Use of cmiet protein and its encoding gene in improving plant resistance to manganese

Abstract

The application discloses application of CMIET protein and a coding gene thereof in improving manganese resistance of plants, and belongs to the technical field of biotechnology, and particularly relates to application of CMIET protein and the coding gene thereof in improving manganese resistance of plants.The CMIET protein can regulate biological manganese resistance, and through regulation of the protein CMIET or regulation of expression of the gene or regulation of activity or content of the protein CMIET, excess manganese in a chlamydomonas chloroplast can be discharged to a cytoplasm, so that balance of manganese ion concentration in the chloroplast is maintained, the chloroplast is not affected by manganese poisoning, normal photosynthesis capacity of the cell is ensured, and normal photosynthesis is ensured.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of the CMIET protein and its encoding gene in improving plant resistance to manganese. Background Technology

[0002] Manganese (Mn) is the second most abundant transition metal in the Earth's crust after iron (Fe), and is widely distributed in soils, sediments, and organisms. In soil, manganese exists in different oxidation states, but the divalent form of manganese (Mn) is the most common. 2+ Manganese is the most readily soluble in water and is absorbed and utilized by plants. It is a trace element required for the growth of all known organisms and plays a rich role in living systems. Manganese participates in a variety of metabolic processes, including photosynthesis, respiration, fatty acid and protein synthesis, and enzyme activation.

[0003] Manganese is a component of plant chloroplasts and directly participates in photosynthesis. The role of manganese in photosynthesis was discovered as early as 1937 in green algae. More than 80% of the manganese in chloroplasts is related to photosystem II (PSII). In photosynthetic organisms, the most important role of manganese is as a component of the calcium manganese cluster in the PSII oxygen-evolving complex. Atmospheric oxygen comes from the splitting of water, a reaction that strictly depends on the tetramanganese cluster (Mn4CaO5) bound to the PSII on the thylakoid membrane lumen side.

[0004] For most plants, at least 20 to 40 milligrams of manganese per kilogram of dry weight are required. Manganese deficiency is a major plant nutrient imbalance. In clover young leaves, manganese deficiency reduces photosynthetic oxygen release by more than 50%, while having a smaller impact on chlorophyll concentration or leaf dry weight. After supplementing manganese-deficient leaves, photosynthetic oxygen release can recover to normal levels within one day. In wheat and corn, manganese deficiency also leads to a reduction in photosynthetic oxygen release. Under manganese deficiency conditions, NPQ (non-photochemical quenching) in plants decreases by 40%-50%. In higher plants, manganese deficiency has the strongest inhibitory effect on photosynthesis, especially the oxygen release process of PSII. Manganese deficiency leads to reduced manganese binding in the PSII complex, causing the decomposition of the PSII supercomplex. The abundance of proteins such as D1 and peripheral proteins PsbP and PsbQ is significantly reduced. The PSII core complex is oxidatively damaged, reducing quantum yield and carbon assimilation efficiency, thus affecting plant growth. Manganese deficiency also leads to changes in the ultrastructure of the thylakoid membrane. Following continuous manganese deficiency in plants, leaf veins become chlorotic, followed by necrotic spots. This is believed to be related to thylakoid system disorder and the absence of PSII reaction centers. Studies on manganese deficiency in *Chlamydomonas reinhardtii* highlight the fundamental role of manganese in photosynthesis and its potential role as a cofactor for MnSOD (Mn superoxide dismutase). The phenomenon that MnSOD activity decreases before PSII activity indicates that the regulation of intracellular manganese supply in *Chlamydomonas reinhardtii* preferentially supports PSII function in chloroplasts, rather than MnSOD function.

[0005] Although manganese is important for plants, their manganese requirements are relatively low. Plants often absorb far more manganese than they need, and this excess is harmful. High manganese toxicity disrupts various physiological processes in plant cells, such as causing oxidative stress, inhibiting enzyme activity, and hindering chlorophyll synthesis and photosynthesis. It also impedes the effective absorption of several elements, including phosphorus, iron, and magnesium. These changes in physiological processes lead to chlorosis and dark brown spots on plant leaves, ultimately resulting in a significant reduction in plant biomass.

[0006] To ensure a balance between manganese absorption and utilization in plants, appropriate manganese transport proteins are needed to efficiently transfer manganese into or out of cells or different organelles. CMIET proteins belong to the UPF0016 protein family, which contains a class of very important manganese transport proteins. In plants, these proteins are located in various organelles, such as the Golgi apparatus, endoplasmic reticulum, and chloroplasts. However, in non-plant eukaryotes, these proteins are only found in the Golgi apparatus. In bacteria, members of the UPF0016 family are located on the plasma membrane, and their main function is to export excess manganese (Mn). 2+ To prevent manganese poisoning. Members of UPF0016 primarily function in cation transport, thereby influencing protein glycosylation in yeast and humans, human lactation, photosynthesis in plants and cyanobacteria, and Mn in bacteria. 2+ Toxicity and other necessary processes. The MneA protein found in Vibrio cholerae also belongs to the UPF0016 family and is responsible for manganese transport. Chlamydomonas reinhardtii typically lives in moist soil or freshwater lakes in nature. These environmental conditions may expose Chlamydomonas reinhardtii to the toxic effects of excessive manganese from external sources. Therefore, Chlamydomonas reinhardtii needs to eliminate the adverse effects of manganese toxicity on its growth through manganese transport regulation mechanisms. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to regulate the resistance of organisms to manganese stress and maintain ion homeostasis in cells.

[0008] To address the problems existing in the prior art, the present invention provides the application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in regulating biological manganese stress resistance.

[0009] The use of the protein or gene expression regulator or substance regulating the activity or content of said protein provided by this invention in any of the following:

[0010] 1) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in regulating biological resistance to manganese stress.

[0011] 2) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the preparation of products that regulate biological resistance to manganese stress;

[0012] 3) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the cultivation of organisms with altered resistance to manganese stress.

[0013] 4) The use of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in the preparation of products that cultivate organisms with altered resistance to manganese stress.

[0014] 5) The application of proteins or substances that regulate gene expression or substances that regulate the activity or content of said proteins in plant breeding;

[0015] The protein is any of the following proteins:

[0016] a1) A protein with the amino acid sequence SEQ ID No. 2;

[0017] a2) A protein having the same function as the amino acid sequence shown in SEQ ID No. 2, by substitution and / or deletion and / or addition of one or more amino acid residues;

[0018] Proteins that have more than 80% identity and the same function of any of the amino acid sequences defined in (a3)a1)-(a2);

[0019] The fusion protein is obtained by attaching a tag to the end of any of the proteins defined in (a4), (a1), and (a3).

[0020] The protein described in a1) above is named CMIET.

[0021] To facilitate the purification or detection of the protein in a1), a tag protein can be attached to the amino or carboxyl terminus of the protein, which consists of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

[0022] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0023] The tagged proteins include, but are not limited to: GST (glutathione thiotransferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose-binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tagged protein, Myc tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomer red fluorescent protein), or AviTag tagged protein.

[0024] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein CMIET of this invention using known methods, such as directed evolution or point mutation. Any artificially modified nucleotides that have 75% or more identity with the nucleotide sequence of the protein CMIET isolated in this invention, as long as they encode and function the protein CMIET, are derived from and equivalent to the nucleotide sequence of this invention.

[0025] The aforementioned 75% or higher degree of identity can be 80%, 85%, 90%, or 95% or higher degree of identity.

[0026] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0027] In this document, the 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0028] In this document, the 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.

[0029] In the above applications, the protein is derived from *Chlamydomonas reinhardtii*.

[0030] In this document, the organism may be a plant or a microorganism. The microorganism may be a fungus. The fungus may be a yeast. The yeast may be a brewer's yeast.

[0031] The breeding mentioned above can refer to plant breeding.

[0032] The plant breeding mentioned above can be used to cultivate plants with high manganese resistance.

[0033] In this article, the substance that regulates the activity and / or content of the protein may be a substance that regulates gene expression, wherein the gene encodes the protein CMIET.

[0034] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0035] In this invention, the regulation can be increased, enhanced, or raised. The regulation can also be decreased, weakened, or reduced.

