Application of CMIET protein and coding gene thereof in improving manganese resistance of plants

Abstract

The invention discloses application of a CMIET protein and a coding gene thereof in improving manganese resistance of plants. The invention belongs to the technical field of biology, and particularly relates to application of CMIET protein and a coding gene thereof to improvement of manganese resistance of plants. The CMIET protein can regulate and control biological manganese resistance, and excessive manganese in Chlamydomonas chloroplast can be discharged to cytoplasm by regulating and controlling expression of the protein CMIET or regulating and controlling gene or regulating and controlling activity or content of the protein CMIET, so that balance of manganese ion concentration in the chloroplast is maintained, and the chloroplast is prevented from being affected by manganese toxicity; the normal photosynthesis capability of the cells is maintained, and the normal photosynthesis is ensured.

Description

Technical Field

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

[0002] Manganese (Mn) is the second most abundant transition metal in the earth's crust after iron (Fe). It is widely distributed in soil, sediments and organisms. In soil, manganese exists in different oxidation states, but divalent manganese (Mn 2+ ) is the most soluble in water and absorbed by plants. Manganese is a trace element required for the growth of all known organisms and plays a rich role in life systems. Manganese is involved 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 is directly involved in plant photosynthesis. As early as 1937, the role of manganese in photosynthesis was discovered in green algae. More than 80% of manganese in chloroplasts is related to photosystem II (PSII). In photosynthetic organisms, the most important role of manganese is to form the calcium-manganese cluster in the PSII oxygen-releasing complex. Oxygen in the atmosphere comes from the splitting of water, and this reaction strictly depends on the four manganese clusters (Mn4CaO5) bound to PSII on the lumen side of the thylakoid membrane.

[0004] For most plants, at least 20 to 40 mg of manganese per kilogram of dry weight is required. Manganese deficiency is a major nutritional disorder in plants. Manganese deficiency reduces photosynthetic oxygen release in young clover leaves by more than 50%, while having little effect on chlorophyll concentration or leaf dry weight. After supplementing manganese-deficient leaves, photosynthetic oxygen release can be restored to the level of normal leaves within one day. In wheat and corn, manganese deficiency also causes a decrease in photosynthetic oxygen release capacity. Under manganese deficiency conditions, NPQ (non-photochemical quenching) of plants will decrease 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, such as a significant decrease in the abundance of D1 protein and peripheral proteins PsbP and PsbQ, and oxidative damage to the PSII core complex, which reduces quantum yield and carbon assimilation efficiency, affecting plant growth. Manganese deficiency can lead to changes in the ultrastructure of the thylakoid membrane. Continuous manganese deficiency in plants leads to chlorosis of leaf veins followed by necrotic spots, which are thought to be related to disorganization of the thylakoid system and loss of PSII reaction centers. Studies on manganese deficiency in Chlamydomonas reinhardtii have highlighted the essential role of manganese in photosynthesis and its role as a cofactor for MnSOD (Mn superoxide dismutase). The fact that MnSOD activity decreases before PSII activity suggests that regulation of intracellular manganese supply in Chlamydomonas reinhardtii preferentially supports PSII function in the chloroplasts rather than MnSOD function.

[0005] Although manganese is important for plants, the amount of manganese required by plants is relatively low. Usually, the manganese absorbed by plants often far exceeds the amount they need. Excessive absorption of manganese is harmful to plants. High manganese toxicity can cause damage to various physiological processes in plant cells, such as causing oxidative stress, inhibiting enzyme activity, and hindering chlorophyll synthesis and photosynthesis. It also hinders the effective absorption of various elements, such as the absorption of phosphorus, iron and magnesium. This series of changes in physiological processes can cause plant leaves to fade, produce dark brown spots, and ultimately lead to a significant reduction in plant biomass.

[0006] In order to ensure the balance of manganese absorption and utilization by plants, corresponding manganese transporters are needed to effectively transfer manganese into or out of cells or different organelles. CMIET protein belongs to the UPF0016 protein family, and the proteins in this family are a very important class of manganese transporters. In plants, proteins in this family are localized in different organelles, such as the Golgi apparatus, endoplasmic reticulum, and chloroplasts. However, in non-plant eukaryotic organisms, proteins in this family are only found in the Golgi apparatus. In bacteria, members of UPF0016 are located on the plasma membrane, and their main function is to export excess Mn 2+ , to prevent manganese poisoning. UPF0016 members mainly play a role in cation transport, thus affecting protein glycosylation in yeast and humans, human lactation, photosynthesis in plants and cyanobacteria, and Mn in bacteria. 2+ The MneA protein found in Vibrio cholerae also belongs to the UPF0016 family and is responsible for the transport of manganese. Chlamydomonas reinhardtii usually lives in moist soil or freshwater lakes in nature. Such environmental conditions may cause Chlamydomonas reinhardtii to be toxic from excessive manganese from the outside world. Therefore, Chlamydomonas reinhardtii needs to eliminate the adverse effects of manganese toxicity on its own growth through the manganese transport regulation mechanism. Summary of the invention

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

[0008] In order to solve the problems existing in the prior art, the present invention provides the use of a protein or a substance for regulating the expression of a gene or a substance for regulating the activity or content of the protein in regulating the manganese stress resistance of an organism.

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

[0010] 1) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in regulating biological manganese stress resistance;

[0011] 2) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the preparation of a product regulating biological manganese stress resistance;

[0012] 3) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in cultivating an organism with altered manganese stress resistance;

[0013] 4) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the preparation of a product for cultivating an organism with altered manganese stress resistance;

[0014] 5) Application of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in plant breeding;

[0015] The protein is any of the following:

[0016] a1) a protein having an amino acid sequence of SEQ ID No. 2;

[0017] a2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 after one or more amino acid residues are replaced and / or deleted and / or added;

[0018] a3) a protein having an amino acid sequence with more than 80% identity with any one of a1) to (a2) and having the same function;

[0019] a4) A fusion protein obtained by connecting a tag to the end of the protein defined in any one of a1) to (a3).

[0020] The name of the protein described in a1) above is CMIET.

[0021] In order to facilitate purification or detection of the protein in a1), a tag protein may be connected to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID No. 2 in the sequence listing.

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

[0023] The tag protein includes but is not limited to: GST (glutathione sulfhydryltransferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

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

[0025] The aforementioned 75% or more identity may be 80%, 85%, 90% or 95% or more identity.

[0026] Herein, identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of an amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST webpage on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively and searching for the identity of a pair of amino acid sequences for calculation, the value (%) of identity can then be obtained.

