured under the same cell culture conditions for about 7 days and observed and screened. It was found that the WT cell line died, and the remaining surviving cell lines were the target tolerant cell lines.

[0038] (2-2) Extraction of mutant genome: Cultivate the Chlamydomonas mutant to the S1 generation according to the Chlamydomonas cell culture method, and determine that the cell activity is good for subsequent experiments. Take the mutant sample and place it on ice, add 100μL preheated CTAB lysis solution, blow it repeatedly with a pipette, mix it thoroughly, then add 400μL CTAB lysis solution, mix it, place the sample in a 60℃ metal bath to fully lyse, and mix it upside down every 10 minutes. Subsequently, prepare 500μL of DNA extraction solution with phenol, chloroform, and isoamyl alcohol in a ratio of 25:24:1 and add it to the sample, slowly mix it upside down for 5 minutes, and centrifuge it at 14000rpm for 10 minutes after sufficient mixing to separate the samples. Take the upper clear liquid into a new 1.5mL EP tube. Prepare chloroform and isoamyl alcohol in a ratio of 24:1, and add a solution equal to the volume of the supernatant to the sample. Slowly invert the sample upside down to mix for 5 minutes. After fully mixing, centrifuge at 14000rpm at room temperature for 10 minutes. Take the upper clear liquid to a new EP tube. Add 2 times the volume of anhydrous ethanol to the sample according to the volume of the upper liquid absorbed. After fully mixing, place the sample at -20℃ for 30 minutes to allow the gDNA to fully precipitate, then centrifuge at 14000rpm at room temperature for 10 minutes, discard the supernatant, and leave the white precipitate. Add 500μL of 70% ethanol to the precipitate, slowly blow to rinse the precipitate, centrifuge at 14000rpm at room temperature for 10 minutes, discard the supernatant, and repeat twice. The gDNA precipitate after two rinses was left to dry at room temperature for 5 min. After the ethanol evaporated completely, 15-25 μL of ddH2O was added to the sample to dissolve it according to the size of the precipitate, and 1 mg / mL of RNase was added to make the concentration 20 μg / mL. The sample was placed at 37°C for 1 h.

[0039] (3) The mutation site sequence was amplified by RESDA-PCR technology, and sequencing and comparison with the Chlamydomonas genome database revealed that the aphVIII fragment was inserted into the 3'UTR region of the Aida gene.

[0040] (3-1) Design of primers required for RESDA-PCR: There are two main types of primers in RESDA-PCR technology. One type is specific primers designed based on the insert fragment aphVIII, and the other type is random primers designed based on the characteristics of the Chlamydomonas genome. Among them, outer-R and inner-R are upstream and downstream nested primers designed based on the insert fragment aphVIII; DegAluI, DegTaq I, DegSac II, and DegPst I are degenerate primers composed of the specific recognition sequences of the four restriction endonucleases AluI, Taq I, Sac II, and Pst I that are widely present in the Chlamydomonas genome; Q0 is a specific second-round primer bound to the degenerate primer.

[0041] (3-2) RESDA-PCR first amplification reaction system: In the first amplification, the flanking fragment of the antibiotic marker gene aphVIII was amplified from the genomic DNA purified from the mutant using the aphVIII gene-specific primers and one of the degenerate primers. Four independent reactions were established using the above four different degenerate primers and one selected outer-R-specific primer.

[0042] (3-3) RESDA-PCR second amplification reaction system: In order to obtain more specific amplification products, the gene-specific primer Q0 at the 5' end of the above-mentioned degenerate primer and the inner specific primer of the nested primer are used to amplify the PCR product of the first amplification as a template (1 μL of the diluted PCR product of the first amplification, the dilution ratio is 1 / 25), which is also 4 PCR reactions. That is, the specific primer inner-R and the primer Q0 on the specific binding site on the degenerate primer constitute one reaction.

[0043] (3-4) Gene sequence analysis and comparison: The amplified PCR products were run on gels, purified, sent for sample delivery, and sequenced to obtain the flanking sequences of the mutant genes. The obtained flanking sequences of the mutants were compared with the genome sequences on the Chlamydomonas website (Chlamydomonas reinhardtii v5.6, https: / / phytozome-next.jgi.doe.gov / blast-search) to obtain the mutant gene information. The specific location of the mutation site is as follows: Figure 1 As shown, the analysis revealed that the aphVIII fragment was inserted into the 3'UTR region of the Aida gene.