[0036] In the above applications, the substance regulating gene expression or the substance regulating protein activity or content can be a biological material related to the protein described above, and the biological material can be any of the following:

[0037] c1) The nucleic acid molecule that encodes the protein described above;

[0038] c2) An expression cassette containing the nucleic acid molecule described in c1);

[0039] c3) A recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2);

[0040] c4) Recombinant microorganisms containing the nucleic acid molecules described in c1), or recombinant microorganisms containing the expression cassette described in c2), or recombinant microorganisms containing the recombinant vector described in c3);

[0041] c5) A transgenic plant cell line containing the nucleic acid molecule described in c1), or a transgenic plant cell line containing the expression cassette described in c2);

[0042] c6) Transgenic plant tissue containing the nucleic acid molecules described in c1), or transgenic plant tissue containing the expression cassette described in c2);

[0043] c7) A transgenic plant organ containing the nucleic acid molecule described in c1), or a transgenic plant organ containing the expression cassette described in c2);

[0044] e1) Nucleic acid molecules that inhibit, reduce, or silence the expression of the protein-encoding genes mentioned above;

[0045] e2) An expression cassette containing the nucleic acid molecule described in e1);

[0046] e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2);

[0047] e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3);

[0048] e5) A transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2);

[0049] e6) Transgenic plant tissue containing the nucleic acid molecules described in e1), or transgenic plant tissue containing the expression cassette described in e2);

[0050] e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0051] In the above applications, the nucleic acid molecule described in c1) can be any of the following DNA molecules:

[0052] d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No. 1;

[0053] d2) The coding region sequence is the DNA molecule shown in SEQ ID No. 1 of the sequence listing;

[0054] d3) has 90% or more identity with the nucleotide sequence defined by d1) or d2) and is a DNA molecule encoding the protein described above;

[0055] d4) Hybridizes under strict conditions to a nucleotide sequence defined by d1) or d2) and encodes a DNA molecule that encodes the protein described above.

[0056] In the above applications, the nucleic acid molecule described in e1) can be a DNA molecule with the nucleotide sequence shown in SEQ ID No. 4.

[0057] The nucleic acid molecules mentioned in this article can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0058] The vectors described herein are well-known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the pYL156 vector.

[0059] Recombinant expression vectors containing the CMIET gene can be constructed using existing plant expression vectors. These plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors suitable for plant microbombardment. The plant expression vectors may also contain the 3' untranslated region of the exogenous gene, i.e., containing a polyadenylate signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylate signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor; similar functions exist for the untranslated regions transcribed at the 3' end of genes including, but not limited to, Agrobacterium crown gall-inducing (Ti) plasmids (such as the Nos gene for lipase synthesis) and plant genes (such as the soybean storage protein gene).

[0060] When constructing recombinant plant expression vectors using the CMIET gene, any enhancing or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes.

[0061] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes or luminescent compounds that produce color changes (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). From a safety perspective, transgenic plants can be screened directly under stress without adding any selective marker genes.

[0062] By using any vector capable of guiding the expression of exogenous genes in plants, the CMIET gene or gene fragments provided in this invention can be introduced into plant cells or recipient plants to obtain transgenic cell lines and transgenic plants with altered manganese stress resistance. The expression vector carrying the CMIET gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants.

[0063] The present invention also provides a method for improving the resistance of organisms to manganese stress, the method comprising step M, wherein step M is to enhance, increase or upregulate the activity and / or content of the aforementioned proteins in the target organism, or / and enhance, increase or upregulate the expression level of the encoding genes of the aforementioned proteins, thereby improving the resistance of organisms to manganese stress.

[0064] The present invention also provides a method for reducing biological manganese stress resistance, the method comprising step P, wherein step P is to inhibit or reduce or silence the activity and / or content of the aforementioned protein in the target organism, or / and, inhibit or reduce or silence the expression level of the gene encoding the aforementioned protein, thereby reducing biological manganese stress resistance.

[0065] In the above method, reducing the expression level and / or activity of the gene encoding the protein CMIET in the target organism can be achieved by using gene mutation, gene knockout, gene editing, or gene knockdown techniques to reduce or inactivate the gene encoding the protein CMIET in the genome of the target organism.

[0066] The present invention provides a method for cultivating plants with reduced resistance to manganese stress, comprising inhibiting or reducing or silencing the expression of the coding gene of the above-mentioned protein and / or the content and / or activity of the above-mentioned protein in the target plant, or / and inhibiting or reducing or silencing the activity and / or content of the coding gene of the above-mentioned protein, thereby obtaining plants with reduced resistance to manganese stress.

[0067] In one embodiment of the present invention, the breeding method for cultivating plants with reduced resistance to manganese stress includes the following steps:

[0068] (1) Construct recombinant expression vectors that suppress, reduce, or silence the proteins described above;

[0069] (2) The recombinant expression vector constructed in step (1) is transferred into the recipient plant to obtain plants with lower resistance to manganese stress than the recipient plant.

[0070] In this invention, the purpose of plant breeding may include cultivating plants with reduced resistance to manganese stress.

[0071] In this invention, the plant may be one of the following:

[0072] N1) Chlorophytes;

[0073] N2) Volvoxales plants;

[0074] N3) Chlamydomycetes;

[0075] N4) Chlamydomonas species;

[0076] N5) Chlamydomonas rhinelandii.

[0077] The *Chlamydomonas rhine* can specifically be *Chlamydomonas 21gr*.

[0078] In this article, the concentration of manganese stress is 250 μM.

[0079] This study identified a novel manganese transporter in *Chlamydomonas*, named CMIET (Chloroplast Manganese Ion Efflux Transporter). CMIET belongs to the UPF0016 family, which has four members in *Chlamydomonas reinhardtii*. Bioinformatics analysis and interspecies homology comparison showed that CMIET has the highest homology with CPLD63 in *Chlamydomonas* and CMT1 in *Arabidopsis thaliana*. Similar to CMT1 and CPLD63, CMIET is also located on the chloroplast membrane and is responsible for manganese ion transport, but in the opposite direction: CMIET transports manganese ions from the chloroplast stroma to the cytoplasm.

[0080] Studies on the CMIET deletion mutant revealed that the deletion of CMIET significantly increased the intracellular manganese ion content, elevated cytoplasmic acidity, and markedly altered intracellular ion homeostasis within Chlamydomonas cells. Under both normal and manganese-deficient culture conditions, the CMIET mutant exhibited stronger growth performance and superior photosynthetic capacity compared to the wild type; the CMIET mutant was also more sensitive to high manganese toxicity stress.

[0081] In summary, the CMIET protein located on the chloroplast membrane of Chlamydomonas is responsible for transporting manganese ions from inside the chloroplast to the cytoplasm, thereby maintaining the balance of manganese ion concentration inside the chloroplast, protecting the photosynthetic organ from the effects of high manganese toxicity, and ensuring the normal functioning of photosynthesis. Attached Figure Description

[0082] Figure 1 To restore the sensitivity of the yeast Δgdt1 mutant to excess manganese in CMIET, A. Δgdt1, Δgdt1 transformed with pYES (empty vector) (hereinafter referred to as Δgdt1+empty vector), and Δgdt1 transformed with pYES-CMIET (hereinafter referred to as Δgdt1+CMIET) were grown on SD plates for 3 days; B. Δgdt1, Δgdt1 transformed with pYES (empty vector) (hereinafter referred to as Δgdt1+empty vector), and Δgdt1 transformed with pYES-CMIET (hereinafter referred to as Δgdt1+CMIET) were grown on SD-U plates (without uracil) supplemented with 5 mM MnCl2 for 3 days.

[0083] Figure 2Subcellular localization of CMIET. A. Venus and CMIET-Venus were expressed in *Chlamydomonas reinhardtii*, and fluorescence was observed using a laser scanning confocal microscope. Venus excitation fluorescence was at 514 nm, and emission fluorescence at 527 nm. Chlorophyll autofluorescence excitation fluorescence was at 488 nm, and emission fluorescence was at 650 nm. Scale bar: 5 μm. B. eGFP and CMIET-eGFP were expressed in *Arabidopsis thaliana* protoplasts, and fluorescence was observed using a laser scanning confocal microscope. eGFP excitation fluorescence was at 488 nm, and emission fluorescence was at 505-550 nm. Chlorophyll autofluorescence excitation fluorescence was at 488 nm, and emission fluorescence was at 650 nm. Scale bar: 5 μm. The white arrows in the figure indicate the protein localization regions.