[0027] Herein, the 80% or greater identity may 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] Herein, the 90% or greater identity may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

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

[0030] Herein, 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 Saccharomyces cerevisiae.

[0031] In the above, the breeding may be plant breeding.

[0032] In the above, the plant breeding may be breeding plants with high manganese resistance.

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

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

[0035] In the present invention, the regulation may be up-regulation, enhancement or increase. The regulation may also be down-regulation, attenuation or reduction.

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

[0037] c1) a nucleic acid molecule encoding 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) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism 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 molecule 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) a nucleic acid molecule that inhibits, reduces or silences the expression of the protein encoding gene 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) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism 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 molecule 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 application, 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 in the sequence listing;

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

[0055] d4) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described above.

[0056] In the above application, the nucleic acid molecule described in e1) may be a DNA molecule whose nucleotide sequence is shown as SEQ ID No.4.

[0057] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule 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, including but not limited to: plasmid, phage (such as lambda phage or M13 filamentous phage, etc.), cosmid (i.e., cosmid), Ti plasmid or viral vector. Specifically, it can be vector pYL156 vector.

[0059] The recombinant expression vector containing the CMIET gene can be constructed using an existing plant expression vector. The plant expression vector includes, but is not limited to, a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment. The plant expression vector may also include a 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as, but not limited to, Agrobacterium crown gall induction (Ti) plasmid genes (such as carmine synthase Nos gene), plant genes (such as soybean storage protein genes) 3' end transcription non-translated regions have similar functions.

[0060] When using CMIET gene to construct recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter (ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct plant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0061] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0062] By using any vector that can guide the expression of foreign genes in plants, the CMIET gene or gene fragment provided by the present invention is introduced into plant cells or recipient plants, and transgenic cell lines and transgenic plants with altered manganese stress resistance can be obtained. The expression vector carrying the CMIET gene can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, and the transformed plant tissues can be cultivated into plants.

[0063] The present invention also provides a method for improving the manganese stress resistance of an organism, the method comprising step M, wherein step M is to enhance, increase or up-regulate the activity and / or content of the protein mentioned above in the target organism, or / and, enhance, increase or up-regulate the expression level of the gene encoding the protein mentioned above, so as to improve the manganese stress resistance of the organism.

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

[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 technology to reduce or inactivate the activity of 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 manganese stress resistance, 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, to obtain plants with reduced manganese stress resistance.

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

[0068] (1) constructing a recombinant expression vector for inhibiting, reducing or silencing the gene encoding the protein described above;

[0069] (2) Transforming the recombinant expression vector constructed in step (1) into a recipient plant to obtain a plant having lower manganese stress resistance than the recipient plant.

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

[0071] In the present invention, the plant may be as follows:

[0072] N1) Chlorophyceae;

[0073] N2) Volvoxales;

[0074] N3) Chlamydomonas;

[0075] N4) Chlamydomonas;

[0076] N5) Chlamydomonas reinhardtii.

[0077] The Chlamydomonas reinhardtii may specifically be Chlamydomonas reinhardtii 21 gr.

[0078] Herein, the concentration of manganese stress is: 250 μM.

[0079] In this study, a new manganese transporter in Chlamydomonas was identified and named CMIET (Chloroplast Manganese Ion Efflux Transporter). CMIET protein belongs to the UPF0016 family, and this family has four members in Chlamydomonas reinhardtii. Through bioinformatics analysis and comparison of homologous proteins among multiple species, the results showed that CMIET has the highest homology with CPLD63 in Chlamydomonas and CMT1 in Arabidopsis. Similar to CMT1 and CPLD63, CMIET protein is also located in the chloroplast envelope and is responsible for the transport of manganese ions, but the transport direction is exactly opposite to theirs. CMIET protein transports manganese ions in the chloroplast matrix to the cytoplasm.

[0080] Through the study of CMIET deletion mutants, the results showed that the loss of CMIET led to a significant increase in the manganese ion content in Chlamydomonas cells, an increase in cytoplasmic acidity, and a significant change in the homeostasis of intracellular ions. Under normal culture conditions and manganese-deficient culture conditions, cmiet mutants showed stronger growth performance and better photosynthesis ability than the wild type; cmiet mutants were 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 inside the chloroplast to the cytoplasm, thereby maintaining the balance of manganese ion concentration inside the chloroplast, protecting the photosynthetic organs from the toxicity of high manganese, and ensuring the normal progress of photosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] Figure 1 CMIET restored the sensitivity of yeast Δgdt1 mutant to excess manganese. A. Δgdt1, Δgdt1 transformed into pYES (empty vector) (hereinafter referred to as Δgdt1+empty vector) and Δgdt1 transformed into pYES-CMIET (hereinafter referred to as Δgdt1+CMIET) were grown on SD plates for 3 days; B. Δgdt1, Δgdt1 transformed into pYES (empty vector) (hereinafter referred to as Δgdt1+empty vector) and Δgdt1 transformed into pYES-CMIET (hereinafter referred to as Δgdt1+CMIET) were grown on SD-U (no uracil added) plates supplemented with 5 mM MnCl2 for 3 days.

[0083] Figure 2The subcellular 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 514nm, and emission fluorescence was at 527nm. Chlorophyll autofluorescence excitation fluorescence was at 488nm, and emission fluorescence was at 650nm. The scale bar is 5μm; B. eGFP and CMIET-eGFP were expressed in Arabidopsis protoplasts, and fluorescence was observed using a laser scanning confocal microscope. eGFP excitation fluorescence was at 488nm, and emission fluorescence was at 505-550nm. Chlorophyll autofluorescence excitation fluorescence was at 488nm, and emission fluorescence was at 650nm. The scale bar is 5μm. The white arrow in the figure indicates the protein localization area.

[0084] Figure 3 The effect of lack of CMIET on the growth of Chlamydomonas. A. Schematic diagram of CMIET gene, including 7 exons (black squares), 6 introns (black line segments between two exons) and UTR region (grey squares). The foreign gene was inserted into the third intron, and P1-P3 marked the position of primers used for genotype identification; B. Genotype identification of cmiet mutants; C. Growth of Chlamydomonas strains under different culture conditions; D. Analysis of chlorophyll concentration of Chlamydomonas strains under different culture conditions. Mixotrophic medium, low light: 25μM Mn 2+ , 50 μmol photons m -2 s -1 ; Mixotrophic medium, high light: 25μM Mn 2+ , 1200 μmol photons m -2 s -1 ; Mixotrophic medium, Mn deficiency, low light: 0 μM Mn 2+ , 50 μmol photons m -2 s -1 ; Mixotrophic medium, Mn deficiency, high light: 0μM Mn 2+ , 1200 μmol photons m -2 s -1 ; Mixotrophic medium, 10x manganese, low light: 250μMMn 2+ , 50 μmol photons m -2 s -1 Asterisks indicate statistically significant differences (t-test), **P<0.01; ***P<0.001.