[0044] Example 2

[0045] This embodiment provides a method for identifying the tolerance and adsorption efficiency of the above-mentioned Aida gene mutant Chlamydomonas reinhardtii to copper ions in aqueous solution, comprising the following steps:

[0046] (1) Chlamydomonas in Cu 2+ Growth status under stress: According to the culture method of Chlamydomonas cells, wild type and mutants were picked from fresh TAP plates and cultured to the S1 generation. Samples were counted in the clean bench, and then centrifuged at 2500rpm for 3 minutes in a sterilized 50mL centrifuge tube. All cells were collected and resuspended in fresh TAP medium to a concentration of 1×10 8 cell / mL cell suspension. Sterilized 0.1M Cu 2+ The solution was added to the blowing bottle at 0, 200, 400, 600, 800, and 1000 μL, respectively. 2 mL of 1×10 8Then, the liquid in each aeration bottle was filled up to 200 mL with fresh TAP liquid medium to obtain 0, 100, 200, 300, 400, and 500 μM Cu 2+ Chlamydomonas cells treated with TAP medium. The treated cells were placed in a light incubator for photoperiodic culture, and then samples were taken and counted at 1, 2, 3, and 4 days, and growth curves were drawn. The results are shown in Figure 2 As shown in the figure, it can be seen that the Aida gene mutant Chlamydomonas reinhardtii has a high growth rate and a high tolerance to copper ions; the wild-type WT algae strain has a slow growth rate and is easily inhibited by copper ions.

[0047] (2)Cu 2+ Adsorption efficiency determination: According to the culture method of Chlamydomonas cells, wild type and mutants were picked from fresh TAP plates and the cells were cultured to the S1 generation. Samples were counted in the clean bench, and then centrifuged at 2500rpm for 3 minutes in a sterilized 50mL centrifuge tube. All cells were collected and washed with pure water three times, each time at 2500rpm and centrifuged for 3 minutes. Finally, the concentration was adjusted to 1×10 7 Cell suspension of 100 μg / mL. Set different adsorption time lengths (0.25, 0.5, 1, 2, 3, 4 h). Transfer the samples at the corresponding adsorption time points to EP tubes, centrifuge at 6000g for 1 min at room temperature, take the supernatant, and mark it. Add the above samples to a new 24-well plate, dilute the samples with a metal liquid concentration exceeding 75 μM, add the corresponding reagent according to the national standard method, wait for the color development to be completed, and then use an enzyme marker to detect the OD 457 Substitute the absorbance value measured by the microplate reader into the standard curve to obtain the Cu 2+ The concentration data was imported into GraphPad software and statistical graphs were drawn. The results are shown in Figure 3 As shown in the figure, it can be seen that under the same adsorption time and copper ion concentration conditions, the Aida gene mutant Chlamydomonas reinhardtii has a higher adsorption efficiency than the wild-type WT algae strain.

[0048] In summary, the present invention relies on the wild-type cell strain of Chlamydomonas reinhardtii, and through copper ion tolerant cell screening and gene identification from the random mutant library constructed from the wild-type cell strain of Chlamydomonas reinhardtii, the gene Aida with copper ion tolerance function regulation is identified, and the gene mutation leads to a significant increase in the biomass of the Chlamydomonas cell strain under copper ion stress and has an improved copper ion adsorption efficiency. Relying on the present invention and its subsequent research, it is expected to analyze how the gene participates in the biological metabolism of heavy metal copper ions and other processes, and preliminarily construct genetically engineered Chlamydomonas cells based on the gene for bioremediation of heavy metal copper ion-type sewage and the recycling of agricultural water resources.

Claims

1. Aida Application of genes in the tolerance of Chlamydomonas reinhardtii to copper ion stress, the Aida The nucleotide sequence of the gene is shown in SEQ ID No 1, Aida Insertion mutation of the gene can improve the tolerance of Chlamydomonas reinhardtii to copper ion stress.

2. according to claim 1 Aida The application of the gene in the tolerance of Chlamydomonas reinhardtii to copper ion stress is characterized in that: Said Aida The coding region of the gene can encode Aida protein, and the nucleotide sequence of the coding region is shown as SEQ ID No 2; the amino acid sequence of the Aida protein is shown as SEQ ID No 3.

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