[0084] Figure 3 The effects of CMIET deficiency on Chlamydomonas growth are shown below. A. Schematic diagram of the CMIET gene, including 7 exons (black squares), 6 introns (black lines between two exons), and the UTR region (gray squares). The exogenous gene is inserted into the third intron. P1-P3 mark the primer positions used for genotyping. B. Genotyping of the CMIET mutant. C. Growth of Chlamydomonas strains under different culture conditions. D. Chlorophyll concentration analysis of Chlamydomonas strains under different culture conditions. The culture medium was multitrophic, and the light intensity was low: 25 μM Mn. 2+ 50 μmol photons m -2 s -1 Dual-trophic culture medium, high light: 25μM Mn 2+ 1200 μmol photons m -2 s -1 Dual-trophic culture medium, manganese deficiency, low light: 0 μM Mn 2+ 50 μmol photons m -2 s -1 Dual-trophic culture medium, manganese deficiency, high light: 0 μM Mn 2+ 1200 μmol photons m -2 s -1 Dual-trophic culture medium, 10 times manganese, low light: 250 μMn 2+ 50 μmol photons m -2 s -1 An asterisk indicates a statistically significant difference (t-test), **P<0.01; ***P<0.001.

[0085] Figure 4 Chlorophyll fluorescence was measured. A.21gr (control algal strain), cmiet, and cmiet complement 1 were cultured in a multitrophic medium under low light (25 μM Mn). 2+50 μmol photons m -2 s -1 The maximum photochemical efficiency, actual photochemical quantum efficiency, electron transport rate, and non-photochemical quenching coefficient of PSII were measured under the following conditions: B.21gr, cmiet, and cmiet complementary 1 in a dual-culture medium and under high light (25 μM Mn) conditions. 2+ 1200 μmol photons m -2 s -1 The maximum photochemical efficiency, actual photochemical quantum efficiency, electron transport rate, and non-photochemical quenching coefficient of PSII were measured under the following conditions: C.21gr, cmiet, and cmiet complementary 1 in a dual-nutrient medium, manganese deficiency, and low light (0 μM Mn). 2+ 50 μmol photons m -2 s -1 The maximum photochemical efficiency, actual photochemical quantum efficiency, electron transport rate, and non-photochemical quenching coefficient of PSII were measured under the following conditions: D.21gr, cmiet, and cmiet complementary 1 in a dual-nutrient medium, manganese deficiency, and high light (0 μM Mn). 2+ 1200 μmol photons m -2 s -1 The maximum photochemical efficiency, actual photochemical quantum efficiency, electron transport rate, and non-photochemical quenching coefficient of PSII were determined under the following conditions: E.21gr, cmiet, and cmiet complementary 1 in a dual-trophic medium, 10 times manganese, and low light (250 μM Mn). 2+ 50 μmol photons m - 2 s -1 The maximum photochemical efficiency, actual photochemical quantum efficiency, electron transport rate, and non-photochemical quenching coefficient of PSII were measured under the following conditions. An asterisk indicates statistically significant differences (t-test), *P<0.05; ***P<0.001.

[0086] Figure 5 Transmission electron microscopy analysis of 21gr (control algal strain) and CMIET under different treatment conditions. A. Cells in a dual-trophic medium under low light (25 μM Mn) conditions... 2+ 50 μmol photons m -2 s -1 A. Treat cells under the same conditions (dual-culture medium, low light) and count the number of thylakoid membrane layers; B. Treat cells under the same conditions (dual-culture medium, low light) and count the number of thylakoid membrane layers; C. Treat cells under the same conditions (dual-culture medium, high light, 25 μM Mn) and high light ( ... 2+ 1200 μmol photons m -2 s-1 D. Select 10 different cell lines under the same treatment conditions (mixed trophic medium, high light) and count the number of thylakoid membrane layers; E. Treat cells under mixed trophic medium, manganese deficiency, and low light (0 μM Mn) conditions. 2+ 50 μmol photons m -2 s -1 F. Select 10 different cell lines under the same treatment conditions (dual-trophic medium, manganese deficiency, low light) and count the number of thylakoid membrane layers; G. Treat cells under dual-trophic medium, manganese deficiency, high light (0 μM Mn) conditions. 2+ 1200 μmol photonsm -2 s -1 H. Select 10 different cell lines under the same treatment conditions (mixed trophic medium, manganese deficiency, high light) and count the number of thylakoid membrane layers; I. Cells were treated under mixed trophic medium, 10 times manganese, and low light (250 μM Mn) conditions. 2+ 50 μmol photons m -2 s -1 Under the same conditions (mixed-culture medium, 10 times manganese, low light), 10 different cell lines were selected, and the number of thylakoid membrane layers was counted. An asterisk indicates statistically significant differences (t-test), *P<0.05; ***P<0.001.

[0087] Figure 6 The accumulation of thylakoid membrane complexes in different algal strains under different treatment conditions was shown. A. Dual-trophic medium, low light (25 μM Mn) 2+ 50 μmol photons m -2 s -1 Thylakoid membranes were extracted from cells treated under the following conditions; after volume enrichment with detergent, protein complexes were separated by blue-green gel electrophoresis; B. Dual-trophic culture medium, high light (25 μM Mn) 2+ 1200 μmol photons m -2 s -1 Thylakoid membranes were extracted from cells treated under the following conditions: [C. Detergent-enriched culture medium, manganese deficiency, low light (0 μM Mn)]; protein complexes were separated by blue-green gel electrophoresis. 2+ 50 μmol photons m -2 s -1 Thylakoid membranes were extracted from cells treated under the following conditions; after volume enrichment with detergent, protein complexes were separated by blue-green gel electrophoresis; D. Dual-trophic culture medium, manganese deficiency, high light (0 μM Mn) 2+ 1200 μmol photons m -2 s -1Thylakoid membranes were extracted from cells treated under the following conditions; after volume enrichment with detergent, protein complexes were separated by blue-green gel electrophoresis; E. dual-trophic medium, 10-fold manganese, low light (250 μM Mn) 2+ 50 μmol photons m -2 s -1 Thylakoid membranes were extracted from cells treated under certain conditions, and after being enlarged with detergent, protein complexes were separated by blue-green gel electrophoresis. PSII SC: PSII supercomplex; PSI: photosystem I; PSIID: PSII dimer; PSII M: PSII monomer; LHC: light-harvesting antenna complex.

[0088] Figure 7 Immunoblot analysis of thylakoid membrane subunit proteins. A. Dutrophic medium, low light (25 μM Mn) 2+ 50 μmol photons m -2 s -1 A. Immunoblot analysis of photosystem complex subunits under the following conditions; B. Dual-trophic culture medium, high light (25 μMn) 2+ 1200 μmol photons m -2 s -1 Immunoblot analysis of photosystem complex subunits under the following conditions: C. Dual-trophic culture medium, manganese deficiency, low light (0 μM Mn) 2+ 50 μmol photons m -2 s -1 Immunoblot analysis of photosystem complex subunits under the following conditions; D. Dual-trophic culture medium, manganese deficiency, high light (0 μM Mn) 2+ 1200 μmol photons m -2 s -1 Immunoblot analysis of photosystem complex subunits under the following conditions: E. Dual-trophic medium, 10-fold manganese, low light (250 μM Mn) 2+ 50 μmol photons m -2 s -1 Immunoblot analysis of photosystem complex subunits under certain conditions.

[0089] Figure 8 The oxygen evolution and proton kinetic potential of 21gr (control algal strain), cmiet, and cmiet complementary strain 1 were measured. A. Multitrophic medium, low light (25 μM Mn) was used. 2+ 50 μmol photons m -2 s -1 A) Oxygen emission determination of different algal strains under the following conditions; B) Dual-trophic culture medium, manganese deficiency, low light (0 μM Mn) 2+ 50 μmol photons m -2 s-1 A. Oxygen release from different algal strains under the specified conditions; B. Evaluation of proton kinetic potential induction using electrochromic shift (ECS) signals; C. Proton kinetic potential of different algal strains; D. pH gradient of different algal strains; E. Potential gradient of different algal strains. An asterisk indicates statistically significant differences (t-test), ***P<0.001.