[0085] Figure 4 Chlorophyll fluorescence was measured by A.21gr (control strain), cmiet and cmiet complement 1 in mixotrophic medium, low light (25 μM Mn 2+, 50 μmol photons m -2 s -1 ) conditions; B.21gr, cmiet and cmiet complementation 1 in mixotrophic medium, high light (25 μM Mn 2+ , 1200 μmol photons m -2 s -1 ) conditions; C.21gr, cmiet and cmiet complementation 1 in mixotrophic medium, manganese deficiency, low light (0 μM Mn 2+ , 50 μmol photons m -2 s -1 ) conditions; D.21gr, cmiet and cmiet complementation 1 in mixotrophic medium, manganese deficiency, high light (0 μM Mn 2+ , 1200 μmol photons m -2 s -1 ) conditions. The maximum photochemical efficiency of PSII, the actual photochemical quantum efficiency of PSII, the electron transfer rate and the non-photochemical quenching coefficient were determined. E.21gr, cmiet and cmiet complement 1 were grown in mixotrophic medium, 10 times manganese, low light (250 μM Mn 2+ , 50 μmol photons m - 2 s -1 ) The maximum photochemical efficiency of PSII, the actual photochemical quantum efficiency of PSII, the electron transfer rate and the non-photochemical quenching coefficient were determined under the conditions of . Asterisks indicate statistically significant differences (t-test), *P<0.05; ***P<0.001.

[0086] Figure 5 Figure 2 shows the transmission electron microscopy analysis of 21gr (control strain) and cmiet under different treatment conditions. 2+ , 50 μmol photons m -2 s -1 ) conditions; B. Select 10 different cells under the same treatment conditions (mixotrophic medium, low light) and count the number of thylakoid membrane layers; C. Cells were treated under mixotrophic medium, high light (25μM Mn 2+ , 1200 μmol photons m -2 s-1 ) conditions; D. Select 10 different cells under the same treatment conditions (mixotrophic medium, high light) and count the number of thylakoid membrane layers; E. Cells were treated under mixotrophic medium, manganese deficiency, low light (0 μM Mn 2+ , 50 μmolphotons m -2 s -1 ) conditions; F. Select 10 different cells under the same treatment conditions (mixotrophic medium, manganese deficiency, low light) and count the number of thylakoid membrane layers; G. Cells under mixotrophic medium, manganese deficiency, high light (0μM Mn 2+ , 1200 μmol photonsm -2 s -1 ) conditions; H. Select 10 different cells under the same treatment conditions (mixotrophic medium, manganese deficiency, high light) and count the number of thylakoid membrane layers; I. Cells were treated under mixotrophic medium, 10 times manganese, low light (250μM Mn 2+ , 50 μmol photons m -2 s -1 ) conditions; J. 10 different cells under the same treatment conditions (mixotrophic medium, 10 times manganese, low light) were selected to count the number of thylakoid membrane layers. Asterisks indicate statistically significant differences (t-test), *P<0.05; ***P<0.001.

[0087] Figure 6 Figure 2 shows the accumulation of thylakoid membrane complexes in different algae strains under different treatment conditions. 2+ , 50 μmol photons m -2 s -1 ) were used to extract thylakoid membranes from cells treated with detergent and separated by blue-green mild gel electrophoresis after volume expansion; B. Mixotrophic medium, high light (25 μM Mn 2+ , 1200 μmolphotons m -2 s -1 ) were used to extract thylakoid membranes from cells treated with detergent and separated by blue-green mild gel electrophoresis after volume expansion; C. Mixotrophic medium, manganese deficiency, low light (0 μM Mn 2+ , 50 μmol photons m -2 s -1 ) were used to extract thylakoid membranes from cells treated with detergent and separated by mild blue-green gel electrophoresis after volume expansion; D. Mixotrophic medium, manganese deficiency, high light (0 μM Mn 2+ , 1200 μmol photons m -2 s -1) were used to extract thylakoid membranes from cells treated with detergent and separated by blue-green mild gel electrophoresis after volume expansion; E. mixotrophic medium, 10 times manganese, low light (250 μM Mn 2+ , 50 μmol photons m -2 s -1 ) were used to extract thylakoid membranes from cells treated with detergent and separated by blue-green mild gel electrophoresis after detergent expansion. PSII SC: PSII supercomplex; PSI: photosystem I; PSIID: PSII dimer; PSII M: PSII monomer; LHC: light-harvesting antenna complex.

[0088] Figure 7 For immunoblotting analysis of thylakoid membrane subunit proteins. 2+ , 50 μmol photons m -2 s -1 ) conditions; B. Mixotrophic medium, high light (25μM Mn 2+ , 1200 μmol photons m -2 s -1 ) conditions; C. Mixotrophic medium, manganese deficiency, low light (0 μM Mn 2+ , 50 μmol photons m -2 s -1 ) under the conditions of immunoblotting analysis of photosystem complex subunits; D. Mixotrophic medium, manganese deficiency, high light (0μM Mn 2+ , 1200 μmol photons m -2 s -1 ) conditions. E. Mixotrophic medium, 10x manganese, low light (250 μM Mn 2+ , 50 μmol photons m -2 s -1 ) conditions.

[0089] Figure 8 The oxygen evolution and proton motive force were determined by 21gr (control strain), cmiet and cmiet complementary 1. A. Mixotrophic medium, low light (25μM Mn 2+ , 50 μmol photons m -2 s -1 ) under different algal strains; B. Mixotrophic medium, manganese deficiency, low light (0 μM Mn 2+ , 50 μmol photons m -2 s-1 ) under different algal strains; C. Evaluation of the induction of proton motive force by electrochromic shift (ECS) signal; D. Proton motive force of different algal strains; E. pH gradient of different algal strains; F. Potential gradient of different algal strains. Asterisks indicate statistically significant differences (t-test), ***P<0.001.

[0090] Fig. 9 Acid pH dye Lysosensor DND-189 can detect the acidic pH region in cells of different algae strains. 2+ , 50 μmol photons m -2 s -1 ) conditions; B. The acid pH dye Lysosensor DND-189 can detect the acidic pH region in cells of different algae strains. 2+ , 50 μmol photons m -2 s -1 ) conditions; C. Cells were grown in a mixotrophic medium, low light (25 μM Mn 2+ , 50 μmol photons m -2 s -1 ) were grown and tested under the same conditions. Positive values ​​indicate H + flow out of the cell, negative values ​​indicate H + flow into the cells; D. cells in a mixotrophic medium, manganese deficiency, and low light (0 μM Mn 2+ , 50 μmol photons m -2 s -1 ) were grown and tested under the same conditions. Positive values ​​indicate H + flow out of the cell, negative values ​​indicate H + Flow into cells; E. Total elemental concentration of manganese in cells. Elemental concentrations were quantified by inductively coupled plasma-optical emission spectrometry and were based on dry weight; F. Total elemental concentration of calcium in cells. Elemental concentrations were quantified by inductively coupled plasma-optical emission spectrometry and were based on dry weight. Asterisks indicate statistically significant differences (t-test), ***P<0.001.

[0091] Fig.10This is the analysis of CPLD63 and CGLD1 gene expression. A. Expression of CPLD63 in 21gr (control algae strain) under different conditions; B. Expression of CPLD63 in cmiet under different conditions; C. Expression of CPLD63 in 21gr and cmiet under normal conditions; D. Expression of CPLD63 in 21gr and cmiet under manganese deficiency; E. Expression of CGLD1 in 21gr under different conditions; F. Expression of CGLD1 in cmiet under different conditions; G. Expression of CGLD1 in 21gr and cmiet under normal conditions; H. Expression of CGLD1 in 21gr and cmiet under manganese deficiency. Normal: 25μM Mn 2+ ,50μmol photonsm -2 s -1 , manganese deficiency: 0μM Mn 2+ ,50μmol photons m -2 s -1 Asterisks indicate statistically significant differences (t-test), *P<0.05; **P<0.01; ***P<0.001. DETAILED DESCRIPTION

[0092] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0093] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0094] The quantitative experiments in the following examples were performed in triplicate unless otherwise specified.

[0095] The vector pYES2 in the following examples was kindly donated by Professor Zhang Zhaoliang's research group at Anhui Agricultural University and has been recorded in: Cloning and Organelle Expression of Bamboo Mitochondrial Complex I Subunits Nad1, Nad2, Nad4, and Nad5 in the Yeast Saccharomyces cerevisiae. The public can obtain the biological material from the applicant. The biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0096] The Δgdt1 yeast mutant in the following examples was kindly provided by Professor Zhang Zhaoliang's research group at Anhui Agricultural University and has been described in: The Yeast Protein Gdt1p Transports Mn 2+ The public can obtain the biological material from the applicant, and the biological material is only used for repeating the experiments of the present invention and cannot be used for other purposes.

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

[0098] The pAN580 vectors in the following examples were purchased from Biorun Biotechnology, the website of which is: http: / / www.biorun.com / bioHosts / hosts.html.

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

[0100] The following examples were processed using SPSS11.5 statistical software, and the experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used, and P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, and P < 0.001 (***) indicated an extremely significant difference.

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

[0102] 1. Obtaining CMIET protein from Chlamydomonas reinhardtii

[0103] DNA from 21gr cells of Chlamydomonas reinhardtii was extracted and used as a template. PCR amplification was performed using primers CMIET gene F: 5'-ATGTTGTCGTCAGCTTGTCT-3' and CMIET gene R: 5'-TCACAGCAGGCTGAAGGCGG-3' and KOD FXDNA 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 encoded amino acid sequence is the CMIET protein of SEQ ID No. 2. In the genomic DNA of Chlamydomonas reinhardtii 21gr, the genomic gene encoding the CMIET protein is shown in SEQ ID No. 3 of the sequence table. The 1st to 22nd positions of SEQ ID No. 3 are the first exon, the 100th to 274th positions are the second exon, the 512th to 605th positions are the third exon, the 840th to 1010th positions are the fourth exon, the 1364th to 1492th positions are the fourth exon, the 1758th to 2014th positions are the fifth exon, and the 2257th to 2464th positions are the sixth exon.

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

[0106] The Δgdt1 yeast mutant cannot normally transfer manganese to the Golgi apparatus, so the mutant cannot grow normally in high manganese medium.

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

[0108] The specific construction steps are as follows:

[0109] 1) Construction of recombinant vector: Use pEASY-Uni Seamless Cloning and Assembly Kit (All-in-one Gold) to connect the coding sequence fragment of CMIET with the pYES2 vector after enzyme digestion. 2×Assembly Mix 5.0μL, vector 1.5μL, target fragment 3.5μL, total system 10μL. After gentle mixing, incubate at 50℃ for 15min to finally obtain the recombinant vector pYES2-CMIET.

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

[0111] Take 20mL yeast solution (OD 5460.6-1.0), centrifuge at 700×g for 5 minutes at room temperature, discard the supernatant; resuspend the precipitate with 1 mL of sterile ddH2O for later use; take 1 μL of the recombinant vector pYES2-CMIET to be transformed and add 300 μL of transformation premix (270 μL 50% PEG4000, 5 μL 5M lithium acetate, 25 μL 2 mg·mL -1 Salmon sperm genomic DNA), vortex to mix; add 100 μL of resuspended yeast solution to the premix, mix gently and place in a 42°C water bath for 45 minutes, and flick to mix several times during the process; centrifuge at 700×g for 5 minutes at room temperature, discard the supernatant, add 100 μL of sterile saline (0.9% NaCl) to resuspend the precipitate; evenly spread the resuspended bacterial solution on the corresponding SD screening plate, invert and culture at 30°C for 2-4 days to obtain a recombinant yeast strain containing the recombinant vector pYES2-CMIET, referred to as strain Δgdt1+CMIET. At the same time, the empty vector pYES2 was introduced into the Δgdt1 yeast mutant strain using the above method to obtain the recombinant yeast strain Δgdt1+empty as a control.

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

[0113] The experimental results showed that the strains Δgdt1, Δgdt1+empty vector and Δgdt1+CMIET could grow normally on SD medium ( Figure 1 The yeast mutant strain transformed with the empty vector Δgdt1 (Δgdt1+empty vector) and the original mutant strain Δgdt1 were unable to grow normally on high manganese medium ( Figure 1 In contrast, the Δgdt1 yeast mutant (Δgdt1+CMIET) transformed with the CMIET expression vector pYES2-CMIET was not affected by high manganese and could grow normally. This result indicates that manganese is a transport substrate of the CMIET protein and can be transferred into the yeast mutant by CMIET to restore the high manganese-sensitive phenotype of the mutant strain.