[0090] Figure 9 This study aimed to detect cellular cation / proton ratios. A. The acidic pH dye Lysosensor DND-189 could detect acidic pH regions within different algal strains. Chlamydomonas cells were cultured in a multitrophic medium under low light (25 μM Mn) conditions. 2+ 50 μmol photons m -2 s -1 B. The acidic pH dye Lysosensor DND-189 can detect the acidic pH region within different algal strains. Chlamydomonas cells were grown under dual-trophic medium, manganese deficiency, and low light (0 μM Mn) conditions. 2+ 50 μmol photons m -2 s -1 C. Cells were grown under the following conditions: (a) dual-trophic medium and low light (25 μM Mn). 2+ 50 μmol photons m -2 s -1 Growth and detection were performed under the specified conditions. Positive values ​​indicate H. + Flowing out of the cell, negative values ​​indicate H + D. Cells in a dual-nutrient medium, manganese deficiency, and low light (0 μM Mn) flow into the cell; 2+ 50 μmol photons m -2 s -1 Growth and detection were performed under the specified conditions. Positive values ​​indicate H. + Flowing out of the cell, negative values ​​indicate H + E. Total manganese concentration in cells. Elemental concentrations were quantified using inductively coupled plasma optical emission spectrometry (ICP-OES) on a dry weight basis; F. Total calcium concentration in cells. Elemental concentrations were quantified using ICP-OES on a dry weight basis. An asterisk indicates statistical significance (t-test), ***P < 0.001.

[0091] Figure 10The expression of CPLD63 and CGLD1 genes was analyzed. The breakdown is as follows: A. CPLD63 expression in 21gr (control strain) under different conditions; B. CPLD63 expression in cmiet under different conditions; C. CPLD63 expression in 21gr and cmiet under normal conditions; D. CPLD63 expression in 21gr and cmiet under manganese deficiency conditions; E. CGLD1 expression in 21gr under different conditions; F. CGLD1 expression in cmiet under different conditions; G. CGLD1 expression in 21gr and cmiet under normal conditions; H. CGLD1 expression in 21gr and cmiet under manganese deficiency conditions. Normal: 25 μM Mn 2+ 50 μmol photonsm -2 s -1 Manganese deficiency: 0 μM Mn 2+ 50 μmol photons m -2 s -1 An asterisk indicates a statistically significant difference (t-test), *P<0.05; **P<0.01; ***P<0.001. Detailed Implementation

[0092] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0093] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0094] Unless otherwise specified, all quantitative experiments in the following examples are performed in triplicate.

[0095] The carrier pYES2 in the following embodiments was kindly provided by Professor Zhang Zhaoliang's research group at Anhui Agricultural University and is described in: Cloning and Organelle Expression of Bamboo Mitochondrial Complex I Subunits Nad1, Nad2, Nad4, and Nad5 in the Yeast Saccharomyces cerevisiae. This biological material is available to the public from the applicant and is intended solely for the purpose of replicating the experiments of this invention and shall not be used for any other purpose.

[0096] The Δgdt1 yeast mutant strains in the following examples were kindly provided by Professor Zhang Zhaoliang's research group at Anhui Agricultural University and are described in: The Yeast Protein Gdt1p Transports Mn 2+ Ions and thereby Regulates Manganese Homeostasis in the Golgi. The biological material is available to the public from the applicant and is intended solely for the purpose of replicating experiments of the present invention and may not be used for any other purpose.

[0097] The pLM005 vector used in the following examples was purchased from Matt Laudon Chlamydomonas Resource Center University of Minnesota 140 Gortner Laboratory, at https: / / www.chlamycollection.org / product / plm005 / .

[0098] The pAN580 vector used in the following examples was purchased from Biorun Biotechnology, and the website address is: http: / / www.biorun.com / bioHosts / hosts.html.

[0099] The Chlamydomonas 21gr used in the following examples was purchased from Matt Laudon Chlamydomonas Resource Center, University of Minnesota 140, Gortner Laboratory (website: https: / / www.chlamycollection.org / , catalog number CC-1690).

[0100] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0101] Example 1: The role of CMIET protein in manganese transport

[0102] 1. Obtaining CMIET protein from Chlamydomonas reinhardtii

[0103] DNA was extracted from Chlamydomonas reinhardtii 21gr cells. Using this DNA as a template, PCR amplification was performed using primers CMIET gene F: 5'-ATGTTGTCGTCAGCTTGTCT-3' and CMIET gene R: 5'-TCACAGCAGGCTGAAGGCGG-3', with the aid of KOD FX DNA Polymerase (Toyobo, KFX-101) to obtain the amplified product (i.e., the coding region of the CMIET gene).

[0104] The coding sequence (CDS) of the CMIET gene in *Chlamydomonas reinhardtii* 21gr is SEQ ID No. 1, and the amino acid sequence encoding the CMIET protein is SEQ ID No. 2. The genomic gene encoding the CMIET protein in the genomic DNA of *Chlamydomonas reinhardtii* 21gr is shown in SEQ ID No. 3 of the sequence listing. In SEQ ID No. 3, positions 1-22 are exon 1, positions 100-274 are exon 2, positions 512-605 are exon 3, positions 840-1010 are exon 4, positions 1364-1492 are exon 5, positions 1758-2014 are exon 5, and positions 2257-2464 are exon 6.

[0105] 2. Construction of yeast CMIET protein mutant strain

[0106] The Δgdt1 yeast mutant cannot properly transfer manganese into the Golgi apparatus, therefore this mutant cannot grow normally on high-manganese medium.

[0107] CMIET was constructed into the yeast transformation vector pYES2 and then transformed into the Δgdt1 yeast mutant, while an empty vector was also transformed as a control.

[0108] The specific construction steps are as follows:

[0109] 1) Recombinant vector construction: The coding sequence fragment of CMIET was ligated to the enzyme-digested pYES2 vector using the pEASY-Uni Seamless Cloning and Assembly Kit. The mixture consisted of 5.0 μL of 2×Assembly Mix, 1.5 μL of vector, and 3.5 μL of target fragment, for a total volume of 10 μL. After gentle mixing, the mixture was incubated at 50°C for 15 min to obtain the recombinant vector pYES2-CMIET.

[0110] 2) The yeast conversion steps are as follows:

[0111] Take 20 mL of yeast culture (OD) 546Centrifuge at 700×g for 5 minutes at room temperature (between 0.6 and 1.0), discard the supernatant; resuspend the precipitate in 1 mL of sterile ddH2O for later use; add 1 μL of the recombinant vector pYES2-CMIET to be transformed into 300 μL of transformation premix (270 μL 50% PEG4000, 5 μL 5M lithium acetate, 25 μL 2 mg·mL⁻¹). -1 Salmon sperm genomic DNA was extracted and vortexed. 100 μL of resuspended yeast culture was added to the premix, gently mixed, and then incubated at 42°C for 45 minutes, swirling several times during incubation. The mixture was centrifuged at 700 × g for 5 minutes at room temperature, the supernatant was discarded, and 100 μL of sterile physiological saline (0.9% NaCl) was added to resuspend the precipitate. The resuspended culture was evenly spread on the corresponding SD selection plates and incubated upside down at 30°C for 2-4 days to obtain a recombinant yeast strain containing the recombinant vector pYES2-CMIET, abbreviated as strain Δgdt1+CMIET. Simultaneously, the empty vector pYES2 was introduced into the Δgdt1 yeast mutant using the same method to obtain the recombinant yeast strain Δgdt1+ empty vector, serving as a control.

[0112] The structure of the CMIET expression vector pYES2-CMIET is described as follows: The recombinant expression vector is obtained by replacing the small fragment between the HindIII and BamHI restriction sites of the pYES2 vector with a nucleotide sequence that contains the coding sequence of CMIET, while keeping the other sequences of the pYES2 vector unchanged.

[0113] Experimental results showed that all strains Δgdt1, Δgdt1+ empty vector, and Δgdt1+CMIET could grow normally on SD medium. Figure 1 (A). Neither the yeast mutant strain Δgdt1 transformed with an empty vector (Δgdt1 + empty vector) nor the original mutant strain Δgdt1 could grow normally on high-manganese medium. Figure 1 (B) In contrast, the Δgdt1 yeast mutant strain (Δgdt1+CMIET) transformed with the CMIET expression vector pYES2-CMIET was unaffected by high manganese levels and could grow normally. This result indicates that manganese is a transport substrate for the CMIET protein and can be transformed into yeast mutant strains by CMIET, restoring the high manganese-sensitive phenotype of the mutant strains.