[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 Middle A); The recombinant vector PAN580-CMIET-eGFP was transformed into Arabidopsis protoplasts ( Figure 2 B) The specific steps are as follows:

[0116] Specific construction method of recombinant vector:

[0117] The coding sequence fragment of CMIET was connected to the pLM005 vector after restriction digestion using pEASY-Uni Seamless Cloning and Assembly Kit (All-in-One). 2×Assembly Mix 5.0μL, vector 1.5μL, target fragment 3.5μL, total system 10μL. After gentle mixing, incubate at 50℃ for 15min, and finally obtain the recombinant vector pLM005-CMIET-Venus.

[0118] The coding sequence fragment of CMIET was connected to the PAN580 vector after restriction digestion using pEASY-Uni Seamless Cloning and Assembly Kit (All-in-One). 2× Assembly Mix 5.0 μL, vector 1.5 μL, target fragment 3.5 μL, total system 10 μL. After gentle mixing, incubate at 50°C for 15 minutes to finally obtain the recombinant vector PAN580-CMIET-eGFP.

[0119] Chlamydomonas reinhardtii electroporation method:

[0120] 1. Take 100 mL of logarithmic growth phase (about 2×10 6 cells·mL -1 ) was transferred to a sterilized 50 mL centrifuge tube, placed on ice for 10 min, and centrifuged at 5000 rpm (3000 × g) at 4 °C for 5 min;

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

[0122] Solution A: 40.0 g NH4Cl, 5.0 g CaCl2·2H2O, 10.0 g MgSO4·7H2O, add ddH2O to make up to 1 L. When preparing, the reagents must be dissolved in order to avoid precipitation.

[0123] Phosphate Buffer II: K2HPO4 108.0g, KH2PO4 56.0g, add ddH2O to make up 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 4H2O 1.1g, add ddH2O to make up to 1L.

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

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

[0127] 2) Heat EDTA-Na2 and dissolve it in 250 mL ddH2O, then add it to the solution, continue stirring and heating until the solution boils.

[0128] 3) Stop heating, allow the solution to cool naturally to 70-75°C, then continue heating and keep the solution at this temperature.

[0129] 4) Use KOH solution to adjust the pH value of the solution to 6.5-6.8. Be careful not to let the solution temperature fall below 70℃ or the pH value rise above 6.8. Add ddH2O to make the volume to 1L.

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

[0131] 6) Filter the precipitate with filter paper and store at 4°C away from light.

[0132] The final concentration of sucrose was 60 mM, and the sucrose solution was sterilized by filtration using a 0.22 μm filter) to fully resuspend the cell pellet and place it on ice for 15 min;

[0133] 3. In a clean bench, add 5 μL of 10 mg mL -1 1000ng of boiled salmon sperm DNA, recombinant vector plasmid (500-1000ng), 250μL of resuspended algae solution, mix well and cover the electroporation cup;

[0134] 4. Place the electric shock cup in a 16°C water bath for 5 minutes;

[0135] 5. Use filter paper to wipe the moisture on the outside of the shock cup, place the shock cup in the shock device of the electroporator (BTX), clamp the shock cup with the metal sheets on both sides, put down the safety cover, set the shock parameters (voltage 800V, resistance 1575Ωresistance, capacitance 50μF capacitance), press the Pulse key to perform the shock, 10-14ms is the normal time range, and immediately transfer the shock cup to ice for 10 minutes after the shock;

[0136] 6. In a clean bench, add 40 mL of TAP sucrose solution to a sterilized 50 mL centrifuge tube, transfer the electroporated Chlamydomonas cells in the electroporation cup to the centrifuge tube and mix with the TAP sucrose solution. Place the centrifuge tube on a shaker with slow and weak light (25°C, 50 rpm, 10 μmol photons·m -2 ·s -1 )Resuscitation for 24 hours;

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

[0138] 8. Pour off the supernatant in the clean bench, add 1 mL of TAP liquid medium to resuspend the precipitated cells, and apply the corresponding resistance TAP plate. After the water is dried, place the plate at 25°C and 50 μmol photons·m -2 ·s -1 The cells were cultured under the appropriate conditions and single clones were grown for identification and 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, 0.4Mmannitol, 20mM KCl, 20mM MES pH 5.7, 55℃ water bath for 10min, add the following reagents after cooling to room temperature, 10mM CaCl2, 0.1% BSA, 5mM β-Mercaptoethanol. Note: The hydrolysate should be prepared and used immediately, and impurities should be filtered out with a 0.45μm membrane filter; before adding R-10 powder, preheat the MES solution in a 70℃ water bath for 3-5min.

[0142] 2. PEG4000 Solution: 40% PEG4000, 0.2 Mannitol, 100 mM CaCl2. Note: Prepare the solution at least 1 hour in advance before use.

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

[0144] 4.MMG Solution: 4mM MES, 0.4M mannitol, 15mM MgCl2, pH adjusted to 5.7 with KOH, sterilized at high temperature and high pressure, stored at room temperature.

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

[0146] Protoplast preparation and transformation:

[0147] 1. Select the 5th, 6th, and 7th rosette leaves of Arabidopsis thaliana that have not bloomed for 3-4 weeks under short-day growth, remove the leaf tips and petioles. Take about 1 / 3 of the middle of the leaf and cut it into 0.5-1mm wide strips with a sharp blade. Transfer it to the enzymatic solution with flat-tip tweezers and use tweezers to help the leaves be completely immersed in the enzymatic solution;

[0148] 2. Vacuum for 10-30 minutes until most of the leaves are submerged in the enzymatic solution. Perform enzymatic hydrolysis in the dark at room temperature for at least 3 hours. When the enzymatic solution turns green, gently shake the culture dish to release the protoplasts.

[0149] 3. Precool 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 100×g for 3 min at 4°C (preferably using a round-bottom centrifuge tube, with a maximum of no more than 200×g), remove the supernatant as much as possible, gently resuspend the protoplasts with 10 mL of ice-cold W5 solution, and incubate vertically on ice for 30 min;

[0152] 6. Centrifuge at 100×g for 3 min at room temperature to remove W5 as much as possible, and resuspend the protoplasts with an appropriate amount of MMG solution (about 1 mL) to a final concentration of about 2×10 5 cells·mL -1 ;

[0153] 7. For each reaction, add 10 μL of purified PAN580-CMIET-eGFP recombinant vector plasmid (about 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 the transformation mixture at room temperature for 5-15 minutes;

[0155] 9. Add 2 times the volume of W5 solution to dilute the conversion mixture and gently invert to mix to terminate the reaction;

[0156] 10. Centrifuge at 100×g for 3 min at room temperature and discard the supernatant;

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

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

[0159] The structure of the recombinant vector pLM005-CMIET-Venus is described as follows: A recombinant expression vector is obtained by replacing the small fragment between the Hpa I restriction recognition sites 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: a recombinant expression vector is obtained by replacing the small fragment between the Sma I and Sma I restriction enzyme recognition sites of 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 in the figure, CMIET protein is concentrated in the membrane near the T-zone of the cup-shaped chloroplast of Chlamydomonas. At the same time, the PAN580-CMIET-eGFP recombinant vector was transferred into Arabidopsis protoplasts, and the fluorescence signal results also showed that CMIET protein is localized in the chloroplast membrane.

[0162] Example 2: Effect of CMIET deficiency on the phenotype of Chlamydomonas mutants

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

[0164] The authenticity of the mutant was verified by PCR. Figure 3 Middle B), primer P1: 5'-ACATACGCACCAATCATGTCAAGC-3', primer P2: 5'-GCGGGTGGGGTAGTTGATACAAT-3', primer P3: 5'-GGGGAAAGGCTCGAGTGGGA-3'.

[0165] The high-fidelity PCR enzyme KOD FX (TOYOBO) was used to complete the PCR amplification of the gene fragment. 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°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 60°C annealing for 30 s (annealing temperature varies according to primer Tm value), 68°C extension for 1.5 min (extension time is set according to target fragment length 1 min / kb), 25-30 cycles (denaturation-annealing-extension is one cycle), 68°C extension for another 5 min

[0168] The results showed that exogenous gene fragments were detected in the cmiet mutant, proving that an exogenous gene (nucleotide sequence is SEQ ID No. 4 in the sequence list) was indeed inserted into the third intron of the cmiet mutant, making it impossible to obtain the correct CMIET gene fragment.

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

[0170] The growth of cmiet mutant, wild type 21gr and complementary algae strain cmiet complementation 1 were analyzed. Each strain was replicated three times and the culture conditions were as follows:

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

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

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

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

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

[0176] The chlorophyll fluorescence determination method of Chlamydomonas liquid culture samples is as follows: Take the algae strain to be tested to determine the chlorophyll concentration, and adjust the chlorophyll concentration to 25 μg mL -1 After 15-20 minutes of dark adaptation, the required chlorophyll fluorescence parameters were measured using the Imaging-PAM (Walz) instrument of the Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences.

[0177] The results show that Figure 3 C and D), in normal manganese (25 μM Mn 2+ ) content or manganese deficiency (0μM Mn 2+ ) culture, the cmiet mutant outperformed the wild type and the complemented strain, and its chlorophyll concentration was significantly higher than that of the wild type and the complemented strain; in contrast, the mutant grew better in high manganese (250 μM Mn 2+ ) The growth of the cmiet mutant in the culture medium was severely inhibited, the algae turned yellow and white, and the chlorophyll concentration was significantly reduced. Compared with the wild type and complementary algae, the cmiet mutant was more susceptible to manganese toxicity. The above results show that the loss of CMIET has an important effect on the growth of mutant algae.

[0178] 2. Effect of CMIET deficiency on photosynthetic capacity of Chlamydomonas mutants

[0179] Imaging-pam was used to measure the light response curves of the algae strains under different culture conditions in step 1. 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 culture conditions in mixotrophic medium ( Figure 4 (A), there is no significant difference in photosynthetic capacity between the cmiet mutant, wild type 21gr and complemented strain cmiet complementation 1. Figure 4 (B), the mutant and the wild type differ only in the non-photochemical quenching coefficient. The non-photochemical quenching coefficient of the cmiet mutant is higher than that of the wild type and the complementary algae strain. Figure 4 Middle C) or Highlight ( Figure 4 Under the conditions of (D), the photosynthetic capacity of cmiet mutants was significantly higher than that of wild and complementary algae strains. Especially after high light treatment, the photosynthetic parameters of cmiet mutants could maintain a high level under adverse conditions. cmiet mutants are extremely sensitive to high manganese toxicity. Figure 4 Middle (E), the mutant completely lost the ability to photosynthesize.

[0181] The above results prove that CMIET protein has a significant effect on the growth, development and photosynthetic capacity of Chlamydomonas, especially plays a vital 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 method of cmiet mutant, wild type 21gr and complementary algae strain cmiet complementation 1 is the same as step 1 in this example.

[0184] The 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 There is no significant difference in the morphological structure between mutant and wild-type cells. The number of thylakoid membrane layers was counted ( Figure 5 B), the results showed that there was no significant difference between the mutant and the wild type. Figure 5 C) and manganese-deficient culture, low light ( Figure 5 Middle E) or after highlight processing ( Figure 5 In the middle G), the chloroplast thylakoid membrane structure of the wild type becomes sparse, the interval between the lamellae increases, and the length becomes shorter. However, the thylakoid membrane of the cmiet mutant can maintain a more complete morphology. By counting the number of thylakoid membrane layers ( Figure 5 D, F, and H), the mutant cells have more thylakoid membrane layers than the wild type, with significant differences. However, after high manganese stress treatment, the mutant cell structure was almost completely destroyed, and the thylakoid membrane could not be observed ( Figure 5I and J). These results indicate 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 loss of CMIET will affect the photosynthetic ability of Chlamydomonas cells and the structure of the thylakoid membrane, the accumulation of the photosynthetic complex and important subunits of the cmiet mutant, wild-type 21gr and complementary algae strain cmiet complementation 1 were detected.