[0114] 3. Subcellular localization of CMIET protein

[0115] To determine the subcellular localization of CMIET, the recombinant vector pLM005-CMIET-Venus was transformed into Chlamydomonas reinhardtii 21gr( Figure 2 (A); The recombinant vector PAN580-CMIET-eGFP was transformed into Arabidopsis protoplasts ( Figure 2 The specific steps for (B) are as follows:

[0116] Specific methods for constructing recombinant vectors:

[0117] The coding sequence fragment of CMIET was ligated to the enzyme-digested pLM005 vector using the pEASY-Uni Seamless Cloning and Assembly Kit. The total volume was 10 μL, consisting of 5.0 μL of 2×Assembly Mix, 1.5 μL of vector, and 3.5 μL of target fragment. After gentle mixing, the mixture was incubated at 50°C for 15 min to obtain the recombinant vector pLM005-CMIET-Venus.

[0118] The coding sequence fragment of CMIET was ligated into the enzyme-digested PAN580 vector using the pEASY-Uni Seamless Cloning and Assembly Kit. The total volume was 10 μL, consisting of 5.0 μL of 2×Assembly Mix, 1.5 μL of vector, and 3.5 μL of target fragment. After gentle mixing, the mixture was incubated at 50°C for 15 min to obtain the recombinant vector PAN580-CMIET-eGFP.

[0119] Electroconversion method for Chlamydomonas reinhardtii:

[0120] 1. Take 100 mL of logarithmic growth phase (approximately 2 × 10⁻⁶) 6 cells·mL -1 The Chlamydomonas algal solution was transferred to a sterile 50mL centrifuge tube, placed on ice for 10min, and centrifuged at 5000rpm (3000×g) at 4℃ for 5min.

[0121] 2. In a clean bench, discard the supernatant and add 1 mL of pre-cooled TAP medium containing sucrose solution (specific formula: 20 mL 1M Tris-base, 10 mL Solution A, 1 mL Phosphate Buffer II, 1 mL Hutner's traceelements solution, 1 mL Acetic acid). Add the appropriate antibiotic according to the recombinant vector's resistance gene (bleomycin resistance was added in this experiment at a concentration of 5 μg / mL). Adjust the pH of the solution to 7.2-7.4 and then add ddH2O to bring the volume to 1 L. When preparing solid medium, add 1.5% (w / v) agar powder and autoclave at 121℃ for 20 min.

[0122] Solution A consists of 40.0 g NH₄Cl, 5.0 g CaCl₂·2H₂O, and 10.0 g MgSO₄·7H₂O, diluted to 1 L with ddH₂O. The reagents must be dissolved sequentially to avoid precipitation.

[0123] Phosphate Buffer II: K2HPO4 108.0g, KH2PO4 56.0g, add ddH2O to bring the volume to 1L.

[0124] Hutner's trace elements solution: EDTA-Na2 50.0g, H3BO3 11.14g, ZnSO4·7H2O 22.0g, MnCl2·4H2O 5.1g, FeSO4·7H2O 5.0g, CoCl2·6H2O 1.6g, CuSO4·5H2O 1.6g, (NH4)6Mo7O 24 Add 1.1g of 4H2O and ddH2O to a final volume of 1L.

[0125] Preparation steps for Hutner's trace elements solution:

[0126] 1) Prepare a 1L glass beaker, add 550mL ddH2O, and then add the other reagents except EDTA-Na2 in sequence. Stir and heat the solution to 70℃.

[0127] 2) After EDTA-Na2 is dissolved in 250mL ddH2O by heating, add it to the solution, continue stirring and heating until the solution boils.

[0128] 3) Stop heating and allow the solution to cool naturally to 70-75℃, then continue heating and maintain the solution at that temperature.

[0129] 4) Adjust the pH of the solution to 6.5-6.8 with KOH solution. Note that the solution temperature should not be lower than 70℃ or the pH value should not be higher than 6.8. Add ddH2O to make up to 1L.

[0130] 5) Transfer the solution to a brown bottle, seal the bottle opening with cotton or breathable sealing film, and place at room temperature away from light for 10-14 days until the solution turns purple-red.

[0131] 6) After filtration and sedimentation using filter paper, store at 4°C away from light.

[0132] The final sucrose concentration was 60 mM. The sucrose solution was filtered through a 0.22 μm filter membrane for sterilization to fully resuspend the cell pellet. The solution was then placed on ice for 15 min.

[0133] 3. In a clean bench, add 5 μL of 10 mg / mL solution to a pre-cooled 0.4 cm bio-Rad electroconvulsive cuvette. -1 Boil salmon sperm DNA, recombinant vector plasmid (500-1000 ng), 250 μL of resuspended algae solution, mix well and cover the electroporation cup.

[0134] 4. Place the electrocution cup in a 16℃ water bath for 5 minutes;

[0135] 5. Wipe the outside of the shock cup dry with filter paper, place the shock cup in the shock device of the BTX device, make the metal plates on both sides clamp the shock cup, lower the safety cover, set the shock parameters (voltage 800V, resistance 1575Ω, capacitance 50μF), press the Pulse button to start the shock, 10-14ms is the normal time range, immediately after the shock, transfer the shock cup to ice and place it for 10 minutes;

[0136] 6. In a clean bench, add 40 mL of TAP sucrose solution to a sterile 50 mL centrifuge tube. Transfer the Chlamydomonas cells that have undergone electrolysis from the electrolysis cuvette to the centrifuge tube and mix them with the TAP sucrose solution. Place the centrifuge tube on a shaker at low speed and dim light (25℃, 50 rpm, 10 μmol photons·m⁻¹). -2 ·s -1 Recovery within 24 hours;

[0137] Centrifuge at 7,5000 rpm (3000 × g) at 25°C for 5 min;

[0138] 8. Discard the supernatant in a clean bench, add 1 mL of TAP liquid medium to resuspend the precipitated cells, spread on the corresponding TAP-resistant plates, and after drying, place the plates at 25°C for 50 μmol photons·m -2 ·s -1 Cultured under specific conditions, monoclonal antibodies were grown and then identified and used for subsequent experiments.

[0139] Arabidopsis protoplast preparation and transformation:

[0140] Preparation of experimental reagents:

[0141] 1. Cellulose hydrolysate: 1.5% Cellulase R-10, 0.4% Mecerozyme R-10, 0.4M mannitol, 20mM KCl, 20mM MES, pH 5.7. Incubate at 55℃ for 10 min, cool to room temperature, then add the following reagents: 10mM CaCl2, 0.1% BSA, 5mM β-Mercaptoethanol. Precautions: Prepare the hydrolysate fresh before use. Filter out impurities using a 0.45μm membrane filter. Before adding R-10 powder, preheat the MES solution at 70℃ for 3-5 min.

[0142] 2. PEG4000 Solution: 40% PEG4000, 0.2% Mannitol, 100mM CaCl2. Precautions: Prepare and use immediately; prepare the solution at least 1 hour in advance.

[0143] 3. W5 Solution: 154mM NaCl, 125mM CaCl2, 5mM KCl, 2mM MES, KOH to adjust pH to 5.7, autoclave, store at room temperature.

[0144] 4. MMG Solution: 4mM MES, 0.4M mannitol, 15mM MgCl2, KOH to adjust pH to 5.7, autoclave, store at room temperature.

[0145] 5. WI Solution: 4mM MES, 20mM KCl, 0.5M mannitol, KOH to adjust pH to 5.7, autoclave, store at room temperature.

[0146] Protoplast preparation and transformation:

[0147] 1. Select the 5th, 6th, and 7th rosette leaves of Arabidopsis thaliana that has not flowered after 3-4 weeks of short-day growth, and remove the leaf tips and petioles. Take about 1 / 3 of the leaf from the middle and cut it into strips 0.5-1mm wide with a sharp blade. Transfer the strips to the enzymatic hydrolysate with flat-tipped tweezers and use tweezers to help the leaves completely immerse in the enzymatic hydrolysate.

[0148] 2. Vacuum for 10-30 minutes until most of the leaf is submerged in the enzymatic hydrolysate. Incubate at room temperature in the dark for at least 3 hours. When the enzymatic hydrolysate turns green, gently shake the culture dish to promote the release of protoplasts.

[0149] 3. Pre-cool a certain amount of W5 solution and examine the protoplasts under a microscope;

[0150] 4. Wet a 100-mesh sieve with W5 solution, dilute the enzyme solution containing protoplasts with an equal amount of W5 solution, and filter to remove undissolved leaves;

[0151] 5. Centrifuge at 4℃ for 3 min at 100×g (preferably using round-bottom centrifuge tubes, not exceeding 200×g), remove as much supernatant as possible, gently resuspend the protoplasts in 10 mL of W5 solution pre-cooled on ice, and incubate vertically on ice for 30 min.