[0188] The Chlamydomonas reinhardtii thylakoid membrane extraction experiment must be carried out on ice and in a weak light environment. If conditions permit, the experiment can be carried out in a low-temperature cold room equipped with a green light. The specific steps are as follows:

[0189] 1) Prepare the experimental buffer according to Table 1 below, and add protease inhibitors (1 mM PMSF, 1 mM benzamidine, 1 mM aminocaproic acid) to each buffer before the experiment;

[0190] Table 1. Thylakoid membrane extraction buffer

[0191]

[0192]

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

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

[0195] 4) Using a high pressure homogenizer (PHD) to break up Chlamydomonas cells, 5000-10000 psi (adjust the pressure according to different algae strains), 2 min;

[0196] 5) The disrupted Chlamydomonas cells were centrifuged at 5000 rpm (3000 × g) at 4°C for 10 min, and the supernatant was transferred to a new high-speed centrifuge tube (BECKMAN COULTER);

[0197] 6) Centrifuge at 40,000 × g at 4°C 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 liquid to a glass homogenizer for grinding and homogenization to remove large agglomerates;

[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 components in the homogenizer and then add them to the high-speed centrifuge tube, centrifuge at 40,000 × g and 4°C 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 transfer the resuspended liquid to the glass homogenizer for grinding and homogenization;

[0201] 10) The homogenate was transferred to an ultracentrifuge tube (BECKMAN COULTER), 2 mL of HEPES 3 buffer was added to the glass homogenizer to rinse the Chlamydomonas component in the homogenizer and then added to the ultracentrifuge tube;

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

[0204] 13) After the centrifuge tube is finely balanced, use an ultracentrifuge (BECKMAN COULTER) to centrifuge at 220,000 × g at 4°C for 60 min, with both the acceleration and deceleration set to the slowest;

[0205] 14) Carefully draw the green component between HEPES 3 and HEPES 4 buffer into a new high-speed centrifuge tube with a syringe, add 10-15 mL HEPES 6 buffer, mix well, centrifuge at 40,000 × g at 4°C for 20 min, and discard the supernatant;

[0206] 15) Add 100 μL HEPES 6 buffer to fully resuspend the precipitate to obtain the thylakoid membrane product, which was quickly frozen in liquid nitrogen and stored at -80°C.

[0207] The above thylakoid membrane products were then verified by BN-PAGE gel electrophoresis and SDS-PAGE gel electrophoresis, and protein immunoblotting.

[0208] The experimental results show that under the low light culture conditions of mixotrophic medium ( Figure 6 Middle A, Figure 7 In A), the loss of CMIET did not cause changes in the accumulation of photosynthetic complexes and important subunits. Under high light culture conditions in mixotrophic medium ( Figure 6 Middle B, Figure 7(B), the accumulation of PSII dimers, PsbO, Cyt f and LHCSR3 proteins increased in the cmiet mutant. Figure 6 Middle C, Figure 7 C), the accumulation of cmiet photosynthetic complex and important subunit proteins increased to varying degrees. Figure 6 Middle D, Figure 7 Middle D) The accumulation of cmiet photosynthetic complex and important subunit proteins also increased, especially PsbO protein. After high manganese stress treatment, cmiet had almost no accumulation of photosynthetic complex, and the proteins were almost all degraded, further indicating that the lack of CMIET caused Chlamydomonas cells to be unable to cope with high manganese toxicity stress ( Figure 6 Middle E, Figure 7 Middle E).

[0209] Through immunoblotting, it was found that the loss of CMIET had a significant effect on the accumulation changes of the main subunits of the oxygen-evolving complex. The changes in the oxygen-evolving complex will directly reflect the photosynthetic oxygen release of Chlamydomonas. Therefore, the oxygen release rate of the corresponding algal strains was measured.

[0210] The steps for determining the oxygen evolution rate are as follows:

[0211] 1) Determine the chlorophyll concentration of the algae strain to be tested, and adjust the chlorophyll concentration of the algae strain to be tested to be consistent;

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

[0213] 3) Take 1.5mL of the algae solution to be tested and add it to the sample channel. Add 50μL of 0.5M NaHCO3, 25℃, 100μmolphotons·m -2 ·s -1 , the oxygen evolution rate was measured at 100 rpm, each sample was measured for 5 min, and the technology was repeated 3 times;

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

[0215] The results show that ( Figure 8 (A and B) Under different treatment conditions, the photosynthetic oxygen evolution rate of the cmiet mutant was significantly better than that of the wild type and the complementary algae strain, which was consistent with the results of immunoblotting.

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

[0217] 5. Loss of CMIET affects cellular ion homeostasis

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

[0219] 1) Take 20 mL and grow to the logarithmic growth phase (about 2×10 6 cells·mL -1 ) strains, centrifuge at 5000×g for 5 min at room temperature, 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 for 5 min at room temperature, discard the supernatant, and repeat the washing 2-3 times;

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

[0222] 4) Lysosensor DND-189 fluorescent dye (1 mM, Shanghai Yisheng) was diluted to a concentration of 2 μM 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 concentration of the dye 1 μM, and incubate at room temperature with slow shaking for 30-120 minutes;

[0224] 6) The cells were observed and photographed using an ultra-high resolution laser confocal microscope 3D-SIM (Nikon).

[0225] B. Non-invasive micro-measurement of H inside and outside cells + The flow determination steps are as follows:

[0226] The samples were sent to the public instrument platform of the Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences, to perform non-destructive measurement of the ion flow inside and outside Chlamydomonas cells using the Chloroplast Material Permeability Micro-Measurement System (Younger Company).

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

[0228] Microwave digestion of samples:

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

[0230] 2) Dry the sample in an oven at 65°C and grind the dried sample into powder using a mortar;

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

[0232] 4) Add 2 mL of hydrogen peroxide solution (super pure), cover the tube, shake well, and let stand in a fume hood at room temperature for 15 minutes;

[0233] 5) Tighten the tube cap and place it in a microwave digester for digestion. The microwave digestion program is shown in Table 2 below.

[0234] Table 2. Microwave digestion procedure

[0235] step Heating time Target temperature Keep Time 1 5min 100℃ 2min 2 5min 150℃ 10min 3 5min 180℃ 30min

[0236] 6) After digestion is completed and cooled, open the lid of the microwave digestion tube and transfer all the solution in the digestion tube to a 50mL plastic volumetric flask. Rinse the wall of the digestion tube several times with a small amount of ddH2O and transfer it to the volumetric flask. Make up to volume with ddH2O, shake well and filter with quantitative filter paper. The filtered solution can be used for determination.

[0237] D. Determination of metal element content:

[0238] 1) The samples were digested by microwave and the metal element content was determined by inductively coupled plasma emission spectrometry (Thermo);

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

[0240] The experimental results show that ( Figure 8 C), the proton motive force of cmiet mutant ( Figure 8 D), pH gradient ( Figure 8 E) were significantly higher than those of the wild type and complementary algae strains, and the potential gradient ( Figure 8 F) was significantly reduced. This indicates that the loss of CMIET leads to an increase in the proton concentration on the lumen side of the thylakoid membrane of the mutant Chlamydomonas cells, which increases the activity of ATP synthase and increases the energy supply to cope with stress such as high light and nutrient deficiency.

[0241] The low pH dye Lysosensor DND-189 can stain the acidic part of the cells. The results of 3D-SIM microscopy showed that there was almost no accumulation of acidic regions in the wild-type cells and the cells of the complementary algae strain grown in a medium with normal manganese content, but there were a large number of acidic regions in the cytoplasm of the cmiet mutant ( Fig. 9 In the case of manganese deficiency, the opposite is true ( Fig. 9 (middle B).

[0242] The non-invasive micro-measurement technique (NMT) was used to measure the intracellular and extracellular H + The results of flow cytometry showed that under normal culture conditions, the extracellular H + Flow into cells, H in wild-type and complementary algal strains + Flow out of the cell ( Fig. 9 In the case of manganese deficiency, the opposite is true ( Fig. 9 The above results show that the lack of CMIET cannot transport Mn normally. 2+ This will cause H + The concentration changed significantly.

[0243] Inductively coupled plasma-optical emission spectroscopy was used to measure the intracellular manganese content. The results showed that the manganese accumulated in the cmiet mutant was significantly higher than that in the wild type and the complementary algae strain ( Fig. 9 E). The results of the test ( Fig. 9 Middle (F) shows 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 envelope, responsible for transporting excess manganese in the chloroplast to the cytoplasm.

[0245] 6. Analysis of changes in other chloroplast-localized manganese transporters in cmiet mutants

[0246] The expression levels of the genes encoding the chloroplast-localized manganese transporters CPLD63 and CGLD1 in the wild-type 21gr and cmiet mutants were detected. The specific steps are as follows:

[0247] Prepare fluorescence quantitative 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, and fluorescence quantitative PCR reaction was performed using LightCycler480 (Roche) PCR instrument. PCR amplification program: 95°C pre-denaturation for 2 min, 95°C denaturation for 5 s, 60°C annealing for 30 s, 72°C extension for 30 s, 40 cycles. The experimental results were analyzed using LightCycler480 analysis software.

[0248] The results are as follows Fig.10As shown in Figures AH: Whether in the wild type or the mutant, manganese deficiency conditions will cause a significant increase in the expression of CPLD63 and CGLD1 genes, indicating that under manganese deficiency conditions, Chlamydomonas cells need to increase the expression of CPLD63 and CGLD1 proteins to transport more manganese ions from the environment to the chloroplasts and thylakoid membranes to maintain cellular photosynthesis; due to the lack of CMIET, the manganese in the chloroplasts of cmiet mutant cells cannot be quickly transported out, so cmiet does not need to significantly increase the expression of CPLD63 and CGLD1 proteins compared to the wild type to enhance the transport of manganese ions to the chloroplasts and thylakoid membranes.

[0249] The volume of chloroplasts in Chlamydomonas cells accounts for more than half of the total volume. Most of the excess manganese absorbed from the outside 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 confirmed that Chlamydomonas can excrete excess manganese in chloroplasts into the cytoplasm through CMIET protein, thereby ensuring that the chloroplasts are not affected by manganese toxicity and maintaining the normal photosynthesis ability of the cells.

[0250] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the expression of a gene or a substance that regulates the activity or content of the protein in any of the following: 1) Use of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in regulating manganese resistance of organisms; 2) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the preparation of a product regulating biological manganese stress resistance; 3) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in cultivating an organism with altered manganese stress resistance; 4) Use of a protein or a substance regulating the expression of a gene or a substance regulating the activity or content of the protein in the preparation of a product for cultivating an organism with altered manganese stress resistance; 5) Application of proteins or substances regulating gene expression or substances regulating the activity or content of the proteins in biological breeding; The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No. 2; a2) a protein having the same function as the amino acid sequence shown in SEQ ID No. 2 after one or more amino acid residues are replaced and / or deleted and / or added; a3) a protein having an amino acid sequence identity of more than 80% with any one of a1) to a2) and having the same function; a4) A fusion protein obtained by connecting a tag to the end of the protein defined in any one of a1) to a3).

2. The use according to claim 1, characterized in that: The protein is derived from Chlamydomonas.

3. The use according to claim 1 or 2, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application of claim 1 or 2, and the biological material is any one of the following: c1) a nucleic acid molecule encoding the protein; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); 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); c6) transgenic plant tissue containing the nucleic acid molecule described in c1), or transgenic plant tissue containing the expression cassette described in c2); 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); e1) a nucleic acid molecule that inhibits, reduces or silences the expression of the protein encoding 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) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); 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); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); 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).

4. The use according to claim 3, characterized in that: c1) The nucleic acid molecule is a DNA molecule as shown in any of the following: d1) the nucleotide sequence is the DNA molecule shown in SEQ ID No.1; d2) the coding region sequence is the DNA molecule shown in SEQ ID No. 1 in the sequence listing; d3) a DNA molecule having 90% or more identity with the nucleotide sequence defined in d1) or d2) and encoding the protein of claim 1; d4) A DNA molecule which hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described in claim 1.

5. A method for improving biological manganese stress resistance, characterized in that: The method comprises step M, wherein step M is to enhance, improve or up-regulate the activity and / or content of the protein in the application of claim 1 or 2 in the target organism, or / and enhance, improve or up-regulate the expression level of the gene encoding the protein in the application of claim 1 or 2, so as to improve the manganese stress resistance of the organism.

6. A method for reducing biological manganese stress resistance, characterized in that: The method comprises step P, wherein step P is to inhibit, reduce or silence the activity and / or content of the protein in the application of claim 1 or 2 in the target organism, or / and, to inhibit, reduce or silence the expression level of the gene encoding the protein in the application of claim 1 or 2, so as to reduce the organism's resistance to manganese stress.

7. A method for breeding organisms with reduced resistance to manganese stress, characterized in that: The invention comprises inhibiting, reducing or silencing the expression level of the gene encoding the protein in the application described in claim 1 or 2 in the target organism, and / or the activity and / or content of the protein 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.

8. The method according to claim 7, characterized in that: The steps include: (1) constructing a recombinant expression vector for inhibiting, reducing or silencing the gene encoding the protein; (2) Transforming the recombinant expression vector constructed in step (1) into a recipient plant to obtain a plant having lower manganese stress resistance than the recipient plant.

9. The protein described in the use according to claim 1 or 2 and / or the biological material described in the use according to claim 3 or 4.

10. The use according to any one of claims 1 to 4, and / or the method according to any one of claims 5 to 8, characterized in that: The plant is any of the following: N1) Chlorophyceae; N2) Volvoxales; N3) Chlamydomonas; N4) Chlamydomonas; N5) Chlamydomonas reinhardtii.

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