[0152] Centrifuge at 100×g at room temperature for 3 min, removing as much W5 as possible. Resuspend the protoplasts in an appropriate amount of MMG solution (approximately 1 mL) to achieve a final concentration of approximately 2×10⁻⁶. 5 cells·mL -1 ;

[0153] 7. For each reaction, add 10 μL of purified PAN580-CMIET-eGFP recombinant vector plasmid (approximately 10-20 μg plasmid DNA) to a 2 mL centrifuge tube, add 100 μL of protoplasts, mix gently, and add 110 μL of 40% PEG4000 solution and mix gently.

[0154] 8. Induce conversion of the mixture at room temperature for 5-15 minutes;

[0155] 9. Dilute the conversion mixture with twice the volume of W5 solution, and gently invert to mix to terminate the reaction;

[0156] Centrifuge at 10.100×g at room temperature for 3 min, then discard the supernatant;

[0157] 11. Add 1 mL of W5 solution to suspend and wash once, centrifuge at 100×g at room temperature for 3 min, and discard the supernatant;

[0158] 12. Add 1 mL of WI solution and gently resuspend the protoplasts in a porous tissue culture dish (the culture dish should be moistened with 5% BSA beforehand to prevent adhesion to the wall), and induce protoplasts at room temperature for 16-18 h.

[0159] The structure of the recombinant vector pLM005-CMIET-Venus is described as follows: The recombinant expression vector is obtained by replacing the small fragment between the Hpa I restriction site of the pLM005 vector with a nucleotide sequence containing the coding sequence of CMIET, while keeping the other sequences of the pLM005 vector unchanged.

[0160] The structure of the recombinant vector PAN580-CMIET-eGFP is described as follows: The recombinant expression vector is obtained by replacing the small fragment between the Sma I and Sma I restriction sites in the PAN580 vector with a nucleotide sequence containing the coding sequence of CMIET, while keeping the other sequences of the PAN580 vector unchanged.

[0161] The results are as follows Figure 2 As shown, the CMIET protein is concentrated on the chloroplast membrane near the T-zone of Chlamydomonas cup chloroplasts. Simultaneously, the fluorescence signal results of transfecting Arabidopsis protoplasts with the PAN580-CMIET-eGFP recombinant vector also indicated that the CMIET protein is located on the chloroplast membrane.

[0162] Example 2: Effect of CMIET deletion on Chlamydomonas mutant phenotype

[0163] The cmiet mutant used in this study was purchased from the Chlamydomonas mutant library (website: www.chlamylibrary.org, mutant number: LMJ.RY0402.051309). Mutant information is as follows: Figure 3In step A, the foreign gene is inserted into the third intron of the CMIET gene.

[0164] Verification of the authenticity of mutants using PCR ( Figure 3 (B) Primer P1: 5'-ACATACGCACCAATCATGTCAAGC-3', Primer P2: 5'-GCGGGTGGGGTAGTTGATACAAT-3', Primer P3: 5'-GGGGAAAGGCTCGAGTGGGA-3'.

[0165] PCR amplification of the gene fragment was performed using the high-fidelity PCR enzyme KOD FX (TOYOBO). The 50 μL PCR system was as follows: 2×PCR buffer 25.0 μL, 2 mM dNTPs 10.0 μL, 10 μM primer-F 1.5 μL, 10 μM primer-R 1.5 μL, DNA template (50 ng / μL) 1.0 μL, KOD FX 1.0 μL.

[0166] ddH2O 10.0μL.

[0167] PCR amplification program: 94℃ pre-denaturation for 2 min, 98℃ denaturation for 10 s, 60℃ annealing for 30 s (annealing temperature adjusted according to primer Tm value), 68℃ extension for 1.5 min (extension time set according to target fragment length 1 min / kb), 25-30 cycles (denaturation-annealing-extension constitutes one cycle), 68℃ final extension for 5 min.

[0168] The results showed that a foreign gene fragment was detected in the cmiet mutant, proving that a foreign gene (nucleotide sequence of SEQ ID No. 4 in the sequence listing) was indeed inserted in the third intron of the cmiet mutant, thus making it impossible to obtain the correct CMIET gene fragment.

[0169] 1. Effects of CMIET deletion on the growth and development of Chlamydomonas mutants

[0170] The growth of the cmiet mutant was analyzed. The cmiet mutant, wild-type 21gr, and complementary algal strain cmiet complement 1 were each replicated in triplicate under the following conditions:

[0171] A. Dual-trophic culture medium, low light: 25μM Mn 2+ 50 μmol photons m -2 s -1 ;

[0172] B. Mixed-culture medium, high light: 25μM Mn 2+1200 μmol photons m -2 s -1 ;

[0173] C. Dual-trophic culture medium, manganese deficiency, low light: 0μM Mn 2+ 50 μmol photons m -2 s -1 ;

[0174] D. Dual-trophic culture medium, manganese deficiency, high light: 0μM Mn 2+ 1200 μmol photons m -2 s -1 ;

[0175] E. Mixed-culture medium, 10x manganese, low light: 250μM Mn 2+ 50 μmol photons m -2 s -1 .

[0176] The method for determining chlorophyll fluorescence in Chlamydomonas sylvestris liquid culture samples is as follows: Take the algal strains to be tested and measure the chlorophyll concentration, adjusting the chlorophyll concentration uniformly to 25 μg·mL. -1 After dark adaptation for 15-20 minutes, the required chlorophyll fluorescence parameters were measured using the Imaging-PAM (Walz) instrument on the bioimaging technology platform of the Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences.

[0177] The results show ( Figure 3 C and D), in normal manganese (25 μM Mn) 2+ Manganese content or deficiency (0 μM Mn) 2+ During cultivation, the cmiet mutant showed superior growth compared to the wild-type and complementary strains, with significantly higher chlorophyll concentrations. Conversely, the mutants grown in high-manganese (250 μM Mn) conditions showed significantly lower chlorophyll concentrations. 2+ The growth of the CMIET mutant in the culture medium was severely inhibited, with the algae turning yellow and white, and the chlorophyll concentration significantly reduced. Compared with the wild type and complementary algal strains, the CMIET mutant was more susceptible to manganese toxicity. These results indicate that the absence of CMIET has a significant impact on the growth of the mutant algal strain.

[0178] 2. Effects of CMIET deletion on the photosynthetic capacity of Chlamydomonas mutants

[0179] The light response curves of algal strains under different culture conditions in step 1 above were measured using imaging-pam. The specific experimental steps are the same as step 1 in this embodiment.

[0180] The results are as follows Figure 4As shown: Under low light conditions in a dual-trophic medium ( Figure 4 In the study of the mixed-trophic medium, after 24 hours of high-light treatment (Central A), there was no significant difference in the photosynthetic capacity of the cmiet mutant, wild-type 21gr, and complementary algal strain cmiet-complementary 1. Figure 4 In the case of B), the mutant and wild type differed only in the non-photochemical quenching coefficient; the cmiet mutant had a higher non-photochemical quenching coefficient than the wild type and the complementary algae. After manganese deficiency treatment, low light ( Figure 4 (C) or highlight ( Figure 4 Under high light conditions (D), the photosynthetic capacity of the cmiet mutant was significantly increased compared to the wild-type and complementary algal strains. Especially after high light treatment, all photosynthetic parameters of the cmiet mutant maintained high levels under adverse conditions. The cmiet mutant is exceptionally sensitive to high manganese toxicity; under high manganese stress conditions (D), the photosynthetic capacity of the cmiet mutant was significantly increased compared to the wild-type and complementary algal strains. Figure 4 In the case of E), the mutant completely lost its ability to photosynthesize.

[0181] The above results demonstrate that the CMIET protein has a significant impact on the growth, development, and photosynthetic capacity of Chlamydomonas, and plays a crucial role in maintaining normal growth of Chlamydomonas under high manganese toxicity stress.

[0182] 3. Effects of CMIET deficiency on Chlamydomonas cell structure

[0183] The culture methods for the cmiet mutant, wild-type 21gr, and complementary algal strain cmiet complement 1 are the same as step 1 in this embodiment.

[0184] Wild-type and mutant cells under different treatment conditions were observed using transmission electron microscopy.

[0185] Under normal manganese content and low light conditions ( Figure 5 In the mutant (A), there was no significant difference in cell morphology and structure between the mutant and wild-type cells. This was determined by statistical analysis of the number of thylakoid membrane layers. Figure 5 (B) The results showed no significant difference between the mutant and the wild type. However, under normal manganese content and high light conditions (B), Figure 5 Medium C) and manganese deficiency culture, low light ( Figure 5 (E) or after highlighting ( Figure 5 In the wild-type chloroplast (G) mutant, the thylakoid membrane structure becomes sparse, with increased lamellar spacing and shortened length. However, the thylakoid membrane of the cmiet mutant maintains a more complete morphology. This can be achieved by statistically analyzing the number of thylakoid membrane layers (G). Figure 5 In the D, F, and H groups, the mutant cells had a significantly higher number of thylakoid membrane layers compared to the wild type. However, after high manganese stress treatment, the mutant cell structure was almost completely destroyed, and the thylakoid membrane could not be observed. Figure 5(I and J). This indicates that CMIET plays a very important role in maintaining the morphological stability of chloroplasts in Chlamydomonas cells.

[0186] 4. CMIET deficiency affects the accumulation of photosynthetic complexes and subunits.

[0187] Since the absence of CMIET affects the photosynthetic capacity of Chlamydomonas cells and the structure of the thylakoid membrane, the accumulation of the CMIET mutant, wild-type 21gr, and complementary strain CMIET complement 1 photosynthetic complex and important subunits was examined.

[0188] The extraction of thylakoid membranes from Chlamydomonas reinhardtii must be conducted entirely on ice under low-light conditions. If possible, the experiment can be performed in a low-temperature cold chamber equipped with a green light lamp. The specific steps are as follows:

[0189] 1) Prepare the relevant buffer solutions according to Table 1 below. Before the experiment, add protease inhibitors (1 mM MPMSF, 1 mM benzoamidine, 1 mM aminocaproic acid) to each buffer solution.

[0190] Table 1. Thylakoid membrane extraction buffer

[0191]

[0192]

[0193] 2) Collect at least 500 mL of logarithmic growth phase (approximately 2 × 10⁻⁶ mL). 6 cells·mL -1 Centrifuge the Chlamydomonas algae solution at 5000 rpm (3000×g) at 4℃ for 10 min, and discard the supernatant;

[0194] 3) Add 10 mL of HEPES1 buffer to fully resuspend the precipitated cells;

[0195] 4) Use a high-pressure homogenizer (PHD) to break Chlamydomonas cells at 5000-10000 psi (adjust the pressure according to different algal strains) for 2 minutes;

[0196] 5) Centrifuge the broken Chlamydomonas cells at 5000 rpm (3000×g) at 4℃ for 10 min, and transfer the supernatant to a new high-speed centrifuge tube (BECKMAN COULTER);

[0197] 6) Centrifuge at 40000×g and 4℃ for 20 min using a high-speed centrifuge (BECKMAN COULTER), and discard the supernatant;

[0198] 7) Add 3 mL of HEPES2 buffer to fully resuspend the cell pellet. Transfer the resuspended solution to a glass homogenizer and homogenize it to remove large clumps.

[0199] 8) Transfer the homogenate to a high-speed centrifuge tube. Add 2 mL of HEPES2 buffer to the glass homogenizer to rinse the Chlamydomonas component in the homogenizer, and then add it to the high-speed centrifuge tube. Centrifuge at 40000×g at 4℃ for 20 min and discard the supernatant.

[0200] 9) Add 3 mL of HEPES 3 buffer to the centrifuge tube to fully resuspend the cell pellet, and then transfer the resuspended solution to a glass homogenizer for grinding and homogenization.

[0201] 10) Transfer the homogenate to an ultracentrifuge tube (BECKMAN COULTER). Add 2 mL of HEPES 3 buffer to the glass homogenizer to rinse the Chlamydomonas component in the homogenizer, and then add it to the ultracentrifuge tube in the same way.

[0202] 11) Carefully and slowly add 2 mL of HEPES 4 buffer to the ultracentrifuge tube, so that it is on top of HEPES 3 buffer;

[0203] 12) Carefully and slowly add 4 mL of HEPES 5 buffer to the ultracentrifuge tube, so that it is on top of HEPES 4 buffer;

[0204] 13) After finely balancing the centrifuge tubes, centrifuge them at 220,000 g at 4℃ for 60 min using an ultracentrifuge (BECKMAN COULTER), with both acceleration and deceleration set to the slowest speed.

[0205] 14) Carefully aspirate the green fraction between HEPES 3 and HEPES 4 buffers into a new high-speed centrifuge tube using a syringe, add 10-15 mL of HEPES 6 buffer, mix well, centrifuge at 40000×g at 4℃ for 20 min, and discard the supernatant.

[0206] 15) Add 100 μL of HEPES 6 buffer to fully resuspend the precipitate, and obtain the thylakoid membrane product. After quick freezing in liquid nitrogen, store at -80°C.

[0207] The thylakoid membrane products were then verified by BN-PAGE gel electrophoresis and SDS-PAGE gel electrophoresis, and further verified by Western blotting.

[0208] Experimental results show that under low-light culture conditions in a dual-trophic medium ( Figure 6 China A, Figure 7 In the case of CMIET (A), its absence did not induce changes in the accumulation of photosynthetic complexes and important subunits. Under high-light conditions in a ditrophic medium (A), Figure 6 B, Figure 7In the cmiet mutant (B), the accumulation of PSII dimers increased, as did the accumulation of PsbO, Cyt f, and LHCSR3 proteins. Under manganese-deficient and low-light conditions in a mixed-nutrient medium (B), Figure 6 C in the middle Figure 7 In medium C), the accumulation of CMIET photosynthetic complex and important subunit proteins increased to varying degrees. Under manganese-deficient and high-light conditions in a fascicular medium (…),… Figure 6 D, Figure 7 The CMIET photosynthetic complex and important subunit proteins also accumulated and increased, especially PsbO protein. After high manganese stress treatment, almost no photosynthetic complex accumulated in CMIET, and almost all proteins were degraded, further indicating that the absence of CMIET leads to Chlamydomonas cells' inability to cope with high manganese toxic stress. Figure 6 E, Figure 7 (E).

[0209] Immunoblotting revealed that the absence of CMIET significantly affected the accumulation and changes of the major subunits of the oxygen-evolving complex. Changes in the oxygen-evolving complex directly reflect the photosynthetic oxygen release of Chlamydomonas. Therefore, the oxygen release rate of the corresponding algal strains was measured.

[0210] The procedure for determining the oxygen release rate is as follows:

[0211] 1) Measure the chlorophyll concentration of the algal strains to be tested, and adjust the chlorophyll concentration of the algal strains to be consistent;

[0212] 2) Assemble the Oxytherm+R liquid oxygen electrode (Hansatech) and complete the standard curve preparation according to the instructions for use;

[0213] 3) Add 1.5 mL of the algal solution to the sample well, add 50 μL of 0.5 M NaHCO3, and incubate at 25 °C with 100 μmol photons·m -2 ·s -1 The oxygen release rate was determined at 100 rpm for 5 min per sample, with 3 technical replicates.

[0214] 4) Create a graph based on the data.

[0215] The results show that ( Figure 8 In both A and B), under different treatment conditions, the photosynthetic oxygen release rate of the cmiet mutant was significantly better than that of the wild type and the complementary algae, and this result is consistent with the immunoblotting results.

[0216] The above results indicate that the changes in CMIET growth, development, and photosynthetic capacity are mainly due to the accumulation and changes in important subunits of the photosynthetic complex.

[0217] 5. The absence of CMIET affects cellular ion homeostasis.

[0218] A. The steps for staining the acidic parts of cells with the low-pH dye Lysosensor DND-189 are as follows:

[0219] 1) Take 20 mL and grow it to the logarithmic growth phase (approximately 2 × 10⁻⁶). 6 cells·mL -1 Centrifuge the algal strain at 5000×g at room temperature for 5 min and discard the supernatant;

[0220] 2) Add 20 mL of Na3PO4 solution (10 mM pH 7.5) to resuspend and wash the precipitate, centrifuge at 5000×g at room temperature for 5 min, discard the supernatant, and repeat the washing 2-3 times;

[0221] 3) Add 10 mL of Na3PO4 solution to resuspend the precipitate;

[0222] 4) Dilute Lysosensor DND-189 fluorescent dye (1mM, Shanghai Yisheng) to a concentration of 2μM using Na3PO4 solution;

[0223] 5) Take 1 mL of resuspended algae solution, add an equal volume of 2 μM DND-189 solution to make the final dye concentration 1 μM, and incubate at room temperature with slow shaking for 30-120 min.

[0224] 6) Observe and photograph the cells using a 3D-SIM (Nikon) ultra-high resolution laser confocal microscope.

[0225] B. Using non-invasive microprobe technology to measure intracellular and extracellular H + The procedure for measuring flow is as follows:

[0226] The samples were handed over to the public instrument platform of the Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, to perform non-destructive measurements of ion flow inside and outside Chlamydomonas cells using the chloroplast material permeability micrometer system (Yange Company).

[0227] C. The steps for determining intracellular manganese content using inductively coupled plasma-optical emission spectrometry (ICP-OES) are as follows:

[0228] Microwave digestion of samples:

[0229] 1) Prepare 500 mL of logarithmic growth phase (approximately 2 × 10⁻⁶) 6 cells·mL -1 Centrifuge the algal solution at 5000×g at room temperature for 5 min, and discard the supernatant;

[0230] 2) Dry the sample in a 65℃ oven, and then grind the dried sample into powder using a mortar and pestle;

[0231] 3) Weigh 0.1500-0.3000g of sample into a microwave digestion tube, add 6mL of concentrated nitric acid (analytical grade), cover the tube, shake well, and let stand in a fume hood at room temperature for 2-4 hours (overnight is better);

[0232] 4) Add 2 mL of hydrogen peroxide solution (analytical grade), cap the tube, shake well, and let stand in a fume hood at room temperature for 15 min;

[0233] 5) Tighten the tube cap and place it in the microwave digester for digestion. Refer to Table 2 below for the microwave digestion procedure.

[0234] Table 2. Microwave Digestion Procedure

[0235] 1 5min 100℃ 2min 2 5min 150℃ 10min 3 5min 180℃ 30min

[0236] 6) After digestion and cooling, open the microwave digestion tube cap and transfer all the solution in the digestion tube to a 50mL plastic volumetric flask. Rinse the walls of the digestion tube several times with a small amount of ddH2O, then transfer it to the volumetric flask. Make up to volume with ddH2O, shake well, and filter with quantitative filter paper. The filtered solution is ready for determination.

[0237] D. Determination of metal element content:

[0238] 1) The metal element content of the microwave digested samples was determined using inductively coupled plasma atomic emission spectrometry (Thermo).

[0239] 2) Perform calculations and draw graphs based on the measured data.

[0240] Experimental results show that ( Figure 8 The proton dynamic potential of the cmiet mutant (C) Figure 8 (middle D), pH gradient ( Figure 8 The potential gradient (E) was significantly higher than that of the wild type and complementary algal strains. Figure 8 The concentration of protons in the thylakoid membrane of mutant Chlamydomonas cells was significantly reduced. This indicates that the absence of CMIET leads to an increase in proton concentration on the thylakoid membrane lumen side of the mutant Chlamydomonas cells, which increases energy supply to cope with abiotic stresses such as high light and nutrient deficiency by promoting increased ATP synthase activity.

[0241] The low-pH dye Lysosensor DND-189 can stain the acidic regions of cells. Observation using a 3D-SIM microscope showed that wild-type cells and complementary algal strains grown in medium with normal manganese content had almost no accumulation of acidic regions, but the cmiet mutant cytoplasm contained a large number of acidic regions. Figure 9 (A). Under manganese-deficient culture conditions, the opposite is true ( Figure 9 (B)

[0242] Using non-invasive microprobe (NMT) to measure intracellular and extracellular H + The flow assay results showed that, under normal culture conditions, extracellular H+ in the cmiet mutant cells... + H flows into cells, in wild-type and complementary algal strain cells + extracellular flow ( Figure 9 (C). The opposite is true under manganese deficiency conditions. Figure 9 (D). The above results indicate that the deletion of CMIET prevents the normal transport of Mn. 2+ This will cause intracellular H + The concentration changed significantly.

[0243] Intracellular manganese content was determined using inductively coupled plasma optical emission spectrometry (ICP-OES). The results showed that the manganese accumulation in the cmiet mutant was significantly higher than that in the wild type and the complementary algal strain. Figure 9 (E). Measurement results ( Figure 9 The results (F) indicate that changes in manganese accumulation in cmiet cells also cause changes in calcium content.

[0244] The above results indicate that CMIET is a manganese ion transporter located on the chloroplast membrane, responsible for transporting excess manganese from the chloroplast into the cytoplasm.

[0245] 6. Analysis of changes in other chloroplast-localizing manganese transporters in the cmiet mutant

[0246] The expression levels of the manganese transport proteins CPLD63 and CGLD1, which encode chloroplast localization, were detected in the wild-type 21gr and cmiet mutants. The specific steps are as follows:

[0247] Preparation of the real-time PCR reaction solution (20 μL system): TB Green Premix Ex Taq (2×) (TliRNaseH Plus), Bulk (Takara) 10 μL, PCR forward primer (10 μM) 0.4 μL, PCR reverse primer (10 μM) 0.4 μL, cDNA template (total amount not exceeding 100 ng) 2 μL, ddH2O 7.2 μL. Three technical replicates were performed for each sample using a LightCycler 480 (Roche) PCR instrument. PCR amplification program: 95℃ pre-denaturation for 2 min, 95℃ denaturation for 5 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 40 cycles. Experimental results were analyzed using LightCycler 480 analysis software.

[0248] The results are as follows Figure 10As shown in the AH diagram, manganese deficiency significantly increases the expression levels of CPLD63 and CGLD1 genes in both wild-type and mutant cells. This indicates that under manganese deficiency, Chlamydomonas cells need to increase the expression of CPLD63 and CGLD1 proteins to transport more manganese ions from the environment to chloroplasts and thylakoid membranes to maintain photosynthesis. Since the absence of CMIET prevents the rapid transport of manganese from chloroplasts in cmiet mutant cells, cmiet cells do not require a significant increase in the expression of CPLD63 and CGLD1 proteins to enhance the transport of manganese ions to chloroplasts and thylakoid membranes compared to the wild-type.

[0249] Chloroplasts account for more than half of the total volume of Chlamydomonas cells. Most of the excess manganese absorbed from the outside world will enter the chloroplasts for storage. Therefore, when the accumulation of manganese exceeds the corresponding threshold, Chlamydomonas cells will activate the corresponding detoxification mechanism. This study confirms that Chlamydomonas can expel excess manganese from chloroplasts into the cytoplasm through the CMIET protein, thereby ensuring that the chloroplasts are not affected by manganese toxicity and maintaining the normal photosynthetic capacity of the cells.

[0250] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of substances that reduce the expression of protein-coding genes in any of the following: 1) Application in reducing biological manganese resistance; 2) Application in the preparation of products that reduce resistance to biological manganese stress; 3) Application in the cultivation of organisms with reduced resistance to manganese stress; 4) Application in the preparation of products from organisms with reduced resistance to manganese stress; The protein is the protein with the amino acid sequence SEQ ID No. 2; The organism in question is Chlamydomonas; The substance is any one of the following: e1) Nucleic acid molecules that inhibit or silence the expression of the protein-coding gene; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3).

2. A method for reducing biological resistance to manganese stress, characterized in that: The method is to reduce the activity and / or content of the protein described in claim 1 in the target organism to reduce the organism's resistance to manganese stress. The organism in question is Chlamydomonas.

3. A breeding method for cultivating organisms with reduced resistance to manganese stress, characterized in that: This includes reducing the expression level of the gene encoding the protein in the application of claim 1 in a recipient organism to obtain an organism with reduced manganese stress resistance, wherein the manganese stress resistance of the organism with reduced manganese stress resistance is lower than that of the recipient organism; the organism is Chlamydomonas.

4. The method according to claim 3, characterized in that: Includes the following steps: (1) Construct a recombinant expression vector that reduces the expression level of the gene encoding the protein; (2) The recombinant expression vector constructed in step (1) is transferred into the recipient Chlamydomonas to obtain organisms with lower resistance to manganese stress than the recipient Chlamydomonas.

Images